NZMS

Annual Conference 2019

25-28 November, Convention Centre, Palmerston North

PROGRAMME & ABSTRACTS

www.nzmsconference.org.nz

CONTENTS

PAGE

Conference Committee 2

While at the Conference… 3

Plenary & Keynote Speakers 3

Exhibitors & Sponsors 5

Oral Programme 7

Poster Programme 11

Abstracts: Plenary & Keynote 15

Oral Presentations 24

Posters 47

CONFERENCE COMMITTEE

Conference Chair: Steve Flint (Massey University)

Organising Committee: Adrian Cookson (AgResearch) Carl Mesarich (Massey University) Caroline Kim (Plant and Food Research) Charles Lee (University of Waikato) Christina Moon (AgResearch) Eteta Trueman (Massey University) Jasna Rakonjac (Massey University) Patrick Biggs (Massey University) Rose Collis (AgResearch) Sandeep Kumar (AgResearch) Sara Burgess (Massey University) Rosemary Hancock (événements Limited) Conference Manager | evenements@xtra.co.nz

WHILE AT THE CONFERENCE…

o Registration desk contact phone number: Rosemary, ph 021 217 8298

o Please wear your name label to all conference sessions and social functions. These badges identify you as a NZMS conference participant. The venue is a public space and you may be asked to leave the catering areas if we cannot identify you!

o Please take note of the location of the exits in all conference rooms and in your accommodation.

Please vacate the building immediately should the alarms sound or when directed to do so by a safety warden.

o Please ask at registration for the free Wi-Fi password

o Please do not leave personal belongings unattended at any time. If you require temporary storage

please ask at the registration desk.

o There are no refunds for non-attendance at sessions or social functions.

PLENARY SPEAKERS

#1 Kevin Keener Iowa State University SPONSOR: Massey University (SOFAT)

#2 Debbie Williamson Microbiological Diagnostic Unit Public Health Laboratory, Australia SPONSOR: NZMS

#3 Natalia Requena Karlsruhe Institute of Technology SPONSOR: Massey University (SFS)

#4 Frank Takken University of Amsterdam SPONSOR: AgMardt

#5 Gisèle LaPointe University of Guelph SPONSOR: Riddet Institute

#6 Scott Chimileski Paul Smith's College of the Adirondacks and Harvard University SPONSOR: NZMS

#7 Philip Pope Norwegian University of Life Sciences SPONSOR: AgResearch

#8 Remco Kort VU University Amsterdam SPONSOR: Massey University (SOFAT)

#9 Jane Mullaney AgResearch SPONSOR: MBIE

#10 Max Kennedy Ministry for Business Employment and Innovation SPONSOR: MBIE

#11 Matthias Horn University of Vienna SPONSOR: MBIE

NZMS ORATOR

Jasna Rakonjac Massey University

KEYNOTE SPEAKERS #1: Barry Scott

Massey University

SPONSOR: NZMS

#2: Sinisa Vidovic University of Minnesota

SPONSOR: Plant & Food Research

#3: Mark Turner University of Queensland

SPONSOR: NZMS

#4: Eddy Smid Wageningen University

SPONSOR: Massey University

(SOFAT)

#5: Karen McIntyre Health Canada

SPONSOR: NZMS

#6: Duncan McMillan TU Delft

SPONSOR: NZMS

EXHIBITORS:

www.as1.co.nz

www.customscience.co.nz

www.dnature.co.nz

www.johnmorrisgroup.com

www.mediray.co.nz

www.ngaio.co.nz

www.thermofisher.co.nz

PRINCIPAL SPONSOR:

MAJOR SPONSORS:

GENERAL SPONSORS:

www.waikato.ac.nz

www.riddet.ac.nz

www.plantandfood.co.nz

POSTER SESSION:

http://direct.dksh.co.nz/

PROGRAMME (s) = in student oral competition

Monday 25th November

Time Session 1 Conference Room

Session 2 The Gallery

Session 3 Seminar Room

2.00 pm Student 3-minute presentation competition

4.00 Conference Opening

Steve Flint Chair NZMS conference

CHAIR Steve Flint

4.10 OPENING ADDRESS Mackenzie Nicol (MBIE)

4.20 Plenary #1 – Sponsor: SOFAT Kevin Keener

4.55 Welcome Mixer (in The Gallery)

6.00 Public Seminar – Sponsor: SOFAT

Museums and Microbes Remco Kort

~7.30 Free Evening – time to experience some of our many restaurants and bars

Tuesday 26th November (s) = in student oral competition

Time Session 1 Conference Room

Session 2 The Gallery

Session 3 Seminar Room

7.00 am Technicians’ Breakfast

8.30 Plenary #2 – Sponsor: NZMS

CHAIR: Patrick Biggs Debbie Williamson

9.15 Plenary #3 – Sponsor: SFS

CHAIR: Barry Scott Natalia Requena

10.00 Morning Tea Plant Microbe Interactions Antimicrobial Resistance Environmental Microbiology

CHAIR Daniel Berry Rose Collis Adrian Cookson 10.30 Keynote #1 – Sponsor: NZMS

609 Barry Scott Keynote #2 – Sponsor: Plant & Food Research

Sinisa Vidovic 548 Lucy Stewart

10.50 672 Simisola Oso 11.10 669 Mercedes Rocafort 606 Attika Rehman (s) 662 Paul Demchick 11.30 650 Ellie Bradley 531 Sophie van Hamelsveld (s) 585 John Mackay 11.50 635 Berit Hassing 581 Van Hung Vuong Le (s) 549 Connor Watson 12.10 Lunch

1.10 Plenary #4 – Sponsor: AgMardt

CHAIR: Christine Voisey Frank Takken

1.55 Plant Microbe Interactions – cont’d Veterinary Microbiology Environmental Microbiology – cont’d CHAIR Taryn Miller Paul Ogbuigwe Lucy Stewart 2.00 567 Linda Johnson 563 Marie Moinet (s) 634 Adrian Cookson 2.20 648 Yanan (Melissa) Guo 664 Manjula Jayasinghe 541 Debolina Sarkar 2.40 626 Yonathan Lukito 610 Manpreet Dhami 3.00 Afternoon Tea

Plant Microbe Interactions – cont’d Zoonotic Diseases Environmental Microbiology – cont’d CHAIR Berit Hassing Shahista Nisa 3.30 668 Daniel Berry 529 Paul Ogbuigwe 642 Elizabeth Buckley 3.50 604 Taryn Miller 641 Nilukshi Liyanagunawardena 687 Xue-Xian Zhang 4.10 593 Cade Fulton 594 Julia Robertson 550 Marion Schoof 4.30 573 Rudolf Schlechter (s) 625 Alexis Risson (s)

4.50 – 6.50 Poster Session #1 (Exhibition Hall) – Sponsored by DKSH 7.00 – 9.00 STUDENT FUNCTION – “Science Feud” (Conference Room)

Free Evening – time to experience some other restaurants and bars in Palmerston North

Wednesday 27th November (s) = in student oral competition

Time Session 1 Conference Room

Session 2 The Gallery

Session 3 Seminar Room

8.30 am Plenary #5 – Sponsor: Riddet Institute

CHAIR: Wayne Young Gisèle LaPointe

9.15 Plenary #6 – Sponsor: NZMS

CHAIR: Sara Burgess Scott Chimileski

10.00 Morning Tea Food Microbiology and safety Medical Microbiology Gut Microbiology

CHAIR Murali Kumar Laryssa Howe Laureen Crouzet 10.30 Keynote #3 – Sponsor: NZMS

611 Mark Turner 559 Stephen On 675 Alex Parker (s)

10.50 597 Marina Harper 646 Kerry Bentley-Hewitt 11.10 619 Aswathi Soni 686 Mike Taylor 592 Marc Bailie 11.30 535 Dong Zhang 607 NicK Heng 643 Wayne Young 11.50 537 David Flossdorf (s) 694 Michael Wyatt 682 Anika Hoogeveen 12.10 Lunch 12.30 NZMS AGM

1.30 Plenary #7 – Sponsor: AgResearch

CHAIR: Graeme Attwood Philip Pope

2.15 Food Microbiology and Safety – cont’d Virology Gut Microbiology – cont’d CHAIR Aswathi Soni Dragana Gagic Nina Butowski 2.20 Keynote #4 – Sponsor: SOFAT

554 Eddy Smid 545 Filomena Ng 684 Christina Moon

2.40 557 Danielle Kok (s) 665 Annie West (s) 3.00 582 Jessica Chiang 615 Joanne Turnbull (s) 560 Linda Ogutu (s) 3.20 Afternoon Tea

Food Microbiology and Safety – cont’d Microbial Physiology and Ecology Biofilms CHAIR Alexis Risson Olin Silinder Jon Palmer 3.50 Keynote #5 – Sponsor: NZMS

Karen McIntyre Keynote #6 – Sponsor: NZMS

568 Duncan McMillan 613 Gary Dykes

4.10 544 Laurine Kaul 4.30 658 Helen Withers and Anne-Marie Perchec

Merien 572 Marketa Vlkova 605 Michal Bernach (s)

4.50 575 Sean Bisset (s) 5.10 - 7.10 Poster Session #2 (Exhibition Hall)

7.10 Yet more free time to check out some different bars and restaurants

Thursday 28th November (s) = in student oral competition

Time Session 1 Conference Room

Session 2 The Gallery

Session 3 Seminar Room

8.30 am Plenary #8 – Sponsor: SOFAT

CHAIR: Steve Flint Remco Kort

9.15 Food Microbiology and Safety – cont’d Microbial Evolution CHAIR David Flossdorf Patrick Biggs 9.20 638 Eden Esteves (s) 678 Heather Hendrickson 9.40 556 Junwen Zhang (s) 589 Farhad Golzar

10.00 569 Georgia Breckell 10.20 Morning Tea

Student Competition FINAL 10.50

Six finalists announced after completion of the 3-minute presentations on Monday afternoon

11.10 11.30 11.50 12.10 12.30 12.50 Lunch + @ 1.00pm, Rob Elshire (GFANZ) "Genomics for Aotearoa New Zealand: Where society and genomics science meet." (Seminar Room) CHAIR Tash Forester

1.40 Plenary #9 – Sponsor: MBIE

Mātai koiora moroiti: Taha Māori Jane Mullaney

2.20 Plenary #10 – Sponsor: MBIE Max Kennedy

3.00 Afternoon Tea CHAIR Mike Taylor

3.30 Plenary #11 – Sponsor: NZMS Matthias Horn

4.15 ORATOR Assoc. Prof. Jasna Rakonjac

5.00 Conference Close Free Time

7.00 pm CONFERENCE DINNER – Distinction Hotel

POSTER PRESENTATIONS (Sorted on last name)Presenting on: Number First Name Last Name Category Title

Tues 654 Catherine Andrews Environmental MicrobiologyReducing Rumen Methanogen Growth in Pure Culture by Inhibitor Compounds

Tues 633 Graeme Attwood Environmental MicrobiologyMicrobial marker genes for predicting methane yield in ruminants

Tues 592 Marc Bailie Molecular MicrobiologyA genetic approach to exploring the phenotypic differences between strains ofLactobacillus fermentum and their effect on the human gastrointestinal tract.

Tues 520 Agata Bancerz-Kisiel Veterinary MicrobiologyCharacterization of Yersinia enterocolitica Strains Isolated from Cattle and Potentially Pathogenic to Humans

Wed 636 Daniel Alexander Bastias Campos Plant-Microbe InteractionsEndosymbiotic bacteria of plant-associated fungi: friends or foes?

Wed 605 Michal Bernach OtherArtificial plant leaf surface as a surrogate for microbial colonization.

Tues 575 Sean Bisset Molecular MicrobiologyUnderstanding the mechanism of action of the bacteriostatic peptide glycocin F

Wed 569 Georgia Breckell OtherSo you want to build a genome? Completing de novo Escherichia coli genomes with long read data

Wed 690 Wenli ChenPhenotypical-genotypical correlation and c-di-GMP turnover ability of genes encoding GGDEF/EAL/HD-GYP domain proteins in Pseudomonas putida

Wed 691 Wenli ChenBroadening the knowledge on c-di-GMP mediated FleQ dependent transcriptional regulation in Pseudomonas putida

Tues 539 Clara Chong Veterinary MicrobiologyThe microbial biogeography of the gastrointestinal tract of preterm and term lambs

Wed 525 Jonathan Chua Food MicrobiologyCharacterising the surface properties of oral probiotic Streptococcus salivarius K12 for designing new food delivery formats

Wed 595 Priya Darshini Environmental Microbiology It takes two to tango, real �me inves�ga�on of endosymbiosis using Pseudomonas fluorescens andDictyostelium discoideum

Tues 651 Silvia de la Rosa Plant-Microbe InteractionsIdentification of avirulence effectors from the apple scab fungus, Venturia inaequalis

Tues 570 Alyssa Earnshaw Molecular MicrobiologyWho is in cow poop? Characterization of the bovine faecal microbiome

Wed 645 Mareike Erdmann Antimicrobial Resistance Colistin Resistance in Pseudomonas aeruginosaTues 637 Rana Fathizargaran Molecular Microbiology 16S rRNA gene PCR contamination: tracking the sourceWed 546 Davide Fraccascia Microbiota Exploring new players in theFaecalibacterium genus

Tues 555 Qifan Fu Antimicrobial ResistanceEffects of herbicides on antibiotic resistance in Serratia marcescens

Wed 580 Alex Grey Antimicrobial ResistanceScreening New Zealand Fungi for Anti-Mycobacterial Compounds using Bioluminescent Organisms

Wed 608 Kelly Hong Antimicrobial ResistanceQuantifying the predictability of the emergence of antibiotic resistance in a model strain and environmental isolates of Escherichia coli

Tues 679 Anika Hoogeveen MicrobiotaThe difference in ileal and caecal microbial composition did not influence organic matter fermentation but the short-chain fatty acid production in the growing pig fed a human-type diet

Tues 666 Parham Hosseini Antimicrobial ResistanceFunctional expression of Aspergillus fumigatus CYP51 isoforms in Saccharomyces cerevisiae

Wed 601 Yiying Huang Food Microbiology The diversity of biofilm sporulation forBacillus cereus

Tues 673 Carel Jobsis MicrobiotaExploring the effects of enteral nutrition on intestinal microbiota using an in vitro model

Tues 524 Renee Johansen Plant-Microbe Interactions Beyond Myrtle Rust: Towards Ecosystem Resilience

Tues 627 Rachel Kaminsky Environmental MicrobiologyResolving broad patterns of prokaryotic community structure and composition in New Zealand pasture soils

Wed 571 Mikhail Keniya Molecular MicrobiologyAmino acid substitutions in lanosterol 14α-demethylase responsible for intrinsic azole resistance in mucormycetes

Wed 652 Sofia Khanum Proteins/Biochem/Structural BioCharacterisation of immunogenic epitopes on a methanogen surface protein

Wed 632 Zoe King Antimicrobial ResistanceGenomic characterisation and comparison of antibiotic resistant and susceptible Helicobacter pylori.

Wed 674 Theerapong Krajaejun Infectious DiseasesProtein A/G-based enzyme-linked immunosorbent assay for detection of anti-Pythium insidiosum antibodies in human and animal subjects

Tues 631 Sandeep Kumar MicrobiotaUsing rumen microbial community composition data to predict methane yield ranks of sheep fed different diets

Tues 581 Van Hung Vuong Le Antimicrobial Resistance Novel Nitrofuran Activating Enzyme in Escherichia coli

Wed 562 Rayen Leon-Quezada Proteins/Biochem/Structural BioProduction of nanorods to solve the end-cap structures of filamentous phage

Tues 689 Xinyue Li Antimicrobial ResistanceExperimental evolution of bacterial pathogens on antimicrobial copper surfaces

Tues 659 Sam Mahoney-Kurpe MicrobiotaRumen isolates of the Christensenellaceae R-7 group are able to utilise hemicellulose and produce hydrogen and ethanol as major end products

Wed 671 Moritz Miebach Plant-Microbe InteractionsThe good, the bad or the ugly - how do plants distinguish beneficial from pathogenic bacteria?

Wed 649 Bhargava Reddy Morampalli Environmental MicrobiologyFull length cDNA sequencing to study transcriptional regulation in bacteria - a proof of concept

Tues 552 Naren Naren Molecular MicrobiologyLocal versus global regulatory roles of the histidine utilization repressor HutC in Pseudomonas fluorescens SBW25

Tues 612 Amila Nawarathna Food MicrobiologyAntimicrobial activity of Clostridium enriched conditioned media

Tues 528 Thu Nguyen Food Microbiology Inactivation aflatoxin M1 by ultra violet light (254 nm)

Tues 565 Stephen On Microbial ImagingElastic Light Scatter pattern analysis of foodborne pathogens: darkness from light?

Wed 523 Nikola Palevich Environmental MicrobiologyPan-genome analysis of Butyrivibrio  and  Pseudobutyrivibrio  provides insight into polysaccharide degradation in the rumen.

Tues 603 Priyali Patel Infectious DiseasesDevelopment of an in vitro competition model for the mouse enteropathogen Citrobacter rodentium

Wed 614 Jasper Perry Molecular MicrobiologyThe Evolution of Endosymbiosis and the Origins of Complex Eukaryotic Life

Wed 693 Lama RahmanThe effects on non-invasive and invasive enteric infections on the microbial ecology of the gut

Wed 622 Ravishankar Rai Antimicrobial ResistanceQuorum sensing inhibitors from endophytic bacteria associated with medicinal Plants of Western Ghats

Wed 623 Ravishankar Rai Plant-Microbe InteractionsQuorum sensing inhibitors from endophytic bacteria associated with Medicinal Plants of Western Ghats

Wed 566 Delphine Rapp Environmental MicrobiologyTemporal detection of Campylobacter jejuni , Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) in calf faeces, calf bedding, birds and flies in a dairy farm environment

Tues 606 Attika Rehman Antimicrobial ResistanceUnderstanding ciprofloxacin resistance in Pseudomonas aeruginosa

Wed 656 Kerri Reilly Molecular MicrobiologyExpression of Cellulosome proteins from a novel New Zealand Ruminococcus sp. HUN007

Wed 661 Xiaoyun Ren Molecular MicrobiologyApplication of selective whole-genome amplification to culture-independent-typing of Neisseria meningitidis

Tues 692 Andrea Sajuthi Rapid CRISPR-based identification of microorganisms

Tues 620 Jaspreet Singh Sidhu Plant-Microbe InteractionsIdentification of the sporidesmin gene cluster in the facial eczema causing fungus, Pithomyces chartarum

Tues 621 Jaspreet Singh Sidhu Plant-Microbe InteractionsIdentification of the sporidesmin gene cluster in the facial eczema causing fungus, Pithomyces chartarum

Wed 583 Kiran Sreeja Jayan Molecular MicrobiologyTowards a molecular mechanism of bacterial recognition of eukaryotic hosts

Wed 680 Rashad Syed Environmental MicrobiologyIsolation and high cell density culture of methanotrophs for use in artificial biofilters to mitigate methane emissions

Tues 647 Mariana Tarallo Plant-Microbe InteractionsCharacterization of two cell death elicitor families from the Dothideomycete fungal phytopathogens Dothistroma septosporum and Cladosporium fulvum

Wed 577 Hwee Sze Tee Environmental MicrobiologyBenthic and planktonic microbial community transitions across a freshwater to marine salinity gradient

Tues 600 Zakieh Vahdati Infectious DiseasesThe impact of non-tuberculous mycobacterial (NTM) infection on the immune response of Galleria mellonella

Tues 578 Shara van de Pas Antimicrobial ResistanceScreening New Zealand fungi for new antibiotics: a modified OSMAC approach to maximise biodiscovery success.

Wed 553 Tianyang Wang BiofilmsEffect of calcium on bacterial attachment and biofilm formation of Geobacillus stearothermophilus.

Wed 628 Alibe Wasa Other "NsARC" The solution to bad bugs

Tues 676 Kathryn Wigley Environmental MicrobiologyIdentifying active biofilm community members participating in methane oxidation using RNA stable Isotope probing

Wed 677 Juliana Yeung Molecular Microbiology Cell envelope-associated proteins in rumen methanogens

PLENARY SPEAKER ABSTRACTS

PLENARY #1 – KEVIN KEENER

Aflatoxin Degradation in Shelled Corn Using High Voltage Atmospheric Cold Plasma Kevin Keener1, Hu Shi2, Kizito Nishimwe2 1. University of Guelph, Guelph, ON, Canada 2. Food Science and Human Nutrition, Iowa State University, Ames, IA, United States Mycotoxins are toxic secondary metabolites produced by filamentous fungi contaminating various food and feed crops posing serious health risks for both human and animals. According to the Food and Agriculture Organization of the United Nations (FAO), 25% of the world's crop are contaminated with mycotoxins. Decontamination of aflatoxin has been a continuing challenge for the food and feed industry. High Voltage Atmospheric Cold Plasma (HVACP) is a novel processing method that has shown potential for reduction and possible elimination of aflatoxin from grains and seeds. HVACP technology is an adaptation of a dielectric barrier discharge APCP with a number of unique features requiring only air and electricity. The unique features of HVACP include: 1) the ability to create plasma inside sealed containers of both simple and complex shapes such as glass jars, plastic bags, cardboard cartons, plastic and wooden totes; 2) a high voltage electrode design capable of generating plasmas in a variety of gases at voltages above 50 kV; 3) unique dielectric barrier configurations which restrict current flow into the plasma gas, resulting in a low power requirement; and 4) low operational costs due to compatibility with single phase electrical power (120 V or 240 V). HVACP direct treatments were performed using room air and 80 kV on a 25 gram sample of shelled corn artificially inoculated with 450 ppb of aflatoxin B1 resulted in an 85% reduction of toxin in five minutes treatment and 24 hours of sealed storage at room temperature. Aflatoxin B1 degradates were separated using high pressure liquid chromatography time of flight mass spectrometry (HPLC-TOF-MS). Based on these results six main degradants were elucidated and two degradation pathways were proposed. Degradation of aflatoxin by HVACP treatment involves addition of H•, OH•, CHO• radicals, epoxidation by HO2• radicals, and oxidation by species OH•, H2O2 and O3. Further studies using biological assays are now underway. These include MTT and DNA fragmentation assay. HVACP treated samples showed no color or smell differences compared to the controls. These results suggest that high voltage atmospheric cold plasma is a viable approach to removing aflatoxin from contaminated grains and seeds.

PLENARY #2 – DEBBIE WILLIAMSON

Genomic technologies have revolutionised public health microbiology, in Australia and globally. One of the major challenges in implementation of this technology is how best to integrate data into public health practice. Here, I will discuss how our group has applied genomics to questions of public health importance, with a specific focus on two major public health themes, namely sexually transmitted infections and foodborne disease.

PLENARY #3 – NATALIA REQUENA

The power of symbiotic fungi to control their plant hosts

Natalia Requena, Meike Hartmann, Sven Heidt and Ruben Betz natalia.requena@kit.edu

Most plants in natural ecosystems live in association with AM fungi what allows them to survive under poor nutrient conditions. In particular, phosphate scarcity is a major limiting factor for plant growth, and plants in symbiosis with AM fungi show under those conditions a significant benefit compared to their non-colonized counterparts. Thus, besides other adaptive responses controlled by the conserved phosphate starvation response (PSR) pathway, that systemically and locally controls plant phosphate homeostasis, plants can also engage in symbiosis to withstand phosphate limitation. AM fungi colonize the root cortex and transfer phosphate to their host roots at specialized structures called arbuscules where dedicated plant phosphate transporters load it into cortical cells. Interestingly, external high phosphate inhibits AM fungal colonization, arbuscule development and symbiosis progression and within a short-term, plants are able to get rid of their fungal symbionts. Therefore, it was unclear why phosphate downloaded at arbuscules does not interfere with symbiosis progression. We have now discovered that AM fungi employ conserved effector proteins that locally modify the plant PSR by intersecting with the splicing factor SR45, a novel negative regulator of this pathway. SP7-like effectors interact with SR45 modulating the fate of specific mRNAs, notably the PSR transcription factor MtPHL7. Our work shows that MtPHL7 accumulates in arbuscule-containing cells and is per se sufficient to induce PSR, while its silencing phenocopies the silencing of SP7-like effectors that impairs arbuscule-development.

We had previously shown that SP7 contributes towards keeping the plant immune response low. These new results extend the function of SP7-like proteins to the control of plant phosphate homeostasis. Although a priori these two functions might appear unrelated, recent studies have demonstrated that the root microbiota is directly responsible for the integration of phosphate nutrition and defense responses in plants. Moreover, SR45 has been shown to be a suppressor of plant immunity. And thus, we propose here that AM fungi employ SP7-like proteins to achieve such integration. The presence of similar proteins in all analyzed families of AM fungi, the conservation of the plant nodes where the SP7-like proteins act, and the mycorrhizal defective phenotype of SP7 and SP5 down-regulated plants, highlight the evolutionary pressure and the importance of these effectors for the outcome of the symbiotic interaction.

PLENARY #4 – FRANK TAKKEN

Good fungi turning bad – what is the difference between beneficial and pathogenic Fusarium oxysporum strains? Frank L.W. Takken1, Maria E Constantin1, Fran J de Lamo1, Mara de Sain1, Like Fokkens1, Petra M Houterman1, Jiming Li1, Martijn Rep1 1. University of Amsterdam, Amsterdam, NH, Netherlands The fungus Fusarium oxysporum(Fo) is a common soil resident. Besides its ability to grow saprophytically it is well known for its intimate contacts with living plant roots. The outcomes of these interactions range from beneficial to deleterious. With a host range of over 100 crop species, Fo is actually one of the most devastating fungal plant pathogens. It is therefore remarkable that the same fungus is used as a biocontrol agent to enhance plant tolerance to (a)biotic stresses. Co-inoculation of tomato roots with pathogenic and biocontrol Fostrains results in colonization of the vasculature of the host by the pathogen, but disease symptoms are strongly reduced. We are interested in how this protection is conferred and what characteristics distinguish a pathogenic strain from a biocontrol strain. Foharbors conditionally dispensable chromosomes that in pathogenic strains are enriched for effector genes that are correlated with host range. Transfer of a pathogenicity chromosome to a biocontrol strain can convert it into a pathogen. Vice versa,loss of a pathogenicity chromosome can convert a pathogen into a biocontrol strain. To determine the genetic requirements distinguishing these strains we sequenced 80 genomes of pathogenic and non-pathogenic Fo isolates and determined their “effectorome” to correlate key effectors with the ability to colonize the tomato plants and/or cause disease. In addition, the xylem proteomes of (co)inoculated plants were determined to identify fungal- and plant-produced proteins involved in this interaction. The results of this ongoing analysis will be presented, along with a few examples of Foeffector proteins and a specific class of plant proteins of which we have obtained some insight into the function in the tri-partite interaction.

PLENARY #5 – GISELE LAPOINTE

Insights into gut microbial interactions through in vitro modelling of the gastrointestinal microbiome

Gisèle LaPointe, University of Guelph, Guelph, Ontario, Canada The human gut is home to 1014 microbial cells. However, it is only in the last decade that culture-independent techniques have allowed us to explore the diversity of this ecosystem, of which many members are difficult to isolate by culture-dependent methods. By 3 years of age, we have acquired our adult profile of gut microbes that generally remains stable (homeostasis) due to our immune system, unless there are perturbations such as antibiotic intake and disease. Gut microbiota can differ according to lifestyle, dietary intake, age, gender, and these changes may be reversible, but the effect of diet depends on the initial gut microbiota of the individual. There is vast opportunity for using our food to help maintain gut homeostasis, but there are also many challenges to understanding the complexity of interactions among gut microbes and with host cells, and how to actually modulate this activity through our food. In vitro digestion (IVD) complements animal and clinical studies, but how well do in vitro gut microbiome models represent real life situations? In vitro gut microbiome modelling is particularly appropriate for testing compounds that may have a negative impact on the health of human subjects. Specific examples are experimentation with foodborne pathogens and food additives. How does food formulation and processing impact the survival and virulence potential of Salmonella in the face of colonization resistance from the gut microbiota? Do food dyes and plasticizers have an impact on microbial metabolism in the gut? This presentation will highlight research addressing these questions using three IVD models: the TIM1 (stomach, small intestine), the PolyFermS (stabilized ileum microbiota) and the ProDigest SHIME®. Metabolite profiling by nuclear magnetic resonance (NMR) can show changes in short chain fatty acids (SCFA: acetate, butyrate and propionate) that reveal how these treatments impact microbial metabolism. From the food microbiologist’s perspective, understanding the impact of food on health requires taking into account the entire production and digestion process to gain knowledge of the factors influencing the microbial quality and safety of food, its transformation into nutrients and delivering bioactive components to the gut ecosystem.

PLENARY #6 – SCOTT CHIMILESKI

In this lecture, Scott Chimileski will share his fascination with the wonders of the microbial world through vignettes and images from their new book, Life at the Edge of Sight: A Photographic Exploration of the Microbial World (Harvard University Press). The presentation will feature a visual exploration of microbes, from the pioneering findings of a seventeenth-century visionary to magnificent close-ups of the inner workings and cooperative communities of Earth’s most prolific—but often invisible—organisms.

PLENARY #7 – PHIL POPE

Custom plant fibres for beneficial microbes: a path to microbiome modulation? Phil B Pope1 1. Norwegian University of Life Sciences, Ås, AKERSHUS, Norway The mammalian gut is an infamous plant fiber-digesting ecosystem, which depending on its host, can harbor a complex mixture of bacteria, archaea, protozoa, fungi and viruses that coordinate breakdown of complex dietary carbohydrates into nutrients. Despite extensive efforts to functionally elucidate the gut microbiome, fiber degradation has so far been attributed to a limited number of cultivable representatives that predominantly originate from the human gut. Moreover, the majority of the gut microbiota, their complex interactions and the enzymatic machineries they employ remain poorly understood. Here, we present an overview of our recent efforts, where we combine traditional culturing, meta-omics, bioinformatics, biochemistry and enzymology to investigate the different saccharolytic mechanisms that beneficial microbiota employ from both ruminants and monogastric animals. We demonstrate key findings from studies on well-known Roseburia intestinalis and Fibrobacter succinogenes isolates, a novel as-yet uncultured Bacteroidetes family (‘Candidatus MH11’), and large-scale ruminant and monogastic (pigs) meta-omics projects that seek to decrypt plant fiber metabolism at a community level. These approaches have revealed new mechanistic information related to the hydrolytic capacity of outer membrane vesicles (OMVs), Polysaccharide utilization loci (PULs) and large multi-modular enzymes. Collectively, these findings have led to new customized diets for production animals, which match specific fibre structures to the enzymatic capabilities of beneficial butyrate-producing populations, and thus stimulate their metabolism in the gut.

PLENARY #8 – REMCO KORT

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The harvest of a decade of microbiological research for functional fermented foods in East-Africa Remco Kort1, 2, Nieke Westerik1, 2, Alex Wacoo1, 3, 2, Wilbert Sybesma2 1. Molecular Cell Biology, VU University Amsterdam, Amsterdam, Netherlands 2. Yoba for Life foundation, Amsterdam, The Netherlands 3. Biochemistry, Makerere University, Kampala, Uganda Background In resource disadvantaged countries, poor hygienic conditions and malnutrition frequently lead to infectious diseases, including respiratory tract infections and diarrheal disorders, in particular in children. In addition, the warm and humid climate allows for contamination with fungi leading to the presence of mycotoxins in frequently consumed cereals such as maize. People in these countries might benefit from consumption of functional fermented foods containing probiotic bacteria, which are able to reduce of the incidence or severity of infectious diseases and the uptake of mycotoxins in the body. Aim The aim of this research is to provide access to functional fermented foods through the development of a bacterial starter culture allowing dairy farmers and small-scale entrepreneurs in East-Africa to convert their locally produced milk into a probiotic yoghurt. A second aim is to assess the health benefits of the probiotic yoghurt on local consumers. Methods A dried probiotic starter culture has been developed, containing the world’s best documented probiotic strain, the human gut isolate Lactobacillus rhamnosus GG. The strain's inability to grow in food matrices like milk was surmounted by the formulation of a dried starter consortium with an adjuvant strain of Streptococcus thermophilus. Results Fermentation of milk and cereals with the starter culture allows propagation of L. rhamnosus up to titers of 1 x 109 cfu ml-1. Fermentation of a maize-based food led to an over 1000-fold reduction of aflatoxins spiked in raw ingredients. Sachets prepared with the two strains retained viability for at least two years. A nutritional trial with 1,116 school children of 3-6 years old suggested reduction of common cold as well as skin disease such as Tinea capitis, among children consuming probiotic yoghurt compared to milk. Conclusion A stable dried starter culture has been developed, which facilitates local and low-cost production of functional fermented foods (currently > 250 small companies in 6 countries produce over 50,000 liters of probiotic yoghurt per week). The initiative led to a successful school feeding program in Uganda with promising effects on health, but more studies are needed to further substantiate health benefits of probiotic yoghurt in favor of milk.

1. Kort R, Westerik N, Mariela Serrano L, Douillard FP, Gottstein W, Mukisa IM, Tuijn CJ, Basten L, Hafkamp B, Meijer WC, Teusink B, de Vos WM, Reid G, Sybesma W. (2015) A novel consortium of Lactobacillus rhamnosus and Streptococcus thermophilus for increased access to functional fermented foods. Microb Cell Fact. 14:195.

2. Kort R, Sybesma W. (2012) Probiotics for every body. Trends Biotechnol. 30:613-5. 3. Westerik, N, Wacoo, AP, Anyimo, E, Matovu, W, Reid, G, Kort, R, Sybesma, W. (2019) Improving health and wealth by

introduction of an affordable bacterial starter culture for probiotic yoghurt production in Uganda. Challenges 10: 2. 4. Wacoo AP, Mukisa IM, Meeme R, Byakika S, Wendiro D, Sybesma W, Kort R. (2019) Probiotic enrichment and reduction

of aflatoxins in a traditional African maize-based fermented food. Nutrients. 11: 2.

PLENARY #9 – JANE MULLANEY

Tikanga and the inner microbiologist Jane Mullaney, AgResearch ‘Vision Mātauranga’ was a descriptor conceived by Charles Royal back in 2005 when describing Māori research. Vision Mātauranga has four strands which cover: Indigenous innovation, environmental sustainability, health and economic benefits. More recently Emeritus Professor Mason Durie observed that there are Māori who are scientists and scientists who are Māori, and asked which one we were. As someone who identifies with being a scientist first, then Māori, this shapes the way I approach research and how I integrate the concept of Vision Mātauranga into the science that I do. I believe that mātauranga, and mātauranga Māori while different approaches, complement one another and together add to the depth of our knowledge and understanding of the world that we live in. I will give an outline of a proposed Vision Mātauranga-centric project that is holistic, practical, Vision Mātauranga and multidisciplinary.

PLENARY #10 – MAX KENNEDY

The History and Future Potential of Industrial Fermentation in Aotearoa New Zealand Max Kennedy PhD (MIT), FIChemE, FIPENZ, FNZIC and Camilla Gardiner, PhD This presentation will look at the history of industrial fermentation in New Zealand. Examples will cover the use of fermentation by Māori, Captain Cook’s spruce beer, whiskey production, Chew Chong, continuous beer fermentation, whey fermentation, cheese and yoghurt, sheep vaccine manufacture, the first large scale GMO fermentation in a contained facility (30 year anniversary in 2020), thermophiles, biopesticides, wine, pharmaceuticals, and greenhouse gases. Next, the current $85M investment by MBIE in industrial fermentation will be reviewed to give a view of the current trajectory of the fermentation sector. Lastly, the opportunity for the future potential of the sector to add value to New Zealand raw materials is postulated.

PLENARY #11 – MATTHIAS HORN

Intracellular symbionts and pathogens in the environment Matthias Horn1 1. University of Vienna, Vienna, VIENNA, Austria Intracellular microbes have a fundamental impact on the life of most animals, including humans. As symbionts they may provide nutrition or affect reproduction; as pathogens they can cause disease, often with severe consequences for their hosts’ health. To better understand how microbes acquired the ability to infect and thrive within eukaryotic host cells, my lab focuses on intracellular microbes in the environment, particularly those associated with protists. As predators free-living amoeba feed on other microbes and shape microbial communities. Yet they also serve as hosts for diverse bacteria able to survive phagocytosis and capable of exploiting eukaryotic cells for intracellular replication. These interactions have evolved over the course of hundreds of millions of years and have contributed to the emergence of bacterial mechanisms for the infection of eukaryotes, which are still used today by pathogens and symbionts alike. In this talk I will summarize what we have learned about the evolution of the intracellular lifestyle from studying chlamydiae that occur as symbionts in amoeba and pathogens of diverse animals. Molecular surveys indicate that these microbes are much more diverse than currently recognized, and I will show how single cell genomics and accumulating metagenomic data revealed surprising insights and contribute to a more comprehensive understanding of their genomic diversity, versatility, and evolutionary origin. Co-infection of protists by multiple symbionts are common under natural conditions in the environment but rarely studied in the lab. I will highlight the importance of investigating intracellular microbes in this context taking Legionella pneumophila as an example, an amoeba pathogen and the causative agent of Legionnaire’s disease in humans. I will show how co-infection with chlamydial symbionts affects this pathogen, and how gene expression analysis by RNA sequencing (dual RNA-seq) can provide insights into the molecular response of both microbes during co-infection. Our data suggest that chlamydial symbionts could be key in shaping environmental survival, abundance, and virulence of the pathogen L. pneumophila, thereby affecting the frequency of human infection. Taken together, studying environmental microbes associated with protists provides a framework and important context for our understanding of biology, ecology and evolution of host-associated lifestyles.

