Bacteriocins of bovine non-aureus staphylococci

University of Calgary

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Graduate Studies The Vault: Electronic Theses and Dissertations

2017

Bacteriocins of bovine non-aureus staphylococci

Carson, Domonique

Carson, D. (2017). Bacteriocins of bovine non-aureus staphylococci (Unpublished master's

thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25092

http://hdl.handle.net/11023/4124

master thesis

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UNIVERSITY OF CALGARY

Bacteriocins of bovine non-aureus staphylococci

by

Domonique Carson

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES

CALGARY, ALBERTA

SEPTEMBER, 2017

© Domonique Carson 2017

ii

Abstract

The non-aureus staphylococci (NAS) species are among the most prevalent isolated from bovine

milk and have been reported to inhibit major mastitis pathogens, likely by producing

bacteriocins. This thesis is comprised of two sections, focusing on in vitro inhibition assays and

in silico identification of bacteriocin gene clusters and bacteriocin resistance genes in NAS and

Staphylococcus aureus, using isolates obtained from the Canadian Bovine Mastitis and Milk

Quality Research Network. The first part determined the inhibitory capability of 441 bovine

NAS isolates (comprising 25 species) against bovine S. aureus and human methicillin-resistant S.

aureus (MRSA) and determined the presence of bacteriocin biosynthetic gene clusters in NAS

whole genomes. Overall, 40 isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis,

S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth

of S. aureus in vitro; of which, 23 isolates (from S. capitis, S. chromogenes, S. epidermidis, S.

pasteuri, S. simulans, and S. xylosus) also inhibited MRSA. 105 putative bacteriocin gene

clusters encompassing 6 different subclasses (lanthipeptides, sactipeptides, lasso peptides, class

IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. The second

part of the thesis determined the susceptibility of 139 bovine S. aureus isolates to a bacteriocin

producing S. chromogenes isolate and identified and described the distribution of genes

potentially associated with susceptibility and resistance in S. aureus whole genomes. Overall, 90

S. aureus isolates (65%) were resistant to inhibition by the S. chromogenes isolate. We identified

77 genes that were associated with an isolate being resistant. We also identified 76 genes that

were associated with an isolate being susceptible to the S. chromogenes. Bacteriocin

susceptibility and resistance seems to be linked to a large number of genes, the majority of which

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are annotated as hypothetical proteins and will need further assessment to determine their role in

S. aureus susceptibility. Overall, bacteriocins may be a potential source of novel antimicrobials

and this thesis represents the foundation to explore novel NAS bacteriocins.

iv

Acknowledgements

Firstly, I would like to thank my supervisors Herman Barkema and Jeroen De Buck for

all of their patience and support during this project. Thank you to Herman for the opportunity to

attend so many conferences and speak about my research. Thank you to Jeroen for all your

direction with my projects. I also would like to thank my committee members for their support

and Dr. John Kastelic for his edits of my manuscript and for his scientific writing courses.

I would also like to thank Uliana Kanevets and Aaron Lucko for their help in the lab.

Thank you to Matthew Workentine for all his help with bioinformatics.

Thank you to the original CNS crew, Larissa Condas and Diego Nobrega for everything.

Larissa, thank you for showing me the CNS ropes, I came into this project with little lab

experience and your expertise was essential for my success. Your endless encouragement and

honest life talks during your time here were also so appreciated- “Everything is AWESOME”.

Diego, thank you for all your help, from whole genome sequencing to talking through absolutely

everything with me, I could not have done it without you. I am also so grateful for the rest of the

CNS crew that has joined us. Thank you Ali Naqvi for helping me with statistics and thank you

Ana Paula Monteiro Alves for being my conference buddy and letting me be the absolute

introvert that I am. Lastly, thank you to Sohail Naushad, without you I would have not been able

to do this. Thank you for the countless conversations about genes, bioinformatics, and how to

interpret my results. Thank you for the coffee dates and the pep talks and for always being on my

team. Thank you for all of your help with organizing genomes, creating trees, running jobs, and

giving me feedback whenever I ask.

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Thank you to all my fellow graduate students. Thank you to Emily Morabito for

beginning this program at the same time as me and becoming such a huge support as we figured

this all out together. Thank you to Caroline Corbett for being a rock in all of this. You were

always there to talk and were always able to provide valuable feedback on my research. Thank

you for being such a good friend to me. Also thank you Casey Jacobs for always being there for

me (usually with a beer and chocolate ready). Our daily dog walks became so special and

without them life would have been much more difficult.

I would also like to thank Dr. Keliesha Roth and Amy Stanley for being the best friends a

girl could ask for in this life. Keliesha, thank you for always making me feel like I could do this.