KEYNOTE SPEAKER ABSTRACTS

KEYNOTE #1 – BARRY SCOTT

Chitin deacetylases mediate cell wall conversion of chitin to chitosan to control hyphal growth and maintenance of a fungal-grass mutualistic symbiotic interaction Barry Scott1, Nazanin Noorifar1, Matthew Savoian1, Yonathan Lukito1 1. Massey University, Manawatu MC, PALMERSTON NORTH, New Zealand Filamentous fungi form mutualistic symbiotic interactions with both below- and above-ground tissues of plants. One of the most well understood above-ground symbiotic associations is that between the endophyte Epichloë festucae and its grass host Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network. However, little is known about how the cell wall of this endophyte is modified to avoid a host defence response and support intercalary hyphal growth. Here we show, using a suite of molecular probes, that chitin in the cell wall of E. festucae is converted to chitosan as the hyphae switch from free-living to endophytic growth. Similarly, when hyphae transition from endophytic to epiphytic growth there is a switch from chitosan back to chitin synthesis. E. festucae has three chitin deacetylases, two of which are expressed in planta. Deletion of either of these genes disrupts both intercalary hyphal growth and the symbiotic interaction, establishing that conversion of chitin to chitosan is required for E. festucae to establish and maintain symbiotic grass associations.

KEYNOTE #2 – SINISA VIDOVIC

Adaptive Stress Physiology of Non-Typhoidal Salmonella: Microbiological Linkages with Food Safety

Sinisa Vidovic, PhD Salmonella Enteritidis is a non-typhoidal serovar of great public health significance worldwide. To gain a better understanding of the within-host adaptation of S. Enteritidis, we performed core genome single nucleotide polymorphism (SNP) phylogeny, population structure, and genome variation analyses on a diverse collection of this pathogen. The core SNP phylogeny revealed the existence of two host-restricted clades positioned at the bottom (human) and the top (avian) of the tree, further indicating their distant phylogeny. Bayesian model showed significant (p < 0.01) associations among invasive and gastrointestinal isolates that formed “human clade” and the isolates that formed “avian – human gastrointestinal” clade. Clinical isolates of the human clade showed the highest degree of genome variation resulting in a mean pairwise distance of 0.00014, followed by the isolates of the avian-human gastrointestinal clade 0.00005. In contrast, the most diverse group of isolates, forming population “C”, showed the least degree of genome variation. Collectively, this study showed that the genome variation of the S. Enteritidis isolates associated with the host-restricted clusters substantially increases compared to their non-host restricted counterparts, further indicating an important role of point mutations in within-host adaptation of this Salmonella serovar. In another study, we aimed to determine the roles of the Cpx and RpoE, two major extracytoplasmic stress response regulators, in the biofilm development of S. Enteritidis. The CpxR had the highly significant, but opposite effects on the auto-aggregation and swarming motility of S. Enteritidis. The auto-aggregation was negatively affected in the ∆cpxR mutant, whereas the same mutant significantly outperformed its wild type counterpart in the swarming motility, indicating that the CpxR plays a role in biofilm-associated phenotypes. Indeed, our biofilm related assays showed that the CpxR is of critical importance in the biofilm development under both static and dynamic media flow conditions. In contrast, the RpoE sigma factor showed no significant role in the biofilm development under dynamic condition. The cpxR deletion negatively affected the constitutive expression of the operons critical for biosynthesis of O – antigen and adherence, whereas positively affected the expression of the virulence genes critical for Salmonella mediated endocytosis. The CpxR induced expression of curli csgAB and fimbrial stdAC operons only during the biofilm development and flagellar motAB and fliL operons exclusively during planktonic phase, indicating a responsive biofilm-associated loop of the CpxR regulator. In conclusion, we determined the genetic basis underlying the CpxR biofilm regulation, which may point to a valuable target for the development of measures to control biofilms generated by this zoonotic pathogen.

KEYNOTE #3 – MARK TURNER

Stress signalling in food fermentation bacteria Mark S Turner1 1. University Of Queensland, BRISBANE, QLD, Australia Lactic acid bacteria (LAB) are used in a wide range of food applications, including as starter and adjunct cultures in food fermentations. In these systems they are exposed to a variety of stressors including heat, osmotic, nutrient starvation and acid. They must be able to sense and overcome these stressors in order to perform efficiently in food fermentation. Our work using a model LAB, Lactococcus lactis, which is used in cheese fermentation, has identified an important signalling nucleotide molecule called cyclic-di-AMP which controls a variety of stress adaptation processes in the cell. Using next generation sequencing and molecular genetics methods we and others have uncovered proteins that are regulated through cyclic-di-AMP binding in Lactococcus. These include the glycine-betaine transport transcriptional regulator BusR, the potassium importer Kup and the central metabolism enzyme pyruvate carboxylase. Cyclic-di-AMP is found in a wide variety of Gram-positive bacteria including Lactobacillus, Streptococcus, Listeria, Staphylococcus and Bacillus and shown to regulate similar processes including osmotic stress and antibiotic resistance. In this talk I will give an overview of the current knowledge of cyclic-di-AMP stress signalling which may ultimately provide a foundation for the development of novel antimicrobials against pathogens and for improving industrial strains used in food fermentations.

KEYNOTE #4 – EDDY SMID

Exploring and exploiting unknown territory in microbial physiology for novel food biotechnology applications Eddy J. Smid1 1. Wageningen University, Wageningen, GELDERLAND, Netherlands The knowledge of microbial physiology is commonly derived from studies of fast growing cultures. In nature, most microbes are continuously exposed to energy limitation. Consequently, they have evolved strategies to combat unfavourable conditions. The use of nutrient rich media and sustained focus on exponential growth has limited our understanding of slow growing microbes coping with severe nutrient limitations. This unknown territory of microbial behaviour potentially offers possibilities for new biotechnological applications. The aim of this study was to explore the properties of cell cultures growing under severe nutrient restriction and to exploit these for developing new biotechnological processes. Here, we focus on the production of dairy related aroma compounds by Lactococcus lactis growing at near zero-growth rates. The metabolic behaviour of L. lactis was studied at extremely low growth rates imposed by prolonged retentostat cultivation. Maintenance energy requirement, metabolite production, stress resistance, viability, plasmid content and morphology of the retentostat cultivated cells were investigated and compared to relevant control cultures. We demonstrated the formation of viable but not culturable L. lactis cells in response to near-zero growth rates. Moreover, the maintenance requirement was found to decrease 7-fold at growth rates below 0.0025 h-1. Finally, we discovered that the production of volatile aroma compounds of retentostat cultures of L. lactis resembled the aroma profiles of ripened cheese, demonstrating new options for biotechnological production of complex aroma blocks.

KEYNOTE #5 – KAREN McINTYRE

Karen McIntyre Director General, Food Directorate, Health Canada Oversight of the microbiological safety of foods is an important area of focus for Health Canada’s Food Directorate. While the responsibility for food safety is shared among government authorities (federal/provincial/territorial), industry partners and stakeholders, the Food Directorate is responsible for establishing food safety policies and standards in Canada. The Directorate also conducts health risk assessments during food safety investigations, conducts research to support policy and regulatory development, and communicates food safety risks to Canadians. Informed by the investigation of foodborne illness outbreaks over the past several years, and building on organizational strengths in these areas, the Directorate’s research priorities encompass the spectrum of microbial hazards (i.e., bacterial, viral, and parasitic organisms). In addition to conducting microbiology research, the Food Directorate also provides national reference services and networks in a number of foodborne pathogen areas. With an estimated 4 million Canadians affected by foodborne illness every year, recent efforts have focused on protecting Canadians from critical food safety issues, such as E. coli 0121 in flour, Salmonella in poultry products, E. coli 0157 in raw beef, and Listeria in foods. Ongoing efforts to strengthen food safety in Canada have predicated on addressing recent and emerging issues in microbiological food safety, including enhancing consumer education on safe food handling and cooking practices, strengthening policies and standards, and leveraging risk assessments to identify areas where interventions could have the greatest impact in reducing risk to Canadians. Our research scientists continue to collaborate with other investigators as part of scientific networks, such as the Food and Environmental Parasitology Network (FEPN), Verotoxigenic E. coli Network (VTEC), Food and Environmental Virology Network (FEVN) and ViroNet Canada. Our Microbiology Research Laboratory is applying emerging Whole Genome Sequencing (WGS) and bioinformatics approaches to study prevalence, virulence and antimicrobial resistance traits in bacteria pathogens. In partnership with FoodNet Canada, the Food Directorate is working towards reducing the burden of enteric disease through sentinel surveillance to allow for reliable assessment of risks posed by enteric pathogens.

KEYNOTE #6 – DUNCAN McMILLAN

The lipid environment shapes a second quinone binding site in an NDH-2, revealing the functional role of quinone isoprenoids Duncan McMillan1, Albert Godoy-Hernandez1 1. Delft University of Technology, Delft, SUD HOLLAND, Netherlands Type-II NADH:quinone oxidoreductases (NDH-2s) play a key role in the metabolism of archaea, bacteria, and eukaryotes. They are an essential element of the electron transport chain, where they generate transmembrane gradients. This process has been widely studied; however, it is poorly understood in a hydrophobic membrane environment, largely due to technical difficulties. Herein, we conducted a thorough study of the NDH-2 from the thermophilic bacterium Caldalkalibacillus thermarum (CthNDH-2), focusing on the influence of the hydrophobic interior of biological membranes, and the role that membrane-bound electron carriers play. We reveal that lipid environment, temperature, and quinone isoprenoid length are crucial for efficient catalysis. Strikingly, a lipid environment harboring menaquione-7 produced a 2-fold faster kcat than menaquione-4 at 37°C. Furthermore, while enzymatic turnover increases with temperature, Km remains unchanged if lipids are present. We also observe that activation energy decreases in vesicle systems, indicating a different catalytic mechanism than previously described in solution-based studies. Among these membranes’ constituents, the critical lipid component is phosphatidylethanolamine, not cardiolipin, unlike for other quinone-utilizing enzymes. Lastly, by using electrochemistry in immobilized vesicles we both eliminated the use of soluble quinones and were able to slow CthNDH-2 turnover. This approach revealed that CthNDH-2 kinetics in a lipid environment follow a Hill-plot profile, indicating a co-operative mechanism involving 2 quinone binding sites per CthNDH-2 monomer. Collectively, we demonstrate that the lipid environment influences the catalytic mechanism of this promising drug target and provides unique insights into how electrons are transferred by oxidoreductases in a natural environment.

ORAL ABSTRACTS – NUMERICAL ORDER

id #529

The role of protozoan genotypic diversity in humans: Implications for the epidemiology of cryptosporidiosis and giardiasis in New Zealand Paul C Ogbuigwe1, David Hayman1, Nigel French1, Patrick Biggs1, Juan Carlos Garcia-Ramirez1, Matthew Knox1, Anthony Pita2 1. Massey University, Palmerston North, MANAWATU, New Zealand 2. MicroAquaTech, Palmerston North, Manawatu, New Zealand Surveillance data from 2016 in New Zealand found that Cryptosporidium and Giardia were responsible for 5.4% and 7.4% respectively, of the total outbreaks in the country. However, there has been an inability to identify the same genotypes in epidemiologically linked cases using Sanger sequencing. Furthermore, the reportedly hyper-transmissible C. parvum genotype IIaA15G2R1 dominant in people and calves in most countries including the USA, UK, Spain and India is present but not dominant in New Zealand. Given the clinical effects of cryptosporidiosis and giardiasis are highly variable and the mechanisms underlying the epidemiological and phenotypic differences within these organisms are poorly understood, there are opportunities to use in vitro and next generation sequencing methods to address some of these questions. We hypothesise that 1) epidemiologically linked cases will share subtypes undetectable with consensus PCR and Sanger sequencing, and 2) that host-pathogen interactions are determined by genetic differences at the between species level and ecological differences at the between genotype level. To address our first hypothesis, we employed amplicon-based metabarcoding to characterise the within-host genetic diversity of Cryptosporidium and Giardia in humans from outbreaks that occurred in New Zealand between 2010 and 2018. To address our second hypothesis, we employ in vitro techniques to understand species and genotypic differences influencing the infectivity of these parasites. To do so we have adopted the newly characterised COLO-680N human cell line for the culture of Cryptosporidium and Caco-2 for Giardia and used flow cytometric analysis to assess levels of infectivity between isolates. Investigating the differences in human cell infectivity between isolates could provide insights into the molecular mechanisms of these diseases, through whole genome and transcriptomic sequencing, thereby informing future studies, guiding policy and leading to a better understanding of protozoa outbreak epidemiology.

id #531

Surface water and mahinga kai as vectors of antibiotic resistance. Sophie van Hamelsveld1, Brigitta Kurenbach1, Gayle C Ferguson2, William Godsoe3, Jack A Heinemann1 1. University of Canterbury, Christchurch, CANTERBURY, New Zealand 2. Institute of Natural and Mathematical Sciences, Massey University, Auckland, Auckland Region, New Zealand 3. Bio-Protection Centre, Lincoln University, Christchurch, Canterbury, New Zealand New Zealanders have a strong affinity for surface water and demand high environmental standards to allow its use for food production and recreation. Unfortunately, these standards are not always met. Our research aims to establish and monitor the levels of the fecal indicator Escherichia coli in Canterbury waterways. We chose E. coli because some strains are human pathogens, and its presence is correlated with other bacteria, such as Salmonella and Campylobacter. Furthermore, E. coli is a sentinel for detecting increases in antibiotic resistance and is also capable of spreading resistance genes to other bacteria. Initially, we focussed on quantifying antibiotic resistance among E. coli in the Ōtākaro/Avon River and its tributaries. This river runs through Christchurch’s iconic Hagley Park and is used for both recreation and food harvesting. To close the loop between the environment and humans, we have also been conducting preliminary research into the potential for traditional Māori kai moana and kai te awa to serve as vectors of drug resistant bacteria from the environment to humans. We isolate E. coli from water and food samples, perform antibiotic minimum inhibitory concentration testing and test their ability to conjugate. We use whole genome sequencing to find antibiotic resistance genes and test the evolutionary relatedness of isolates. In most water samples, there were between 10-1000 E. coli per 100ml. A high prevalence of multiple drug resistance was observed, including ciprofloxacin resistance and extended spectrum beta-lactamase resistances, which are frequently transferred through conjugation. These phenotypes confirm the antibiotic resistance genes and plasmid-diagnostic genes found in the whole genome sequences. The numbers of E. coli in our samples class these waterways as unsafe for skin contact or ingestion. Because we have also detected high levels of E. coli in stream-grown watercress, this mahinga kai may be a vector to transfer drug resistant E. coli from the environment to humans, but these data are preliminary.

id #535

Milkfat influences proteolytic enzyme activity of dairy Pseudomonas species Dong Zhang1, Jon Palmer1, Koon Hoong Teh2, Miranda Maru Angeli Calinisan3, Steve Flint1 1. The School of Food and Nutrition, Massey University, Palmerston North, New Zealand 2. Yashili New Zealand Dairy Co., Ltd, Auckland, New Zealand 3. Institute of Food Sciences, Singapore Institute of Technology, Singapore This study investigated the effect of growth conditions on production of proteases of six Pseudomonas strains, (Pseudomonas fragi DZ1, Pseudomonas koreensis DZ138, Pseudomonas rhodesiae DZ351, Pseudomonas fluorescens DZ390, Pseudomonas synxantha DZ832 and Pseudomonas lundensis DZ845), isolated from raw milk. Protease activity of all Pseudomonas strains in dairy media (skim milk and whole milk) was significantly higher (p< 0.05) than in non-dairy media (TSB), with most activity from

Pseudomonas grown in whole milk. The protease activity from P. lundensis DZ845 grown in TSB with the addition of 5% (w/v) butter was higher than other dairy ingredients added to TSB and the amount of protease activity increased with increasing concentrations of butter (from 5-15%). P. rhodesiae DZ351 showed little protease activity in all TSB supplemented with dairy ingredients. Only four of the six strains produced one protease of 47 kDa when grown in TSB. However, all six strains were able to produce at least one type of proteases in milk medium. For P. lundensis DZ845, a 12% casein zymography gel revealed that the presence of butter could induce protease activity. This is the first study showing the effect of milkfat (butter) on protease activity of Pseudomonas. This highlights the greater vulnerability of whole milk compared to skim milk to protease activity

id #536

Role of abiotic factors- temperature, water activity and oxygen concentration on spore formation within the biofilm of Geobacillus stearothermophilus Murali Kumar1, Steve Flint1, Jon Palmer1 1. Massey University, Palmerston North, MANAWATU, New Zealand Obligate thermophiles such as Anoxybacillus flavithermus and Geobacillus stearothermophilus are known to be major contaminants during milk powder manufacture1. G. stearothermophilus is capable of forming biofilms within pipelines in the evaporator section of the milk powder manufacturing plant. Upon maturation of the biofilm, spores are produced within the biofilm which are released into the end product1. Spore formation within the biofilm is known to be influenced by several abiotic factors i.e. sporulation temperature, water activity and oxygen concentration2. In this study, the effect of temperature on sporulation within the biofilm of G. stearothermophilus A1, D1, P3 and ATCC 12980 in UHT skim milk was studied using a microtiter plate biofilm assay. We observed that temperatures exceeding 60°C induced sporulation as early as 4h whereas sporulation at 55°C was delayed until 8h for all the strains isolated from the dairy environment. A significant variation in the spore forming potential was observed between the dairy isolates and the reference strain. The outcome of this study can be beneficial in the design of thermal processing steps involved in the manufacture of commercially sterile dairy products.

1. Characterization of thermophilic bacilli from a milk powder processing plant, Burgess et al., 2014. Journal of Applied Microbiology, 116, 350-359 2. Sporulation boundaries and spore formation kinetics of Bacillus spp. as a function of temperature, pH and aw, Baril et al., 2012. Food Microbiology, 32, 79-86

id #537

Spore forming bacterial populations in New Zealand dairy feeds David Flossdorf1, Steve Flint1, Jon Palmer1, Paul Jamieson 2, Nick Sneddon2, Bridget Maclean2 1. Massey Institute of Food Science and Technology, Massey university, Palmerston North, New Zealand 2. Fonterra Research Centre, Palmerston North, New Zealand Since the early 2000s forages and supplementary feeds utilised on New Zealand dairy farms have changed but the potential consequences of those changes on the spore populations in the raw milk are unknown. To investigate the potential effect that the bacterial spore populations within the feeds can have on the bacterial spore populations in raw milk, samples of raw milk and the feeds consumed were analysed for their spore populations. Bacterial spore selection and detection was based on standard microbiological methods, with a heat treatment at 80°C for 12 min to select for spores, followed by detection using two agars and three different incubation temperatures, to capture a wide spectrum of psychrotrophic, mesophilic and thermophilic spores. From this analysis, the highest counts of bacterial spores were found in tuber-feeds (fodder beet (Beta vulgaris) and turnips (Brassica rapa)) and palm kernel expeller (made from Elaeis guineensis) followed by pasture silage (Lolium Perenne + Trifolium Repens) and feed concentrates. Low counts of spores were present in raw milk samples. The bacterial spores found were identified using a MALDI-TOF-MS Biotyper (Bruker). These data showed that ~ 70 % of the bacterial spore species identified in milk, could also be identified in feeds from the same farm. The results also showed variations in the diversity of the spore populations between different feed samples, milk samples and different farms.This study indicates that the bacterial spore populations within cow feeds could influence the spore populations present in the raw milk.

id #541

Can we prevent Kauri dieback disease through the modification of electric fields? Debolina Sarkar1 1. University of Canterbury, Ilam, Christchurch, New Zealand The oomycete Phytophthora can cause serious threats to native flora and agriculture and food biosecurity, causing devastating diseases to plants such as kauri, oak, avocado and soy. In New Zealaand, Phytophthora agathidicida is the causative agent in kauri dieback and is receiving much coverage in the mainstream press as it can kill a mature kauri tree within two years. These organisms can infect their host plants via motile zoospores. These can travel between sites of infection via water in the soil. My research will focus on mimicking the natural environment around the roots of the plant using microfluidic Lab-on-a-Chip (LOC) devices to try to investigate the electrical parameters that allow zoospores to infect plant roots. Plant roots can generate electric fields which may attract zoospores. Different LOC devices have been designed that contain anodes and cathodes, that enable the study of the swimming behaviour of Phytophthora zoospores in the presence of electric fields. Preliminary data indicates that zoospores show a tendency to aggregate close to the anode. In the future this research may allow us to design devices that modify electric fields around vulnerable plants and attract zoospores away from roots and thus inhibit their infective capability

id #544

Toxic cocktail for bacteria – staphylococci party ends deadly Laurine Kaul1, Andrew Zannettino2, Regine Suess3, Katharina Richter1 1. Richter Lab, Surgery Department, University of Adelaide, Adelaide, SA, Australia 2. Faculty of Health and Medical Sciences, University of Adelaide, Precision Medicine Theme, South Australian Health & Medical Research Institute, Adelaide, SA, Australia 3. Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany Background and Aims: Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) are associated with hernia mesh infections and surgical site infections. These major infectious diseases frequently lead to clinical complications and increased healthcare costs, particularly when bacteria establish antibiotic-resistance and form biofilms (i.e. bacterial clusters embedded in a protective matrix). While the resistance of bacteria is steadily on the rise, the development of new antibiotics has declined. Therefore, new antibacterial treatments are urgently needed. The aim is to investigate the antibacterial activity of a toxic cocktail comprising diethyldithiocarbamate (DDC) and copper(II) (Cu) against staphylococci. Methods: The minimal inhibitory concentration (MIC) of DDC and Cu was determined in 3 methicillin-resistant S. aureus (MRSA 1, 2 and 3) and 2 S. epidermidis (SE 1 and 2) strains. To assess the potential synergy between the two compounds and antibiotics, checkerboard assays were performed in biofilms. The fractional inhibitory concentration index was calculated and used to define synergy, near synergy or additive effects of the compounds. Results: The MIC of DDC was 64 µg/ml for MRSA 1 and 2 and 3, and 32 µg/ml for SE 1 and 2. The MIC of Cu was >256 µg/ml in all strains tested. The combination of DDC and Cu resulted in a MIC reduction to 2 and 1 µg/ml in the MRSA strains and to 4 and 2 µg/ml in SE strains. In biofilms DDC-Cu reached synergy in MRSA 1 and 2, near synergy in MRSA 3 and additive effects in SE 1 and 2. Furthermore, in MRSA biofilms DDC-Cu showed synergy with the antibiotic classes fluoroquinolones, tetracyclines and glycopeptides, near synergy with aminoglycosides and additive effects with penicillins and cephalosporins. Conclusion: Synergistic and additive effects against MRSA and S. epidermidis make DDC-Cu a new treatment strategy against staphylococci, with the potential of enhancing the activity of multiple classes of antibiotics. Future research will focus on determining the mode of action of DDC-Cu and on the development of drug-delivery approaches for surgical applications

id #545

Development of a real-time TaqMan RT-PCR assay for the sensitive detection of diverse Raspberry ringspot virus isolates Filomena Ng1, Joe Tang1, Lisa Ward1 1. Plant Health and Environment Lab (MPI), St. Johns, AUCKLAND, New Zealand Raspberry ringspot virus (RpRSV) belongs to subgroup A of the Nepovirus genus, and was first identified in raspberry in the 1950s. Since then, RpRSV has been isolated from a diverse number of hosts, including economically important plants such as grapevine, cherry, and berry fruits. This virus can be transmitted mechanically, through seed, or by a nematode vector, and infection can result in plant yield reduction and plant death. Due to its risk to the horticultural industry, stringent and efficient measures are required to detect RpRSV in plant material being imported into New Zealand. Here we report the development of a one-step TaqMan real-time RT-PCR assay for the rapid detection of RpRSV. This assay was demonstrated to have greater specificity and sensitivity than existing conventional RT-PCR methods for the detection of diverse RpRSV isolates. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate fast and efficient phytosanitary testing of nursery stock that require RpRSV testing as an import requirement, and will also be used for the general detection of RpRSV in a wide range of hosts.

id #548

Methane oxidation rates at Hikurangi margin cold seeps Lucy Stewart1, Tyla Hill-Moana2, Julie Brown3, Sarah Bury3, Gareth Crutchley4 1. GNS Science, Lower Hutt, WELLINGTON, New Zealand 2. School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand 3. Ecological Stable Isotope Laboratory, National Institute of Water and Atmospheric Research, Wellington, New Zealand 4. GEOMAR Helmholtz Centre for Ocean Research , Kiel, Germany Cold methane seeps on the Hikurangi subduction margin off the eastern coast of Te Ika a Māui(North Island of New Zealand) host abundant microbial and macrofaunal communities supported by escaping methane from subsurface gas hydrate deposits. Notably, these seeps show high levels of aerobic methane oxidation (Sommer et al. 2010). In this study we show that at an active New Zealand seep site, aerobic gammaproteobacterial Methylococcales are the dominant methanotrophic groups well into the anoxic zone. A nearby extinguished seep site retains anaerobic, but not aerobic methanotrophs. Oxic and anoxic incubations of methane seep sediments show that anaerobic methane-oxidising sediment communities are less sensitive than aerobic communities to methane deprivation and can adapt to higher oxygen inflow. Acidification also promotes anaerobic methane oxidation. These results suggest anaerobic methane-oxidising communities may be more resilient to the effects of climate change than aerobic communities. Consequently, Hikurangi margin seeps may experience significant community shifts as a result of climate change or proposed commercial gas production from the underlying methane hydrates.

1. Sommer, S., Linke, P., Pfannkuche, O., Niemann, H., & Treude, T. (2010). Benthic respiration in a seep habitat dominated by dense beds of ampharetid polychaetes at the Hikurangi Margin (New Zealand). Marine Geology, 272(1–4), 223–232. https://doi.org/10.1016/j.margeo.2009.06.003

id #549

The social ecology of Serratia entomophila - pathogen of the New Zealand grass grub Connor Watson2, 1, Travis Glare1, Maureen O'Callaghan2, 1, Mark Hurst2, 1, Ben Raymond3 1. Lincoln University, Bioprotection Research Center, Lincoln, Canterbury, New Zealand 2. AgResearch, Lincoln, Canterbury, New Zealand 3. University of Exeter, Penryn, Cornwall, United Kingdom The endemic New Zealand grass grub (Costelytra giveni) is an economically significant grassland pest within New Zealand. Due to their impact on agricultural production, several methods are being used to control or prevent the establishment of new grass grub populations within the soil. One such method involves the use of biopesticide products derived from the bacterial genus Serratia, specifically the entomopathogen S. entomophila, which causes amber disease within the grubs; this results in gut clearance and feeding cessation, leading to death via septicaemia after approximately 2 months. The ability to cause amber disease is dependent upon the presence of the amber disease associated plasmid (pADAP), which encodes the required virulence determinants. Following the collapse of these grass grub populations after several years, non-pathogenic plasmid-free S. entomophila strains predominate within the soil. Whilst the interactions between S. entomophila and the grass grubs are well studied, the interactions between these plasmid-bearing and plasmid-free S. entomophila strains are not understood, particularly the potential impact of these interactions upon the efficacy of an applied biopesticide. Using a series of strains with antibiotic tags, various in vitro and in vivo experiments were conducted using inoculants of differing ratios of tagged pathogenic and non-pathogenic strains, enabling the relative growth and competition between strains in laboratory broth culture and in soil to be assessed. Within natural soil, where the strains were kept free-living within soil microcosms, no competition was observed, and the levels of both pathogenic and non-pathogenic strains remained stable over a two-month period. In nutrient-rich broth, the non-pathogenic strain outgrew the pathogenic strain within several days, where by the fifth day the non-pathogenic strain comprised at least 75% of the sample population. Additionally, grass grub co-infection assays with varying ratios of the tagged strains revealed the horizontal transfer of pADAP from the pathogenic to the non-pathogenic strain within the larval gut. Further testing is required to determine whether this competition and the transfer of pADAP will have any influence of the efficacy of the plasmid-bearing strain, and how this may impact upon the ability of S. entomophila to cause amber disease within grass grubs

id #550

Identifying the regulator of the Yersinia entomophaga exoproteome Marion Schoof1, 2, Maureen O'Callghan1, 2, Campbell R. Sheen3, Travis R. Glare2, Mark R.H. Hurst1, 2 1. AgResearch, Lincoln, Canterbury, New Zealand 2. Bio-Protection Research Centre, Lincoln University, Lincoln, Canterbury, New Zealand 3. Protein Science and Engineering, Callaghan Innovation, Christchurch, Canterbury, New Zealand Bacterial protein secretion, which can be triggered by environmental factors such as pH, oxygen level and nutrient availability, is crucial to maintenance of viability and pathogenicity. Tight regulation of gene expression in response to these stimuli allows the cell to adapt, react to environmental changes and induces pathogenicity. The bacterium Yersinia entomophaga, isolated from a diseased grass grub, shows pathogenicity against a broad host range including the New Zealand grass grub Costelytra giveni, the South African black beetle Heteronychus arator and other insect species. The Y. entomophaga MH96 genome encodes for a range of virulence factors such as the main virulence factor Yen-TC, putative toxins (Vip2, CdtB, LopT, PirAB), degradative enzymes such as proteases, chitinases, esterases, and lipases. The bacterium releases a wide range of these proteins at 25°C into culture but not at 37°C. In this study a new method combining transposon-mediated mutagenesis with a modified Bradford assay was used to develop a high-throughput exoproteome screening assay (HESA) to identify genes regulation the Y. entomophaga exoproteome. Using the HESA a genome cluster was identified, which regulates global protein secretion in Yersinia entomophaga. The cluster comprises of a DNA-binding protein (RoeA), an 1kb intergenic region and a predicted phage-like cell wall lysis cassette. Three transposon insertions that caused almost complete abolition of exoprotein production were located within the intergenic region between roeA and the lysis cluster. Additionally, the mutations of RoeA and the lysis cassette abolished protein secretion and resulted in morphological changes of the cell. Further complementation and overexpression studies show the crucial role of RoeA as a regulator for global protein secretion in Y. entomophaga.

id #556

Diversity of winemaking yeast species analysed by MALDI-TOF MS in an organic vineyard, New Zealand Junwen Zhang1, Bin Tian1, Jeffrey Plowman2, Stefan Clerens2, Stephen L. W. On1 1. Department of Wine, Food and Molecular Biosciences, Lincoln University, P O Box 7647, Canterbury, New Zealand 2. AgResearch Ltd., 1365 Springs Rd, Lincoln 7674, New Zealand Objectives:Rapid yeast identification is of particular importance in monitoring wine fermentation and strain application in winemaking. The objectives of this study were to 1) establish MALDI-TOF MS as a robust and rapid method for yeast identification and differentiation, 2) assess the diversity of culturable yeast species in an organic winery. Methods and Results:Pinot Noir grape juice sourced from Greystone vineyard at harvest (Waipara, New Zealand) was allocated in two fermentation tanks located in vineyard and winery, from which a total of 82 unknown yeast isolates were purified.14 known reference yeast species related to winemaking, and the 82 field isolates were submitted to MALDI-TOF MS analysis at high mass range (2,000-20,000 Da) and low mass range (500-4,000 Da) using optimized sample preparation (3-day-old colony and matrix suspension method). Cluster analysis using profiles at low mass, high mass or combined low-high range were compared using Pearson correlation and UPGMA algorithm. For cross-validation, 26S rDNA sequencing was used to determine the identity of selected field isolates. Nine species belonging to seven genera were identified: Pichia kluyveri, Pichia membranfaciens, Pichia terricola, Starmerella bacillaris, Hansenispora uvarum, Candida californica, Metschnikowia pulcherrima,Aureobasidium pullulans,and Saccharomyces cerevisiae. Species-specific biomarkers for each species were defined combining automatic peak matching and manually check. High-mass spectra provided the most robust species identifications.

Conclusions:MALDI-TOF MS is a robust and rapid proteomic approach for yeast identification at species level; optimised sample preparation is critical for best results. To our knowledge, this is the first paper to describe MALDI-TOF MS for yeast identification in New Zealand. MALDI profiles in the range 2,000 to 20,000 Da have best taxonomic resolution. Some differences were seen in the species isolated from the different wine environments, illustrating the dynamic nature of wine fermentation microbiology. The methods and database established here should be a useful tool for future research on diversity and behaviour of fermentative yeast in New Zealand.

id #557

From soil to solution: isolating bacteriophages from the environment to combat AFB Danielle Kok1, Heather Hendrickson1 1. Massey University, Albany, AUCKLAND, New Zealand American Foulbrood (AFB) is a disease of honey bee larvae and is caused by the bacterial pathogen Paenibacillus larvae. AFB is the most serious disease that infects honey bees and is present in almost all countries where honey bees are found. AFB has been present in New Zealand since 1877 and spread to all parts of the country within 10 years. Unlike other countries, the use of antibiotics in hives infected with P. larvae is prohibited under New Zealand law and infected hives must be destroyed immediately. Our research is looking into the use of Bacteriophages as a preventative measure against AFB. Bacteriophages (phages for short) are simple viruses that kill specific bacteria. Phages are highly abundant in the environment with an estimated census of 1031 globally. Work carried out in other laboratories around the world has shown that phages can be isolated from healthy hives and nearby soil and that AFB pathogens are susceptible to destruction by these phages. We aim to isolate phages that are destructive to P. larvae and combine them into a bio-protective phage cocktail for field-testing as a prophylactic treatment. In the first year of this project we obtained 7 separate samples of the pathogen from infected hives and collections in New Zealand. In order to obtain soil and bee debris samples to screen for phages, we used a citizen science approach to collecting 400+ samples from hives throughout New Zealand. We have confirmed that 23 of these samples have novel native phages by establishing plaque formation on one or more of the pathogen samples. Electron microscopy of a set of these bacteriophages has demonstrated that they have prolate capsids (109-120μm) and tails (166-190μm) and morphologies that are consistent with classification as Siphoviridae. The bacteriophages have similar host ranges on the pathogen isolates we have collected to date. Future work will include continued sample screening, complete sequencing and genome annotation and safety testing to assemble a safe and effective prophylactic phage cocktail. This project: ABAtE (Active Bacteriophages for AFB Elimination), provides the groundwork study for an innovative approach to naturally protecting NZ beehives against AFB.

id #559

A critical rebuttal of the proposed division of the genus Arcobacter into six genera Stephen L.W. On1, William G. Miller2, Patrick Biggs3, Angela J. Cornelius4, Peter Vandamme5 1. Lincoln University, Lincoln, CANTERBURY, New Zealand 2. Produce Safety and Microbiology Research Unit, , US Department of Agriculture, Albany, California, USA 3. School of Fundamental Sciences, Massey University, Palmerston North, New Zealand 4. Institute of Environmental Science and Research, Christchurch, Canterbury, New Zealand 5. Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium Objective: to critically examine the proposal by Pérez-Cataluña et al. (2018) to restructure the genus Arcobacter into six distinct genera. Our reasons for doing so were based on the facts that this proposal excluded other members of the Epsilonproteobacteria as references to provide context, and described a phenotyping scheme that did not clearly differentiate most of the genera it delineated. Methods and results: We used a comprehensive polyphasic taxonomic approach to reassess the restructuring proposal. This included comparative analyses of complete genome sequences of 82 Epsilonproteobacteria (including 26 arcobacters); phylogenetic analyses of three housekeeping genes and also 342 core genes; and phenotypic criteria. Genome sequences were analysed with tools to calculate average nucleotide identity, in silico DNA-DNA hybridisation values and G+C ratios. All genome analyses revealed Arcobacter (as originally defined) to be relatively homogenous and phylogenetic analyses clearly distinguished the group from other Epsilonproteobacteria. In contrast, distinction of the groups proposed by Pérez-Cataluña et al. (2018) was questionable. Similarly, phenotypic analyses supported the delineation of Arcobacter as originally defined, but did not justify the position of the genera proposed by the aforementioned authors. Arcobacter anaerophilus was not found to be an obligate anaerobe as described previously. Conclusion: our polyphasic taxonomic study supports the continuance of the previous classification of “aerotolerant campylobacters” as Arcobacter and refutes the proposed subdivision of Pérez-Cataluña et al. (2018). Emendments to descriptions of the genus Arcobacter and to A. anaerophilus are proposed.

id #560

Quantifying abundance of segmented filamentous bacteria from rat ileum pre- and post-weaning Linda A Ogutu1, 2, 3, Rachel C Anderson1, 2, Jasna Rakonjac4, Nicole C Roy1, 2, Warren C McNabb2 1. Food Nutrition & Health Team, AgResearch, Palmerston North, Manawatu, New Zealand 2. Riddet Institute, Palmerston North, Manawatu, New Zealand 3. School of Food & Advanced Technology, Massey University, Palmerston North, Manawatu, New Zealand 4. School of Fundamental Sciences, Massey University, Palmerston North, Manawatu, New Zealand Segmented filamentous bacteria (SFB) are Gram-positive, obligate anaerobic, spore-forming, commensals suggested to play a role in infant small intestine barrier maturation. This is due to the observed link between SFB abundance and concentrations of immunoglobulin A and interleukin 17, though it is unclear whether these immune factors influence SFB abundance. SFB have been identified from ileum tissue and faeces of mice pre- and post-weaning (3-4 weeks) and from infants’ faeces at weaning (6-24 months).

This suggests SFB are present in the infant ileum at weaning. Ethical challenges of infant research mean in vitro culturing of SFB is desirable, but has been unsuccessful as SFB require epithelial cell attachment. Therefore, in vivo SFB time-course studies are currently performed; this study sought to determine the abundance of SFB in pre- and post-weaning rats. So far, SFB time-course studies have only been performed on 3-week postnatal mice that have small ileum tissue size. This study used rats which provided larger sample sizes. Additionally, two previously used techniques of isolating SFB from the ileum, whole tissue homogenisation and ileal mucosal scraping, were compared. The abundance of SFB from the ileum tissue, ileum contents and faeces was quantified by qPCR using SFB-specific primers. The concentrations of IL-17 and IgA were also measured through antibody-specific ELISA assays. A consistent time-course pattern of SFB in both whole tissue and mucosal scrapes was observed pre- and post-weaning over 5 weeks. A similar SFB pattern was observed in the ileum contents and faeces. Contrary to previous reports, the concentration of IL-17 and IgA did not show a clear correlation with SFB abundance. Significant differences in SFB abundance from whole ileum tissue favour tissue homogenisation for isolating SFB. A similar SFB time-course pattern in the faeces and ileum contents suggests faecal SFB can represent ileal SFB, hence reducing animal numbers; this also suggests that infants’ faeces can be a valid medium to assess ileal SFB. These results will allow for the design of future studies investigating the relationship between diet, SFB abundance and the infant immunological barrier. Such studies could increase understanding of how weaning diet constituents promote SFB functionality.

id #563

Can sequencing methods help decipher transmission pathways of Leptospira within a community of hosts in New Zealand ? Marie Moinet1, Shahista Nisa1, Julie Collins-Emerson1, Jackie Benschop1, David A Wilkinson2, 1 1. mEpiLab, School of Veterinary Science, Massey University, Palmerston North, New Zealand 2. New Zealand Food Safety Science & Research Centre, Palmerston North, New Zealand Methods like amplicon sequencing, Multi-Locus Sequence Typing (MLST) or whole genome sequencing (WGS) allow a deeper understanding of Leptospira diversity and evolution than serotyping. We applied all of these methods to investigate reservoir dynamics in farm environments, progressively incrementing the theoretical resolution of isolate classification by increasing the total proportion of the organism’s genome included in the analysis. Kidneys from wildlife captured in two farms, and urine from sympatric livestock were sampled for culture and PCR. Samples were initially screened using a lipL32 PCR. Those positive without an isolate were screened with a second PCR, targeting the glmU gene, and amplicons sequenced. This gene allows the differentiation of all endemic strains known to circulate in NZ. Genomic DNA was extracted and sequenced from 79 isolates using Illumina HiSeq. Raw read data was assembled de novo using skesa, and contigs of assembled draft genomes were used to classify each isolate using different genotyping methodologies: These included i) three recognised MLST schemes, ii) a core-genome MLST scheme (Pasteur Institute Paris), and iii) single nucleotide polymorphism (SNP) analysis from core gene components. Four genotypes were detected by glmU, MLST and cgMLST with total concordance between methods: Leptospira borgpetersenii serovar Ballum, (mice, hedgehogs, rats, cattle), Hardjobovis (cattle) and Balcanica (possum, deer), and L. interrogans serovar Pomona (cattle, hedgehog). The intra-genotype diversity was too low for source attribution. For instance among the 73 Ballum isolates, the SNP analysis showed 0 to 6 SNPs despite covering >99.8% of the genome. Ballum strains were most similar to an isolate from the Netherlands, in agreement with the European origin of invasive mice in New Zealand. All techniques showed issues of sensitivity, with 50 lipL32-positive samples not assigned to a serovar and 7 samples lipL32- and glmU-negative for which leptospire-shaped bacteria were seen by dark-field microscopy but not isolated, and therefore not typed. Sequencing methods are a valuable addition to serotyping for eco-epidemiological studies, but are limited by within-strain low variation and a low sensitivity. They are currently dependent on successful isolation, and therefore better isolation and enrichment methods would improve how genomics could be applied to leptospirosis research.

id #567

Epichloë bromicola effector screening in Nicotiana spp Pranav Chettri1, Benjamin Moody1, Christine Voisey1, Rosie E Bradshaw2, Carl H Mesarich3, Linda J Johnson1 1. AgResearch (Grasslands Research Centre), Palmerston North, Manawatu, New Zealand 2. School of Fundamental Sciences, Massey University, Palmerston North, Manawatu, New Zealand 3. School of Agriculture and Environment, Massey University, Palmerston North, Manawatu, New Zealand The fungal endophyte Epichloë bromicola forms mutualistic symbioses with grasses of the Elymus complex. Artificial inoculation of Epichloë endophytes into modern wheat (Triticum aestivum) and wheat-alien experimental lines to form novel symbioses has produced a range of infected plant phenotypes (from normal to severe plant stunting and/or death). To understand the molecular basis of endophyte-host compatibility, a transcriptome analysis was conducted with E. bromicola in association with its native host, a modern wheat host and a wheat-alien host from the tribe Hordeae that exhibited contrasting endophyte compatibility outcomes. Many E. bromicola putative effector genes were highly up-regulated during these interactions. Effectors of fungal pathogens play crucial roles in actively suppressing host defence responses or preventing their recognition by corresponding plant immune receptor proteins; however, little is known about their roles in mutualistic symbioses. To evaluate the role of E. bromicola effectors in a non-host plant, 29 candidate effector genes were identified using a bioinformatics pipeline and transiently expressed by Agrobacterium-mediated transformation in leaves of Nicotiana tabacum. The first 16 (of 29) cloned putative effector genes were sub-cloned into two independent plant expression vectors to facilitate their expression in either the apoplast or in the plant cytoplasm. Significantly, two of the putative effectors triggered cell death in N. tabacum when expressed in the apoplast, suggesting that they are recognised as potential invasion patterns by these non-host plants. This result suggests the existence of an evolutionarily-conserved effector recognition mechanism in plants in response to pathogenic and symbiotic fungi. To our knowledge, these are the first Epichloë effectors identified to date. Future work will focus on confirming their roles in regulating host compatibility by performing gene deletion and overexpression studies of the two effector candidates and inoculating the mutants into hosts of different compatibilities.

id #569

So you want to build a genome? Completing de novo Escherichia coli genomes with long read data Georgia L Breckell1, Olin K Silander1 1. Massey University, Albany, AUCKLAND, New Zealand Single molecule, long read sequencing (eg Oxford Nanopore or PacBio) have changed the landscape of genome assembly and it is now possible to routinely sequence and assemble bacterial genomes to single contig, reference level. Despite increased read lengths, next generation sequencing platforms have lower accuracy than short read Illumina data, making it advantageous to combine the two data types to produce a hybrid assembly. Several “Hybrid” assembly methods are available to combine short and long read data for assembly, however they each approach the assembly problem uniquely and do not always result in identical assemblies. Here we use short and long read data from 55 Escherichia coli natural isolates to test the assembly accuracy of several popular assemblers. As hybrid assemblies are regularly a single contig, metrics developed for the assessment of highly fragmented short read assemblies, such as N50, are not suitable for near complete hybrid assemblies. We implement a series of 6 metrics to evaluate the accuracy of highly contiguous assemblies: contiguity, read coverage, concordant read mapping, accurate rRNA region arrangement, fraction of short ORFs and accurate plasmid assignment. On the basis of these results, we highlight the advantages and disadvantages of different genome assemblers.

id #572

Effect of single SNPs on expression from E. coli lac operon Marketa Vlkova1, Olin K Silander1 1. Massey Univesity, Albany, AUCKLAND, New Zealand Bacteria often respond to dynamically changing growth conditions by altering transcription. However, little is known about how natural selection has shaped these plastic responses and downstream transcriptional changes. To better understand how selection has shaped these traits we have measured transcriptional responses of E. coli strains isolated from soil samples to exposure to diverse sets of conditions. We use fluorescent reporters together with flow cytometry to quantify changes in responses at the level of the single cell. We characterize mean expression levels among naturally occurring promoter variants in different E. coli genotypic backgrounds. We find that even a single SNP in downstream ORF can have a significant impact on gene expression levels. These results give us a better view on how different aspects of transcriptional plasticity has evolved within a single bacterial species.

id #573

The effect of resource demands in the spatial distribution of phyllosphere-associated bacterial communities Rudolf O Schlechter1, 2, Mitja NP Remus-Emsermann1, 2 1. University of Canterbury, Christchurch CBD, CANTERBURY, New Zealand 2. Biomolecular Interaction Centre, University of Canterbury, Christchurch, Canterbury, New Zealand Plants host a diverse microbiota, in which bacteria are predominant. Although the composition of bacterial communities has been characterised, the mechanisms and factors shaping the structure of these communities are still unclear. Bacteria are able to successfully colonise the inside of plants as well as the surface of below- and above-ground organs, the latter is referred to as the phyllosphere. The phyllosphere encompasses the microbial habitats formed by all the above-ground plant parts, e.g., stems, flowers and leaves. Leaves feature a heterogeneous topography at the micrometre scale, composed of elevations and grooves formed by epidermal cells, which are interspersed with other cell types such as guard cells, trichomes, and hydathodes, offering a set of microsites for bacteria to live in. However, to colonise the phyllosphere, bacteria need to cope with low water and resource availability, UV radiation, and high-temperature fluctuations throughout the day. The fact that the phyllosphere is densely populated by bacteria despite the restricted nature of this environment suggests that deterministic factors, particularly species-species interactions such as resource competition, influence the structure of bacterial communities. To determine the potential of leaf-colonising bacteria to compete for leaf-derived resources, the resource utilisation profiles of bacterial leaf-colonisers from Arabidopsis thaliana were determined in silico via flux balance analysis and validated in vitro. We hypothesise that similarities in resource utilisation are driven by phylogenetic relatedness and these relations could restrict species coexistence at a micrometre scale on the leaf surface. Bacterial strains equipped with different fluorescent proteins were used to design binary communities to determine the effect of resource competition on population development in vitro and on Arabidopsis leaves. Additionally, the spatial distribution of each species was determined on leaves to study coexistence at the micrometre resolution. By studying factors shaping the structure of bacterial leaf communities, we will guide the rational design of biocontrol communities to protect crop and native plants from pathogens.

id #575

Understanding the mechanism of action of the bacteriostatic peptide glycocin F Sean Bisset1, 2, Mark Patchett1, Sung Yang3, Margaret Brimble3, 2, Gillian Norris1, 2 1. Massey University, Palmerston North, MANAWATU, New Zealand 2. Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand 3. School of Chemical Sciences, The University of Auckland, Auckland, New Zealand Bacteriocins are a class of bacterially-produced peptides that possess an inhibitory effect on closely-related strains or species of bacteria. Glycocin F (GccF) is a di-glycosylated, 43 amino acid bacteriocin (glycocin) produced by a strain of the probiotic bacteria Lactobacillus plantarum. GccF contains two N-acetylglucosamine (GlcNAc) moieties required for its anti-bacterial activity, one O-linked through serine 18, and the other S-linked through the C-terminal cysteine located at the end of a flexible tail. Two nested

disulfide bonds are also required for maintaining the folded structure. Whereas most bacteriocins exhibit bactericidal effects on a narrow range of species, GccF displays a very potent and reversible bacteriostatic activity towards a wide range of gram positive bacteria, including vancomycin-resistant strains, making it a potentially useful scaffold for developing new compounds to combat antibiotic resistant bacteria. However, the growth-inhibiting mechanism of action of GccF remains to be elucidated. The recent development of a complete chemical synthesis scheme for GccF enabled the production of GccF analogues, not accessible by recombinant methods, to study the role of different structural features of the peptide. Bioassays were carried out with these GccF analogues using a susceptible strain of L. plantarum. Altogether, these results provide additional evidence for a role of a GlcNAc transporter in GccF’s activity, as well as highlighting the structural aspects of this peptide that are critical for inducing stasis, including specific roles of the Ser18-O- and Cys43-S- linked sugars. In addition to this, transcriptomic analysis of cells treated with GccF were carried out on a susceptible strain of Enterococcus faecalis to identify the molecular cause of this bacteriostasis, along with interaction studies with GccF’s immunity factor to find complexes that are targeted by this peptide. The results of this work will be presented, along with a model mechanism of action.

id #581

Novel Nitrofuran Activating Enzyme in Escherichia coli Van Hung Vuong Le1, Patrick J. Biggs1, David Wheeler2, Ieuan Davies3, Jasna Rakonjac1 1. School of Fundamental Sciences, Massey University, Palmerston North, MANAWATU, New Zealand 2. NSW Department of Primary Industries, Orange, Australia 3. New Zealand Pharmaceuticals Ltd., Massey University, Palmerston North, MANAWATU, New Zealand Widespread prevalence of multi-drug resistant bacteria has necessitated employment of multiple strategies besides conventional discovery and development of novel small-molecule antimicrobial agents. A promising approach is revival of “old” antimicrobials, such as synthetic 5-nitrofuran drugs. Recent epidemiological data reported a very low resistance frequency amongst Escherichia coli isolates to this antimicrobial class, forecasting the increasing importance of its uses to combat antibiotic resistant enterobacteria. Typical examples of this group are furazolidone (FZ), nitrofurantoin and nitrofurazone. Though they had a long history of clinical uses, the understanding about the mechanism of action of 5-nitrofurans remains elusive. They are known as prodrugs that are activated in E. coli by reduction catalyzed by two type-I oxygen-insensitive nitroreductases, NfsA and NfsB. Our preliminary data show that FZ still retained relatively significant antibacterial activity in the nitroreductase-deficient ΔnfsA ΔnfsB E. coli strain, indicating the presence of additional activating enzymes and/or the antibacterial activity of the unreduced form. In this study, we employed genome sequencing, bioinformatic and genetic approaches to characterize the FZ-resistant E. coli mutants which were isolated from spontaneous mutations of ΔnfsA ΔnfsB E. coli cultures and described the involvement of ahpF mutations in FZ resistance. Antimicrobial susceptibility assays indicated that overexpression of ahpF sensitized the nitrofuran-resistant ΔnfsA ΔnfsB ΔahpF strain to all three 5-nitrofurans to the level of the drug-sensitive wild-type strain in both aerobic and anaerobic conditions. The data from enzymatic assays confirm the activation role of AhpF in which this enzyme catalyzes 5-nitrofuran reduction in a manner different from that of two well-studied nitroreductases NfsA and NfsB. The findings open new avenues to counteract E. coli isolates resistant to existing commercial nitrofuran antibiotics, by enhancing cellular expression/availability of AhpF or designing nitrofuran analogues with higher affinity for this enzyme.

id #582

Rapid detection of microbes using fibre fluorescence spectroscopy systems Jessica Chiang2, 1, Julia Robertson2, 1, Cushla McGoverin3, 1, Frederique Vanholsbeeck3, 1, Simon Swift2 1. The Dodd-Walls Centre for Photonic and Quantum Technologies, Auckland, New Zealand 2. Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand 3. Department of Physics, University of Auckland, Auckland, New Zealand Microbial contamination caused by foodborne pathogens leads to a large number of infections in both developed and developing countries. As part of the common goal to meet food safety standards, there is a need to develop a method to rapidly and accurately detect, identify and enumerate pathogenic microbes. A crucial characteristic for rapid enumeration is the ability to detect bacteria in a near real-time manner. Therefore, in pursuit of an innovative method, the utilisation of fluorescence spectroscopy has been investigated using a portable fibre-optic fluorescence spectroscopic device (optrode). We propose the full fluorescence spectra collected by the optrode contains adequate information to report on the bacterial growth phase, viability and species. Previous work has investigated the fluorescence dyes, SYTO 9, propidium iodide, and acridine orange, for their potential to distinguish between live and dead cells mixed at different ratios. The results suggested that live and dead cell ratios can be detected; however, there are still certain limitations, such as for reliable bacterial speciation, a relatively high limit of detection, and the free unbound dyes affecting the fluorescence signal to noise ratio. The aim of this project is to investigate commercially available fluorescent dyes like 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride (CTC) and 5(6)-carboxyfluorescein diacetate (CFDA) that inform on the respiration activity and esterase activity, respectively, of bacteria. These two viability-determining dyes are assessed to establish their efficacy in reporting on viability and speciation. Preliminary results suggest that CTC can be used to quantify viable cells in a mixture of live and dead cells without the help of a secondary dye to identify the dead cells. Three 4-methylumbelliferyl (4-MU) fluorescent dyes are also examined to target the activities of particular bacterial enzymes to assist the identification of bacterial species. Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Bacillus cereus were stained with the above fluorescent dyes and the optrode used to collect fluorescence emission spectra covering the full visible region (350-800 nm) and analysed for species specific signatures. In summary, by using dyes that target specific bacterial enzymes, we obtained encouraging preliminary results to assess bacterial viability and differentiate bacteria.

id #585

Sampling and testing for American Foulbrood in bees and other sample types using novel eDNA and multi-target qPCR methods John Mackay1, Tammy Waters2, Rebecca Hewett1 1. dnature diagnostics & research Ltd, Gisborne, New Zealand 2. Scion Ltd, Rotorua, New Zealand American Foulbrood (AFB) is the most devastating of honeybee diseases and is only one of three diseases subject to national pest management law in New Zealand. It can be estimated that AFB has a minimum direct cost of over 15 million dollars per year to beekeepers. Worse, the incidence of the disease is increasing at an estimated 15% per year. We have developed a triplex qPCR for AFB (two targets plus an internal control) and are using this to screen various samples including bees, wax debris and honey. We are also testing new strategies for rapidly sampling beehives to reduce the time and labour involved in dismantling beehives to collect bee samples. This work aims to predict the development of this disease before clinical signs are apparent and thus help prevent its further spread.

id #589

Protozoan Predation as a Driver of Novel Bacterial Traits; Experimental Evolution Approach Farhad Golzar1, Heather Hendrickson2 1. School of Natural and Computational Sciences, Massey University, Auckland, New Zealand 2. School of Natural and Computational Sciences, Massey University, Auckland, New Zealand Protozoans are common and considered to be a major mortality factor of bacteria in many environments. Protozoan predators have an important role in bacterial ecology and the evolution of adaptive traits such as biofilm-formation, encapsulation and intracellular growth. It has been hypothesised that this predator-prey interaction can select for bacterial pathogenicity towards eukaryotes more generally. To investigate the evolution of novel interactions between organisms that initially had a simple prey-predator relationship, we have established an experimental co-evolution and allowed it to progress for 90-days. The first hypothesis we evaluate using this system is the degree to which predation selects for the evolution of bacterial defence-traits. We chose Pseudomonas fluorescens SBW25 and protozoan predator Naegleria gruberi and serially propagated these together on enriched solid media. Both organisms were periodically preserved and counted by CFU or plaque assays. During the course of co-evolution, we observed the development of many novel bacterial colony morphotypes; distinct wrinkly spreaders (WS), mucoid, volcano and mountain-type etc. To assess how these phenotypes correlate to grazing resistance and bacterial virulence towards the protozoa, we measured the ability of the ancestral protozoa to consume the bacteria in line-tests and plate-test assays. Our preliminary results suggest that at least three of our co-evolved strains; WS, mountain and mucoid-type compare to ancestral SBW25 are highly and six of the other isolates are partially resistant to predation. In twelve cases, we observed that bacteria limit the growth of amoebae and three of these strains; mountain-type (Fold change > 4.6; P < 0.004), WS (Fold change > 2.3; P < 0.01) and fried-egg-type (Fold change = 2.5; P < 0.03) showed higher virulence than WT SBW25 as measured by protozoan mortality against ancestral protozoa. To identify the genetic changes that allowed the bacteria to evolve novel strategies in the face of predation, we conducted population and clone sequencing and I will discuss our ongoing work towards dissecting the molecular basis of these changes in symbiotic state. In the future, I will characterise the primary mutations in the bacteria and determine whether these evolved traits contribute to virulence against multicellular hosts.

id #592

A genetic approach to exploring the phenotypic differences between strains of Lactobacillus fermentum and their effect on the human gastrointestinal tract. Marc Bailie3, 1, 2, Eric Altermann1, 4, Wayne Young1, 4, Nicole Roy1, 2, 4, Warren McNabb3, 2, 4 1. Food Nutrition & Health Team, AgResearch, Palmerston north, New Zealand 2. Riddet Institute, Palmerston north, New Zealand 3. Massey University, Palmerston North, PALMERSTON NORTH, New Zealand 4. The High-Value Nutrition National Science Challenge, Palmerston north, New Zealand Microbes are intrinsic to the wellbeing and development of more complex organisms such as humans. Strain level diversification of microbial species is a naturally occurring phenomenon. These strain level changes can alter the interaction these microbes have with their host, in turn altering the host phenotype. Lactobacillus fermentum is a beneficial but transient member of the human microbiome and is “generally regarded as safe” (GRAS). However, two oral isolates sampled from healthy human volunteers here in Palmerston North exhibit divergent phenotypes. L. fermentum AGR1487 exhibits greater tolerance for bile acids and low pH and utilises different carbohydrates than L. fermentum AGR1485. AGR1487 also increases colon inflammation in germ-free mice and decreases barrier integrity in human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers, an accepted model of the intestinal barrier. Neither inflammation nor disruption of barrier integrity is observed when AGR1485 is cultured in these same environments. We aim to use a genetic approach to unravel the differences between these strains that give rise to their observed phenotypic differences. Finished genomes for both strains were achieved using hybrid assembly, that utilised a combination of PacBio long reads and Illumina short reads. Both genomes were annotated using GAMOLA and manually curated for accuracy. The two genomes have a similar structure, but AGR1487 (1,939,032bp) appears to have a series of deletions resulting in a genome that is 287 kbp smaller than AGR1485 (2,226,862 bp). An analysis of the gene content of AGR1485 and AGR1487 along with 12 other strains of L. fermentum is currently underway. The gene expression of the two strains will be assessed over time while they are in contact with Caco-2 cells in vitro. The role of the gene(s) candidates will be studied using CRISPR/Cas9 to inactivate or complement gene candidates from AGR1485 and AGR1487, and the resulting mutant strains will be evaluated for loss or acquisition of the tight-junction weakening phenotype.

This work will enhance the understanding of bacterial-host interactions and their impact on human well-being and enable screening for these gene(s), should they exist in other “good” bacteria.

1. Anderson, R., Ulluwishewa, D., Young, W., Ryan, L., Henderson, G., & Meijerink, M. et al. (2016). Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons. Scientific Reports, 6(1). doi: 10.1038/srep20318

2. Sengupta, R., Anderson, R., Altermann, E., McNabb, W., Ganesh, S., & Armstrong, K. et al. (2015). Lactobacillus fermentumAGR1487 cell surface structures and supernatant increase paracellular permeability through different pathways. Microbiologyopen, 4(4), 541-552. doi: 10.1002/mbo3.260

3. Anderson, R., Young, W., Clerens, S., Cookson, A., McCann, M., Armstrong, K., & Roy, N. (2013). Human Oral Isolate Lactobacillus fermentum AGR1487 Reduces Intestinal Barrier Integrity by Increasing the Turnover of Microtubules in Caco-2 Cells. Plos ONE, 8(11), e78774. doi: 10.1371/journal.pone.0078774

id #593

Fight or blight: the role of early reactive oxygen species production in the resistance and susceptibility of Camellia blooms to fungal petal blight disease Cade D Fulton1, Nikolai Kondratev1, Matthew Denton-Giles1, Paul P Dijkwel1 1. Massey University, Palmerston North, MANAWATU, New Zealand Petal blight disease is caused in Camellia blooms by the fungus Ciborinia camelliae (Sclerotiniaceae). However, some species of Camellia are resistant to the blight, such as Camellia lutchuensis, while others, including the Camellia ‘Nicky Crisp’ hybrid, are extremely susceptible. To further investigate the mechanism of disease resistance in Camellia, we are investigating an early defence response to pathogen invasion - the rapid accumulation of reactive oxygen species (ROS). ROS is a group of highly reactive signalling molecules that can alter the cellular redox environment of a plant under pathogen attack. ROS can also affect the infectiveness and virulence of the invading pathogen. Specifically, our research quantifies and compares ROS production in C. lutchuensis blooms and in ‘Nicky Crisp’ blooms in their response to C. camelliae infection. Hydrogen peroxide staining assays show that localised ROS production occurs approximately 18 hours earlier in C. lutchuensis, than in the ‘Nicky Crisp’ hybrid after infection. To better understand the role of this difference in the timing of ROS production we scavenged ROS with antioxidants and found that C. lutchuensis’ susceptibility to petal blight slightly increases while ‘Nicky Crisp’ becomes more resistant. This is demonstrated by a comparison of petal lesion sizes after ROS scavenging. These results point toward the role that early ROS production plays in the resistance of Camellia lutchuensis to petal blight and that delayed ROS production in the ‘Nicky Crisp’ hybrid contributes to its susceptibility. To further elucidate the role of early ROS production in petal blight resistance, we hypothesise that upregulation of early ROS producing genes, such as NADPH-oxidases and apoplastic peroxidases, will also occur earlier in C. lutchuensis than in ‘Nicky Crisp’. The elucidation of the cellular oxidative state, effects of antioxidant treatments and associated gene expression in naturally disease-resistant species of Camellia, will provide a new avenue of research to assist with the development of future management strategies of petal blight.

id #594

Detection of bacterial cell death using absorbance spectra Julia Robertson1, 2, Brendan Darby3, Matthias Meyer3, Cushla McGoverin1, 2, Frederique Vanholsbeeck1, 2, Simon Swift1 1. University of Auckland, Auckland 2. The Dodd-Walls Centre for Photonic and Quantum Technologies, Auckland, New Zealand 3. Victoria University of Wellington, Wellington, New Zealand Biophotonics is a multidisciplinary research area encompassing application of light-based technologies to the life sciences and medicine. In the realm of infectious disease research, the majority of the work has focused on utilisation of fluorescent dyes to detect and quantify viability and other physiological states with measurements taken using a fluorometer, flow cytometer, or fluorescent microscope. The available viability fluorescent dyes inform on specific cell processes that reflect a basic aspect of bacterial cell life, such as an intact cell membrane as indicated by membrane integrity. However, application of these dyes to bacteria exposed to a wide range of inimical processes has demonstrated that they are not always adequate for live/dead determination. Investigation of bacterial physiology using absorbance is an under-exploited biophotonics approach due to the limitations imposed by the associated scattering of light from turbid bacterial samples. We have employed a novel, next-generation spectroscopy instrument called the CloudSpec, developed at the Victoria University of Wellington, which permits turbid media to be analysed quickly and accurately. The CloudSpec eliminates the influence of scattering allowing for a direct measurement of absorbance and generation of an absorbance spectrum. We have explored the potential to use CloudSpec derived absorbance spectra to detect near real-time loss of Escherichia coli viability accrued from lethal challenges, which include 70 % isopropanol and the antibiotics, ampicillin and ciprofloxacin. We have also investigated the influence of staining with fluorescent membrane integrity dyes allowing for comparison with measurements taken with a fluorometer developed at The University of Auckland called the Optrode. Preliminary results suggest that there are spectral differences between the absorbance of lethally challenged cells and viable cells for both unstained and stained cell samples. The collected spectra also gave insight into dye excitation and emission characteristics, which complement and supplement corresponding fluorescence measurements taken with the Optrode.

id #597

Identification of the Type VI Secretion System delivery determinants that allow the nosocomial pathogen Acinetobacter baumannii strain AB307-0294 to kill prey bacteria Marina Harper2, 1, John Boyce2, 1 1. Infection and Immunity Program, Monash Biomedicine Discovery Institute , Melbourne, Victoria 2. Monash University, Melbourne, VIC, Australia Antibiotics save millions of lives every year and underpin world healthcare systems at multiple levels. However, infections caused by multi-drug resistant (MDR) bacterial pathogens are now a world-wide medical emergency. Thus, novel antimicrobials are urgently needed. In order to give them a competitive advantage, many predatory bacteria produce unique proteinaceous antimicrobials that are injected into other strains/species via the Type VI Secretion System (T6SS), a complex syringe-like nanomachine with a piercing tip containing three VgrG proteins. The nosocomial pathogen Acinetobacter baumannii strain AB307-0294 encodes a functional T6SS apparatus and we have recently shown that it encodes three different VgrG proteins that each deliver a specific antimicrobial effector. The T6SS effectors produced by A. baumannii strain AB307-0294 include a peptidoglycan hydrolase, a DNase and an effector with unknown antimicrobial action. In this study, we aimed to investigate how each of the Acinetobacter baumannii strain AB307-0294 VgrG proteins precisely interacts with its cognate effector protein to allow efficient delivery. Our experimental methods included the use of bacterial two hybrid reporter assays, interbacterial killing assays and alanine-scanning mutagenesis. Using a combination of these methods, we have identified the regions in the VgrG1 protein and its cognate effector protein required for these two proteins to directly interact and kill prey cells. A complete understanding of the precise interaction mechanisms between the T6SS VgrG tip proteins and their cognate antimicrobial effectors will allow us to in future design hybrid effector molecules with novel antimicrobial activity, delivered by a prey of our choosing to specific target species.

id #604

Gene cluster diversity for indole-diterpene biosynthesis in the grass endophyte Epichloë festucae Taryn Miller1, Wade Mace 1, Paul Maclean1, Barry Scott2, Richard Johnson 1 1. Grasslands AgResearch, Palmerston North, MANAWATU, New Zealand 2. School of fundamental sciences , Massey University , Palmerston North, Manawatu, New Zealand Indole-diterpenes (IDT) are an important class of bioprotective metabolites synthesized by Epichloë festucae in association with Festuca and Lolium spp. The most abundant product in L. perenne infected with strain Fl1 is lolitrem B, a potent tremorgenic mycotoxin responsible for the mammalian syndrome ‘ryegrass staggers’. Molecular cloning and functional analysis has shown that the genes responsible for lolitrem B biosynthesis are organized in three sub-telomeric gene clusters interspersed with transposon relics. These 11 idt genes comprise a set of core genes for the biosynthesis of the first stable intermediate, paspaline, and additional genes that encode products for further decoration of the core molecule. Genome sequence and PCR analysis has shown that there is remarkable diversity in the idt gene profile within E. festucae, resulting in a diversity of IDT products synthesized in planta. The aim of this work is to analyse the idt gene cluster structure in an agriculturally important E. festucae var loli (asexual) strain AR48 and determine whether this explains the observed IDT chemical phenotype. The genome of AR48 was sequenced using Illumina technology and the idt gene cluster analysed. AR48 was found to have all 11 idt genes with conserved synteny to strain Fl1. However, idtP and idtQ (cytochrome P450 monooxygenases) had a lower than expected sequence identity when compared with Fl1. In addition, idtF (DMAT synthase) and idtK (P450 monooxygenase) have SNPs that introduce frame-shift mutations and premature termination of translation. In planta synthesis of lolitriol, terpendole B and lolitrem K, which all lack an additional prenylated cyclic ring found in lolitrem B and E, is consistent with IdtF and IdtK being non-functional. Interestingly, one novel IDT compound was uniquely identified in AR48 infected ryegrass with structure analysed by mass spectrometry. It is the combination of the decorated groups on the paspaline core structure that is novel rather than the placement. The working hypothesis is that IdtP and IdtQ from AR48 have broader substrate binding capabilities due to the sequence divergence that enables the production of a compound with a novel modification combination.

id #605

Artificial plant leaf surface as a surrogate for microbial colonization. Michal Bernach1, 2, Rebecca Soffe2, Volker Nock2, Mitja Remus-Emsermann1 1. School of Biological Sciences, University of Canterbury, Christchurch, Canterbury, New Zealand 2. Department for Electrical and Computer Engineering, University of Canterbury, Christchurch, Canterbury, New Zealand Different surrogate surfaces, varying in complexity and origin, have been used to investigate aspects of microbial life on the plant leaves. However, none of them allows to delve far into the intricacies of plant-microbe and microbe-microbe interactions occurring on leaves. Here, we present consecutive steps undertook, in order to create an artificial leaf platform that can be applied in the studies focused on microbial life of plant leaves. We demonstrate that polydimethylsiloxane (PDMS) can be employed to recreate the topography of leaves of the model plant Arabidopsis thaliana. Our results show that the physicochemical properties of PDMS are comparable to live leaves of Arabidopsis. PDMS neither deteriorates over time, nor influenced bacterial viability compared to isolated leaf cuticles, used as a biological control. Additionally, the quality of surface feature replication is at nanometer precision. PDMS is therefore a suitable material to mimic plant leaf surfaces. Subsequently, we modified PDMS to increase its permeability to water and nutrients by adding excipients at different concentrations. We tested how the modification of PDMS influences pattern resolution, surface properties and characterised the changes in permeability to selected particles (water, fructose and tryptophan) using whole-cell fluorescent bioreporter technology. When fully validated, artificial leaves will enable the implementation of new experimental conditions, that can be precisely controlled. This bottom-up approach will help to deconstruct the interdependencies of physical, chemical and biological events taking place on plant leaves and can help to answer important questions of plant-microbiology and microbial ecology.

id #606

Understanding ciprofloxacin resistance in Pseudomonas aeruginosa Attika Rehman1, Wayne M Patrick2, Iain L Lamont1 1. Biochemistry, University of Otago, Dunedin, New Zealand 2. School of Biological Sciences, Victoria university , Wellington, New Zealand Publish consent withheld

id #607

Characterisation of bacteriocin resistance development by Streptococcus mitis strain I18 Nicholas CK Heng1, Jo-Ann L Stanton2, Jackson Treece2, Muriel Dufour3, Geoffrey R Tompkins1 1. Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand 2. Department of Anatomy, University of Otago, Dunedin, New Zealand 3. University of Otago, Dunedin, OTAGO, New Zealand Members of the genus Streptococcus inhabit a variety of sites in the oral cavity and many species produce proteinaceous antibiotics (bacteriocins). Whilst much is known about bacteriocins and their genetic determinants, less is understood about the mechanisms of bacteriocin resistance. Streptocin STH2, produced by Streptococcus gordonii, is a competence-induced, narrow-spectrum bacteriocin that targets closely-related organisms including strains of Streptococcus mitis and Streptococcus oralis. The primary objective of this study was to understand the proteomic and genetic basis of resistance development by S. mitis I18 to STH2. Total protein extracts from S. mitis I18 and its STH2-resistant derivative I18** were analysed by SDS-polyacrylamide gel electrophoresis, protein bands of interest excised, and their N-terminal amino acid sequences determined. Expression of several proteins was increased in strain I18** of which four were characterised further. Whereas two proteins (12- and 35-kDa) matched cytoplasmic “housekeeping” proteins (histone-like protein HU and ribosomal protein L6, respectively), the other two (20- and 45-kDa) exhibited similarity to putative cell surface components (oligopeptide permease and cellobiose phosphotransferase IIC, respectively). The genomes of I18 and I18** were also sequenced using the newly-available ultra-long read length Oxford Nanopore MinION DNA sequencer. At time of writing, the genome of S. mitis I18 was sequenced fully and comprises a single 2,043,053-bp chromosome. However, annotation of the genome sequence revealed many fragmented genes, e.g. dnaA (the first gene of all bacterial genomes) is split into four reading frames. Homopolymer-related sequencing errors are the likely cause of this gene fragmentation, indicating that nanopore technology may have significantly higher error rates than other next-generation DNA sequencing technologies. The S. mitis I18 genome will be re-sequenced using the Ion Torrent sequencing system, and re-assembled with both MinION and Ion Torrent datasets. This sequencing strategy will also be used with the I18** genome. In conclusion, bacteriocin resistance in S. mitis I18 is associated with overexpression of certain cell surface proteins, and identification of their structural genes will facilitate mutational studies to confirm their involvement. Further direct comparison of the I18 and I18** genomes may reveal more STH2-related resistance factors.

id #610

Species turnover as a mechanism for microbial adaptation to warming Manpreet K Dhami1, Gabriel Moinet2, Ana Podolyan1, John Hunt2, Pete Millard2 1. Biodiversity & Conservation, Manaaki Whenua Landcare Research, Lincoln, New Zealand 2. Ecosystems and Global Change, Manaaki Whenua Landcare Research, Lincoln, New Zealand Microbial respiration, via soil organic matter (SOM) decomposition, is central to global CO2 release. However, little attention has been given to the underlying microbial processes and how they will respond to the projected scenarios of anthropogenic warming. Models that explicitly include microbial processes offer markedly different future SOM stock predictions than traditional models where microbial processes are implicit. Understanding temperature sensitivity of soil microorganisms is therefore a critical prerequisite to accurately estimate future atmospheric CO2. Through empirical short-term shifts in ambient temperature, we tested whether (a) soil microbes exhibit sustained respiration under scenarios of warming, and whether (b) species turnover maybe the prevailing mechanism underlying community-level adaptation to temperature shifts. We measured respiration and RNA activity of microbial communities (bacteria and fungi) across a 7-day period simulating rapid warming and cooling across soils from SOM root exclusion plots (3-month new- and 30-year old). First, we confirmed that the two SOM plots harboured distinct microbial communities, with higher species diversity observed in soil with new SOM. Despite differences in community structure, we found that microbial respiration in both the plots increased with temperature and was explained by increased microbial RNA abundance in soils. Furthermore, we confirmed that while total operational taxonomic unit (OTU) diversity did not shift significantly across our study (DNA profiles), RNA activity varied across different groups of OTUs. We classified these OTUs as “psychrophiles”, “mesophiles” and “thermophiles” based on their temperature responses. Psychrophiles and thermophiles constituted only 5% of total OTU diversity but contributed disproportionately to respiratory function. Our results suggest that temperature-sensitive functional diversity exists within soil microbial communities and that community-level responses may be mediated via species-turnover.

id #613

Biofilm formation by Campylobacter jejuni: complex or simple? Gary Dykes1 1. Curtin Univerity, Bentley, WA, Australia Campylobacter jejuni is well established as a foodborne pathogen of public health concern in most developed nations. While substantial work has been conducted on understanding sources and transmission of C. jejuni, fewer studies have investigated the

strategies used by this species to survive in the environment. Biofilm formation has been touted as being an important mechanism by which C. jejuni do this. With its fastidious growth requirements, the fact that many strains are poor biofilm formers, and a lack of evidence of biofilm formation in the environment, however, this is not a given. We have investigated attachment and biofilm formation by C. jejuni in a number of static and flow model systems which suggest that this pathogen is unlikely to “grow” biofilm in the environment. This despite the fact that some strains are able to grow and form biofilm under aerobic conditions in the laboratory. We have also been developing and using an individual-based cellular automata mathematical model to further investigate the theoretical and practical limits of C. jejuni biofilm formation under a range of conditions. This model also points towards the strong limitations imposed on biofilm formation by environmental conditions. Using data from these systems together with new and available molecular data we suggest an overall framework for the role attachment and biofilm formation may, or may not, play in C. jejuni survival in the environment.

id #615

Superhero Sidekicks: Alternative Host Bacteriophages to prevent American Foulbrood Joanne Turnbull1, Danielle Kok1, Heather Hendrickson1 1. School of Natural and Computational Sciences, Massey University, Auckland, New Zealand American Foulbrood (AFB) is a devastating disease in beehives caused by the bacterial pathogen Paenibacillus larvae. Currently, the only accepted response to AFB in New Zealand is the destruction of the entire hive by burning. The significance of honey bees to export and agricultural industries makes it important for apiarists to avoid AFB infection. We are looking to bacteriophages for an alternative solution in preventing this disease. Bacteriophages (known as phages), the viruses that parasitise bacteria, are generally highly host-specific. They could therefore be key in a preventative measure to protect hives from P. larvae infection without damaging bacterial species that are important to hive health. Previous studies suggest that phage therapy can prevent American Foulbrood, both prophylactically and in treatment of an existing infection. The ABAtE project (Active Bacteriophages for American FoulBrood Eradication) at Massey University is finding and characterising P. larvae bacteriophages that can be used to combat American Foulbrood infection. My contribution to the ABAtE project involves finding related bacterial species to act as alternative hosts for discovering and propagating bacteriophages that can also infect P. larvae. My objectives are to cultivate five alternative species and prepare them for long-term storage, before commencing a protocol for isolating bacteriophages specific to Paenibacillus Ash 1994 (NZ isolate). A draft genome for this host will also be created for comparison with P. larvae. To date, four of these bacterial isolates have been grown on agar streak plates using media specific to each species, and single colony isolates frozen at -80°C. P. Ash 1994 is currently being used for bacteriophage screening via enrichment of filtrate from environmental samples and full-plate plaque assays. I will present my progress on phage discovery and whole genome sequencing of this alternate host strain. Research into these related alternative host species may provide additional resources in preventing P. larvae infection and providing a solution to American foulbrood for New Zealand’s apiculture.