Thank you for letting me come work at your house, for taking such good care of me after my

surgery this spring, for always taking the dogs for me whenever they needed somewhere to go,

and for bringing Took into my life. You are one of the most amazing people I’ve ever met and a

huge reason that I am the person I am today. Amy, thank you for being the most understanding

and amazing human. You supported me through this every way a person possibly could… you

ran away to the mountains with me when I needed that, you exercised the dogs for me when I

couldn’t, you provided hours of deep conversations about life, and you have always just wanted

what is best for me. Thank you to you both.

Thank you to Reid Anderson. You came into my life on that airplane at the exact moment

I needed you to. You have been my strength in times of weakness, my never ending support

system, my inspiration to keep working towards my goals, and (most importantly) the best dog

dad I could ever ask for. You mean more to me than you’ll ever know.

Lastly, thank you to my family for the endless support and understanding. Thank you for

answering all of my phone calls and always giving me words of encouragement. It has been hard

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to be so far apart but it never felt too far. Thank you for instilling in me a passion for learning

and for sticking things out.

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Dedication

To my family,

in particular my parents

and my chosen family

who have always supported me

and to my dogs

who have kept me sane.

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Table of Contents

Abstract .......................................................................................................................................... ii

Acknowledgements ...................................................................................................................... iv

Dedication .................................................................................................................................... vii

Table of Contents ....................................................................................................................... viii

List of Tables ................................................................................................................................ xi

List of Figures and Illustrations ................................................................................................ xii

List of Symbols, Abbreviations and Nomenclature ................................................................ xiv

Preface .......................................................................................................................................... xv

Chapter One: General Introduction ........................................................................................... 1

1.1 Mastitis in the Canadian dairy industry ........................................................................... 1

1.2 Non-aureus staphylococci ................................................................................................... 2

1.3 Bacteriocins ......................................................................................................................... 4

1.4 Classification of bacteriocins ............................................................................................. 5

1.4.1 Lanthipeptides ................................................................................................................ 7

1.4.2 Sactipeptides ................................................................................................................ 12

1.4.3 Lasso Peptides .............................................................................................................. 14

1.4.4 Class IIa Bacteriocins .................................................................................................. 15

1.4.5 Class IIb Bacteriocins .................................................................................................. 16

1.4.6 Class IIc Bacteriocins .................................................................................................. 17

1.4.7 Class IId Bacteriocins .................................................................................................. 18

1.5 Immunity genes and cross immunity .............................................................................. 19

1.6 Bacteriocin discovery and purification ........................................................................... 20

1.7 In silico screening .............................................................................................................. 23

1.8 Applications of bacteriocins ............................................................................................. 26

1.9 Bacteriocin resistance ....................................................................................................... 31

1.10 Thesis outline ................................................................................................................... 34

1.10.1 Bacteriocins of non-aureus staphylococci isolated from bovine milk ....................... 34

ix

1.10.2 Identifying putative bacteriocin resistance genes in Staphylococcus aureus whole

genomes ................................................................................................................................ 35

Chapter Two: Bacteriocins of non-aureus staphylococci isolated from bovine milk ........... 36

2.1 Abstract .............................................................................................................................. 36

2.2 Introduction ....................................................................................................................... 38

2.3 Materials and methods ..................................................................................................... 40

2.3.1 Isolates ......................................................................................................................... 40

2.3.2 Phenotypic testing ........................................................................................................ 41

2.3.3 Effect of proteinase K on inhibition ............................................................................. 41

2.3.4 Whole genome sequencing, assembly, and annotation ................................................ 42

2.3.5 Screening of genomes for bacteriocin clusters ............................................................ 43

2.3.6 BLAST ......................................................................................................................... 44

2.3.7 Genome comparison .................................................................................................... 44

2.3.8 Precursor gene alignments ........................................................................................... 45

2.4 Results ................................................................................................................................ 45


2.4.2 Effect of proteinase K on inhibition ............................................................................. 46


2.5 Discussion .......................................................................................................................... 51

2.6 Conclusions ........................................................................................................................ 58

Chapter Three: Identifying putative bacteriocin resistance genes in Staphylococcus aureus

whole genomes ............................................................................................................................. 79

3.1 Abstract .............................................................................................................................. 79

3.2 Introduction ....................................................................................................................... 81

3.3 Materials and methods ..................................................................................................... 83

3.3.1 Isolates ......................................................................................................................... 83


3.3.3 Whole genome sequencing, assembly, and annotation ................................................ 84


3.3.5 Screening of genomes for immunity related genes ...................................................... 86

x

3.4 Results ................................................................................................................................ 87

3.4.1 Phenotypic test ............................................................................................................. 87


3.4.3 Screening of genomes for putative resistance related genes ........................................ 88

3.5 Discussion .......................................................................................................................... 90

3.6 Conclusions ........................................................................................................................ 95

Chapter Four: Summarizing discussion ................................................................................. 102

4.1 NAS phenotypic testing .................................................................................................. 102

4.2 Bacteriocin clusters in NAS ............................................................................................ 105