1. Beims, H., et al., Paenibacillus larvae-Directed Bacteriophage HB10c2 and Its Application in American Foulbrood-Affected Honey Bee Larvae. Applied and Environmental Microbiology, 2015. 81(16): p. 5411-5419.

2. Brady, T.S., et al., Bacteriophages as an alternative to conventional antibiotic use for the prevention or treatment of Paenibacillus larvae in honeybee hives. Journal of Invertebrate Pathology, 2017. 150: p. 94-100.

3. Ghorbani-Nezami, S., et al., Phage Therapy is Effective in Protecting Honeybee Larvae from American Foulbrood Disease. Journal of Insect Science 2015. 15(1): p. 84.

4. Yost, D.G., P. Tsourkas, and P.S. Amy, Experimental bacteriophage treatment of honeybees (Apis mellifera) infected with Paenibacillus larvae, the causative agent of American Foulbrood Disease. Bacteriophage, 2016. 6(1).

id #619

Development of methods to identify the cold spots in food treated by Coaxially Induced Microwave Pasteurization and Sterilization (CiMPAS) Aswathi Soni1, Gale Brightwell1, Jeremy Smith2, Richard Archer2, Kris Tong2 1. AgResearch, Palmerston North, NEWZEALAND, New Zealand 2. Massey Institute of Food Science and Technology, Massey University, Palmerston North, NewZealand Coaxially Induced Microwave Pasteurization and Sterilization (CiMPAS) technology is a thermal regime, where food is treated above 120 °C in pressurized cavities using hot water along with microwave treatment at 915 MHz before being rapidly cooled. CiMPAS has the potential to be a successful sterilization method to eliminate pathogens, including bacterial spores, and extend the shelf life of food products while the nutritive qualities and flavours are maintained better than other sterilization regimes like retorting. The process is expected to vary across different food products depending on the composition and other intrinsic factors. This project funded by the food industry enabling technology (FIET) aims to develop rapid validation methods to support regulatory approval of CiMPAS. Mashed potato spiked with D-ribose (1%) and lysine (0.5%) was used as a model food system to see the development of the chemical marker M-2 (4-hydroxy-5-methyl-3(2H)-furanone), which attributes to the browning due to the time and temperature-dependent Maillard reaction. Post-treatment, each tray was divided into 9 parts and the browning development was measured with a colourimeter, after being standardized against a white tile. Statistical analysis was conducted using one-way ANOVA (Post hoc Tukey’s test) to identify the statistically different colourimetric values (M-2 assay). The difference in concentration of M-2 was also confirmed using high-pressure liquid chromatography. The coldest location on each tray was inoculated with 104 CFU/ml of Geobacillus stearothermophilus ATCC 12980 spores followed by CiMPAS treatment. The number of spores before and after each treatment were obtained and an enrichment assay was also conducted to improve the detection limit. The coldest spot on each tray was identified using the extent of browning where M-2 accumulation was always significantly (p<0.05) lower in the cold spot as compared to the hot spot in every tray. Cold spots or the least thermally treated spot on most of the trays showed inactivation equivalent to 4 Log CFU/mL of G. stearothermophilus spores. The results showed that the combined method of M-2 estimation and microbial inactivation can be used as an effective tool for regulatory acceptance and industrial applications of CiMPAS.

id #625

Prevalence and diversity of spoilage bacteria in old and new bedding materials used in dairy sheep farms Alexis N Risson1, 2, Anne Midwinter1, Craig Prichard3, Gale Brightwell2, Tanushree B Gupta2 1. mEpiLab, Hopkirk Research Institute - School of Veterinary Science, Massey University, Palmerston North, Manawatu, New Zealand 2. Food Assurance team, Hopkirk Research Institute, AgResearch, Palmerston North, Manawatu, New Zealand 3. School of Management, Massey University, Palmerston North, Manawatu, New Zealand In a country where cow milk is one of the main pillars of the economy, ewe milk production has been rapidly expanding in Aotearoa. Owing to its nutritional value and gastrointestinal gentleness, ewe milk is considered a high-quality product. In New Zealand, dairy sheep are mostly grazed on pasture but also kept inside under special conditions for periods of time. When housed, sheep lay on bedding which get contaminated with soil or faeces and act as reservoirs of spoilage bacteria and further drivers of raw milk contamination. Two groups of bacteria mainly involved in the quality modulation of dairy are spore-forming and psychrotrophic non-spore-forming bacteria. The former can withstand stringent processing conditions whilst the latter can grow at temperatures lower than 5°C. Moreover, exoenzymatic activity of spoilage bacteria can lead to the development of sensory defects in dairy products. The present study was carried out to investigate the prevalence and diversity of spoilage bacteria in bedding materials used in the sheds. Composite samples from pens having new bedding material (3 weeks old, n=11) and old beddings (3 months old, n=9), were analysed using culture-dependent methods to isolate bacteria growing at different temperatures which were identified by partial 16S rRNA gene sequencing. The study showed the presence of a variety of psychrotrophic non-spore-forming and spore-forming bacteria in the beddings tested, including thermoduric Bacillus and Clostridium species as well as psychrotrophic Pseudomonas. Thermoactinomyces species were the dominant thermophilic bacteria in both bedding types. Protease and lipase activities were screened using plate-based assays. Of the 264 isolates screened, 37% and 48% were proteolytic and lipolytic respectively. These results indicate the presence of known spoilage bacteria in bedding materials, however, the prevalence was found to be less than that documented for bovine studies. This study shows the importance of investigating the diversity of bacterial contaminants from bedding, to help deducing interventions to reduce these bacteria in the farm environment.

id #626

Epigenetic regulation of endophyte infection ability. Yonathan Lukito1, 2, Kate Lee1, Nazanin Noorifar1, Kimberly A Green1, David J Winter1, Tracy K Hale1, Tetsuya Chujo1, Linda J Johnson2, Murray P Cox1, Barry Scott1 1. Massey University, Palmerston North, New Zealand 2. Grasslands Research Centre, AgResearch Limited, Palmerston North, New Zealand We report in this study the first description of Epichloë festucae mutants with an entirely novel phenotype, the inability to colonise its host. E. festucae is a filamentous fungus that is an agriculturally important symbiont of the perennial ryegrass due to the many host fitness enhancements it provides. Non-infectious mutants lack genes for the histone H3K9 and H3K36 methyltransferases, clrD and setB, respectively. The ΔclrD mutant is defective in all forms of H3K9 methylation, while the ΔsetB mutant is specifically impaired in H3K36 trimethylation. Domain analysis and complementation studies revealed that the histone methylation defects in both mutants are responsible for the non-infectious phenotypes of the strains. We did not find a stronger induction of host defence genes by the mutants, indicating that rather than being repelled by the host, these mutants lack the expression of infection-enabling factors. This was supported by the finding that both mutants are capable of infecting the host if co-inoculated with the wild-type strain of E. festucae. We thus hypothesized that the wild-type strain provides the missing infection factors and conducted an RNAseq analysis which identified four small (<200 aa) secreted and entirely uncharacterised E. festucae proteins; a hydrophobin, a carbohydrate-binding protein, and two other proteins with no known domains as strong candidates for these infection factors. The genes for these proteins are located either adjacent to repeats- or AT-rich regions in the genome. A recent study by our group (1) suggests that these regions in the E. festucae genome are enriched for symbiotic and secondary metabolite genes. Functional characterisations are currently underway to confirm if these genes directly confer infection ability in E. festucae and the results will be presented.

Winter, D.J., Ganley, A.R., Young, C.A., Liachko, I., Schardl, C.L., Dupont, P.Y., Berry, D., Ram, A., Scott, B. and Cox, M.P., 2018. Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae. PLoS genetics, 14(10), e1007467.

id #634

Redefining Escherichia coli as a faecal indicator bacterium for water quality assessment purposes. Adrian L Cookson1, 2, Lynn E Rogers1, Rose M Collis1, Megan Devane3, David A Wilkinson2, Rebecca Stott4, Patrick J Biggs2, 5, Jonathan C Marshall2, 5, Janine Kamke6, Gale Brightwell1 1. AgResearch Limited, Palmerston North, New Zealand 2. mEpiLab, Massey University, Palmerston North, New Zealand 3. Institute of Environmental Science and Research Limited, Christchurch, New Zealand 4. National Institute of Water and Atmospheric Research, Hamilton, New Zealand 5. Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand 6. Horizons Regional Council, Palmerston North, New Zealand Escherichia coli naturally occurs in the gastro-intestinal tract of warm-blooded animals and is the preferred indicator of faecal contamination for water quality monitoring. Recent studies however have identified that non-pathogenic cryptic Escherichia species, indistinguishable from E. coli using standard culture methods, may survive and grow in soil and water. Preliminary evidence indicates that these naturalised Escherichia species from environmental sources may contribute to the failure of waterways to meet water

quality standards in the absence of faecal contamination, but standard culture-based methods for E. coli enumeration are unable to delineate naturalised strains. To provide an indication of the prevalence of naturalised Escherichia species, water, sediment, soil, periphyton and faecal samples (n=188) were obtained from environments with contrasting anthropogenic impacts. Sample sites included a headwater stream of the Manawatū River catchment, situated amongst native bush at Pūkaha Mount Bruce, an intensively pest-managed mainland island in Southern Tararua; the confluence of the Mākirikiri Stream and the Mangatera River near Dannevirke; and a constructed wetland intercepting drainage flow from intensive agricultural land use in Waikato. In total 238 separate E. coli isolates underwent whole genome sequencing, including 23 naturalised Escherichia (1 cryptic Escherichia clade IV and 22 cryptic Escherichia clade V). Core and accessory genome analysis separated cryptic Escherichia from E. coli sensu stricto whilst functional analysis indicated that proteins involved in ‘cell motility’ were over-represented in cryptic Escherichia compared to E. coli, whereas those involved in ‘transcription’, and ‘energy production and conversion’ were underrepresented. Amplicon sequencing of the hypervariable gnd gene was also undertaken to provide an indication of the E. coli communities associated with individual environmental samples. Cryptic Escherichia-associated gnd amplicons were found in samples from at least 65% of the natural freshwater sites, but only 27% of samples obtained from the constructed wetland. Cryptic Escherichia species are common components of the environmental microbiome and are readily distinguishable from E. coli sensu stricto at the core and accessory genome level. Subsequent analysis at the functional level provides some indication of traits that may prove advantageous for the maintenance and growth of cryptic Escherichia in environmental/extra-intestinal niches.

id #635

Phosphatidic acid produced by phospholipase D is a key signaling molecule for hyphal cell-to-cell fusion and the mutualistic interaction between Epichloë festucae and perennial ryegrass Berit Hassing1, David Winter1, Ulrike Brandt2, Kimberly Green1, Carl Mesarich1, Carla Eaton1, André Fleissner2, Barry Scott1 1. Massey University, Palmerston North, MANAWATU, New Zealand 2. Technische Universität Braunschweig, Braunschweig , Lower Saxony, Germany Epichloё festucae is a filamentous fungus that forms mutually beneficial symbiotic associations with perennial ryegrass. The fungus systemically colonises the intercellular spaces of aerial tissues to form a highly structured and interconnected hyphal network. In addition, hyphae are able to exit the leaf and form a network on the leaf surface. This interaction is of great agricultural interest due to an increased tolerance of the infected grass to biotic and abiotic stress. Consequently, many studies have been carried out to identify fungal proteins that are required for mutualism. Among those identified to date are proteins involved in chromatin structure, mitogen-activated kinase and superoxide signaling pathways. However, the role of lipid signaling - a process in which the localized production of certain lipid species influences protein activity and localization - in fungal growth and mutualism has not been explored. Here we focus on the role of phosphatidic acid (PA) produced by E. festucae phospholipase D (PLD). Deletion of one of the PLD isoforms, pldB, severely reduced growth of the fungus in axenic culture and microscopic analysis revealed the formation of deformed hyphae and a lack of hyphal cell-to-cell fusion. Employment of a PA biosensor, a fluorescent probe able to detect PA in vivo, demonstrated that PldB is the main producer of PA in E. festucae as it mislocalised in pldB deletion strains. Inoculation of deletion strains into ryegrass seedling resulted in a reduction of plant height accompanied by a significant increase of hyphal biomass. Interestingly, deletion strains were also unable to form hyphal exit structures and thus colonized the plant surface at a reduced rate. Many of the observed phenotypic characteristics resembled those made for E. festucae strains with a disruption in the superoxide producing NADPH-oxidase (Nox) complex. Indeed, staining of superoxide making use of a fluorescent probe and a superoxide sensitive dye revealed differences in the distribution of superoxide. These results highlight the importance of lipid signaling via PA for normal growth and mutualism and indicate a connection to the Nox complex.

id #638

The effect of lactic acid bacteria on the spoilage characteristics of vacuum packaged beef inoculated with Clostridium estertheticum and Clostridium gasigenes Eden Esteves1, 2, 3, Declan J Bolton2, Paul Whyte3, Gale Brightwell1, John Mills1, Tanushree B Gupta1 1. AgResearch, Palmerston North, PALMERSTON NORTH, New Zealand 2. Food Safety, Teagasc Food Reseach Centre, Ashtown, Dublin, Ireland 3. UCD Centre For Food Safety, UCD, Belfield, Dublin, Ireland Vacuum packaging extends the shelf life of meat by 80 to 90 days at -1.5°C. Blown pack spoilage (BPS) of vacuum packaged chilled meat caused by Clostridium estertheticum (C. estertheticum) and Clostridium gasigenes (C. gasigenes) results in ‘blowing’ of the pack, usually with a foul smell and a metallic sheen on the surface of the meat. The main gas produced is carbon dioxide but other gases, such as hydrogen sulphide, are also present. Lactococcus lactis (L. lactis) and Lactobacillus sakei (L. sakei) are homofermentative lactic acid bacteria (LAB) that produce lactic acid as their final fermentation product, that may prevent the development of spoilage bacteria. This study determined the inhibitory effects of high (106 CFU/mL) and low (103 CFU/mL) concentrations of L. lactis 75, L. sakei 44, and L. sakei 63 (previously isolated from vacuum packaged beef) against BPS spores. Beef striploins were inoculated with 10 spores/ml of C. estertheticum and C. gasigenes, vacuum packaged and stored at -1.5 and 2 °C for 90 days. At -1.5°C, vacuum packs inoculated with spores only (control), showed initial signs of spoilage after 21 days with fully blown packs after 63 days of storage. In packs treated with L. sakei 44 (high and low inoculum), spoilage was extended by 28 days and ‘blowing’ of packs extended by 7 days compared to the control. At 2°C, vacuum packs inoculated with spores only, showed initial signs of BPS after 14 days with fully blown packs after 56 days of storage. In packs treated with L. sakei 63 (low inoculum), the time to spoilage was extended by 21 days with packs fully blown after 56 days. Vacuum packs treated with a high inoculum of L. sakei 63 however, showed no delay in spoilage. There was no difference observed in packs treated with L. lactis 75 and BPS spores at both temperatures. These findings suggest L. sakei may have a protective effect against BPS Clostridia on beef.

id #641

Diversity of Salmonella isolates sourced from poultry in Sri Lanka Nilukshi Liyanagunawardena2, 1, Anne Midwinter1, Ji Zhang1, Palika Fernando2, Ruwani Kalupahana3, Nigel French1, Jackie Benschop1 1. mEpiLab, School of Veterinary Science, Massey University, New Zealand 2. Veterinary Research Institute , Peradeniya, Sri Lanka 3. Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka The present study was designed to understand the population structure of Salmonella including phenotypic and genotypic diversity of isolates, in order to inform control strategies for poultry salmonellosis in Sri Lanka. The study included a cross-sectional survey conducted from July to December 2017 in broiler farms and associated hatcheries from poultry-dense areas as well as an outbreak study (from 2010 to 2018), based on isolates and metadata from poultry salmonellosis outbreaks. Altogether 164 pure Salmonella isolates; 128 from the cross-sectional survey and 36 from farm outbreaks, were included following PCR. High-quality genomic DNA was extracted, sequencing libraries prepared and assessed for quality before being sent for Illumina sequencing. The Salmonella in Silico Typing Resource (Sistr) identified fifteen different serovars of Salmonella enterica belonging to diverse serogroups such as B, C1, C2-C4, D1, E1 and E4. The majority of Isolates comprised Kentucky (44, 27%), Enteritidis (37, 23%) and Typhimurium (22, 13%) while Bareilly, Gallinarum, Corvallis, Braenderup, Butanatan, Senftenberg, Paratyphi B var. Java 1, Chester, Durban, Virchow, Weltevreden and Tennessee were present in low numbers. Notably, Enteritidis, Gallinarum and Typhimurium were responsible for more than 83% (30/36) of outbreaks while serovars Tennessee and Gallinarum could be seen only among outbreak isolates. According to Multi Locus Sequence Typing (MLST) there are 18 different sequence types (STs) while three isolates clustered as a new ST. The latest version of the program fast-GeP is underway to perform whole-genome MLST analysis for the acquired genomic assemblies and the study is being continued with further bioinformatics-based analysis on the genetic distances of the isolates. The level of antimicrobial resistance in Salmonella will also be determined against important human and veterinary antibiotics using disk diffusion method.

id #642

Genomic features of UVC resistant and UV sensitive bacteria from the Namib Desert Elizabeth Buckley1, Brent Seale1, Colleen Higgins1, Kevin Lee1 1. AUT, Auckland, AUCKLAND, New Zealand Ultra-violet C (UVC) is a mutagen that causes microbial deoxyribose nucleic acid (DNA) damage through the formation of photoproducts between adjacent thymine, or cytosine bases. To survive this damage, microorganisms require mechanisms to remain active during, and following, UV exposure. Top-soil samples were collected from high UV locations in the Namib Desert. Culturable bacteria were isolated onto nutrient and Reasoner’s 2A agar and screened for UVC resistance. Two UVC resistant (B2 and B4) and two UVC sensitive (E2 and E5) organisms were selected for further analysis based on 16S rRNA phylogeny. Through 16S rRNA Sanger sequencing, B2 was identified as Arthrobacter, and B4, E2 and E5 were identified as Pseudarthrobacter. To better understand how these organisms have adapted to the harsh environmental conditions of the Namib Desert, these isolates were characterised metabolically and at the whole genome level. Draft genomes of the Namib isolates were assembled using SPAdes v3.12.0 and annotated using RAST and Prokka. A bioinformatic pipeline utilising Mauve, OrthoANI and PhyloPhlAn revealed challenges with assigning accurate taxonomy based on the 16S rRNA gene with the Arthrobacter and Pseudarthrobacter genera. Based on a combination of phenotypic and phylogenetic analysis, it appears that all four Namib isolates are genetically distinct from the available reference genomes.

id #643

Soothing tummy bugs with… bugs? Edible NZ insects modify the human gut microbiome in an in vitro model Wayne Young1, 2, 3, Sai Arojju4, Elizabeth Rettedal1, Mark McNeill5, Nigel Bell6, Jessica Gathercole7, Penny Payne8 1. Food Nutrition & Health, AgResearch, Palmerston North, New Zealand 2. High-Value Nutrition, National Science Challenges, Auckland, New Zealand 3. Riddet Institute, Massey University, Palmerston North, New Zealand 4. Forage Genetics, AgResearch, Palmerston North, New Zealand 5. Biocontrol and Biosecurity, AgResearch, Lincoln, New Zealand 6. Soil Biology, AgResearch, Hamilton, New Zealand 7. Proteins and Metabolites, AgResearch, Lincoln, New Zealand 8. People and Agriculture, AgResearch, Hamilton, New Zealand In the search for alternative sources of protein, edible insects present a viable substitute or supplement to meat and dairy. Insects are composed of 30-70% protein on a dry matter basis and are more efficient in converting feed to bodyweight than traditional livestock. However, beyond protein, insects are also composed of chitin, which may have the potential to improve gut health by acting as a prebiotic. Therefore, we investigated the ability of different NZ insects to modify the human gut microbiome using an in vitro model. Black field cricket nymphs, grass grub larvae, and wax moth larvae were subjected to an in vitro digestion and then dialysed to simulate removal of nutrients through upper gastrointestinal tract digestion. The retentate, representing the left-over digestion resistant constituents, was then fermented in triplicate batch cultures inoculated with pooled faecal material from three adult donors for 4h, 7h and 15h. Batch cultures inoculated with faecal material, but without added substrates were also set up to act as PBS controls. At the end of each incubation period, the culture microbiome was analysed by 16S rRNA gene amplicon sequencing. Analysis of faecal cultures showed striking differences in community composition (permutation MANOVA P<0.01). Each type of digested insect led to significant differences in a wide range of taxa. Among these differences, black field cricket and wax moth

increased the prevalence of the Bacteroides three-fold compared to controls (black field cricket 14%, wax moth 11%, PBS 4%; FDR <0.01), and increased Lachnoclostridium over four-fold (black field cricket 3.44%, wax moth 5.07%, PBS 0.78%; FDR <0.01). Grass grub also increased Faecalibacterium proportions 1.6-fold over controls (grass grub 16%, PBS 10%, FDR <0.01). Our results clearly show that edible NZ insects have the potential to modify the human gut microbiome. The increase in Faecalibacterium from digested grass grub is noteworthy as this bacterium is widely regarded to be beneficial for health. Hence, further studies to assess impacts of insects on gut health are warranted.

id #646

Sleeping sweetly with kiwifruit Kerry L Bentley-Hewitt1, Carel Jobsis1, Duncan I Hedderely1, Janine Cooney2, Tania Trower2, Shanthi Parkar1 1. The New Zealand Institute for Plant & Food research, Palmerston North, MANAWATU, New Zealand 2. Physiological Chemistry, The New Zealand Institute for Plant & Food research, Hamilton, Waikato, New Zealand Consumption of two kiwifruit one hour before sleep has been shown to improve sleep quality and duration in adults. The gut has been referred to as a "second brain" due to the extensive vagal neural connections between the gut and the brain. Neuroactive bioamines such as serotonin, gamma-aminobutyric acid and melatonin that accumulate in the gut are known to modulate gut-brain pathways that are associated with sleep. New evidence indicates that gut microbiota harbour the tryptophan decarboxylase gene which contributes up to 17% of the human gut metagenome. The end-product tryptamine is a precursor for sleep-inducing serotonin. This project has used an in vitro approach to investigate if kiwifruit can influence the regulation of bioaminergic pathways. We have investigated the effects of gut microbial metabolism on bioaminergic pathways by analysing bioamine concentrations following kiwifruit fermentation. We have collected 16S sequencing data from bacteria post fermentation and will present research on correlations between bacterial profile and bioamine data. We will also present gene expression data from a colon cell line that was exposed to kiwifruit fermenta to investigate effects on bioaminergic pathways, inflammatory-related genes and circadian genes. Lastly, we will present research on correlations between bacterial profile, gene expression and bioamine concentrations.

id #648

Characterisation of RXLR effectors from the kauri dieback pathogen Phytophthora agathidicida Yanan Guo1, Pierre-Yves Dupont2, Carl H Mesarich3, Preeti Panda4, Nari M Williams4, Rebecca L McDougal4, Rosie E Bradshaw1 1. School of Fundamental Science, Massey University, Palmerston North, MANAWATU, New Zealand 2. Institute of Environmental Science and Research, Christchurch, New Zealand 3. School of Agriculture and Environment, Massey University, Palmerston North, New Zealand 4. Scion, Rotorua, New Zealand Kauri (Agathis australis), an iconic native tree species of New Zealand, is threatened by dieback disease, caused by the oomycete pathogen Phytophthora agathidicida. Preliminary screening results suggest that disease tolerance to dieback is present in kauri populations. However, little is known about how P. agathidicida interacts with kauri at the molecular level. In this study, RXLR effectors from P. agathidicida, which are small proteins secreted from the pathogen and translocated into the host cell cytoplasm to promote host colonization, typically by modulating host immune responses, were identified and characterised. Altogether, 75 RXLRs were analysed using an Agrobacterium-based screening assay in Nicotiana spp. Eight RXLRs triggered cell death, suggesting that they are recognized by cognate immune receptors in Nicotiana. Of these, PaRXLR24, an orthologue of the Phytophthora sojae RXLR Avh238, was studied further and amino acids required for cell death-inducing activity were identified. Another effector, PaRXLR40, was found to suppress PaRXLR24-triggered cell death and Phytophthora infestans Avr3a-potato immune receptor R3a-triggered cell death. Both PaRXLR24 and PaRXLR40 are highly expressed in planta. Potential NB-LRRs involved in PaRXLR24 recognition in N. benthamiana were identified using a RNA interference hairpin-based library. In future, RXLRs will be screened in kauri to identify their host targets and, if present, their corresponding immune receptors. This will improve our understanding of how P. agathidicida and kauri interact at the molecular level, and will ultimately help inform dieback resistance breeding or selection programmes.

id #650

Identifying molecular invasion patterns from the kauri dieback pathogen, Phytophthora agathidicida Ellie L Bradley1, 2, Preeti Panda3, Rosie Bradshaw4, 2, Carl H Mesarich1, 2 1. School of Agriculture and Environment, Massey University, Palmerston North, New Zealand 2. The Bio-Protection Research Center, Massey University, Palmerston North, New Zealand 3. New Zealand Forest Research Institute (Scion), Rotorua, New Zealand 4. School of Fundamental Sciences, Massey University, Palmerston North, New Zealand Kauri, Agathis australis, is an ancient conifer species endemic to New Zealand, where it is both culturally and ecologically important. Kauri is considered a threatened species due to the emergence and spread of kauri dieback disease. Kauri dieback is caused by the pathogenic oomycete, Phytophthora agathidicida, which infects trees through the roots and restricts nutrients to the upper parts of the tree, causing bleeding lesions, leaf chlorosis, and eventual crown death. As the disease continues to spread throughout kauri forests in the northern North Island, encouraging research has indicated there may be natural tolerance to the disease within the kauri population. This tolerance is likely governed, in part, by the plant immune system, which is activated upon recognition of pathogen invasion patterns. These invasion patterns include microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and effectors (proteins required for host colonisation), which are recognised at the plant cell surface by plant immune receptors. My research focuses on a group of extracellular P. agathidicida proteins called carbohydrate-active enzymes (CAZymes). While CAZymes are known to have a vast array of roles in species of all kingdoms of life, work in other pathogenic systems has identified potential roles in pathogenicity and virulence. My research seeks to catalogue the CAZymes present in P. agathidicida, then determine whether they may be recognised by immune receptors in model plant species as well as kauri, using

Agrobacterium- and protein-based infiltration assays, respectively. Several CAZymes have now been identified that activate the plant immune system in the model plant Nicotiana benthamiana, and these will be functionally characterised to understand their role in the plant–pathogen interaction. The CAZymes with the highest level of expression during infection of kauri roots and leaves will be used to screen for resistant kauri plants, identify the plant immune receptors responsible for recognition, and subsequently inform kauri breeding or selection programs to help ensure long-term survival of the species.

id #658

Food Safety Microbiology: Under the MPI-scope Helen Withers1, Anne-Marie Perchec Merien1 1. MPI, Wellington, (SELECT), New Zealand High quality science-based evidence underpins and, in fact, is pivotal for Ministry for Primary Industries (MPI) decision-making. To do this MPI not only relies on existing data derived from research studies performed locally and internationally, but on specific research projects that are commissioned and coordinated by MPI scientists. Microbiologists can be found throughout MPI and within New Zealand Food Safety (NZFS) in a wide range of roles, including critically in the Food Risk Assessment (FRA) team, alongside toxicologists and risk analysts. The FRA team uses both academic-based and industry-focused research to undertake food safety risk assessments to inform governmental risk managers and industry sector decision making. This work ensures that food produced or imported into New Zealand is safe to consume, protecting public health. In addition, FRA research activities support not only the development of domestic standards but also those required for export ensuring that our food exports comply with overseas market requirements. Importantly, FRA science can be used to defend New Zealand industries against unwarranted requests from overseas markets. Although the lines are somewhat blurred, science-based research can be divided into three broad areas: academic, industry or applied and regulatory. To illustrate how regulatory-based research works and how microbiological data from academic and industry-focused research can inform MPI risk management decisions, we will present two case studies from different food sectors, namely meat and produce. For red meat, we will show how different types of microbiological data are used to support Good Hygienic Processing (GHP) down on the processing floor and support approximately $8 billion worth of exports annually. While for produce, we will describe the process used to rank the microbiological risks associated with the consumption of imported fruit and vegetables according to a semi-quantitative multi criteria model. As well as outlining the risk assessment parameters, insight will also be given on how scientific uncertainty is quantified.

id #660

Metagenomic analysis and culture-based methods to examine the prevalence and distribution of antibiotic resistance on two New Zealand dairy farms Rose M Collis1, 2, Patrick J Biggs2, 3, Sara A Burgess1, 2, Anne C Midwinter2, Adrian L Cookson1, 2 1. AgResearch Ltd, Hopkirk Research Institute, Palmerston North, New Zealand 2. Molecular Epidemiology and Veterinary Public Health Laboratory, School of Veterinary Science, Massey University, Palmerston North, New Zealand 3. School of Fundamental Science, Massey University, Palmerston North, New Zealand Antimicrobial resistance is a global threat to human and animal health, with the misuse and overuse of antimicrobials being suggested as the main drivers of resistance. In a global context, New Zealand is a relatively low user of antimicrobials in animal production. However, antimicrobial usage in livestock is a growing consumer and regulator concern that impacts both public health and New Zealand’s food production systems. Currently, antimicrobial usage on dairy farms and its potential for driving the maintenance and spread of antimicrobial resistance genes within the environment, is unknown. This study, focusing on two New Zealand dairy farm environments over an 18-month period, aims to determine the prevalence and distribution of (i) AmpC and extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E), utilising culture-based methods, and (ii) antimicrobial resistance genes, utilising a metagenomic-approach. Here we present our preliminary results based on screening farm environmental samples including bulk tank milk, farm dairy effluent, faeces and soil for AmpC and ESBL-E. A key question is whether the use of antibiotics on the two targeted New Zealand dairy farms will affect the prevalence of antimicrobial resistance genes associated with dairy cattle, animal products and the farm environment. The outcomes from this research will improve our understanding of the current baseline levels of antimicrobial resistance on two New Zealand dairy farms which is essential for assessing the burden of antimicrobial resistance in agricultural environments.

id #662

Motile organism in and near exclusion zone water Paul Demchick1 1. Real World Education, Palmerston North, New Zealand Others have reported properties of water on the order of 1 mm away from certain interfaces that are substantially different from the properties of bulk water. One of the readily observable properties is that small particles are excluded from that area. Therefore, some have referred to this area as an “exclusion zone”, and have referred to the water in that zone as “exclusion zone water” (“EZ water”). Physical chemistry models proposed to account for this observation have been controversial. The exclusion zone excludes particles of diverse chemical types. However, previous work has been conducted with particles for which the exclusion required very low amounts of energy (i.e., there was little force opposing exclusion). This study used motile microbes in addition to non-living particles to explore the nature of the exclusion zone. Diverse motile organisms (eukaryotic and prokaryotic) were convincingly, but not

absolutely, excluded by the exclusion zone. Interestedly, those cells that did enter the exclusion zone had very similar motility patterns (including speed) as those same organisms in bulk water. This would not be expected if the exclusion zone was a material of uniform properties that were different from the properties of bulk water, but is consistent with the exclusion zone having gaps in which bulk-type water is present. For these studies, exclusion zones were formed using various interfaces. These included Nafion 117 (sulfonated tetrafluoroethylene) and other materials in water. The geometry of the interface was found to strongly influence the effect on the micro-organisms near that interface. In short, a straight, smooth interface did more to exclude motile organisms than other interfaces of the same materials.

1. Hwang SG, Hong JK, Sharma A, Pollack GH, Bahng G. Exclusion zone and heterogeneous water structure at ambient temperature. PLoS One. 2018 Apr18;13(4):e0195057.

2. Chen CS, Chung WJ, Hsu IC, Wu CM, Chin WC. Force field measurements within the exclusion zone of water. J Biol Phys. 2012 Jan;38(1):113-20.

id #664

Erysipelothrix rhusiopathiae in islands used for conservation translocations, New Zealand Manjula Jayasinghe1, Anne C Midwinter1, Emilie Vallee1, Wendi Roe1, Charlotte Bolwell1, Brett Gartrell1 1. School of Veterinary Science, Massey University, Palmerston North, New Zealand Erysipelothrix rhusiopathiae, the causative agent of the disease erysipelas is a gram-positive bacillus, ubiquitous in nature, and a commensal in diverse species of animals. Erysipelas in birds is characterised by either acute, fulminating infection or more rarely as a chronic infection. In New Zealand, E. rhusiopathiae has killed endangered birds on offshore islands, including kākāpō, takahē and kiwi. The source of infection is uncertain and the prevalence of E. rhusiopathiae among wild birds is currently unknown. The specific objective of this project was to conduct targeted surveys of seabirds that visit two of New Zealand’s offshore islands used for kākāpō conservation with the goal of determining the prevalence of E. rhusiopathiae. Live and dead seabirds on two islands were sampled and investigated for the presence of E. rhusiopathiae from October to December 2018. From the dead birds, the organism was isolated by culturing the bone marrow on selective agar and identified using, Matrix-assisted Laser Desorption/Ionization with Time-of-flight Mass Spectrometry (MALDI–TOF-MS). Prevalence of E. rhusiopathiae was calculated and compared between predominant species in each island and also between the islands. Blood samples from live birds were subjected to PCR with Erysipelothrix spp. specific primers. Prevalence of E. rhusiopathiae in dead seabirds of Whenua Hou (Codfish Island) and Hauturu (Little Barrier Island) were 3.38% (n=86) and 11.3% (n=44) respectively. On Whenua Hou, E. rhusiopathiae was detected in sooty shearwater (6.0%, n=34) and mottled petrel (2.7%, n=36) while it was detected only in Cook’s petrels (13.51%, n=37) on Hauturu. Two species of live seabirds were sampled and one out of 50 mottled petrels (2.0%) was positive by PCR for the presence of Erysipelothrix spp. Findings indicate the presence of E. rhusiopathiae in of burrowing seabirds from both islands studied. Observed prevalence was different between locations and among different species of seabirds. Further investigations will focus on comparing the genomic diversity of these E. rhusiopathiae isolates with the isolates from endangered native bird species to identify the reservoir diversity of this pathogenic bacterium in islands used for conservation purposes in New Zealand.

id #665

The microbiome in threatened species conservation Annie G West1, Mike W Taylor1 1. School of Biological Sciences, University of Auckland, Auckland, New Zealand As global biodiversity continues to reduce at an alarming rate, threatened species are increasingly being brought under intensive management or into captivity. However, current conservation management programmes are often impeded by poor animal health and low reproductive success. Microorganisms play vital roles in the growth and maintenance of healthy multicellular organisms, including neurological and immune system development, gut nutrition, and pathogen defence. The microbiome refers to the vast community of micro-organisms, and their collective genes, that live on and within a host organism. An imbalance or breakdown of the microbiome may in some cases be associated with severe negative health consequences for the host. Factors such as habitat degradation and transition into captive breeding programmes can significantly alter the microbiome of threatened species, though the effects of such microbial community changes on health, fitness and ultimately survival of the animals remain poorly understood. New Zealand is currently making significant headway in this area with our research on the gut microbiome of the critically endangered kakapo, a flightless, endemic parrot. 16S rRNA gene short-read amplicon sequencing revealed a low-diversity bacterial community that is frequently dominated by what appears to be a single species of Escherichia/Shigella. Currently, nothing is known about the strain-level diversity of this dominant bacterial lineage within the kakapo gut, which may vary with host genomic traits and is important to document if we wish to understand how these organisms may influence kakapo health. Though community composition differed significantly between individual kakapo, we have yet to find any concrete significant effects despite testing for geographic location, age, medical treatment, captivity and hand rearing, and supplemental feeding habits. The abundance of some key operational taxonomic units (OTUs) was correlated with antibiotic treatment and captivity which remain to be explored. Microbiome research is now more pressing than ever after the recent outbreak of Aspergillosis that tragically resulted in the deaths of several adult and juvenile birds. This talk aims to provide an overview of global trends in conservation-related microbiome research, and how we are applying these principles locally to help save the enigmatic kakapo.

id #668

Initiation of the pathogenic sexual cycle of grass-endophytic Epichloë fungi is characterised by conserved changes to gene expression and production of pyrrolodiazepine alkaloids Daniel Berry1, 2, Kate Lee1, 2, Yan-Ni Shi3, Katrin Grage1, 2, Arvina Ram1, Padmaja Nagabhyru4, Christopher L Schardl4, Carolyn A Young5, Paul P Dijkwel1, 2, Helge B Bode3, Barry Scott1 1. School of Fundamental Sciences, Massey University, Palmerston North, New Zealand 2. Bioprotection Research Centre, Massey University, Palmerston North, New Zealand 3. Fachbereich Biowissenschaften, Molekulare Biotechnologie, Goethe Universität Frankfurt, Frankfurt am Main, Germany 4. Department of Plant Pathology, University of Kentucky, Lexington, KY, USA 5. Noble Research Institute, LLC., Ardmore, OK, USA Fungal species from the genus Epichloë are symbionts of grasses that form systemic networks of endophytic and epiphytic hyphae throughout host aerial tissues. Infections by Epichloë spp. are usually asymptomatic, and are generally regarded as mutualistic due to the production of fungal bio-active secondary metabolites that protect the host against biotic stresses. Many Epichloë spp. transmit vertically through the host seed, and this is the only method of dissemination for asexual Epichloë isolates. However, sexual Epichloë spp. are not entirely mutualistic – initiation of the Epichloë sexual cycle is characterised by formation of a pre-sexual “stroma”, which is a dense mycelium that encases and sterilises an emerging host inflorescence. This is a pathogenic interaction, with host nutrients intended for inflorescence development subverted to fuel the fungal sexual cycle. The Epichloë sexual cycle culminates in ejection of ascospores from the mature stroma, which mediate horizontal transfer to uninfected plants. Stroma formation frequency varies between different sexual Epichloë spp., and many can undergo both horizontal and vertical transmission simultaneously on different reproductive tillers from the same plant. We used RNAseq to generate fungal transcriptomes for emerging stromata and vegetative stem tissue samples taken from three different Epichloë-host associations. By comparing differentially-expressed genes across these three associations, we define a robust set of co-regulated orthologs that characterise the switch from mutualistic to pathogenic growth. Interestingly, the secondary metabolism gene clusters that encode synthesis of known bio-protective molecules are strongly and universally downregulated in stromata, as are a number of other uncharacterised secondary metabolism gene clusters. However, a single biosynthetic cluster was identified that exhibited conserved stromata-specific expression. This five-gene “SMT” cluster, which is anchored by a NRPS-like adenylate-forming reductase, was successfully expressed in the heterologous host Penicillium paxilli. This enabled identification of the SMT pathway product, which belongs to a novel class of pyrrolodiazepine alkaloids, and also allowed characterisation of the SMT reaction order and pathway intermediates.

id #669

The role of cell surface modifications in the morphological differentiation of infection structures by the apple scab fungus, Venturia inaequalis Mercedes Rocafort1, Joanna K. Bowen2, Kim M. Plummer3, Rosie E. Bradshaw4, Carl H. Mesarich1 1. Bio-protection Research centre, School of Agriculture and Environment, Massey University, Palmerston North, Manawatu, New Zealand 2. New Zealand Institute for Plant and Food Research, Auckland, New Zealand 3. Department of Animal, Plant and Soil Sciences, Centre for AgriBiosciences, La Trobe University, Melbourne, Victoria, Australia 4. Bio-protection Research centre, School of Fundamental Science, Massey University, Palmerston North, Manawatu, New Zealand Scab disease, caused by the biotrophic fungal pathogen Venturia inaequalis (Vi), is the most economically important disease of apple worldwide. Vi colonizes the sub-cuticular space of apple leaves and fruits, where it develops the infection structures runner hyphae and stromata. Runner hyphae are wider than regular tubular hyphae found on the plant surface, and are often fused lengthwise, while stromata are multi-layered pseudoparenchymatous structures. Both give rise to asexual conidia, but are also likely required for nutrient acquisition and the secretion of effectors, which are molecules required to promote host colonization. As some fungal cell wall components (e.g. chitin and β-glucan) are well known elicitors of plant defence, and because fungal cell surface modifications required for successful colonization of the sub-cuticular space are unknown, we are particularly interested in understanding how Vi, through changes in its cell surface, is able to differentiate sub-cuticular runner hyphae and stromata in absence of strong host immune system activation. Notably, Vi also produces runner hyphae and stromata in the absence of the host, inside cellophane membranes (CMs). Thus, we are also interested in determining whether CMs can act as an artificial host environment for studying these changes. We have used carbohydrate-specific probes and antibodies, in conjunction with confocal microscopy, to study the localization of chitin, β-1,3-glucan, chitosan and α-1,3-glucan on the cell wall surface of infection structures developed in planta and CMs. So far, we have observed that chitin is restricted to fungal septa and appressoria (in planta and CMs), chitosan is present on the surface of infection structures (in planta and CMs), and that around infection structures (in CMs) and tubular hyphae (in planta and CMs), β-1,3-glucan forms part of a mobile matrix. Using antibodies in conjunction with confocal microscopy, we have also tracked the localization of a highly expressed candidate effector protein, Cin1. Strikingly, Cin1 is present in a mobile matrix around infection structures (in CMs) and tubular hyphae (in planta and CMs). Cin1 will be investigated for its role in host colonization by gene deletion. Ultimately, it is hoped that our research can be used to inform durable scab control programmes.

id #672

Surfactant production by epiphytic bacteria and its contribution to hydrocarbon degradation Simisola O OSO1, Mitja Remus-Emsermann1 1. University of Canterbury, Christchurch, CANTERBURY, New Zealand Leaves are covered by waxy cuticles composed of very long hydrocarbons and are colonised by a variety of so-called epiphytic bacteria. In the here presented study, phylogenetically different epiphytic bacteria were tested for their ability to grow on diesel and also produce surfactants. Our results indicated that more than 50% of the strains tested were able to utilise diesel for growth and 70% producing surfactant. All strains that were able to utilise diesel were also able to produce surfactants i.e. 75% of the strains producing surfactants are also able to degrade hydrocarbons. Our findings suggest that a high proportion of epiphytic bacteria are able to degrade hydrocarbons and also produce surfactants. However, it is thus far unclear those bacteria utilise hydrocarbons originating from the cuticle of the living leaves. We selected the genetically amenable strains Pseudomonas citronellolis P3B5 and performed a

random transposon mutagenesis approach to knock out its surfactant genes. The effect of the knockout on hydrocarbon degradation was characterised using growth assays, gas chromatography-flame ionization detector and gas chromatography-mass spectrometry.

id #675

The gut microbiota in New Zealand children with autism spectrum disorder Alex Parker1, Giselle Wong1, Sally Graham2, Amanda Retter2, Johanna Martin2, Pratima Giri2, Mike Taylor1 1. University of Auckland, Grafton, AUCKLAND, New Zealand 2. Kidz First Hospital, Auckland, New Zealand Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that typically involves social impairment and restricted, repetitive behaviours. In many cases, ASD will also present with gastrointestinal (gut) problems. Our study investigates the association between gut microbiota (bacteria and fungi) composition and ASD in a cohort of New Zealand children. Neurotypical siblings were also recruited as a control cohort. DNA was extracted from faecal samples and PCR was used to amplify the 16S rRNA gene and ITS2 genomic region from bacteria and fungi, respectively. Sequencing was conducted using Illumina MiSeq and the data were analysed to reveal the bacterial and fungal communities present. While overall bacterial and fungal community compositions were similar between the ASD and neurotypical sibling cohorts, there were some differences in specific bacterial and fungal taxa. These findings were collated with metadata concerning factors which may influence microbial communities, such as diet and antibiotic use, to provide greater indication of whether there is a microbial component associated with ASD. We also investigated the functional potential of ASD and neurotypical gut microbiotas using PiPhillin and FUNGuild software, with significant similarities and differences emerging. Our 16S rRNA gene sequence data were then analysed together with comparable overseas data using a common bioinformatics pipeline. This demonstrated overall compositional associations with ASD but also highlighted differences between studies. Overall this project provides novel insights about the ASD microbiota in New Zealand children; an improved understanding of the gut microbiota in children with ASD should ultimately help shape future gastrointestinal treatments such as prebiotics, probiotics and faecal microbiota transplants.

id #678

The evolution of spherical cell shape can be recapitulated in the lab using an experimental evolution approach. Heather Hendrickson1, Richard Yulo1 1. School of Natural and Computational Sciences, Massey University , Auckland , New Zealand Phylogenetic analysis of bacterial lineages suggests that rod-like shape is ancestral in bacteria. In most cases, the ancient actin structural homolog in bacteria, MreB appears to be lost early on during this transition. Investigating the causes and consequences of this loss is made difficult in many bacterial model systems because this gene is essential. MreB deletion is possible however in Pseudomonas fluorescens SBW25, though relative fitness of this population is decreased. Using this unfit MreB deletion population as a starting point, an experimental evolution approach was used in 10 separate lineages for approximately 1000 generations. This was sufficient to allow all of these lineages to reach fitness that is on par with the ancestral wild type cells and the resulting bacteria are spherical P. fluorescens SBW25 cells. I will describe the genetic underpinnings of the most common mutations that we observed in this experiment and how these mutations relate to the changes inferred by comparative genomics in extant spherical lineages.

id #682

Ileal microbiota and fermentative capacity can be modified through dietary intervention in a pig model Anika Hoogeveen1, 2, Paul Moughan2, Warren McNabb2, Elizabeth Rettedal2, 3, Suzanne Hodgkinson2, Carlos Montoya2, 3 1. School of Food & Advanced Technology, Massey Univeristy, Palmerston North, New Zealand 2. Riddet Institute, Palmerston North, New Zealand 3. Food Nutrition & Health Team, Grasslands Research Centre, AgResearch limited, Palmerston North, New Zealand Diet has a major impact on the large intestinal microbiota and recent studies have revealed the potential of the small intestinal microbiota to ferment dietary fibre. However, the effect of diet on the small intestinal microbiota and its capacity to ferment fibre has been scarcely studied. The aim of this study was to determine how different foods can modify ileal microbial composition and fibre fermentation. Ten experimental diets containing All Bran, black beans, bread, chickpeas, collagen, peanuts, pigeon peas, sorghum, whey protein isolate, or zein as the sole source of protein were fed to ten ileal-cannulated pigs over six periods according to an incomplete Latin Square design (10x6; i.e. n=6/treatment). On day 7, ileal digesta were collected and stored at -80°C under near anaerobic conditions for microbial analysis and in vitro fermentation. For each diet, an inoculum was prepared with pooled ileal digesta. This inoculum was used to ferment (in vitro) different purified fibre sources (cellulose, pectin, arabinogalactan, inulin, fructooligosaccharides, and high-amylose corn starch) for 2 hours at 37°C. The fermentability was then measured by the disappearance of organic matter, and organic acid concentrations were determined using gas chromatagraphy. The microbial composition of the ileal digesta varied between diets. Pigs fed the All Bran diet resulted in a 2-fold higher abundance of the phylum Bacteroidetes, mainly the genus Prevotella. The chickpea diet resulted in a 2- to 10-fold higher abundance of the phylum Actinobacteria. Pectin was the most fermented in vitro substrate (on average 26%) and cellulose the least (on average 1%) (P<0.05). Microbiota for the black bean diet fermented cornstarch 2-fold more compared to microbiota for the All Bran diet (15% and 7% respectively; P<0.05). The arabinogalactan resulted in the greatest organic acid production, mainly formate (on average 250 μmol/g DM substrate; P<0.05). In general, cellulose and pectin resulted in the lowest organic acid production. Microbiota for the collagen diet produced on average a 4.5-fold greater amount of lactate. The results show the potential for dietary intervention to modify the ileal microbiota and fermentative capacity.

id #684

Anti-parasitic compounds with long-acting, broad spectrum activities impact the rumen microbial communities of grazing sheep Christina D Moon1, Luis Carvalho1, Michelle R Kirk1, Peter H Janssen1, Dave Leathwick1 1. AgResearch Limited, Palmerston North, MANAWATU, New Zealand Anthelmintic treatment of adult ewes is widely practiced by sheep farmers in New Zealand to remove the burden of parasites, and in the expectation that this will result in greater livestock growth rates. Recent trials have shown that the results of anthelmintic use on sheep is variable. Moreover, given the broad spectra of activities of anthelmintic products, active compounds are predicted to impact organisms beyond the parasites targeted. The dense and complex microbial communities of the rumen are essential for feed digestion and provide hosts with the majority of their energy needs. To investigate the impact of anthelmintics on the rumen microbiota, 300 ewes from a Waikato farm were randomly assigned to treatment groups comprising a 100-day controlled release capsule (CRC) containing albendazole and abamectin, a long-acting moxidectin injection, and a non-treated control group. Rumen contents were collected from the sheep immediately before the treatments were administered, and again at 35 and 77 days, over which time the sheep were grazed together on pasture. The microbial community profiles of each sheep on day 0 were analysed via high throughput sequencing of partial 16S rRNA bacterial and archaeal gene amplicons, and partial 18S rRNA protozoal gene amplicons. Bray-Curtis dissimilarities of the community structures were used to select a subgroup of 108 sheep (36 per treatment) that had highly similar community compositions. The community profiles of these animals were examined on days 35 and 77. The treatments had the largest impact on the archaeal community (P ≤ 0.001, ANOSIM), particularly for the CRC-treated compared to the control group at both days 35 and 77. This was associated with increased relative abundances of Methanobrevibacter ruminantium, Methanosphaera sp. ISO3-F5 and Methanomassiliicoccaceae Group 12 sp. ISO4-H5, and decreased relative abundances of Methanobrevibacter gottschalkii in the CRC-treated animals. The bacterial and protozoal communities had subtle but significant differences due to the treatments, and these were no longer significant for protozoa by day 77. Further investigation is underway to uncover the causes of these changes in the microbiota, which will contribute to our understanding of their implications for the host animal.

id #686

Multiomic analysis of polyp biopsies in chronic rhinosinusitis over time and in response to systemic corticosteroids Michael Hoggard1, Bincy Jacobs1, David Wheeler2, Melissa Zoing1, Kevin Chang1, Kristi Biswas1, Martin Middleditch1, Richard Douglas1, Mike Taylor1 1. University of Auckland, Auckland, AUCKLAND, New Zealand 2. Nextgen Bioinformatic Services, Palmerston North Background. The pathophysiology and temporal dynamics of affected tissues in chronic rhinosinusitis (CRS) remain poorly understood. Objective. Here we present a multiomics-based time-series assessment of polyp biopsies from three patients with CRS with nasal polyps, assessing natural variability over time and local response to systemic corticosteroid therapy. Methods. Polyp tissue biopsies were collected over three consecutive weeks. Patients were prescribed prednisone (30 mg daily) for one week between collections two and three. Polyp transcriptome, proteome, and microbiota were assessed via RNAseq, SWATH mass spectrometry, and 16S rRNA and ITS2 amplicon sequencing. Baseline inter-patient variability, natural intra-patient variability over time, and local response to systemic corticosteroid therapy, were investigated. Results. Overall, the highly abundant transcripts and proteins were associated with pathways involved in inflammation mediated by chemokine and cytokine, FAS, cadherin, integrin, Wnt, apoptosis, and cytoskeletal signaling, as well as coagulation and B and T cell activation pathways. Transcripts and proteins that naturally varied over time included those involved with inflammation- and epithelial-mesenchymal transition-related pathways, and a number of common candidate target biomarkers of CRS. Ten transcripts responded significantly to corticosteroid therapy, including downregulation of TNF, CCL20, and GSDMA, and upregulation of OVGP1, and PCDHGB1. Members of the bacterial genus Streptococcus positively correlated with immunoglobulin proteins IGKC and IGHG1. Conclusion. Understanding natural dynamics of CRS-associated tissues is essential to provide baseline context for all studies on putative biomarkers, mechanisms, and subtypes of CRS. These data further our understanding of the natural dynamics within polypoid tissue, as well as local changes in response to systemic corticosteroid therapy.

id #687

One governor, two two-component systems: how carbon/nitrogen metabolic balance is maintained in Pseudomonas Naren Naren1, Xue-Xian Zhang1 1. Massey University at Albany, Albany, AUCKLAND, New Zealand Histidine is a good source of nutrients for Pseudomonas, but its utilization poses a significant challenge as it produces excess nitrogen over carbon. The rate of histidine utilization (hut) must thus be carefully regulated. When growing in nutrient-complex conditions such as a minimal salt medium supplemented with succinate and histidine, bacteria face a physiological dilemma to increase hutgene expression as histidine is the sole source of nitrogen; but at the same time to reduce hutactivities due to the presence of succinate (the most preferred carbon source for Pseudomonas). Using the plant growth-promoting bacterium P. fluorescensSBW25 as a model, we show that the cellular carbon/nitrogen metabolic balance is primarily maintained by a complex interplay between two two-component signal transduction systems CbrAB and NtrBC at the hutoperator sites. A combination of genetic and biochemical analysis indicates that CbrAB directly activates transcription of hut genes but represses their expression at the translational levels via the CbrAB-CrcYZ-Crc/Hfq cascade. NtrBC directly activates huttranscription in response to nitrogen starvation, but this requires the assistance of a regulator that functions as a governor to control the rate of histidine metabolism. Together, our data provide a molecular explanation on the coordination of carbon and nitrogen metabolism in the case of histidine utilization by Pseudomonas.

id #694

Implant materials and prosthetic joint infection: the battle with the biofilm

Donald J. Davidson1,2, David Spratt2, Alexander D. Liddle2,3

1Research Department of Orthopaedics and Musculoskeletal Sciences, University College London, London, UK 2Department of Microbial Diseases, UCL Eastman Dental Institute, University College London, London, UK 3MSK Lab, Imperial College London, London, UK

PRESENTED BY: Michael C Wyatt (Hauora Orthopaedics Ltd)

Prosthetic joint infection (PJI) is associated with poor clinical outcomes and is expensive to treat. Although uncommon overall (affecting between 0.5% and 2.2% of cases), PJI is one of the most commonly encountered complications of joint replacement and its incidence is increasing, putting a significant burden on healthcare systems. Once established, PJI is extremely difficult to eradicate as bacteria exist in biofilms which protect them from antibiotics and the host immune response. Improved understanding of the microbial pathology in PJI has generated potential new treatment strategies for prevention and eradication of biofilm associated infection including modification of implant surfaces to prevent adhesion of bacteria. Much research is currently ongoing looking at different implant surface coatings and modifications, and although most of this work has not translated into clinical medicine there has been some early clinical success. Keywords

1. biofilm 2. implant 3. material 4. microbiology 5. prosthetic joint infection

surface coating

POSTER ABSTRACTS – NUMERICAL ORDER

id #520

Characterization of Yersinia enterocolitica Strains Isolated from Cattle and Potentially Pathogenic to Humans Piotr Lada1, Klaudia Konczyk1, Karolina Lipczynska-Ilczuk1, Wojciech Szweda1, Agata Bancerz-Kisiel1 1. University of Warmia and Mazury in Olsztyn, Olsztyn, WARMINSKO-MAZURSKIE, Poland

Y. enterocolitica as a causative agent of yersiniosis – zoonosis with growing epidemiological significance, poses a significant threat to public health. Concerning the negligible literature data on the occurrence of Y. enterocolitica in cattle, the aim of the study was to characterize Y. enterocolitica strains isolated from this species. A total of 660 samples was examined. Two swabs were taken from each cattle due to the psychrophilic properties of Y. enterocolitica and necessity of application two different culturing methods. Bacteriological examinations, biotyping, serotyping, molecular examinations and a standardized disk diffusion method to assess the sensitivity of the tested strains to antibiotics were used. Y enterocolitica strains were isolated from 3 out of 15 studied herds. Prevalence rate ranged from 0 to nearly 32%. In the herds from which Y. enterocolitica strains were isolated, this rate was 2.2%, 31.8% and 2.9%, respectively for herds No. 1, 2 and 5. In total, 29 Y. enterocolitica strains were isolated, which is 4.4% of taken samples; over 65% of these strains were obtained from cold culturing. All isolated strains belonged to biotype 1A, in majority were not serologically typable, and all were characterized by the presence of ystB gene amplicons – no amplicons of ail and ystA genes were found. Phylogenetic analysis showed the presence of five different phylogenetic groups, genotype 2 was prevailed, the remaining genotypes were represented much less frequently. All tested strains showed resistance to ampicillin and cefalexin, and the vast majority also to amoxicillin with clavulanic acid. The phenomenon of multidrug resistance of isolated strains was also observed – over 90% demonstrated resistance to at least three antibiotics used in the study. The results show that ystB-positive, multidrug resistant Y. enterocolitica strains are present in cattle. Regarding this fact, it should be noted that carcasses of these animals, and consequently also beef, may poses a potential threat to humans. Project financially co-supported by Minister of Science and Higher Education in the rangę of the program entitled "Regional lnitiative of Excellence" for the years 2019-2022, Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN.

1. Bottone, E.J. Yersinia enterocolitica: revisitation of an enduring human pathogen. Clin. Microbiol. Newsl. 2015, 37, 1-8. 2. Jamali, H.; Paydar, M.; Radmehr, B.; Ismail, S. Prevalence, characterization, and antimicrobial resistance of Yersinia

species and Yersinia enterocolitica isolated from raw milk in farm bulk tanks. J. Dairy Sci. 2015, 98, 798-803. 3. Liang, J.; Duan, R.; Xia, S.; Hao, Q.; Yang, J.; Xiao, Y.; Qiu, H.; Shi, G.; Wang, S.; Gu, W.; Wang, C.; Wang, M.; Tian, K.;

Luo, L.; Yang, M.; Tian, H.; Wang, J.; Jing, H.; Wang, X. Ecology and geographic distribution of Yersinia enterocolitica among livestock and wildlife in China. Vet. Microbiol. 2015, 178, 125-131.

4. Mayrhofer, S.; Paulsen, P.; Smulders, F.J.; Hilbert, F. Antimicrobial resistance profile of five major food-borne pathogens isolated from beef, pork and poultry. Int. J. Food Microbiol. 2004, 97, 23-29.

5. McNally, A.; Cheasty, T.; Fearnley, C.; Dalziel, R.W.; Paiba, G.A.; Manning, G.; Newell, D.G. Comparison of the biotypes of Yersinia enterocolitica isolated from pigs, cattle and sheep at slaughter and from humans with yersiniosis in Great Britain during 1999-2000. Lett. Appl. Microbiol. 2004, 39, 103-108.

6. O'Grady, D.; Kenny, K.; Power, S.; Egan, J.; Ryan, F. Detection of Yersinia enterocolitica serotype O:9 in the faeces of cattle with false positive reactions in serological tests for brucellosis in Ireland. Vet. J. 2016, 216, 133-135.

7. Schmid, A.; Messelhäusser, U.; Hörmansdorfer, S.; Sauter-Louis, C.; Mansfeld, R. Occurrence of zoonotic clostridia and Yersinia in healthy cattle. J. Food Prot. 2013, 76, 1697-1703.

id #523

Pan-genome analysis of Butyrivibrio and Pseudobutyrivibrio provides insight into polysaccharide degradation in the rumen. Nikola Palevich1, 2, William Kelly1, Jasna Rakonjac1, 2, Sinead Leahy1, Eric Altermann1, Graeme Attwood1 1. AgResearch Ltd., Palmerston North, MANAWATU, New Zealand 2. Massey University, Palmerston North, Manawatu, New Zealand Rumen bacterial species belonging to the genera Butyrivibrio and Pseudobutyrivibrio are important degraders of plant polysaccharides, particularly hemicellulose and pectin. To investigate their genes required for polysaccharide degradation, the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains, isolated from the plant-adherent microbiome of New Zealand dairy cattle (Fig. 1), have been sequenced via the Hungate1000 project supported by the Joint Genome Institute. Comparative genomics of the Butyrivibrio and Pseudobutyrivibrio pan-genomes have been used to define their conserved and unique gene features and to examine their gene complements encoding polysaccharide-degrading activities. Based on these observations, growth studies were conducted on two Butyrivibrio strains, examining their abilities to compete for the insoluble substrates, xylan and pectin.

id #524

Beyond Myrtle Rust: Towards Ecosystem Resilience Renee Johansen1, Mahajabeen Padamsee1 1. Landcare Research, St Johns, AUCKLAND, New Zealand Many of New Zealand’s best-known, highly-valued native trees - pōhutukawa, rātā, mānuka - are in the plant group Myrtaceae. They urgently need protection from a recently-arrived exotic fungal disease called myrtle rust (MR), which can cause plant/tree death. The causal agent, Austropuccinia psidii, arrived in New Zealand from Australia via wind dispersal in 2017. Significantly, MR has never been eradicated from any country, despite significant effort to control its spread. MR has now been recorded throughout the North Island and is making inroads into the top of the South Island. It is probable that MR is now part of the story of New Zealand, and we have a narrow window of opportunity to reduce the disease’s impact on our forested landscapes. This poster introduces the programme ‘Beyond Myrtle Rust: Towards Ecosystem Resilience’. Hosted by Manaaki Whenua – Landcare Research, this multi-disciplinary multi-year research effort draws upon expertise from eight research institutions. It has four key interlinking elements: pathogen dynamics, ecosystem impacts, novel mitigation techniques, and Kaitiakitanga & Māori lead solutions. Laboratory, greenhouse, and field studies will be engaged. Altogether, this research will provide tools to engineer landscape resilience in the face of this serious threat. Through proactive stakeholder engagement, on-the-ground adoption of tools and techniques will be optimised. In addition, the approach provides a model for future responses to plant pathogen incursions into New Zealand.

id #525

Characterising the surface properties of oral probiotic Streptococcus salivarius K12 for designing new food delivery formats Jonathan Chua1, John Hale2, Phil Bremer1, Pat Silcock1 1. Food Science, University of Otago, Dunedin, New Zealand 2. Blis Technologies, Dunedin, New Zealand The surface properties (zeta potential and surface energy) of oral probiotic bacteria will impact on the way they interact with different components in food, which could then subsequently influence their ability to colonise the oral cavity and confer health benefits. In this work, we looked at characterising the zeta potential and surface energy of oral probiotic Streptococcus salivarius strain K12. Zeta potential was assessed over six pH levels (pH 3, 4, 5, 6, 7 and 8) in potassium phosphate buffer with an ionic strength of either 0.0446M, 0.1M or 0.166M. Contact angle measurements were carried out using three solvents (water, formamide and 1-bromonapthalene) to calculate surface energy, based on the surface tension-component theory. S. salivarius K12 has an overall negative zeta potential, ranging from +1 to -8, depending on pH and ionic strength. As pH increased and ionic strength decreased, zeta potential was found to increase more negatively. The surface energy of S. salivarius K12 was calculated to be 53.32 mJ m-2. Bacterial-food component interactions can be known to occur when zeta potential and surface energy tend to be lower than the surrounding medium. This information will allow us to understand the delivery of S. salivarius K12 through selected food formats as an oral probiotic, which will create new product development opportunities for conferring oral health benefits.

id #528

INACTIVATION AFLATOXIN M1 BY ULTRA VIOLET LIGHT (254 nm) Thu Nguyen1, Steve Flint1, Jon Palmer1 1. School of Food and Advanced Technology, Massey University, Palmerston North, New Zealand Aflatoxin is one of the most toxic mycotoxins, produced mainly by Aspergillus flavus and Aspergillus parasiticus which contaminate food especially in tropical climates. Feed contaminated with aflatoxins consumed by dairy cattle leads to the contamination of milk with aflatoxin M1, which poses a risk to human health. Traditional control involves ensuring the cattle food is kept dry to avoid the growth of toxin producing fungi. However, in some countries, such as Vietnam, where the climiate is particularly damp and technology is limited, alternative methods are needed. The aim of this study is to inactivate AFM1 in milk using UV. This study used UV at 254 nm to reduce AFM1 from a standard solution of 2 ppb added to in water and milk. Treatment was tested by sampling at 1, 10 and 30 min. The impacts of stirring and sample size on AFM1 reduction were investigated. UV at 254 nm was able to reduce aflatoxin in water 1.53, 94.86 % and 100% after 1, 10 and 30 min respectively. AFM1 in milk was also reduced by 78.02% after 10 min of UV treatment with the depth sample of 2 mm. Stirring improved the AFM1 reduction efficacy from 38.71 to 53.53% with sample size of 5 ml and 10 min of treatment. This is the first report of UV light at the wavelength of 254 nm to degrade AFM1 in milk. These results show the potential of UV treatment for reducing AFM1 in milk.

id #538

Elucidation of the genes required for epoxy-janthetrem biosynthesis Debbie Hudson1, Wade J Mace1, Richard D Johnson1 1. AgResearch, Palmerston North, MANAWATU, New Zealand Epichloë endophytes are filamentous fungi, belonging to the family Clavicipitaceae, which live in symbiotic associations with grasses in the sub family Poöideae. In New Zealand, E. festucae var lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and as such is an essential component of pastoral agriculture, where ryegrass is the major forage species. AR37, a strain of E. festucae var lolii, produces in planta a range of unique bioactive secondary metabolites, including janthitrems, which have demonstrated broad insecticidal activity against important pasture pests, such as porina, but can also cause mammalian toxicosis. Previous attempts to make gene modifications in AR37 by

homologous recombination techniques have failed, so a new approach was required to elucidate the genes involved in the janthitrem branch of the indole diterpene (IDT) pathway. E. festucae var lolii AR6 is a strain which contains the genes involved in the early steps of the IDT pathway but does not contain genes to activate any janthitrem production. We genetically modified AR6 by the ectopic insertion of up to three genes (and their native promoters) from AR37, which we hypothesised were responsible for the production of janthitrems. The genes inserted were: idtD, a prenyl transferase, idtO an oxidase and idtA an acyltransferase. The insertion of these genes and their various combinations were screened for by PCR. The modified strains were inoculated into perennial ryegrass, which was harvested for a full chemical analysis of the IDT compounds produced. Insertion of each of idtD, idtO and idtA alone into AR6 did not produce any janthitrems, however, with the addition of both idtD and idtO together, epoxy-janthitriol as well as epoxy janthitrems II and III were produced and the addition of all three AR37 genes (idtD, idtO and idtA) led to the production of all five epoxy janthitrems (triol, I, II, III and IV) in AR6.

id #539

The microbial biogeography of the gastrointestinal tract of preterm and term lambs Clara Yieh Lin Chong1, Tommi Vatanen1, 2, Mark Oliver1, Frank Bloomfield1, Justin O'Sullivan1 1. Liggins Institute, The University of Auckland, Auckland, New Zealand 2. Broad Institute, MIT and Harvard , Cambridge, MA, USA Background The microbiome in the rumen, the first fermentation chamber of ruminants, has been widely studied. In contrast, the microbiome of the small intestine, where the majority of nutrient metabolism and absorption takes place, has not been described in lambs. Similarly, the impact of nutritional supplementation and preterm birth on the Ovis aries (sheep) microbiome remain to be defined. Objectives

• To define the differences between the mucosal and luminal microbiota along the gastrointestinal tract (GIT) of term and preterm sheep.

• To characterise the effect of protein and carbohydrate supplementation on the microbial community in preterm lambs. Methods Mucosal scrapings and luminal samples were collected from the anatomical sites of the GIT (duodenum, jejunum, ileum and colon) of 31 lambs (7 term and 24 preterm) at six weeks post-term age. Genomic DNA was extracted from each sample using the QIAamp PowerFecal DNA kit (QIAGEN). Samples were analysed by 16S rRNA gene amplicon sequencing on Illumina Miseq platform. Amplicon sequence variants and alpha (Shannon) and beta (Bray Curtis) diversities were determined using QIIMEv2019.4. Results The microbiome composition of both the mucosal lining and luminal content varied along the length of the GIT (p<0.0001) and was significantly different between mucosa and lumina in all small anatomical sites except the colon. Anatomical site explained 40% (p<0.0001) of the variation in microbial profiles. Differences between the mucosal lining and luminal content explained a further 12% (p<0.0001). The colon was enriched in Firmicutes, Bacteroidetes, and Proteobacteria for both mucosal and luminal samples, while gram-negative bacteria (i.e. Actinobacteria, Firmicutes, and TM7) and Euryarchaeota were more abundant in ileum compared to colon luminal content. Preterm birth was associated with microbial composition variation (p=0.0022) but 2 weeks of nutritional supplementation after birth with either maltodextrin or branch chain amino acid did not alter the microbiome community in preterm lambs. Discussion There are distinct microbial populations in the mucosal and luminal contents of term and preterm lambs. Microbial populations also differed along the length of the GIT. However, nutritional intake in early life had no effect on microbial populations.

id #546

Exploring new players in the Faecalibacterium genus Davide Fraccascia3, 1, 2, Rachel C Anderson3, 1, Eric Altermann3, 4, 1, Nicole C Roy3, 4, 1, Warren C McNabb4, 1 1. Riddet Institute, Palmerston North, New Zealand 2. School of Food & Advanced Technology, Massey University, Palmerston North, New Zealand 3. Food Nutrition & Health Team, AgResearch, Palmerston North, New Zealand 4. High-Value Nutrition, National Science Challenge, , New Zealand Until recently, Faecalibacterium prausnitzii (FP) was considered the only bacterial species in the Faecalibacterium genus. FP is generally accepted as one of the most abundant species present in the colon of healthy individuals and a significant contributor to short-chain fatty acids (SCFAs) production. Since its characterisation in 2002, FP has been the focus of many studies aiming to elucidate its role in the interaction between the host, the resident microbiota, and health conditions. However, the exact mechanisms by which FP influences gut function have still to be fully understood. Recently, a study established the presence of at least one more species in the Faecalibacterium genus, previously catalogued as a F. prausnitzii strain. It is logical, then, to hypothesise the presence of more species within this genus, rather than diverse strains, given the different nutrient growth requirements reported in the literature between these strains. Genome to genome comparisons will be needed to test the multiple species hypothesis, but currently, only four complete annotated genomes are available. This limitation hinders the capacity to explore the different functional impacts of specific Faecalibacterium species and, consequently, the understanding of the overall role of Faecalibacterium prausnitzii in gut health. This study will expand the basic knowledge of the Faecalibacterium genus through the isolation of novel New Zealand strains. When appropriately characterised, these new Faecalibacterium isolates will increase the information available on the genus, allowing more in-depth pathway and functional analysis. Those analyses will then enable the creation of more refined media for selective isolation and provide insight into food interventions targeted at increasing the abundance of beneficial members of the genus, with a view of improving gut function for the host.

id #552

Local versus global regulatory roles of the histidine utilization repressor HutC in Pseudomonas fluorescens SBW25 Naren Naren1, Xue-Xian Zhang1 1. Institute of Natural and Computational Sciences, Massey University, Auckland Publish consent withheld

id #553

Effect of calcium on bacterial attachment and biofilm formation of Geobacillus stearothermophilus. Tianyang Wang1, Jon Palmer1, Steve Flint1 1. Massey University, Hokowhitu, Palmerston North, MANAWATU, New Zealand This study investigated the effect of calcium ions on bacterial attachment and biofilm formation of Geobacillus stearothermophilus dairy isolates. Biofilms are surface associated bacterial aggregates encased in extracellular polymeric substances (EPS). Crystal violet staining, conductance measurements, EPS extraction followed by colorimetric quantification and total organic carbon (TOC) measurements were used to quantitatively characterize biofilms. Fluorescence microscopy and SEM were used to study the EPS composition and morphology of biofilms. Three time points, 30 min, 10 h and 24 h were chosen to monitor biofilm development. In the presence of 7 mM and 40 mM calcium, bacterial cells showed a dose-dependent increase in attachment to polystyrene. Cells attached in 40 mM calcium were significantly (p-value<0.05) higher than without calcium. Most isolates showed higher biofilm biomass at 24h in the presence of calcium compared to the control. Only one strain showed more biofilm biomass at 10 h and this level remained at 24 h. The SEM images confirmed these observations. Calcium altered EPS composition, the protein and polysaccharide content of the EPS were slightly increased, although not significantly, in the presence of calcium. Therefore, the presence of calcium increased the bacterial cell initial attachment and might also affect later biofilm development by changing EPS composition and biofilm cell viability.

id #555

Effects of herbicides on antibiotic resistance in Serratia marcescens Qifan Fu1, Brigitta Kurenbach1, William Godsoe2, Jack Heinemann1 1. School of Biological Science, University of Canterbury, Chirstchurch, New Zealand 2. BioProtection Centre, Lincoln University, Lincoln, New Zealand Exposure to herbicides and antibiotics has been associated with increased honeybee mortality and colony loss on a global scale. These chemicals disturb the commensal bacteria in honeybee guts, compromising the immunity of honeybees and allowing infection by opportunistic pathogens. The focus of my research is on the recently discovered link between herbicides and the development of antibiotic resistance. The sequential encounter of antibiotics and herbicides by honeybees potentially facilitates the development of antibiotic resistance of bee pathogens, making them harder to treat with antibiotics in the future. Such effects could conflict with efforts to halt honeybee decline. In this study, I am testing test whether exposure to herbicides at sublethal concentrations changes adaptive antibiotic responses and ultimately acquisition of antibiotic resistance by Serratia marcescens, an important opportunistic pathogen responsible for honeybee mortality. Commercial formulations of three herbicides ― dicamba (Kamba), 2,4-dichlorophenoxyacetic acid (2,4-D), and glyphosate (Roundup and Number 8) ― will be tested in combination with relevant clinical and agricultural antibiotics. S.marcescens are inoculated on plates with different concentrations of antibiotics, supplemented with or without herbicides. The survival of S. marcescens on plates containing increasing antibiotic concentrations in the presence and absence of herbicides at sublethal concentrations are compared. This allows the determination of the impact of herbicides. Previous studies using other organisms have shown increases, decreases, or no change in antibiotic susceptibility. Acquired resistance development will be investigated using short term evolution experiments. As other organisms in previous studies, S. marcescens shows different responses to different combinations of antibiotics and herbicides. When exposed to 9000 ppm glyphosate of the Number 8 formulation, the concentration of ciprofloxacin needed to kill the bacteria increased 32-fold and for streptomycin, 4-fold. Meanwhile, ampicillin susceptibility is increased 8-fold, and no susceptibility changes were observed for tetracycline and chloramphenicol. Results of other herbicide-antibiotic combinations will be reported. Increased tolerance to streptomycin under herbicide treatment is a serious concern given its wide use in agriculture. I will further investigate this effect to see if it applies to other antibiotics of the same class and determine the mechanism of resistance.

1. Kurenbach, B., Marjoshi, D., Amábile-Cuevas, C. F., Ferguson, G. C., Godsoe, W., Gibson, P., & Heinemann, J. A. (2015). Sublethal exposure to commercial formulations of the herbicides Dicamba, 2, 4-Dichlorophenoxyacetic acid, and Glyphosate cause changes in antibiotic susceptibility in Escherichia coli and Salmonella enterica serovar Typhimurium. MBio, 6(2), e00009-00015.

2. Kurenbach, B., Gibson, P. S., Hill, A. M., Bitzer, A. S., Silby, M. W., Godsoe, W., & Heinemann, J. A. (2017). Herbicide ingredients change Salmonella enterica sv. Typhimurium and Escherichia coli antibiotic responses. Microbiology, 163(12), 1791-1801. doi:10.1099/mic.0.000573

3. Kurenbach, B., Hill, A. M., Godsoe, W., van Hamelsveld, S., & Heinemann, J. A. (2018). Agrichemicals and antibiotics in combination increase antibiotic resistance evolution. PeerJ, 6, e5801. doi:10.7717/peerj.5801

id #562

Production of nanorods to solve the end-cap structures of filamentous phage Rayen Leon-Quezada1, Jasna Rakonjac1, Andrew Sutherland-Smith1, Vicki Gold2, Becky Conners2 1. Massey University, Palmerston North, MANAWATU / WANGANUI, New Zealand 2. Living Systems Institute, University of Exeter, Exeter, United Kingdom Ff (f1, fd and M13) filamentous phage of E. coli have been the workhorse of phage display technology over the past few decades. Their use has expanded in recent years into nanotechnology, where they serve as filament-like templates (≥ 890 nm x 6 nm) for assembly of nanostructures, from nanowires and nanorings, to nano-scale batteries. The filament end-caps are the key to improving the control of the nanostructure geometries, and for understanding the molecular mechanisms of Ff assembly and infection that occur through pseudo-symmetrical processes involving translocation of end-cap proteins pIII and pVI from membranes into a lipid-free virion and vice versa. While the atomic resolution structure of the Ff filament shaft has been determined, the structure of the phage end-caps has remained unresolved, as they constitute only 2% of the virion mass. To enable the end-caps’ structural analysis we assembled short Ff-derived rods (50 nm in length) in which their end-caps account for 40% of the total virion mass. We further developed a novel nanorod purification method based on CsCl density gradient centrifugation and anion exchange chromatography. Purified nanorods were examined by transmission electron microscopy. Images of the negatively stained nanorods were analysed and five 2D classes of end-caps were determined, possibly corresponding to variations of the two asymmetric nanorod ends, pIII/pVI (pointy) and pVII/pIX (blunt). Taken together, our preliminary work demonstrates that it is possible to analyse the Ff phage end-cap structure by electron microscopy of short Ff-derived nanorods.

id #565

Elastic Light Scatter pattern analysis of foodborne pathogens: darkness from light? Stephen L.W. On1, Yuwei Zhang1, Andrew Gehring2, Jim Bono3, Steve Flint4, Haoran Wang4, James Lindsay5, Craig Billington6, Graham Fletcher7, Euiwon Bae8, Valery Patsekin9, J. Paul Robinson9 1. Lincoln University, Lincoln, CANTERBURY, New Zealand 2. Eastern Regional Research Center, US Department of Agriculture, Pennsylvania, USA 3. Meat Animal Research Center, US Department of Agriculture, Nebraska, USA 4. Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand 5. Office of National Programs, US Department of Agriculture, Agricultural Research Service, Washington, USA 6. Institute of Environmental Science and Research, Christchurch, Canterbury, New Zealand 7. Plant and Food Research, Auckland, New Zealand 8. School of Mechanical Engineering, Purdue University, West Lafayette, Indiana, USA 9. Dept. of Basic Medical Science, Purdue University, West Lafayette, Indiana, USA Objectives: Image analysis of patterns derived from elastic laser-light spectra of bacterial colonies has been shown to be an effective bacterial identification tool. This study investigated the potential of this method to detect “the dark side” of microorganisms: differences between individual strains exhibiting divergence in pathogenic potential that can be accounted for by cell surface characteristics. Methods and Results: Previously characterised strains of Escherichia coli O157 (n=10) and Yersinia enterocolitica (n=8) were used. The E. coli strains had been found to possess either high- or low-colonising potential previously in an ex vivo model (Brandt & Paulin 2010). The Y. enterocolitica isolates comprised four pairs of strains, otherwise identical to each other save for the presence or absence of the characteristic virulence plasmid that codes for the surface-expressed adhesin. Strains were cultured on TSA for 12 – 22 h, and elastic light scatter (ELS) patterns determined from bacterial colonies using a custom-built light scatter based detection instrument. Statistical and Principal Component analysis was undertaken using Baclan analysis software. In each of the analyses, high and low-colonising strains of E. coli, and plasmid-containing or cured strains of Y. enterocolitica were distinguishable based on scatter patterns. Conclusions: Elastic light scatter analysis of bacterial strains seems capable of identifying differences between certain bacterial pathotypes that are determined by surface-expressed characteristics.

id #566

Temporal detection of Campylobacter jejuni, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) in calf faeces, calf bedding, birds and flies in a dairy farm environment Delphine Rapp1, Colleen Ross1, Gale Brightwell1 1. Agresearch Ltd, Palmerston North, PRIVATE BAG 11008, New Zealand Campylobacter jejuni, Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) are important foodborne bacterial pathogens, with cattle a significant reservoir for human infection. This study evaluated the presence of C. jejuni, Salmonella spp. and STEC O26 and O157:H7 in calf-shed bedding (e.g. woodchips), flies, and birds on a pasture-based dairy farm over time using molecular and culture-based methods. A total of 150 composite faecal and environmental samples (including calf faeces and bedding, bird droppings and flies) were collected over 14 months, with at least one sampling visit per month. Overall, C. jejuni was the most prevalent pathogen, detected at every visit in the calf and bird faeces (overall detection 24/28 and 43/61 samples, respectively), at half of the visits in the calf bedding (10/20 samples), and at seven out of nine visits for which flies were collected (13/32 samples). Salmonella spp. were detected at a third of the visits in the calf faeces, calf bedding, bird droppings, and flies, found in 6/28, 5/20, 10/61 and 2/32 samples, respectively. The presence of O157:H7 was confirmed at four visits, while STEC O26 was not detected during the study. Whole genome sequence and phylogenetic analysis of core genome single nucleotide polymorphisms are currently underway to confirm the dissemination of clonal C. jejuni, Salmonella spp. and STEC strains and to provide indications of on-farm transmission control strategies.

Overall, our longitudinal study will provide data to better understand the endemic presence or emergence of these clinically important pathogens on farms, and to inform mathematical models on persistence and transmission dynamics in dairy herds.

id #567

Epichloë bromicola effector screening in Nicotiana spp Pranav Chettri1, Benjamin Moody1, Christine Voisey1, Rosie E Bradshaw2, Carl H Mesarich3, Linda J Johnson1 1. AgResearch (Grasslands Research Centre), Palmerston North, Manawatu, New Zealand 2. School of Fundamental Sciences, Massey University, Palmerston North, Manawatu, New Zealand 3. School of Agriculture and Environment, Massey University, Palmerston North, Manawatu, New Zealand The fungal endophyte Epichloë bromicola forms mutualistic symbioses with grasses of the Elymus complex. Artificial inoculation of Epichloë endophytes into modern wheat (Triticum aestivum) and wheat-alien experimental lines to form novel symbioses has produced a range of infected plant phenotypes (from normal to severe plant stunting and/or death). To understand the molecular basis of endophyte-host compatibility, a transcriptome analysis was conducted with E. bromicola in association with its native host, a modern wheat host and a wheat-alien host from the tribe Hordeae that exhibited contrasting endophyte compatibility outcomes. Many E. bromicola putative effector genes were highly up-regulated during these interactions. Effectors of fungal pathogens play crucial roles in actively suppressing host defence responses or preventing their recognition by corresponding plant immune receptor proteins; however, little is known about their roles in mutualistic symbioses. To evaluate the role of E. bromicola effectors in a non-host plant, 29 candidate effector genes were identified using a bioinformatics pipeline and transiently expressed by Agrobacterium-mediated transformation in leaves of Nicotiana tabacum. The first 16 (of 29) cloned putative effector genes were sub-cloned into two independent plant expression vectors to facilitate their expression in either the apoplast or in the plant cytoplasm. Significantly, two of the putative effectors triggered cell death in N. tabacum when expressed in the apoplast, suggesting that they are recognised as potential invasion patterns by these non-host plants. This result suggests the existence of an evolutionarily-conserved effector recognition mechanism in plants in response to pathogenic and symbiotic fungi. To our knowledge, these are the first Epichloë effectors identified to date. Future work will focus on confirming their roles in regulating host compatibility by performing gene deletion and overexpression studies of the two effector candidates and inoculating the mutants into hosts of different compatibilities.

id #569

So you want to build a genome? Completing de novo Escherichia coli genomes with long read data Georgia L Breckell1, Olin K Silander1 1. Massey University, Albany, AUCKLAND, New Zealand Single molecule, long read sequencing (eg Oxford Nanopore or PacBio) have changed the landscape of genome assembly and it is now possible to routinely sequence and assemble bacterial genomes to single contig, reference level. Despite increased read lengths, next generation sequencing platforms have lower accuracy than short read Illumina data, making it advantageous to combine the two data types to produce a hybrid assembly. Several “Hybrid” assembly methods are available to combine short and long read data for assembly, however they each approach the assembly problem uniquely and do not always result in identical assemblies. Here we use short and long read data from 55 Escherichia coli natural isolates to test the assembly accuracy of several popular assemblers. As hybrid assemblies are regularly a single contig, metrics developed for the assessment of highly fragmented short read assemblies, such as N50, are not suitable for near complete hybrid assemblies. We implement a series of 6 metrics to evaluate the accuracy of highly contiguous assemblies: contiguity, read coverage, concordant read mapping, accurate rRNA region arrangement, fraction of short ORFs and accurate plasmid assignment. On the basis of these results, we highlight the advantages and disadvantages of different genome assemblers.

id #570

Who is in cow poop? Characterization of the bovine faecal microbiome Alyssa Earnshaw1, Nikki Freed1, Olin Silander1, Chad Harland2, Christine Couldrey2 1. Massey University, Auckland, New Zealand 2. Livestock Improvement Corporation, Hamilton, New Zealand As the costs of DNA sequencing are reducing it is now possible to sequence all of the DNA from a microbial community to identify which microbes are present. This is called metagenomics. However, as a new and emerging field, there is no consensus for what the best method is to link DNA sequence data to species identification (Kim et al., 2017), a process called taxonomic identification. Here we sequence a known mock community of microbes using two different DNA sequencing approaches. We then compare two taxonomic classifiers to determine which classifier identifies the species in our mock community the best. We also assess the impact of using different databases by testing four different databases using the best classifier to investigate which gives the most accurate representation of the microbial species actually present. We find that the kraken2 classifier with gtdb database works best, however, all perform similarly. Future work will include using the pipeline established here to apply to microbial communities in faecal matter from cows.

id #571

Amino acid substitutions in lanosterol 14α-demethylase responsible for intrinsic azole resistance in mucormycetes Brian C Monk1, Michaela Lackner2, Joel DA Tyndall3, Mikhail V Keniya1, 4 1. Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, New Zealand 2. Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria 3. School of Pharmacy, University of Otago, Dunedin, New Zealand 4. Department of Oral Sciences, Faculty of Dentistry, University of Otago, Dunedin, New Zealand Objectives. Fungal infections represent a significant challenge for modern healthcare. The widespread use of azole drugs favours acquired resistance and the selection of intrinsically resistant fungal pathogens. The mucormycetes are intrinsically resistant to short-tailed azole drugs including voriconazole (VCZ) but remain susceptible to the long-tailed azole posaconazole (PCZ). We have determined the molecular basis of intrinsic azole resistance in the mucormycete Rhizopus arrhizus using a Saccharomyces cerevisiae expression system. Methods. Codon optimized and C-terminal hexahistidine-tagged ORFs of lanosterol 14α-demethylase (LDM) isoforms F1 and F5 and their cognate NADPH-cytochrome P450 reductase (NCP1) from R. arrhizus were constitutively expressed or co-expressed in a hypersensitive S. cerevisiae host under control of the PDR5 promoter. In some cases expression of the native ScLDM was repressed using the GAL1 promoter. Susceptibility to azole drugs was assessed using broth microdilution assays. The relative expression of recombinant proteins was evaluated using Western blots of crude membrane preparations. Results. Codon optimized R. arrhizus LDM F1 was expressed at twice the level of the F5 isoform while co-expression of the cognate reductase gave a modest (1.7 to 2.5-fold) increase in the amounts of LDM isoforms. Co-expression with RaNCP1 increased resistance to azole drugs from 2 to 8-fold. Strains expressing F1 or F5 isoforms showed similar sensitivity to PCZ but the F5 isoform alone was responsible for resistance to VCZ. A combination of F129Y and A291V mutations in LDM F5 was required to revert the enzyme to a VCZ-sensitive form with azole susceptibilities equivalent to the LDM F1. Conclusion. Our yeast expression system produces functional recombinant LDM isoforms +/- their cognate reductase from the pathogen R. arrhizus, a representative of the evolutionally distant mucormycetes. Intrinsic resistance of R. arrhizus to VCZ is associated with F129Y and A291V substitutions in the F5 isoform of LDM. Use of the S. cerevisiae expression system provides a basis for screening and structural studies that are facilitating the design and discovery of broad-spectrum azole drugs that will overcome target-mediated intrinsic azole resistance.

id #575

Understanding the mechanism of action of the bacteriostatic peptide glycocin F Sean Bisset1, 2, Mark Patchett1, Sung Yang3, Margaret Brimble3, 2, Gillian Norris1, 2 1. Massey University, Palmerston North, MANAWATU, New Zealand 2. Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand 3. School of Chemical Sciences, The University of Auckland, Auckland, New Zealand Bacteriocins are a class of bacterially-produced peptides that possess an inhibitory effect on closely-related strains or species of bacteria. Glycocin F (GccF) is a di-glycosylated, 43 amino acid bacteriocin (glycocin) produced by a strain of the probiotic bacteria Lactobacillus plantarum. GccF contains two N-acetylglucosamine (GlcNAc) moieties required for its anti-bacterial activity, one O-linked through serine 18, and the other S-linked through the C-terminal cysteine located at the end of a flexible tail. Two nested disulfide bonds are also required for maintaining the folded structure. Whereas most bacteriocins exhibit bactericidal effects on a narrow range of species, GccF displays a very potent and reversible bacteriostatic activity towards a wide range of gram positive bacteria, including vancomycin-resistant strains, making it a potentially useful scaffold for developing new compounds to combat antibiotic resistant bacteria. However, the growth-inhibiting mechanism of action of GccF remains to be elucidated. The recent development of a complete chemical synthesis scheme for GccF enabled the production of GccF analogues, not accessible by recombinant methods, to study the role of different structural features of the peptide. Bioassays were carried out with these GccF analogues using a susceptible strain of L. plantarum. Altogether, these results provide additional evidence for a role of a GlcNAc transporter in GccF’s activity, as well as highlighting the structural aspects of this peptide that are critical for inducing stasis, including specific roles of the Ser18-O- and Cys43-S- linked sugars. In addition to this, transcriptomic analysis of cells treated with GccF were carried out on a susceptible strain of Enterococcus faecalis to identify the molecular cause of this bacteriostasis, along with interaction studies with GccF’s immunity factor to find complexes that are targeted by this peptide. The results of this work will be presented, along with a model mechanism of action.

id #577

Benthic and planktonic microbial community transitions across a freshwater to marine salinity gradient Hwee Sze Tee1, David Waite1, Kim M. Handley1 1. School of Biological Sciences, The University of Auckland, Auckland, New Zealand Much of the world’s expanding human population lives in coastal areas, resulting in increasing anthropological pressures on connected riverine, estuarine and coastal habitats. Microorganisms play an important ecological role in carbon break-down and nutrient cycling in aquatic habitats. Yet, we lack a fundamental understanding of how microbial communities function in these

various aquatic habitats, and the relative extent to which major environmental factors (e.g., salinity or benthic versus planktonic habitats) drive compositional and functional differences. Here, we present our findings of microbial dynamics across a freshwater to coastal marine transect, and we contrast the effect of salinity change on water column and benthic habitats. We collected sediments and planktonic biomass at nine subtidal sampling sites, including the Waiwera river, estuary and beach (Auckland). We profiled microbial communities via metagenomics sequencing, and generated a suite of complementary geochemical data. Through the reconstruction of 12,592 full-length 16S and 18S ribosomal subunit genes, we found little overlap between planktonic and benthic microbial communities, and observed higher diversity in the benthic environment compare to the water column. Our results suggest that these habitats strongly shape community composition and structure, and the magnitude of this difference is stronger than that imparted by changes in salinity. While salinity is a major controller of microbial communities, benthic and planktonic environments are subject to differences in oxygen and carbon availability, and community residence times. We also found the distribution of dominant taxa varied along the freshwater-to-marine salinity gradient. Both freshwater sediment and water communities were mainly dominated by Betaproteobacteria, whereas the relative abundances of Flavobacteriales and Rhodobacterales increased with salinity. Interestingly, among all study sites, freshwater benthic and planktonic communities exhibited the largest differences in terms of alpha diversity, possibly due to differences in resource availability. Analyses further indicated that members of the Betaproteobacteria, Bacteroidia, and Actinobacteria were the main contributors to differences in community composition and structure between freshwater sediment and water habitats. Genomic and transcriptomic data will be explored to determine the extent to which community compositional differences drive distinct biogeochemical cycles across these major chemical and physical divides.

id #578

Screening New Zealand fungi for new antibiotics: a modified OSMAC approach to maximise biodiscovery success. Shara van de Pas1, Alex Grey2, Yiwei Diao2, Daniel Mulholland2, Soeren Geese2, Siouxsie Wiles2, Melissa Cadelis3, Brent Copp3, Bevan Weir4 1. University of Auckland, Auckland, AUCKLAND, New Zealand 2. Bioluminescent Superbugs Lab, University of Auckland, Auckland, New Zealand 3. School of Chemistry, University of Auckland, Auckland, New Zealand 4. Mycology and bacteriology department, Manaaki Whenua (Landcare Research), Auckland, New Zealand The world is on the verge of an antimicrobial crisis. Disease-causing Enterobacteriaceae, namely Escherichia coli and Klebsiella pneumoniae are becoming increasingly resistant to therapeutics and the world needs new antibiotics to treat these resistant infections. New Zealand has immense biodiversity with over 6,000 reported fungal species and another 15,000 yet to be discovered. The International Collection of Microorganisms from Plants (ICMP) contains over 10,000 fungal species that have not been screened for their antibiotic potential. In 2002, Bode and colleagues described a novel method for screening called One Strain Many Compounds (OSMAC), based on the finding that microorganisms produce unique compounds when subjected to different environments. I have developed a derivative of this method, called Many Strains Some Conditions (MASSOC) which allows for the screening of multiple strains while retaining the testing variables vital to the success of OSMAC, including media type, age of fungus and light-conditions. Using this method, I screened 40 ICMP isolates from which we identified three unique compounds. Two of these compounds are novel sesquiterpenoids made by ICMP 17554 (Hypochnicium vellereum), a native wood-rot fungus, when cultured on Potato Dextrose Agar. The other compound is a novel macrolide made by ICMP 16864 (Xylariales order) when cultured on Oatmeal Agar. While I did not identify any ideal conditions to screen all fungi under, I did observe some general trends which can be used in a high-throughput screening program, to improve the chances of identifying fungi secreting antibacterial compounds. These include using Oatmeal Agar as a culture media, and testing fungi when they are at 50% radial growth size. My findings support my hypothesis that New Zealand fungi are an ideal source of new antibiotic compounds. Using my results will allow the screening method to be further developed to reduce costs and labour while retaining the benefits of the MASSOC approach.

id #580

Screening New Zealand Fungi for Anti-Mycobacterial Compounds using Bioluminescent Organisms Alex Grey1, Yiwei Diao1, Soren Geese1, Daniel Mulholland1, Shara van de Pas1, Bevan Weir2, Melissa Cadelis3, Brent Copp3, Siouxsie Wiles1 1. Bioluminescent Superbugs Lab, University of Auckland, Auckland, New Zealand 2. Manaaki Whenua, Auckland, New Zealand 3. School of Chemistry, University of Auckland, Auckland, New Zealand Antibiotic regimens for mycobacterial infections such as tuberculosis (TB) can require months to years to fully clear an infection. Additionally, strains of Mycobacterium tuberculosis resistant to nearly all available antibiotics are emerging, fuelled particularly by the HIV/AIDS epidemic, and are effectively immune to treatment. Native New Zealand microorganisms collectively possess an untapped abundance of biochemical wealth, some of which may be provide a novel chemical structure to design more efficacious anti-mycobacterial compounds. We investigated the anti-mycobacterial capacity of fungi from the International Collection of Microorganisms from Plants (ICMP) maintained by Manaaki Whenua (Landcare Research). We screened fungi against strains of M. abscessus and M. marinum which express a plasmid that make them constitutively bioluminescent. We cultured fungi on eight agar types in parallel on a 24-well plate to diversify the secondary metabolites each fungus produced. After inoculating fungal cultures in each well with our glowing mycobacteria diluted in an agar solution, we incubated the plate at 37 °C for several days. We measured the luminescence emitted from each well each day using a Victor X1 Light (PerkinElmer) plate reader to quantify inhibition of mycobacterial growth. After screening, we selected a proportion of active fungi for susceptibility testing of fractionated chemical extracts. 38 of the 40 fungi we screened reduced the bioluminescence of at least one of the two mycobacteria strains by at least 99% when cultured in at least one medium. While most active fungi supressed mycobacterial growth in several media concurrently, some displayed medium-specific activity.

27 out of 30 extracts retained their anti-mycobacterial activity after fractionation. Much of this inhibitory activity was caused by unsaturated fatty acids such as linoleic acid, whose activity is already documented, but two of our screened isolates produced novel compounds which were present in active extract fractions. These compounds were only present when cultured in one medium. The 24-well plate assay successfully identified fungi capable of producing anti-mycobacterial compounds. Despite the ubiquity of inhibitory fatty acids in some fractions, we were still able to discover novel compounds that likely possess anti-mycobacterial activity, suggesting this biological resource warrants further investigation.

id #581

Novel Nitrofuran Activating Enzyme in Escherichia coli Van Hung Vuong Le1, Patrick J. Biggs1, David Wheeler2, Ieuan Davies3, Jasna Rakonjac1 1. School of Fundamental Sciences, Massey University, Palmerston North, MANAWATU, New Zealand 2. NSW Department of Primary Industries, Orange, Australia 3. New Zealand Pharmaceuticals Ltd., Massey University, Palmerston North, MANAWATU, New Zealand Widespread prevalence of multi-drug resistant bacteria has necessitated employment of multiple strategies besides conventional discovery and development of novel small-molecule antimicrobial agents. A promising approach is revival of “old” antimicrobials, such as synthetic 5-nitrofuran drugs. Recent epidemiological data reported a very low resistance frequency amongst Escherichia coli isolates to this antimicrobial class, forecasting the increasing importance of its uses to combat antibiotic resistant enterobacteria. Typical examples of this group are furazolidone (FZ), nitrofurantoin and nitrofurazone. Though they had a long history of clinical uses, the understanding about the mechanism of action of 5-nitrofurans remains elusive. They are known as prodrugs that are activated in E. coli by reduction catalyzed by two type-I oxygen-insensitive nitroreductases, NfsA and NfsB. Our preliminary data show that FZ still retained relatively significant antibacterial activity in the nitroreductase-deficient ΔnfsA ΔnfsB E. coli strain, indicating the presence of additional activating enzymes and/or the antibacterial activity of the unreduced form. In this study, we employed genome sequencing, bioinformatic and genetic approaches to characterize the FZ-resistant E. coli mutants which were isolated from spontaneous mutations of ΔnfsA ΔnfsB E. coli cultures and described the involvement of ahpF mutations in FZ resistance. Antimicrobial susceptibility assays indicated that overexpression of ahpF sensitized the nitrofuran-resistant ΔnfsA ΔnfsB ΔahpF strain to all three 5-nitrofurans to the level of the drug-sensitive wild-type strain in both aerobic and anaerobic conditions. The data from enzymatic assays confirm the activation role of AhpF in which this enzyme catalyzes 5-nitrofuran reduction in a manner different from that of two well-studied nitroreductases NfsA and NfsB. The findings open new avenues to counteract E. coli isolates resistant to existing commercial nitrofuran antibiotics, by enhancing cellular expression/availability of AhpF or designing nitrofuran analogues with higher affinity for this enzyme.

id #583

Towards a molecular mechanism of bacterial recognition of eukaryotic hosts Kiran Sreeja Jayan1, Naren Naren1, Xue-Xian Zhang1 1. School of Natural and Computational Sciences, Massey University, Albany, AUCKLAND, New Zealand Host perception is a crucial step in infectious diseases pathogenesis, which allows pathogenic bacteria to recognise suitable niches and deploy appropriate phenotypes for successful colonization and immune evasion. However, the underlying perception mechanisms remain largely unknown. Our work with the nosocomial pathogen Pseudomonas aeruginosa has led to the hypothesis that urocanate, a histidine metabolite that accumulates in host tissues such as skin, serves as a signalling molecule that elicits bacterial infection via interactions with the bacterial regulatory protein HutC. In Gram negative bacteria, HutC functions as a transcriptional repressor of hut genes for the utilization of histidine (and urocanate) as sources of carbon and nitrogen. Surprisingly, HutC plays a significant role in the global control of cellular metabolism, cell motility, antibiotic induced biofilm formation and expression of virulence factors. Molecular analysis of HutC shows that HutC is capable of binding to promoters of NtrBC, a global regulator for cellular nitrogen metabolism, and the aminoglycoside response regulator Arr gene (among others). Together, our data suggest that in addition to being a valuable source of carbon and nitrogen, urocanate may be central to elicitation of bacterial pathogenesis.

id #592

A genetic approach to exploring the phenotypic differences between strains of Lactobacillus fermentum and their effect on the human gastrointestinal tract. Marc Bailie3, 1, 2, Eric Altermann1, 4, Wayne Young1, 4, Nicole Roy1, 2, 4, Warren McNabb3, 2, 4 1. Food Nutrition & Health Team, AgResearch, Palmerston north, New Zealand 2. Riddet Institute, Palmerston north, New Zealand 3. Massey University, Palmerston North, PALMERSTON NORTH, New Zealand 4. The High-Value Nutrition National Science Challenge, Palmerston north, New Zealand Microbes are intrinsic to the wellbeing and development of more complex organisms such as humans. Strain level diversification of microbial species is a naturally occurring phenomenon. These strain level changes can alter the interaction these microbes have with their host, in turn altering the host phenotype. Lactobacillus fermentum is a beneficial but transient member of the human microbiome and is “generally regarded as safe” (GRAS). However, two oral isolates sampled from healthy human volunteers here in Palmerston North exhibit divergent phenotypes. L. fermentum AGR1487 exhibits greater tolerance for bile acids and low pH and utilises different carbohydrates than L. fermentum AGR1485. AGR1487 also increases colon inflammation in germ-free mice and decreases barrier integrity in human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers, an accepted model of the intestinal barrier. Neither inflammation nor disruption of barrier integrity is observed when AGR1485 is cultured in these same environments.

We aim to use a genetic approach to unravel the differences between these strains that give rise to their observed phenotypic differences. Finished genomes for both strains were achieved using hybrid assembly, that utilised a combination of PacBio long reads and Illumina short reads. Both genomes were annotated using GAMOLA and manually curated for accuracy. The two genomes have a similar structure, but AGR1487 (1,939,032bp) appears to have a series of deletions resulting in a genome that is 287 kbp smaller than AGR1485 (2,226,862 bp). An analysis of the gene content of AGR1485 and AGR1487 along with 12 other strains of L. fermentum is currently underway. The gene expression of the two strains will be assessed over time while they are in contact with Caco-2 cells in vitro. The role of the gene(s) candidates will be studied using CRISPR/Cas9 to inactivate or complement gene candidates from AGR1485 and AGR1487, and the resulting mutant strains will be evaluated for loss or acquisition of the tight-junction weakening phenotype. This work will enhance the understanding of bacterial-host interactions and their impact on human well-being and enable screening for these gene(s), should they exist in other “good” bacteria.

1. Anderson, R., Ulluwishewa, D., Young, W., Ryan, L., Henderson, G., & Meijerink, M. et al. (2016). Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons. Scientific Reports, 6(1). doi: 10.1038/srep20318

2. Sengupta, R., Anderson, R., Altermann, E., McNabb, W., Ganesh, S., & Armstrong, K. et al. (2015). Lactobacillus fermentumAGR1487 cell surface structures and supernatant increase paracellular permeability through different pathways. Microbiologyopen, 4(4), 541-552. doi: 10.1002/mbo3.260

3. Anderson, R., Young, W., Clerens, S., Cookson, A., McCann, M., Armstrong, K., & Roy, N. (2013). Human Oral Isolate Lactobacillus fermentum AGR1487 Reduces Intestinal Barrier Integrity by Increasing the Turnover of Microtubules in Caco-2 Cells. Plos ONE, 8(11), e78774. doi: 10.1371/journal.pone.0078774

id #595

“It takes two to tango”, real time investigation of endosymbiosis using Pseudomonas fluorescens and Dictyostelium discoideum B Priyadarshini ~1, Elizabeth Ostrowski2, Heather Hendrickson1 1. School of Natural and Computational Sciences, Massey university, Auckland, New Zealand 2. School of Natural and Computational Sciences, Massey university, Auckland, New Zealand Endosymbiosis occurs when a free living organism gives up its independence and forms a partnership with a host. These partnerships can stabilise over time, in some cases leading to the evolution of complex organisms. There has been an abundance of endosymbiotic relationships across diverse host lineages.The classic examples of mitochondrial and chloroplast endosymbiosis have proven to be beneficial, however endosymbiosis may develop from initially pathogenic relationships to beneficial ones. One example of a relationship that may develop into an endosymbiosis is that of prey bacteria and their amoeboid predators. In 1960, Jeong reported endosymbiosis that arose de novo in his laboratory between an uncharacterised bacterium and amoeba which initially started as a virulent relationship, later becoming a beneficial endosymbiont. In this case, the first step towards establishment of a novel endosymbiosis was engulfment of the prey by the predator. In my project I aim to use a predator-prey relationship as a starting point to evolve a novel endosymbiosis using an experimental evolution approach. I will use the social amoeba Dictyostelium discoideum as the predator and the bacterium Pseudomonas fluorescens as the prey. Ultimately, I am interested in what if any key molecular changes produce and stabilise novel endosymbioses. Some D. discoideum display a unique characteristic known as ‘farming’ in which the protozoa carry bacteria within their spores during food scarce conditions for dispersal and prudent harvesting of their ‘bacterial crop’. Interestingly, this behaviour in the amoeba is conferred by second bacterium Burkholderia sp. which itself remains safe from being eaten by the amoeba. Istvan Zachar et.al (2018) has proposed that ‘farming’ facilitates establishment of endosymbiosis if prey rich and prey poor conditions alternate. We aim to test this hypothesis by evolving our prey bacteria and predator amoeba under a set of laboratory conditions and monitoring the transitions in real time using microscopy, genomic techniques, and flow cytometry. I will discuss my early progress in establishing the conditions for experimental evolution,strain selection and experimental design. Studying the establishment of a novel endosymbiosis will allow us to establish the early mutational steps that allow such transitions to occur and to stabilise.

id #600

The impact of non-tuberculous mycobacterial (NTM) infection on the immune response of Galleria mellonella Zakieh Vahdati2, 1, Hannah Read2, 1, Siouxsie Wiles2, 1 1. Department of Molecular Medicin and Pathology, University Of Auckland, Auckland, NZ 2. Bioluminescent Superbugs Lab , University Of Auckland, Auckland, New Zealand Over the past decade, larvae of the Greater Wax Moth Galleria mellonella have become widely used as a surrogate host for studying pathogenic microorganisms. The larvae can be housed at 37°C (human body temperature) and have an innate immune system that has much in common with mammals. We have recently established G. mellonella as a host for the opportunistic human bacterium Mycobacterium marinum, which is frequently used to model M. tuberculosis, the causative agent of the lung disease tuberculosis (TB). The aim of this project was to examine the immune responses of G. mellonella to M. marinum infection. We injected larvae with 106-107 colony forming units (CFU) of M. marinum. At various time points during infection, we euthanized groups of larvae to determine their bacterial burden. We performed histological examinations to visualise the interactions between the host (cellular immunity) and M. marinum. In addition, we collected haemolymph to measure a variety of innate immune parameters, including the numbers of haemocytes present, phagocytosis, and melanisation. Our results indicate that M. marinum is pathogenic to G. mellonella, with bacterial numbers steadily rising until the death of the larvae. Histological examination revealed that immune cells are recruited to the site of infection and form nodules, a process known as nodulation. Microscopic examination showed G. mellonella has at least 3-4 subpopulation of immune cells called haemocytes circulating in their haemolymph. Furthermore, we identified that the total number of circulating haemocytes changes during infection with M. marinum and that the host’s phagocytic and melanisation systems are activated.

id #601

The diversity of biofilm sporulation for Bacillus cereus Yiying Huang1 1. Massey University, PALMERSTON NORTH, New Zealand Bacillus cereus is a well-known food pathogen widely distributed in various environments, causing two types of foodborne diseases, diarrhoea and emesis. B. cereus is a particular concern for the food industry causing food safety and quality issues, mainly due to the formation of spores, biofilms and various toxins. This study investigated the diversity of B. cereus isolates from different sources in terms of growth, sporulation, biofilm formation and sporulation within biofilms. It reveals that, in general, biofilms provide a supportive environment for B. cereus to generate spores compared to their counterpart planktonic status, although considerable diversity between isolates was found. Shaking seems to be a favorable condition for B. cereus to form biofilm and spores within biofilms. This work provide a better understanding of the diversity of B. cereus, and more importantly, to provide an idea of the influence of biofilm formation of B. cereus in terms of sporulation. Understanding the conditions in the dairy industry that result in sporulation may help in designing strategies to control sporulation.

id #603

Development of an in vitro competition model for the mouse enteropathogen Citrobacter rodentium. Priyali Patel1, Hannah Read1, Siouxsie Wiles1 1. University of Auckland, Grafton, NA, New Zealand We have previously studied the evolution of a bioluminescent derivative of the enteric pathogen Citrobacter rodentium (ICC180) in its natural host, the laboratory mouse. After natural transmission through 20 successive hosts, a ‘hyper-transmissible’ isolate of C. rodentium (designated as N4P20) was isolated. It is required by New Zealand law that all efforts be made to replace the use of animals in scientific research. We are developing an in vitro assay to further study the ‘hyper-transmissible’ phenotype of N4P20. 3T3 Swiss albino embryonic fibroblasts were seeded onto coverslips in 24-well plates 48 hours prior to infection. C. rodentium was grown in LB overnight at 37°C with shaking and then transferred into high glucose DMEM supplemented with 10% iron-fortified foetal bovine serum. DMEM cultures were grown overnight at 37°C without shaking. 3T3 cells were infected with 100uL of the appropriate bacterial culture and incubated at 37°C with 5% CO2 without shaking for 4 hours. At 3 hours, coverslips were washed with PBS to remove any non-adherent cells and media was changed. Bioluminescence was measured using a multimodal plate reader prior to incubation, at 3 hours and 4 hours post infection. For each infection group, 2 wells were enumerated for bacteria using 1% Triton X-100 and 2 wells were fixed with methanol and stained with Giemsa for microscopy. We are investigating the use of this assay to investigate the ability of N4P20 to compete with other bacteria in vitro in an effort to identify the basis of N4P20 hyper-transmissibility in mice.

id #605

Artificial plant leaf surface as a surrogate for microbial colonization. Michal Bernach1, 2, Rebecca Soffe2, Volker Nock2, Mitja Remus-Emsermann1 1. School of Biological Sciences, University of Canterbury, Christchurch, Canterbury, New Zealand 2. Department for Electrical and Computer Engineering, University of Canterbury, Christchurch, Canterbury, New Zealand Different surrogate surfaces, varying in complexity and origin, have been used to investigate aspects of microbial life on the plant leaves. However, none of them allows to delve far into the intricacies of plant-microbe and microbe-microbe interactions occurring on leaves. Here, we present consecutive steps undertook, in order to create an artificial leaf platform that can be applied in the studies focused on microbial life of plant leaves. We demonstrate that polydimethylsiloxane (PDMS) can be employed to recreate the topography of leaves of the model plant Arabidopsis thaliana. Our results show that the physicochemical properties of PDMS are comparable to live leaves of Arabidopsis. PDMS neither deteriorates over time, nor influenced bacterial viability compared to isolated leaf cuticles, used as a biological control. Additionally, the quality of surface feature replication is at nanometer precision. PDMS is therefore a suitable material to mimic plant leaf surfaces. Subsequently, we modified PDMS to increase its permeability to water and nutrients by adding excipients at different concentrations. We tested how the modification of PDMS influences pattern resolution, surface properties and characterised the changes in permeability to selected particles (water, fructose and tryptophan) using whole-cell fluorescent bioreporter technology. When fully validated, artificial leaves will enable the implementation of new experimental conditions, that can be precisely controlled. This bottom-up approach will help to deconstruct the interdependencies of physical, chemical and biological events taking place on plant leaves and can help to answer important questions of plant-microbiology and microbial ecology.

id #606

Understanding ciprofloxacin resistance in Pseudomonas aeruginosa Attika Rehman1, Wayne M Patrick2, Iain L Lamont1 1. Biochemistry, University of Otago, Dunedin, New Zealand 2. School of Biological Sciences, Victoria university , Wellington, New Zealand Publish consent withheld

id #608

Quantifying the predictability of the emergence of antibiotic resistance in a model strain and environmental isolates of Escherichia coli. Kelly Hong1, Olin Silander1, Nikki Freed1 1. School of Natural and Computational Sciences, Massey University, Auckland, New Zealand Laboratory evolution experiments can empirically address a fundamental question in biology: Is evolution predictable? Steven J. Gould popularized the idea that evolution is not predictable; that “replaying the tape of life” would result in the evolution of completely different organisms. However, similar traits that have evolved independently in closely related, as well as taxonomically diverse groups of organisms, suggest that evolution is reproducible and predictable, both phenotypically and genotypically. One approach to test this reproducibility of adaptation is to, “replay the tape of life”; using experimental evolution- evolving hundreds or thousands of independent microbial populations and observating evolution in real-time. Previous studies have attempted to assess predictability of both phenotypic and genotypic evolution using laboratory-based evolution, yet much of this work has been done using model organisms. Little is known about at what level of genetic divergence this predictability breaks down. To address this limitation, replicate populations of fourteen genetically diverged environmental isolates of E. coli were evolved alongside the model lab strain E. coli K-12 in increasing levels of antibiotic.We use adaptation to antibiotics as this experimental technique is well defined and allows for investigation of the precise order of genetic and phenotypic changes that enable bacterial populations to adapt. Fourty daily transfers were performed, with a 40% increase in antibiotic concentration per day. After this evolutionary regime, surviving isolates were able to grow in media with drug concentrations much higher than their original minimum inhibitory concentration (MIC). We find strong evidence that resistance emerged in a predictable fashion.

id #612

Antimicrobial activity of Clostridium enriched conditioned media Amila S Nawarathna1, 2, Steve Flint2, Jon Palmer2, Gale Brightwell1, Tanushree B Gupta1 1. Food Assurance team, AgResearch Ltd, Palmerston North, Manawatu-Wanganui, New Zealand 2. School of Food and Advanced Technology, Massey University, Palmerston North, Manawatu-Wanganui, New Zealand Antimicrobial producing potential of bacteria has been investigated in numerous studies. However, the discovery of antimicrobials has been biased towards aerobic and facultative anaerobes, and strict anaerobes such as Clostridium spp. have been largely neglected. In recent years, genomic studies have indicated the genetic potential of strict anaerobes to produce putative bioactive secondary metabolites and this has encouraged the exploration of Clostridium spp. for their antimicrobial production. So far, only a limited number of antimicrobial compounds have been isolated, identified and characterized from the genus Clostridium. One of our previous studies has shown that when microbial spores isolated from farm environmental samples such as soil, feed, dairy effluent as well as milk, are enriched in cooked meat glucose starch (CMGS) broth under anaerobic growth conditions for 2 days, only Clostridium spp. can thrive; the growth of other facultative anaerobes is suppressed or inhibited. We hypothesize that this growth inhibition could be due to antimicrobial molecules produced by Clostridium spp. in CMGS medium. Conditioned media (CMs) were prepared by enriching bacterial spores extracted from soil, feed and dairy effluent of three farms and inhibitory activity of CMs against Bacillus mycoides ATCC 6462, Bacillus cereus NZRM 5, Salmonella Typhimurium NZRM 3970, Escherichia coli O157:H7 NCTC 12900 and Pseudomonas aeruginosa ATCC 25668 was assessed using microplate turbidimetric growth inhibition assay and disk-diffusion assay. Clostridium spp. were isolated from Farm 4 soil CM (F4SCM) and identified by 16S rRNA gene amplification and sequencing. CMs prepared from soil samples were found to show higher antimicrobial activity than feed and dairy effluent samples and subsequent growth inhibition analyses of soil CMs prepared from five farms demonstrated a higher growth inhibition activity from F4SCM. Tested Gram-positive, Bacillus spp. were found to be more sensitive to soil CMs than Gram-negative, Escherichia coli and Salmonella Typhimurium except Pseudomonas aeruginosa. In future work, five different Clostridium spp. isolated and identified from F4SCM will be evaluated for their antimicrobial compound synthesis potential by mono and co-culture approaches. Metabolomic and proteomic studies will be performed with the CM of interest to identify, purify and characterize putative antimicrobial metabolites.

id #614

The Evolution of Endosymbiosis and the Origins of Complex Eukaryotic Life Jasper J Perry1 1. School of Natural and Computational Sciences, Massey University, Auckland, ALBANY, New Zealand Endosymbiosis, the partnering of two organisms wherein one organism lives inside the other, is the mechanism by which complex multicellular life began. A fundamental question in evolutionary biology therefore is, how do such relationships form and become stable and what, if any, key molecular changes are required for this to occur? In my research we will aim to answer such questions by studying the early stages in the establishment of endosymbiosis using experimental evolution. Although fairly common in nature, the evolution of endosymbiosis had not been demonstrated in a laboratory setting until the 1960s when a team of researchers showed that a prey bacterium evolved intracellular virulence toward a protozoan predator and later became a beneficial endosymbiont. Such co-evolution has previously been established by the Hendrickson laboratory between the bacterial prey Pseudomonas fluorescens SBW25 and protozoan predator Naegleria gruberi. Similarly, my research will use these two organisms in a co-evolution experiment that will aim to establish endosymbiosis over the course of 10,000 generations. By performing the experiment in an environment that is specifically harmful to the bacteria, we will create a selective pressure for the bacteria to seek refuge inside the protozoa. The cells will be monitored for cooperation and fluorescent microscopy will be used to identify stable endosymbionts. To do this we will create a fluorescent strain of Pseudomonas fluorescens SBW25 for introduction into Naegleria gruberi. Successful candidates for endosymbiosis will be sequenced for novel evolved traits that may be linked to cooperativity and/or intracellular survival. Frequent sampling and cryogenic or liquid preservation of cells throughout this process will enable later reanimation and investigation using flow cytometry and genomic analysis. This approach will provide insight into the biological requirements for establishing endosymbiosis in nature and allow us to better understand the evolutionary pathways that formed the basis of complex eukaryotic life.

id #620

Identification of the sporidesmin gene cluster in the facial eczema causing fungus, Pithomyces chartarum Jaspreet Singh Sidhu1, Ruy Jauregui1, Abdul Baten1, Christine Voisey1, Paul Maclean1, Jan Sprosen2 1. AgResearch Ltd., Palmerston North, MANWATU, New Zealand 2. AgResearch Ltd., Ruakura Research Centre, Hamilton, New Zealand Facial eczema (FE), a debilitating liver syndrome in livestock, is caused by ingestion of sporidesmin, a compound belonging to the epipolythiodioxopiperazine (ETP) class of toxins. Sporidesmin is found in spores of the fungus Pithomyces chartarum. It permanently damages the liver, resulting in peeling skin, abortions and substantial reductions in animal productivity. New Zealand has a high rate of FE compared to other countries as our animals are fed predominantly on ryegrass which supports high levels of P. chartarum. The climate is also particularly conducive for P. chartarum growth. The aim is to identify and annotate the P. chartarum sporidesmin gene cluster. The information gained will be used to support the production of high purity sporidesmin in a heterologous host for FE vaccine production and to develop an automatable spore counting method based on the sporidesmin gene cluster sequence. The P. chartarum genome was sequenced using single molecule real time (SMRT) Sequencing-PacBio and the genome was annotated by the MAKER gene annotation pipeline equipped with Augustus and SNAP gene finding methods. RNA sequencing was performed on two different timepoints of P. chartarum spores from cultures that were 5 and 6 weeks old and transcripts were mapped to the genome. A putative sporidesmin gene cluster has been identified based on BLASTX similarity with the genomes of the fungus Leptosphaeria maculans which produces sirodesmin, a phytotoxin belonging to the ETP class of toxins.

id #621

Identification of the sporidesmin gene cluster in the facial eczema causing fungus, Pithomyces chartarum Jaspreet Singh Sidhu1, Paul Maclean1, Ruy Jauregui1, Abdul Baten1, Christine Voisey1, Jan Sprosen2 1. AgResearch Ltd., Palmerston North, MANWATU, New Zealand 2. AgResearch Ltd., Ruakura Research Centre, Hamilton, New Zealand Facial eczema (FE), a debilitating liver syndrome in livestock, is caused by ingestion of sporidesmin, a compound belonging to the epipolythiodioxopiperazine (ETP) class of toxins. Sporidesmin is found in spores of the fungus Pithomyces chartarum. It permanently damages the liver, resulting in peeling skin, abortions and substantial reductions in animal productivity. New Zealand has a high rate of FE compared to other countries as our animals are fed predominantly on ryegrass which supports high levels of P. chartarum. The climate is also particularly conducive for P. chartarum growth. The aim is to identify and annotate the P. chartarum sporidesmin gene cluster. The information gained will be used to support the production of high purity sporidesmin in a heterologous host for FE vaccine production and to develop an automatable spore counting method based on the sporidesmin gene cluster sequence. The P. chartarum genome was sequenced using single molecule real time (SMRT) Sequencing-PacBio and the genome was annotated by the MAKER gene annotation pipeline equipped with Augustus and SNAP gene finding methods. RNA sequencing was performed on two different timepoints of P. chartarum spores from cultures that were 5 and 6 weeks old and transcripts were mapped to the genome. A putative sporidesmin gene cluster has been identified based on BLASTX similarity with the genomes of the fungus Leptosphaeria maculans which produces sirodesmin, a phytotoxin belonging to the ETP class of toxins.

id #622

Quorum sensing inhibitors from endophytic bacteria associated with medicinal Plants of Western Ghats Ravishankar V Rai1 1. University of Mysore, Mysore, KARNATAKA, India Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms through the production and sensing of N-acyl homoserine lactone (AHL) signal molecules. Enzymatic degradation to disrupt quorum-sensing in these bacteria could pave the way for the new development in decreasing resistance strains and are of significant interest for clinical, agricultural, and industrial applications. There is considerable interest in agents that selectively interfere with the QS systems of pathogenic bacteria, in order to target bacterial virulence and to develop new anti-infective therapies.QS interference or “quorum quenching may be achieved by inhibiting the biosynthesis of the signal molecules, by inhibiting signal detection by blocking the signal receptor, or by enzyme- catalysed degradation or modification of the signal molecules. QS interference is generally believed to less likely select for resistance, because it usually does not directly affect growth. The endophytic bacteria from medicinal plants of Western Ghats of India were screened for quorum quenching activity using the biosensor Chromobacterium violaceum. The cell free lysates of the bacterial endophytes were tested for QQ enzymes and the percentage of AHL hydrolysis was quantified by HPLC. The aiiA homologous genes in these isolate were identified and sequenced. The production of AHL-lactonase by Enterobacter sp. was optimized using RSM. The AHL-lactonase was produced and purified using AKTA pure chromatography to achieve maximum purity of the enzyme. The AHL-lactonase was tested for its ability to inhibit virulence factors expression and biofilm formation in Pseudomonas aeruginosa PAO1.The aiiE gene from Enterobacter sp. was cloned into pMAL-c2X and pET-19m plasmid expressed in E. coli JM109 and Rosetta DE3, respectively, to obtain pure AHL-lactonase. Cell-free lysates of endophytic bacteria showed AHL degrading ability by inhibiting violacein production by 80 % in C. violaceum. The identification of the aiiA homologous gene confirmed the presence of AHL-lactonase in cell-free lysate. Under optimized culture condition, AHL-lactonase isolated from E. aerogenes had a molecular weight of 30 k Daltons. In P. aeruginosa PA01, the AHL-lactonase significantly inhibited biofilm formation and virulence expression such as pyocyanin production by more than 80%, rhamnolipid production by more than 70% and protease production by 80%.%.

1. 1. Quorum quenching activity of AiiA lactonase KMMI17 from endophytic Bacillus thuringiensis KMCL07 on AHL- mediated pathogenic phenotype in Pseudomonas aeruginosa.. Kanmani Anandana , Ravishankar Rai V ( 2019) Microbial Pathogenesis. 132, 230-242 2. Biofilm inhibitory activity of metallo-protein AHL-lactonase from cell-free lysate of endophytic Enterobacter species isolated from Coscinium fenestratum Gaertn , PS Rajesh, PD Rekha and V Ravishankar Rai,( 2019) Biocatalysis and Agricultural Biotechnology, 18, https://doi.org/10.1016/j.bcab.2019.01.047

id #623

Quorum sensing inhibitors from endophytic bacteria associated with Medicinal Plants of Western Ghats Ravishankar V Rai1 1. University of Mysore, Mysore, KARNATAKA, India Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms through the production and sensing of N-acyl homoserine lactone (AHL) signal molecules. Enzymatic degradation to disrupt quorum-sensing in these bacteria could pave the way for the new development in decreasing resistance strains and are of significant interest for clinical, agricultural, and industrial applications. There is considerable interest in agents that selectively interfere with the QS systems of pathogenic bacteria, in order to target bacterial virulence and to develop new anti-infective therapies.QS interference or “quorum quenching may be achieved by inhibiting the biosynthesis of the signal molecules, by inhibiting signal detection by blocking the signal receptor, or by enzyme- catalysed degradation or modification of the signal molecules. QS interference is generally believed to less likely select for resistance, because it usually does not directly affect growth. The endophytic bacteria from medicinal plants of Western Ghats of India were screened for quorum quenching activity using the biosensor Chromobacterium violaceum. The cell free lysates of the bacterial endophytes were tested for QQ enzymes and the percentage of AHL hydrolysis was quantified by HPLC. The aiiA homologous genes in these isolate were identified and sequenced. The production of AHL-lactonase by Enterobacter sp. was optimized using RSM. The AHL-lactonase was produced and purified using AKTA pure chromatography to achieve maximum purity of the enzyme. The AHL-lactonase was tested for its ability to inhibit virulence factors expression and biofilm formation in Pseudomonas aeruginosa PAO1.The aiiE gene from Enterobacter sp. was cloned into pMAL-c2X and pET-19m plasmid expressed in E. coli JM109 and Rosetta DE3, respectively, to obtain pure AHL-lactonase. Cell-free lysates of endophytic bacteria showed AHL degrading ability by inhibiting violacein production by 80 % in C. violaceum. The identification of the aiiA homologous gene confirmed the presence of AHL-lactonase in cell-free lysate. Under optimized culture condition, AHL-lactonase isolated from E. Aerogenes had a molecular weight of 30 k Daltons. In P. aeruginosa PA01, the AHL-lactonase significantly inhibited biofilm formation and virulence expression such as pyocyanin production by more than 80%, rhamnolipid production by more than 70% and protease production by 80%

1. 1. Quorum quenching activity of AiiA lactonase KMMI17 from endophytic Bacillus thuringiensis KMCL07 on AHL- mediated pathogenic phenotype in Pseudomonas aeruginosa.. Kanmani Anandana , Ravishankar Rai V ( 2019) Microbial Pathogenesis. 132, 230-242. 2. Biofilm inhibitory activity of metallo-protein AHL-lactonase from cell-free lysate of endophytic Enterobacter species isolated from Coscinium fenestratum Gaertn , PS Rajesh, PD Rekha and V Ravishankar Rai,( 2019) Biocatalysis and Agricultural Biotechnology, 18, https://doi.org/10.1016/j.bcab.2019.01.047

2. 1. Quorum quenching activity of AiiA lactonase KMMI17 from endophytic Bacillus thuringiensis KMCL07 on AHL- mediated pathogenic phenotype in Pseudomonas aeruginosa.. Kanmani Anandana , Ravishankar Rai V ( 2019) Microbial Pathogenesis. 132, 230-242. 2. Biofilm inhibitory activity of metallo-protein AHL-lactonase from cell-free lysate of endophytic Enterobacter species isolated from Coscinium fenestratum Gaertn , PS Rajesh, PD Rekha and V Ravishankar Rai,( 2019) Biocatalysis and Agricultural Biotechnology, 18, https://doi.org/10.1016/j.bcab.2019.01.047

id #627

Resolving broad patterns of prokaryotic community structure and composition in New Zealand pasture soils Rachel A Kaminsky1, 2, Steve A Wakelin3, 4, Matthew P Highton2, Md Sainur Samad2, 5, Sergio E Morales2 1. AgResearch, Palmerston North, MANAWATU, New Zealand 2. Microbiology and Immunology, University of Otago, Dunedin 3. Forest Systems, Scion Research, Christchurch 4. Forage Science, AgResearch, Christchurch 5. Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen The importance of pasture soils to the New Zealand economy and global food security cannot be understated. Additionally, the importance of soil biodiversity to soil ecosystem functioning and, consequently, food production is becoming apparent. Despite this, our understanding of soil biodiversity is still developing. Here, we review the literature on soil microbial communities inhabiting pasture soils across New Zealand in order to summarize the key properties that impact microbial distributions. This work also includes an examination of 16S rRNA sequencing data from 92 pasture sites. The goal of this examination is to re-test the properties identified in the review on a prokaryote-focused dataset generated by contemporary techniques.Results from this work indicate that pH strongly influences soil microbial communities on a landscape scale, and that this conclusion can be drawn using a range of experimental methods. Geographic region, land use and soil classification serve as secondary modifiers, indicating that these factors may interact resulting in the broad impact of pH.

id #628

"NsARC" The solution to bad bugs Alibe Wasa1, Jack Heinemann1, Catherine Bishop1, William Godsoe1, 2 1. University of Canterbury, Christchurch/Riccarton, CANTERBURY, New Zealand 2. Biological Sciences, Lincoln University, Christchurch, Canterbury, New Zealand A major vehicle for the spread of infectious diseases is contaminated surfaces. Photo-activated titania (TiO2) well known for its antimicrobial activity and self-cleaning properties could be used to coat door handles and similar surfaces, reducing viability and colonization by pathogens and reducing their spread. We tested the survival of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Saccharomyces cerevisiae on the surface of a thin film deposition of nanostructured anatase rutile and carbon (NsARC) under a variety of light wavelengths and intensities (high intensity visible, UV and ambient light) adopting a modified version of the standard ISO 27447:2009 “test method for antimicrobial activity of photocatalytic materials”. We also tested to see how E. coli will respond to antibiotics after being exposed to NsARC. There was significantly less survival (P<0.001) of all the organisms tested on NsARC compared to the inert uncoated stainless steel (304) under all conditions. Attachment of E. coli and S. aureus onto the surface of NsARC was used as a model to evaluate biofilm formation. There was significantly less attachment (P<0.001) of both bacteria on NsARC compared to stainless steel (304). Scanning electron microscopy (SEM) was used to corroborate the results. Interestingly, Escherichia coli were found to be more susceptible to the antibiotic kanamycin following NsARC exposure, compared to unexposed control strains. A phenomenon that would not have been predicted from knowledge of the material alone, was that we observed that unlike TiO2, NsARC had antimicrobial activity in the dark. These results suggest that NsARC can be used as a self-cleaning and self-sterilizing antimicrobial surface coating for the prevention and reduction in the spread of infections.

id #631

Using rumen microbial community composition data to predict methane yield ranks of sheep fed different diets Sandeep Kumar1, Poppy P Miller1, Christina D Moon1, Arjan Jonker1, Melanie K Hess2, Paul H Maclean1, Michelle R Kirk1, Siva Ganesh1, Sharon M Hickey3, Paul Boma1, 4, Edgar Sandoval1, Sarah V Maclean1, Sai Arojju1, Alan F McCulloch2, Suzanne J Rowe2, John C McEwan2, Peter H Janssen1 1. AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand 2. AgResearch Limited, Invermay Agricultural Centre, Mosigiel, New Zealand 3. AgResearch Limited, Ruakura Research Centre, Hamilton, New Zealand 4. Nabuin Zonal Agricultural Research and Development Institute, National Agricultural Research Organization, Moroto, Uganda Agriculture plays a major role in New Zealand’s economy, with ruminant products contributing about one third of total export earnings. Ruminants produce methane, a major greenhouse gas that is a by-product of feed fermentation by the complex rumen microbial community. Several strategies are being explored to reduce methane yields from ruminants. Of these, breeding for low emitting ruminants shows promise and relies on identifying low methane-emitting individuals. The aim of this project was to investigate whether rumen microbial community composition data could be used to identify low methane-emitting sheep. Methane yields (g methane per kg dry matter intake) were measured in five measurement periods on a cohort of 96 yearling ewes fed lucerne pellets and fresh-cut pasture. Methane yield prediction models were built from microbial community structure data consisting of partial bacterial and archaeal 16S rRNA and protozoal 18S rRNA gene sequences. Partial least squares regression, random forest and ridge regression-based methods were used to test predictive abilities with training and test datasets from different measurement periods. Methane yield predictions based on bacterial community structure data alone were similar to those that combined bacterial, archaeal, and protozoal data. Training models on data from lucerne-fed sheep to identify low methane emitting sheep also fed lucerne performed better than corresponding models trained on data from pasture-fed sheep. Uncertainties were greater when predicting between diets, especially when using data from lucerne-fed sheep to identify low-methane emitters in pasture-fed sheep. Adding more data from the same animals to the training data reduced uncertainty both within and between diets. Bacterial genera that were strong predictors of methane yield varied by diet. Among these, Clostridiales vadinBB60 and the Family XIII AD3011 group were the strongest positive predictors of methane yield on lucerne pellets, while Rikenellaceae RC9 gut group and Victivallis were strong positive predictors on fresh-cut pasture. Negatively-correlated predictors included Selenomonas and Kandleria for lucerne and Ruminococcus and multiple genera of Lachnospiraceae for pasture. Overall, this study demonstrated that rumen bacterial 16S rRNA gene sequence data can be used to identify low methane yield sheep with moderate ability, even in the presence of significant dietary effects.

id #632

Genomic characterisation and comparison of antibiotic resistant and susceptible Helicobacter pylori. Zoe King1, Colleen Higgins1, Kevin Lee1, Brent Seale1 1. School of Science, AUT, Auckland, New Zealand Helicobacter pylori is a pathogenic bacterium found in 50 % of the world’s population. This significant human pathogen can cause stomach ulcers and, in some cases, gastric cancer. Recent studies have found increasing resistance to first-line treatment with antibiotics; clarithromycin, metronidazole and in some cases amoxicillin. This increasing resistance calls for an in-depth look into the mechanisms of antibiotic resistance. Four H. pylori isolates from biopsies of patients undergoing gastroscopy were selected for antibiotic susceptibility trials and whole genome comparison. Resistance was determined through minimum inhibitory

concentration (MIC) strips with clarithromycin, amoxicillin and metronidazole. Phylogenetic analysis was conducted using maximum-likelihood method with near full-length 16S rRNA gene sequences amplified with “universal” prokaryotic primers 27F and 1492R. Whole genome sequencing was conducted using Illumina HiSeq systems. The genomes were assembled, annotated, and the resulting annotated assemblies were comparatively analysed. Experimental progress is still ongoing, but initial resistance levels show: Two isolates were resistant to >256 μg/mL of clarithromycin and metronidazole; one isolate was resistant to 24 μg/mL clarithromycin; while one final isolate is susceptible to the all antibiotics tested. Comparative genomics of the isolates is still to be conducted with focus on identifying single nucleotide polymorphisms (SNPs) and other genetic changes that could confer antibiotic resistance. The results of this research aim to increase the knowledge of antibiotic resistance genes in H. pylori that can be used further for more effective treatment regimens.

id #633

Microbial marker genes for predicting methane yield in ruminants Graeme T Attwood1, Yang Li2, Jacinda Aplin1, Priya Soni1 1. AgResearch Ltd, Palmerston North, MANAWATU, New Zealand 2. Department Umweltsystemwissenschaften, Animal Nutrition, ETH, Zurich, Switzerland The rumen microbiome is diverse and its many types of microbes interact to digest feed and drive animal production and greenhouse gas emissions. Ruminant livestock are responsible for ~30% of New Zealand’s anthropogenic greenhouse gases, mainly because of their eructation of methane, which is 25 times more potent than carbon dioxide in climate warming. These emissions are also relevant to animal production as around 6-12% of their energy intake is lost as methane. Many factors control methane output, including diet and host genetics, but the abundance and function of microorganisms in the rumen make strong contributions. We are working on developing a reliable biomarker gene quantification method to allow prediction of ruminant methane emissions from rumen or buccal samples. Studies of sheep selected for high or low methane-yield has identified clear differences between the microbes, genes and transcripts found in their rumen microbiomes. There are at least two different microbiomes that support low methane yield in sheep, and there are strong correlations between both gene and transcript abundances with methane yield measurements. Statistical analyses for the sheep metagenome data identified 185 genes with significant correlations to methane yield; and these genes have served as the candidates for the development of methane yield biomarkers. The genes were ranked according to their methane yield predictive strength, and PCR primers were designed for the 10 top-ranked biomarker candidates. Primer specificity and sensitivity has been tested against rumen DNAs and the three best performing primer sets have been adapted to allow high throughput amplicon sequencing. DNAs have been extracted from rumen contents and buccal swabs collected from pasture-fed sheep with matching methane yield measurements and we are currently assessing biomarker gene abundances via amplicon sequencing to determine the predictive ability of the assay from the two sample sources. The results obtained so far indicate that marker genes are reliably amplified from rumen contents using the amplicon-sequencing format but buccal samples are less consistent, presumably due to the much lower concentration of rumen microbes found in the buccal cavity from rumination events.

id #636

Endosymbiotic bacteria of plant-associated fungi: friends or foes? Daniel A. Bastias 1, Linda J. Johnson 1, Stuart D. Card1 1. Agresearch, Palmerston North, MANAWATU, New Zealand Many bacteria form symbiotic associations with plant-associated fungi. The effects of these symbionts on host fitness usually depends on symbiont or host genotypes and environmental conditions. However, bacterial endosymbionts, i.e. those living within fungal cells, may positively regulate host performance as their survival is heavily dependent on host fitness. A meta-analysis of published results was conducted to evaluate this hypothesis. In addition, the biology of bacterial endosymbionts of fungi was reviewed, with the focus on functional aspects of bacterial-fungal symbiosis. Our analysis supports the hypothesis that endosymbiotic bacteria of fungi are beneficial symbionts. Ecological scenarios, where the presence of beneficial bacterial endosymbionts of fungi could be compromised, are also discussed.

id #637

16S rRNA gene PCR contamination: tracking the source Rana Fathizargaran1, Cara L Brosnahan1, Diana Jaramillo1 1. Ministry for primary industry (MPI), Upper Hutt, WELLINGTON, New Zealand Amplification of no-template controls (NTCs) is a common issue when using universal PCR primers as in the case of 16S rRNA PCR for bacterial identification. We sought to investigate the source of contamination of sporadically occurring amplification of NTCs. DNA sequencing of the NTC PCR products were identified as Bacillus sp. We examined the effect of laboratory surfaces, equipment, plastic ware and PCR reagents including water, primers and master mixes. Results showed that the only factor that had an impact on the amplification of the 16S NTCs were different PCR master mixes. No NTC amplification was observed when the PCR was performed using MTP Taq polymerase (DNA-free). This was regardless of the PCR primer diluent, water or buffer template or plastic ware used. Results were also consistent between operators. In contrast, all variables tested in the presence of other commercial master mixes resulted in consistent amplification of the NTCS. From this, we have reached a good level of certainty about the commercial master mix containing traces of Bacillus sp. Bacillus species are commonly used for the production of enzymes including polymerase. However, it is not known why this occurs sporadically, whether it is an issue dependent on the manufacturing or differences between PCR runs remains to be determined.

id #645

Colistin Resistance in Pseudomonas aeruginosa Mareike B Erdmann1, Paul Gardner1, Ian Lamont1 1. University of Otago, Dunedin, OTAGO, New Zealand Multidrug resistance in Gram-negative bacteria such as Pseudomonas aeruginosapresents a serious burden for public health. P. aeruginosais one of the world’s most common hospital-acquired bacteria with high mortality rates and intrinsic resistance to a wide range of antibiotics. The polymyxin antibiotic colistin is used as ‘last line of defence’ antibiotic for infections in people with cystic fibrosis. This study aims to further understand colistin resistance inP. aeruginosa by comparing resistance of experimentally evolved resistant mutants derived from a sensitive laboratory strain with clinical P. aeruginosaisolates. During the experimental evolution the bacteria were exposed to increasing levels of colistin on agar gradient plates. The phenotypic resistance of experimentally evolved P. aeruginosaand clinical isolates was determined using the minimal inhibitory concentration (MIC). A total of 15 colistin resistant strains were evolved. The MIC levels of these strains were up to 128 times higher than the parental PAO1 strain. Some clinical isolates were resistant to colistin at levels up to 128 higher than the PAO1 strain as well. Whole genome sequencing (WGS) and Single Nucleotide Polymorphism (SNP) analysis to identify novel mutations and other genetic differences that lead to resistance will be performed. These data show that experimental evolution of a sensitive laboratory strain can lead to levels of colistin resistance comparable to those of clinical isolates of P. aeruginosaand will provide a strong base for understanding how P. aeruginosa resists colistin.

id #647

Characterization of two cell death elicitor families from the Dothideomycete fungal phytopathogens Dothistroma septosporum and Cladosporium fulvum Mariana Tarallo1, Lukas Hunziker,1, Carl H Mesarich2, Rebecca L McDougal3, Rosie E Bradshaw1 1. School of Fundamental Sciences, Massey University, Palmerston North 2. School of Agriculture and Environment , Massey University, Palmerston North 3. Scion (New Zealand Forest Research Institute, Ltd), Rotorua Understanding how plants interact with fungal pathogens leads to improvements in plant health. At the molecular level, invasion patterns, termed effectors and pathogen-associated molecular patterns (PAMPs), can be recognized by plant receptors to activate the plant immune system, causing a localized cell death response that prevents further growth of the pathogen. Dothistroma septosporum (Ds) and Cladosporium fulvum (Cf) are two closely related fungal species belonging to the class Dothideomycetes that cause Dothistroma Needle Blight in pine and leaf mould in tomato, respectively. We have identified two families of effectors, Ecp20 and Ecp32, which are conserved between Ds and Cf. The Ecp20 family has predicted structural homology to the Alt a 1 allergen of Alternaria alternata, which has known functions in pathogen virulence, while Ecp32 has homology to proteins of unknown function. Both families have members that are highly expressed during host infection. The Ecp20 family has four paralogues in both species, while the Ecp32 family has five paralogues in Cf and four in Ds. The gene encoding each family member was cloned for Agrobacterium-mediated transient transformation assays in leaves of non-host species Nicotiana tabacum and N. benthamiana to determine whether they trigger a cell death response, and thus are likely to be recognized by cognate immune receptors. For the Ecp20 family, only DsEcp20-3 triggered cell death in both non-host species, while CfEcp20-2 triggered cell death only in N. benthamiana. For the Ecp32 family, most members triggered some degree of cell death in both Nicotiana spp. Importantly, across both families, some members failed to trigger cell death in either species. Based on this result, we will generate chimeric proteins, swapping regions of the cell death and non-cell death elicitors, to identify the region(s) required for cell death elicitation. We will also generate gene deletion strains of Ds and Cf to determine whether members of the Ecp20 and Ecp32 families are required for pathogenicity or full virulence. Collectively, this project will provide a better understanding of how Ds and Cf interact with their hosts at the molecular level, and deliver knowledge that could be used in disease resistance breeding programmes.

id #649

Full length cDNA sequencing to study transcriptional regulation in bacteria - a proof of concept Bhargava Reddy Morampalli1, Olin Silander1 1. Massey University, Albany, AUCKLAND, New Zealand Bacterial transcriptional regulation can be complex, with condition dependent transcription start sites (TSS) and termination sites. Short read cDNA sequencing is the most common technique used to study this in bacteria, often combined with TEX to find TSS. This can give an accurate picture of transcript levels and TSS, but due to fragmentation of cDNA before sequencing, the underlying transcript structure is lost. Long read sequencing allows sequencing of entire transcripts along with the transcription levels. Here we use full length cDNA sequencing from Oxford Nanopore Technologies which allows us to 1) sequence full length transcripts 2) avoid PCR bias 3) provide information about operon structures. We compare transcript levels and transcript structure between E. coli K12 and a natural isolate of E. coli to investigate changes in transcriptional regulation. This will allow us to gain deeper understanding of operon complexity of bacteria and the evolution of transcriptional regulation.

1. Yan, Bo, Matthew Boitano, Tyson A. Clark, and Laurence Ettwiller. “SMRT-Cappable-Seq Reveals Complex Operon Variants in Bacteria.” Nature Communications 9, no. 1 (September 10, 2018): 3676. https://doi.org/10.1038/s41467-018-05997-6.

2. Ju, Xiangwu, Dayi Li, and Shixin Liu. “Full-Length RNA Profiling Reveals Pervasive Bidirectional Transcription Terminators in Bacteria.” Nature Microbiology, July 15, 2019, 1. https://doi.org/10.1038/s41564-019-0500-z.

id #651

Identification of avirulence effectors from the apple scab fungus, Venturia inaequalis Silvia de la Rosa1, Joanna Tannous 2, Bruno Le Cam2, Vincent G.M. Bus3, Rosie E. Bradshaw4, Joanna K. Bowen 5, Kim M. Plummer6, Carl H. Mesarich7 1. Bio-Protection Research Centre, School of Agriculture and Environment, Massey University, Palmerston North, New Zealand 2. Ecologie Évolutive chez les Champignons, Institut de Recherche en Horticulture et Semences, Institut National de la Recherche Agronomique, Beaucouzé, France 3. New Zealand Institute for Plant and Food Research, Havelock North, New Zealand 4. Bio-Protection Research Centre, School of Fundamental Sciences, Massey University, Palmerston North, New Zealand 5. New Zealand Institute for Plant and Food Research, Auckland, New Zealand 6. Department of Animal, Plant and Soil Sciences, Centre for AgriBiosciences, La Trobe University, Melbourne, Australia 7. Bio-Protection Research Centre, School of Agriculture and Environment, Massey University, Palmerston North, New Zealand Scab, caused by the fungus Venturia inaequalis (Vi), is the most economically important disease of apple (Malus  ×  domestica) worldwide. Under favourable conditions, this disease can result in the loss of 70% or more of the crop. Scab disease is predominantly controlled through extensive fungicide application. However, fungicide-resistant strains of Vi have now emerged, and there is growing concern for the risks that fungicides pose to human health and the environment. For these reasons, alternative control strategies are required. Disease-resistant apple varieties represent the best alternative. Disease resistance relies on the recognition of fungal virulence factors, termed effector proteins, by corresponding plant immune receptor proteins. This recognition leads to a hypersensitive response at the site of infection, which renders the pathogen avirulent (hence, recognized effectors are termed avirulence effectors). Unfortunately, in some instances, recognition by immune receptors can be overcome, and thus, plant resistance can be broken. To better understand which immune receptors are more likely to provide durable resistance against scab disease, a better understanding of how Vi overcomes plant resistance at the molecular level is first required. Notably, strains of Vi have been identified in New Zealand that overcome resistance mediated by the Rvi4 immune receptor. We have approximately 100 progeny, derived from a sexual cross between two strains of Vi that differ in their ability to trigger Rvi4-mediated resistance. Using comparative genomics, we aim to identify the AvrRvi4 effector of Vi, which is recognized by the Rvi4 immune receptor, by association with a resistance-breaking mutation. In doing so, we will ascertain how, at the molecular level, strains of Vi overcome Rvi4-mediated resistance. To fulfil our aim, we are currently sequencing the genomes of the two parents and 50 of the progeny in conjunction with pathogenicity tests on Rvi4 apple. I will present our progress on this project, and will report on our ‘effectoromics’ research, where we are seeking to identify Vi effectors that activate the immune system in the non-host model plants Nicotiana benthamiana and Nicotiana tabacum. Ultimately, it is hoped that our research can inform durable scab disease resistance breeding or selection programmes in apple.

id #652

Characterisation of immunogenic epitopes on a methanogen surface protein Sofia Khanum1, Juliana Yeung1, Dairu Shu1, Sandeep Gupta1, Tania Wilson1, Natalie Parlane1, Vincevincenzo carbone2, Eric Altermann2, 3, Axel Heiser1, Peter Janssen2, Neil Wedlock1 1. Animal Health , Hopkirk Research Institute, Agresearch Grasslands, Palmerston North, New Zealand 2. Rumen Microbiolgy, Agresearch Grasslands, Palmerston North, New Zealand 3. Riddet Institute, Massey University, Palmerston North, New Zealand An improved knowledge of immunogenic B and T-cell epitopes of candidate vaccine antigens will help inform better vaccine design. In silico prediction of epitopes is a potentially time saving alternative for experimental epitope identification but is often subject to misidentification of epitopes. As part of our studies developing a vaccine against rumen dwelling methanogens to reduce methane emissions in ruminants we conducted this study to better understand the rules of epitope prediction for methanogen proteins. A model methanogen protein from Methanobrevibacter ruminantium M1, the adhesin-like protein (AdLP, Mru_1499) was selected to map B and T cell epitopes. A series of 17 overlapping 20-mer peptides were selected to cover the Big_1 domain (amino acids 19-198), a part of the extracellular domain of the protein involved in protein-to-protein interactions. Groups (n=4) of BALB/c mice were vaccinated with these peptides. Peptide-specific antibodies were measured by ELISA and an in vitro splenocyte re-stimulation assay used for determining specific T cell responses. Six peptides, A4, A7, A9, A10, A11 and A14 stimulated strong IgG2a or IgG1 antibody responses, suggesting that amino acids within these peptides act as B cell epitopes. Eleven peptides, A3, A5, A6, A7, A8, A10, A11, A12, A13, A14, A15 and A17 stimulated IFN-γ and IL-17A production to varying levels. Overall, three peptides, A7, A10, A11 were shown to be major immunological epitopes of AdLP Big_1 domain. The empirical data on identifying epitopes was compared with epitope predictions made by programmes based on a range of algorithms, including Bepipred, ABCpred, Bcepred, SVMtrip, IEDB, SYFPEITHI, Rankpep, PREDEP and MMBPRED. To achieve greater accuracy, we combined predictions from several algorithms to produce a consensus prediction. In general, the T-cell epitopes on AdLP, identified by in silico predictions were broadly comparable to those determined empirically but in silico epitope predictions were not useful for predicting B-cell epitopes.

id #654

Reducing Rumen Methanogen Growth in Pure Culture by Inhibitor Compounds Catherine Andrews1, Linley Schofield1, Peter Janssen1, Kerri Reilly1, Rosemary Heathcott1, vincenzo carbone1, Ron Ronimus1 1. AgResearch, Hokowhitu, Palmerston North, NEW ZEALAND, New Zealand The rumen is the largest component of a ruminant's digestive tract and is where microbial fermentation occurs to break down feed that the animal cannot. The rumen contains a complex microbiome, including rumen methanogens. These methanogens take advantage of the anaerobic environment of the rumen, and the hydrogen gas produced as a by-product of fermentation. They use the hydrogen gas along with carbon dioxide to produce methane and water. The methane is then eructated into the atmosphere and is a large contributor to New Zealand’s greenhouse gas emissions. We seek to identify inhibitory compounds which show high efficiency in reducing methanogens to low ruminal concentrations with the aim to mitigate methane emissions

from ruminant animals. Culture-based experiments were performed to determine the minimum inhibitory concentration (MIC) of three potential inhibitors. Two pure rumen methanogen cultures, Methanobrevibacter ruminantium strain M1 (DSM1093) and Methanobrevibacter boviskoreani JH1, were tested. Small-molecule inhibitors were added at a range of concentrations. This involved monitoring anaerobic growth via culture optical density in glass culture tubes in quadruplicate over time. Inhibitors were added after growth had been confirmed by OD600 at values between 0.08-0.1. The growth continued for approximately 48 hours post-inhibitor addition. Treated cultures were compared to the following control cultures: untreated, solvent-only, and a positive control (the latter containing a known methanogen inhibitor). The concentration range was determined for each methanogen strain and showed the concentrations at which growth was inhibited, partially inhibited and not inhibited. Overall, Mb. ruminantium M1 was more sensitive to the three inhibitors than Mb. boviskoreani JH1. These results were compared to results from other methods of inhibitor testing used within the wider project. These include a 96-well plate format using pure methanogen cultures, and a rumen fluid-based in-vitro assay. This work is continuing, in the search to discover and test further inhibitors of rumen methanogens.

id #656

Expression of Cellulosome proteins from a novel New Zealand Ruminococcus sp. HUN007 Kerri Reilly1 1. Agresearch, Palmerston North, MANAWATU, New Zealand New Zealand’s ruminants are fed a primarily pasture based diet that is broken down and fermented by the microorganisms found in the rumen. The resulting volatile fatty acids and microbial protein provides the animal with most of the nutrients and energy required for growth and production. The plant material which comprises ruminants diet contains many polysaccharides which are highly resistant to enzymatic degradation. Some fibrolytic bacteria have developed a specialised multi-component complex of proteins and enzymes called a cellulosome. Cellulosomes are composed of structural proteins (scaffoldins) that provide the backbone to anchor various cellulolytic enzymes, such as endoglucanases, cellobiohydrolases, which act synergistically to degrade cellulose. Scaffoldin proteins possess cohesin domains that bind tightly to dockerin modules found on enzyme subunits to form the final architecture of the complex. Ruminococcus sp. HUN007 is a novel putative cellulolytic bacterium isolated in New Zealand from a cow rumen. The HUN007 genome contains genes for 12 cohesin-containing proteins and 169 dockerin-containing proteins, suggesting it can form a cellulosome. In this project, four cohesin and four dockerin-containing genes from Ruminococcus sp. HUN007 were selected for further study. The scaffoldin proteins contained both cohesin and dockerin binding sites, suggesting the possibility for self-adhesion or binding to other scaffoldin molecules. The dockerin-containing proteins included a putative glycosyl hydrolase family 9 (95.1KDa), a glycosyl hydrolase family 48 (90.5KDa), an endoglucanase Cel9P (93.4KDa) and a peptidoglycan binding domain containing protein (34.4KDa). Genes were codon-optimised, synthesized and subcloned into either the pET-28a (+) vector (dockerins) or pET-15b vector (cohesins). These were subsequently expressed in LOBSTR (low background strain) Escherichia coli. The four cohesin-containing proteins and the smaller peptidoglycan dockerin-containing protein were expressed producing proteins of expected molecular weight. In order to express the remaining three dockerin-containing proteins it was necessary to perform dual transformations with both scaffoldin and dockerin containing plasmids and co-express the proteins. This demonstrates the close relationship between some of the cohesin and dockerin-containing proteins. Cellulosomes play a role in the degradation of cellulose in the rumen. Understanding how these complexes fit together could unlock potential improvements to rumen fermentation.

id #659

Rumen isolates of the Christensenellaceae R-7 group are able to utilise hemicellulose and produce hydrogen and ethanol as major end products Sam Mahoney-Kurpe1, 2, Dragana Gagic2, 3, Patrick J Biggs2, 3, Nikola Palevich1, Satoshi Koike4, Christina Moon1, Graeme Attwood1 1. Animal Science, Agresearch Limited, Palmerston North, Manawatu, New Zealand 2. School of Fundamental Sciences, Massey University, Palmerston North, Manawatu, New Zealand 3. School of Veterinary Sciences, Massey University, Palmerston North, Manawatu, New Zealand 4. Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan The microbiota in the rumen, the fermentative forestomach of ruminant animals, drives the conversion of dietary lignocellulosic biomass into a variety of economically valuable bio-products, by providing their hosts the majority of their energy requirements. Despite decades of study, some abundant rumen microbial groups remain poorly characterised. One such group, shown to be part of the core rumen microbiota, and one of the most abundant taxa in forage-fed animals, is the Christensenellaceae R-7 group (phylum Firmicutes). In this study, two Christensenellaceae R-7 group strains from the Hungate1000 culture collection were morphologically and metabolically characterised. Transmission electron microscopy confirmed their cells as rod-shaped, lacking appendages, and possessing cell wall ultra-structures characteristic of gram-negative bacteria. The strains were tested for their ability to grow on a range of 23 soluble and 9 insoluble carbohydrate substrates. Both strains could utilise a variety of the soluble substrates tested, with both growing best on cellobiose, but showing no growth on any of the tested sugar-alcohols. The strains also degraded the hemicelluloses xylan and pectin, but not cellulose. Acetate, ethanol and hydrogen were the major fermentation end products, and were produced continuously during batch-culture growth in rich media containing 30% rumen fluid and cellobiose as the carbon source. The addition of bacto-peptone and casamino acids promoted the growth of both strains, though neither grew solely on these substrates in the absence of a carbon source. Their growth was also further enhanced in media containing rumen fluid collected from fresh forage-fed cows or sheep compared to that from a hay-fed cow. The hemicellulolytic activity of the Christensenellaceae R-7 group strains suggest a role in ruminal fibre degradation. However, as a consequence of their hydrogen and ethanol production, this group may also play an important role in providing substrates to rumen methanogens for methane production.

id #661

Application of selective whole-genome amplification to culture-independent-typing of Neisseria meningitidis Xiaoyun Ren1, Heather Davies1, Philip Carter1, Audrey Tiong1 1. Meningococcal Reference Laboratory, Special Pathogens Unit, Health & Environment, ESR, Porirua, Wellington, New Zealand ESR provides national surveillance for pathogens of public health importance in New Zealand. Increased use of culture independent diagnostic methods has resulted in less access to isolates required to generate typing information for surveillance. The Meningococcal Reference Laboratory, within ESR’s Special Pathogen Unit, is now receiving 40 to 50% of case samples in the form of genomic DNA purified from blood or cerebrospinal fluid. We are unable to type these samples according to international surveillance standards due to the amount and quality of DNA received. Furthermore, for about 2% of the samples, we are unable to obtain any typing information, which can affect clinical and public health decision-making. The purpose of this study is to improve meningococcal typing from clinical DNA samples. We tested human DNA removal and total DNA amplification on clinical DNA samples from meningococcal cases, but found these techniques to yield inconsistent results, and therefore unsuitable for public health surveillance. Selective Whole Genome Amplification uses pathogen specific primers that are less likely to bind to host DNA to selectively amplify pathogen DNA. Using swga (https://github.com/eclarke/swga) with 14 meningococcal reference genomes as foreground and human and mitochondria genomes as background we designed three sets of amplification primers to selectively amplify meningococcal DNA. We found the primer sets preferentially amplified meningococcal DNA in mixtures that contain both human and meningococcal DNA. We are in the process of testing these primers on clinical samples. We hope this approach will allow for the whole-genome sequencing of Neisseria meningitidis directly from clinical samples.

id #666

Functional expression of Aspergillus fumigatus CYP51 isoforms in Saccharomyces cerevisiae Parham Hosseini1, Mikhail V. Keniya1, Joel D.A. Tyndall1, Brian C. Monk1 1. University of otago, Dunedin, OTAGO, New Zealand Objectives Aspergillus fumigatus infections are a serious medical problem, especially for patients undergoing organ transplantation or cancer treatment. Therapeutic options for invasive aspergillosis are limited due to the intrinsic resistance of A. fumigatus to fluconazole and acquired drug resistance due to azole exposure. Furthermore, the use of voriconazole as the primary treatment for invasive aspergillosis has only modest success. The cytochrome P450 enzyme lanosterol 14α-demethylase (Cyp51) is required for the fungal-specific production of ergosterol and is the target of the azole drugs. Unlike humans and yeast, which have a single sterol 14α-demethylase and one cognate NADPH-cytochrome P450 reductase (CprA), A. fumigatus has two isoforms of Cyp51 (AfCyp51A and AfCyp51B) and two paralogs of CprA (AfCprA1 and AfCprA2). Our goal is to better understand AfCyp51A and AfCyp51B structure and function by elucidating their interaction with cognate CprAs. Methods Codon optimised AfCyp51A or AfCyp51B with a C-terminal 6xHis-tag were constititively expressed from the PDR5 locus in a Saccharomyces cerevisiae host strain. AfCprA1 or AfCprA2 with a C-terminal 6xHis-tag were also co-ordinately, constitutively co-expressed from the PDR15 locus in strains expressing AfCyp51A or AfCyp51B. Results PCR and DNA sequence analysis confirmed the correct location and sequences of the heterologous genes inserted in the S. cerevisiae genome. Expression of the AfCpy51 isoforms and AfCprA2 were confirmed using western blots and by mass spectrophotometry. Despite transcription of AfCprA1 mRNA, AfCprA1 was not expressed. MIC80 assays with azole drugs (fluconazole, posaconazole, and voriconazole) and the echinocandin micafungin confirmed the functionality of the AfCyp51s and the impact of AfCprA2 co-expression on AfCyp51 function. Conclusion Functional heterologous expression of AfCyp51A and AfCyp51B has been achieved in yeast, with NADPH-cytochrome P450 reductase AfCprA2 acting as the cognate reductase. A codon optimised construct of AfCprA1 is transcribed but not translated. Use of the S. cerevisiae expression system provides a basis for screening and structural studies that will facilitate the discovery of azole drugs designed to overcome target-mediated intrinsic and acquired azole resistance in A. fumigatus.

id #671

The good, the bad or the ugly - how do plants distinguish beneficial from pathogenic bacteria? Moritz Miebach1, Paula E Jameson1, Mitja NP Remus-Emsermann1, 2 1. School of Biological Sciences, University of Canterbury, Christchurch, Canterbury, New Zealand 2. Biomolecular Interaction Centre, University of Canterbury, Christchurch, Canterbury, New Zealand A rich diversity of microorganisms colonises plants in nature, some promoting plant growth and health, whereas some others cause disease. Studies on plant pathogens showed that plants perceive microbes by detecting highly conserved microbe-associated molecular patterns (MAMPs). Such MAMPs are inherent to microbes irrespective of their relationship to plants, raising the question how and if plants discriminate between beneficial and pathogenic bacteria. Thus far, our knowledge of plant responses to non-pathogenic natural leaf colonisers is sparse. To gain a deeper understanding of plant-bacteria interactions found on healthy plants in nature, we established an axenic plant growth system. Axenically grown Arabidopsis thaliana plants

inoculated with different non-pathogenic bacteria naturally found on A. thaliana leaves were studied and their impact on the plant was assessed. Plant colonisation was studied at several time points after inoculation to gain insights into bacterial colonisation kinetics. To elucidate whether plant responses to natural bacterial leaf colonisers can be predicted by the spectrum of MAMPs being presented, the genomes of the studied bacteria were searched for the presence of known MAMPs. Our results are a starting point for an in-depth elucidation of plant-microbe interactions in the context of healthy plants in nature and how some bacterial leaf colonisers protect plants against pathogens by stimulating the plant’s immune system.

id #673

Exploring the effects of enteral nutrition on intestinal microbiota using an in vitro model Carel M H Jobsis1, Caroline C Kim1, Halina M Stoklosinski1, Sarah B Cordiner1, Duncan I Hedderly1, Paul A Blatchford2, Pramod Gopal1, Andrew S Day3, Shanthi G Parkar1 1. The New Zealand Institute for Plant and Food Research Limited, Palmerston North, New Zealand 2. Zespri International Limited, Mt Maunganui, New Zealand 3. Department of Paediatrics, University of Otago, Christchurch, Christchurch, New Zealand Exclusive enteral nutrition (EEN) is a well-established therapy utilised to induce remission in children with Crohn’s Disease (CD). The objective of this study was to evaluate the effects of enteral formula (EF) fortified with curcumin on the intestinal microbiome of CD children. The effect of EF + curcumin was assessed using six in vitro fermentation vessels simulating the proximal colon. First, all vessels were brought to a standardised steady state using a complete growth medium inoculated with faeces from children with CD. Then three vessels were treated with EF + curcumin and three with EF + curcumin + fibre. Short-chain fatty acids (SCFAs) and polyphenolic metabolite concentrations were quantified throughout the fermentations. Microbial composition and functional changes were assessed by whole genome sequencing followed by bioinformatics and statistical analysis. The proportions of different bacterial species were influenced by the composition of the media. The complete growth medium increased the proportions of beneficial Faecalibacterium and Bifidobacterium compared to the inoculum but also increased the potentially pathogenic Paenibacillus. The EF + curcumin increased the variety and richness (alpha-diversity) of bacteria versus complete medium. However, comparison between the two different curcumin-fortified EF did not show significant bacterial changes, indicating that patients on such treatments might not be negatively affected by a lack of fibre. In comparison to the complete medium, bacterial functions associated with the metabolism of vitamins, some key amino acids and carbohydrates were significantly altered by both test treatments. There was an increase in the number of genes responsible for synthesis of butyrate (a key SCFA) and vitamin B7. The presence of curcumin appeared to increase bacterial genes associated with degradation of xenobiotic compounds, suggesting the curcumin was bioavailable and affecting microbial metabolism. This model may be suitable to further explore the effects of nutritional interventions and advance understanding of the interactions between diet and the intestinal microbiota. EF + curcumin appears to beneficially modulate gut microbial metabolism and the absence of fibre of most EF formulations does not appear to negatively impact microbial structure.

id #674

Protein A/G-based enzyme-linked immunosorbent assay for detection of anti-Pythium insidiosum antibodies in human and animal subjects Chalisa Jaturapaktrarak1, Penpan Payattikul1, Tassanee Lohnoo1, Yothin Kumsang1, Aree Laikul2, Watcharapol Pathomsakulwong3, Chompoonek Yurayart4, Walaiporn Tonpitak5, Theerapong Krajaejun6 1. Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand 2. Department of Large Animal and Wildlife Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand 3. Equine Clinic, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom, Thailand 4. Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand 5. Department of Microbiology, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, Thailand 6. Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Pythiosis is a life-threatening infectious disease caused by the pathogenic oomycete Pythium insidiosum. Reports of both human and animal pythiosis are on the rise worldwide. No effective drugs are currently available for this disease. Surgical removal of P. insidiosum-infected organs is the main option for cure. Prognosis of the pythiosis patients relies on accurate diagnosis and prompt treatment. There are needs for an immunodiagnostic test that can detect the disease in both humans and animals. This study aims at developing an Enzyme-Linked Immunosorbent Assay (ELISA) for immunodiagnosis of pythiosis in multiple host species. A key feature of our ELSIA is the use of protein A/G which binds the immunoglobulins from humans and other animals. A total of 21 pythiosis and 48 control sera, obtained from humans and various animals, were recruited for an assay evaluation. The protein A/G-based ELISA with a proper cutoff point can ultimately discriminate the pythiosis sera from the control sera, providing 100% detection sensitivity, specificity, and accuracy. The successfully-developed protein A/G-based ELISA can detect the anti-P. insidiosum antibodies in serum samples of both humans and animals. It is a versatile, feasible-to-develop, and effective immunodiagnostic assay for pythiosis. Because ELISA is a highly sensitive and specific technique, the A/G-based ELISA can be used to epidemiologically study the seroprevalence of the anti-Pythium insidiosum antibodies in human and animal populations.

id #676

Identifying active biofilm community members participating in methane oxidation using RNA stable Isotope probing Emmanuel O Egbadon1, Kathryn Wigley1, Louise Weaver2, Kim Baronian1, Peter A Gostomski1 1. Chemical and Process Engineering, University of Canterbury, Christchurch, New Zealand 2. ESR, Christchurch, New Zealand Methane (CH4) driven denitrification is a process that could be utilised to remove nitrate from contaminated aquifers, however neither the process nor the microorganisms involved are fully understood. Identifying active biofilm community members that are oxidising methane is one of the first steps required to understand the process of methane driven denitrification. The objective of this study was to use RNA stable isotope probing to identify the active members within an aerobic methane consuming community. A microbial biofilm was established in a packed bed reactor under continuous supply of 4% (v/v) methane gas as the sole carbon source and nitrate as the nitrogen source following inoculation with Lowcliffe, NZ aquifer sediments. Amplicon 16S rRNA gene sequencing (Illumina MiSeq) revealed a community dominated by Methylocystis spp. (40%); all other genera had a relative abundance < 15%. RNA stable isotope probing was performed in a series of batch experiments to identify active community members. Briefly, biofilm from the reactor was inoculated into serum bottles containing either 10% 13C-labelled CH4 or 10% 12C-unlabelled CH4. After incubating for 96 hours, total RNA was extracted from the biofilm, ultracentrifuged in a CsTFA density gradient and fractionated according to RNA buoyant density. The % of total RNA in the heavier fractions (> 1.77 g/ml) from the 13C experiments was greater than from the 12C (unlabelled) controls (39% vs. 13%). qPCR targeting the pmoA gene (diagnostic for methanotrophic bacteria), similarly showed that the relative abundance of pmoA in the heavier fractions was greater in 13C-labelled RNA compared with unlabelled RNA (73% vs. 9%). Finally, isotope ratio mass spectrometry of extracted RNA confirmed assimilation of 13C; increasing from 1% in unlabelled RNA to 11% in 13C experiments. Collectively, these preliminary results indicate that RNA stable isotope probing was able to resolve members that were actively participating in methane oxidation from the rest of the (non-active) community. Future work will focus on using high throughput sequencing of the labelled and unlabelled fractions to determine exactly which bacteria are being labelled and if any cross feeding is occurring. Cross feeding of heterotrophs is one hypothesis for methane-driven denitrification.

id #677

Cell envelope-associated proteins in rumen methanogens Juliana Yeung1, Dairu Shu1, Timothy Ferguson1, Eric Altermann2, 3, Sofia Khanum1, Sandeep Gupta1, Trevor Loo4, Andrew Sutherland-Smith4, Axel Heiser1, Peter H Janssen5, Neil Wedlock1 1. Animal Health, AgResearch Grasslands, Palmerston North, New Zealand 2. Riddet Institute, Massey University, Palmerston North, New Zealand 3. Food Nutrition and Health, AgResearch Grasslands, Palmerston North, New Zealand 4. School of Fundamental Sciences, Massey University, Palmerston North, New Zealand 5. Rumen Microbiology, AgResearch Grasslands, Palmerston North, New Zealand The rumen microbiota is complex and almost totally composed of anaerobic organisms, including methanogenic archaea (methanogens). These occupy a unique niche in the rumen, using hydrogen, formate and methyl compounds to produce methane. Many of the proteins predicted from analysis of genome sequences of rumen-dwelling methanogens, in particular cell envelope-associated proteins, have no known biological function. To elucidate the role of these proteins in methanogen activity in the rumen and in the interaction of these archaea with other ruminal microbes, we have produced antibodies against selected methanogen proteins. The proteins were expressed in a standard bacterial expression system. An important first step was to determine if recombinantly produced proteins can generate antibodies that bind and recognise the native methanogens proteins. Bioinformatic analysis of the genome of Methanobrevibacter ruminantium M1 identified suitable cell surface-located proteins for the studies. Predicted extracellular domains of these proteins were synthesised in Escherichia coli and circular dichroism analysis was performed to check that each recombinant protein had an ordered structure. Antibodies were produced against each recombinant protein and used in ELISA and Western blotting on lysates and hydrophobic membrane fractions prepared from M1 cells. Proteins in the fractions were further separated by 2D gel electrophoresis and specific binding of the antibodies to the native targets was confirmed by LC-MS/MS. Binding of the antibodies to methanogen cells was demonstrated using ELISA and flow cytometry. Our results to date suggest that expression of some of the cell surface proteins in bacteria produced recombinant proteins that sufficiently mimic the native proteins, with cross-reactivity of antibodies to methanogen cell extracts. Our immunological studies, together with new information on gene expression from transcriptomic analysis of M1 in cultures, can be used to further understand the biological function of cell surface proteins.

id #679

The difference in ileal and caecal microbial composition did not influence organic matter fermentation but the short-chain fatty acid production in the growing pig fed a human-type diet Anika Hoogeveen1, 2, Paul Moughan2, Edward de Haas2, Paul Blatchford3, Warren McNabb2, Carlos Montoya2, 4 1. School of Food & Advanced Technology, Massey Univeristy, Palmerston North, New Zealand 2. Riddet Institute, Palmerston North, New Zealand 3. The New Zealand Institute for Plant and Food Research Limited, Palmerston North, New Zealand 4. Food Nutrition & Health Team, Grasslands Research Centre, AgResearch limited, Palmerston North, New Zealand Gastrointestinal fermentation in humans and monogastric animals is considered to occur mainly in the hindgut. Recently, several studies demonstrate that the microbiota in the lower small intestine have the ability to ferment both dietary and non-dietary materials.This study aimed to determine the microbial composition in ileal and caecal digesta and their fermentation capacity. Five pigs (23 ± 1.6 kg body weight) were fed a human-type diet. On day 15, pigs were euthanized and digesta from the terminal

jejunum and terminal ileum were collected as substrates for fermentation. Ileal and caecal digesta were collected for preparing microbial inocula and to perform microbial analysis (qPCR and Illumina sequencing). Terminal jejunal digesta were fermented in vitro with a pooled ileal inoculum for 2 hours, while terminal ileal digesta were fermented in vitro with a pooled caecal inoculum for 24 hours. The total numbers of bacteria, based on 16S rRNA gene copies, in ileal and caecal digesta did not differ (P>0.05). However, differences (P<0.05) were found in the taxonomic composition. Ileal digesta contained higher (P<0.05) numbers of the genera Enterococcus (32-fold greater), and Leuconostoc (55-fold greater), while caecal digesta contained higher numbers of the genera Methanosphaera (15-fold greater), Blautia (65-fold greater), Coprococcus (17-fold greater), Ruminococcaceae (227-fold greater), and Ruminococcus (82-fold greater). The ileal organic matter fermentability (28%) was similar (P>0.05) to its hindgut counterpart (35%). Acetate and iso-valerate production were higher (P<0.05) for ileal fermentation than caecal fermentation, but propionate, butyrate, and valerate production lower. In conclusion, despite differences in microbial composition, quantitatively similar degrees of fermentation were found in the ileum and hindgut of the growing pig fed a human-type diet.

id #680

Isolation and high cell density culture of methanotrophs for use in artificial biofilters to mitigate methane emissions Rashad Syed1, Shuguang Zhang1, Surinder Saggar2, Thilak Palmada2 1. Callaghan Innovation, Lower Hutt, WELLINGTON, New Zealand 2. Landcare Research, Palmerston North, Manawatu, New Zealand Agriculture is the main source of methane emissions (greenhouse gas) in New Zealand. The concentration and rates of methane emission from majority of the NZ dairy effluent ponds and herd-homes may not be suitable for flaring or for use in heating. Mitigating methane emissions using methanotrophs (methane eating bacteria), is proposed as the only eco-friendly option currently available. Previous research at Manaaki Whenua - Landcare Research (MWLR) and Massey University demonstrated the ability of soil biofilters to remove methane emissions coming out from a research dairy farm in Palmerston North. However, transportation of soils (enriched with methanotrophs) is a challenge for scaling-up this technology. This partnership project between Callaghan Innovation and MWLR aims to isolate the methanotrophs from soils primed with methanotrophs, and culture them in bioreactors to produce microbial biomass that can be used in artificial biofilters for the abatement of fugitive methane emissions from various sources nationally and internationally.

id #689

Experimental evolution of bacterial pathogens on antimicrobial copper surfaces Xinyue Li1, Xue-Xian Zhang1 1. Massey University at Albany, Albany, AUCKLAND, New Zealand Healthcare-associated infections (HAIs) affect ~10% hospitalized patients in New Zealand public hospitals. It has been estimated that at least one third of HAIs can be prevented if appropriate infection control protocols (hand hygiene in particular) are fully followed. However, this goal cannot be achieved by merely improving the compliance rates of hygiene practices. It requires new strategies that can help maintain high standards of hospital environmental hygiene. One such emerging strategy for HAI prevention is to apply self-sanitizing copper surfaces to commonly touched places within hospital facilities, which can provide sustained protection against microbial contamination. This is owing to the fact that a wide range of microorganisms can be rapidly killed on the surfaces of metallic copper; a process termed “contact killing”. However, with the increased use of copper in the immediate living environments, there is serious concern that bacterial pathogens may become resistant to copper, and, more significantly, develop overlapping resistance to metallic copper and antibiotics. To address this crucial safety issue, we first designed experiments to unravel the mechanisms of bacterial contact killing on copper. Specifically, we tested the hypothesis that bacterial cells are predominantly killed by a burst release of toxic copper ions, resulting from chemical reactions between bacterial cell surface components and metallic copper. Next, we performed experimental evolution to systematically assess the potential of bacterial pathogens to evolve resistance to metallic copper, and examine the possible impacts on the current medical use of antibiotics.

id #690

Phenotypical-genotypical correlation and c-di-GMP turnover ability of genes encoding GGDEF/EAL/HD-GYP domain proteins in Pseudomonas putida Hailing Nie, Yujie Xiao, Jinzhi He, Huizhong Liu, Liang Nie, Qiaoyun Huang, Wenli Chen* State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China. *Correspondence to: W Chen, Tel: +86-27- 87282730; Fax: +86-27-87280670. E-mail addresses: wlchen@mail.hzau.edu.cn Cyclic diguanylate (c-di-GMP) is a broadly conserved bacterial signaling molecule that modulates diverse cellular processes, such as biofilm formation, colony morphology, and swimming motility. The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs) with GGDEF domain and phosphodiesterases (PDEs) with either EAL or HD-GYP domain. Genome analysis revealed that Pseudomonas putida KT2440 has a large c-di-GMP turnover system, with 42 genes encoding GGDEF and EAL/HD-GYP domain containing proteins on its genome, indicating a vital role of c-di-GMP in physiological adaptation of the strain to various conditions. However, phenotypical-genotypical correlation and c-di-GMP turnover ability of these genes were largely unknown. Herein, by systematically constructing deletion mutants/overexpression strains of the 42 predicted c-di-GMP metabolism–related genes and analyzing the phenotypes, we preliminarily revealed the role of each gene in biofilm formation, colony morphology, and swimming motility. Subsequent results from protein sequence alignments and cellular c-di-GMP assessment indicated that 25 out of the 42 genes were likely to encode DGCs, nine genes were predicted to encode PDEs, four genes encoded bifunctional enzymes, and the other four genes encoded enzymatically inactive proteins. This study firstly systematically analyzed the phenotypical-genotypical correlation and c-di-GMP turnover ability of all the 42 genes in Pseudomonas putida KT2440, which offers a basic understanding of the roles of these 42 genes and can serve as a

toolkit for investigators to further elucidate the functions of these GGDEF and EAL/HD-GYP domain-containing proteins. It can also serve as a helpful tool for further studying c-di-GMP–mediated regulation and response of c-di-GMP to various environmental signals by means of this microbe.

id #691

Broadening the knowledge on c-di-GMP mediated FleQ dependent transcriptional regulation in Pseudomonas putida Yujie Xiao1, Meina He1, Liang Nie, Haozhe Chen1, Wenjing Zhu1, Hailing Nie1, Wenli Chen1*, Qiaoyun Huang1,2* 1State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China. 2Key Laboratory of Arable Land Conservation (Middle and Lower Reaches of Yangtze River), Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan, China. *Correspondence to: W Chen, wlchen@mail.hzau.edu.cn; Q Huang, qyhuang@mail.hzau.edu.cn The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) modulates diverse cellular processes through its receptors. FleQ is a widely distributed transcriptional regulatory c-di-GMP effector in Pseudomonas species. By inversely regulating expression of biofilm and flagellar genes in response to c-di-GMP, FleQ helps to control planktonic to biofilm lifestyle transition. Here by transcriptome sequencing and qRT-PCR verifying, we found 50 new target genes that were co-regulated by c-di-GMP and FleQ in P. putida. Further in vitro binding analysis confirmed that seven out of the 50 genes were under the direct regulation of FleQ in response to c-di-GMP, including PP_0681, PP_0788, PP_4519 (lapE), PP_4858, PP_5222 (cyaA), PP_5496 and PP_5586. Among these, c-di-GMP inhibited expression of PP_0788 and cyaA, and promoted expression of other five genes. Both ATPase activity and RpoN binding ability of FleQ were not required for regulating of the seven target genes. Preliminary study on function of three target genes (lapE, cyaA, PP_5586) revealed the role of c-di-GMP/FleQ in related processes. LapE is a membrane pore responsible for retention of adhesin LapA, and regulation of lapE by c-di-GMP/FleQ favored the secretion of LapA and biofilm formation under high c-di-GMP level. CyaA is an adenylate cyclase responsible for synthesis of cAMP, and inhibited of cyaA by high c-di-GMP led to a lower cAMP level. Besides, we revealed that PP_5586 was necessary for the c-di-GMP mediated exopolysaccharide Pea dependent wrinkly colony morphology. Results of this study extended the knowledge on c-di-GMP mediated FleQ dependent transcriptional regulation in P. putida.

id #692

Rapid CRISPR-based identification of microorganisms Andrea Sajuthi1, Olin Silander1, and Nikki Freed1

1School of Natural and Computational Sciences, Massey University Identifying organisms is important for many reasons such as finding disease etiologies, determining antibiotic resistance patterns, and slowing spread of infection. The identification of microorganisms tends to be slow, imprecise, expensive, or a combination of these. Here we demonstrate a proof of principle experiment using a Cas9/CRISPR system in combination with nanopore DNA sequencing that allows for rapid identification of microorganisms along with important genetic elements such as its antibiotic resistance profile. We designed CRISPR RNAs (crRNAs) specifically targeting the Escherichia coli MG1655 gene gnd, which is known to be highly variable between Escherichia coli subspecies. Using crRNAs we enrich for specific genomic regions and then sequence using a portable long read DNA sequencer (Nanopore) MinION. Our proof of principle results show that in samples of pure E. coli MG1655 genomic DNA spiked with varying ratios of human:MG1655 genomic DNA, we are able to CRISPR enrich a specific genomic locus (gnd) by a factor of more than 100,000-fold. This CRISPR enrichment has several significant advantages over PCR, including the potential for massive multiplexing, the ability to sequence very long genomic regions (> 20Kbp), and the fact that CRISPR-enrichment is an isothermal reaction, in contrast to PCR. This data shows that we are able to identify specific strains of E. coli through the CRISPR-enrichment with nanopore sequencing. This illustrates the feasibility of using CRISPR coupled to nanopore sequencing for other rapid-identification applications, such as pathogen and resistance pattern detection.

id #693

The effects of non-invasive and invasive enteric infections on the microbial ecology of the gut Lama Abdel Rahman1, Xochitl Morgan1, Bruce Russell1, James Ussher1 1Department of Microbiology and Immunology, University of Otago, New Zealand Objectives: Infectious diarrhea is caused by parasites and bacteria and kills approximately 2,195 children every day globally. Diarrhea alters the gut microbiota. Numerous parasites, including Giardia, Dientamoeba fragilis, Cryptosporidium and Entamoeba histolytica have been known to cause diarrhea, but neither the lack of symptoms in some infected individuals nor the effects of these pathogens on the host gut microbiome community are yet fully understood. This study aimed to understand the different effects of non-invasive (e.g. Giardia and D. fragilis) and invasive (e.g. Cryptosporidium and E. histolytica) diarrhea-causing parasites, and diarrhea-causing invasive bacteria (e.g. Salmonella and Campylobacter) on host gut microbial ecology. Methods: 16S rRNA amplicon sequencing data from 467 fecal samples from diarrhea-suffering patients was categorized as pathogen-negative (diarrhea without diagnosis / DWD), or positive for invasive bacteria, invasive parasites or non-invasive parasites. This data was then used to compare alpha and beta diversity of the gut based on the infection type. Results: Non-invasive parasite infection significantly increased the alpha diversity of their host microbial communities. Both invasive parasites and invasive bacteria decreased alpha diversity of infected samples and altered the overall structure of the microbial communities of the infected sample groups. Conclusion: Invasive and non-invasive infections have different effects on the gut microbiome ecology. The effects of these infections have potential implications for optimizing and understanding gut microbiome recovery from infection.

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