4.3 Staphylococcus aureus susceptibility ............................................................................. 107

4.4 Potential immunity genes ............................................................................................... 107

4.5 Conclusions and future research ................................................................................... 108

xi

List of Tables

Table 2-1 Bacteriocin gene clusters identified in bovine non-aureus staphylococci genomes

and inhibitory phenotypes tested against Staphylococcus aureus and MRSA. .................... 60

Table 3-1 Bacteriocin gene clusters identified in bovine Staphylococcus aureus and in vitro

susceptibility to a lanthipeptide-producing S. chromogenes. ................................................ 96

Table 3-2 The number and percentage of S. aureus isolates containing a putative bacteriocin

resistance gene associated with an isolate being resistant to a lanthipeptide encoding S.

chromogenes. ........................................................................................................................ 98

Table 3-3 The number of and percentage of S. aureus isolates containing genes associated

with an isolate being susceptible to a lanthipeptide encoding S. chromogenes. ................... 99

xii

List of Figures and Illustrations

Figure 2-1 Distribution of bacteriocin biosynthetic gene clusters in species of non-aureus

staphylococci isolated from milk of Canadian dairy cows displayed on the phylogenetic

tree from Naushad et al (2017). ............................................................................................ 63

Figure 2-2 Biosynthetic gene clusters and LanA alignments of type A lanthipeptides

identified in non-aureus staphylococci isolated from milk of Canadian dairy cows. .......... 64

Figure 2-3 Phylogenetic tree of Staphylococcus epidermidis isolates from bovine milk

indicating growth inhibition against Staphylococcus aureus and genomically identified

bacteriocin clusters. ............................................................................................................... 66

Figure 2-4 Biosynthetic gene clusters and LanA alignments of type B lanthipeptides with a

single LanM identified in non-aureus staphylococci isolated from milk of Canadian

dairy cows. ............................................................................................................................ 68

Figure 2-5 Biosynthetic gene clusters and LanA alignments of type B lanthipeptides with

dual lanM enzymes identified in non-aureus staphylococci isolated from milk of

Canadian dairy cows. ............................................................................................................ 70

Figure 2-6 Biosynthetic gene clusters and alignments of precursor peptides from

sactipeptides identified in non-aureus staphylococci isolated from milk of Canadian

dairy cows. ............................................................................................................................ 72

xiii

Figure 2-7 Biosynthetic gene clusters and alignments of precursor peptides from the lasso

peptide identified in non-aureus staphylococci isolated from milk of Canadian dairy

cows. ..................................................................................................................................... 73

Figure 2-8 Biosynthetic gene clusters of Class II double glycine leader peptide bacteriocins

identified in non-aureus staphylococci isolated from milk of Canadian dairy cows. .......... 74

Figure 2-9 Biosynthetic gene clusters and precursor alignments of Class IIc circular

bacteriocins identified in non-aureus staphylococci isolated from milk of Canadian

dairy cows. ............................................................................................................................ 75

Figure 2-10 Biosynthetic gene clusters and precursor alignments of Class IId lactococcin-like

bacteriocins identified in non-aureus staphylococci isolated from milk of Canadian

dairy cows. ............................................................................................................................ 77

Figure 3-1 Maximum Likelihood phylogenetic tree of Staphylococcus aureus isolates with

isolates resistant to a bacteriocin producing S. chromogenes indicated with a black dot. .... 97

Figure 3-2 LanA alignments and phylogenetic tree of type A lanthipeptides identified in S.

aureus isolated from milk of Canadian dairy cows. ........................................................... 100

xiv

List of Symbols, Abbreviations and Nomenclature

Symbol Definition aa Amino acid ABC transporter ATP binding cassette transporter BMSCC Bulk milk somatic cell count C-terminal Carboxyl-terminus of protein C-domain Carboxyl-terminus of signal peptide CBMQRN Canadian Bovine Mastitis and Milk Quality Network CNS Coagulase negative staphylococci CM Clinical mastitis GRAS Generally regarded as safe IMI Intramammary infection kDa Molecular mass unit, kilo Dalton LAB Lactic acid bacteria MIC Minimal inhibition concentration MDR Multi drug resistant NAS Non-aureus staphylococci OD600 Optical density measured at a wavelength of 600nm NSR Nisin resistance protein PBS Phosphate buffered saline PCR Polymerase chain reaction pH Potential Hydrogen SCC Somatic cell count TMD Transmembrane domain WGS Whole genome sequencing

xv

Preface

This dissertation consists of two manuscripts – the first manuscript is in press, planned

for issue 17 of Applied and Environmental Microbiology in September, 2017. For both

manuscripts, the first author was involved with study concept and design, acquisition of isolates

and data, laboratory analysis, analysis and interpretation of data, drafting of the manuscript, and

critical revision. This was done under the guidance of the senior author, supervisor and co-

supervisor. All authors provided critical reviews of the manuscripts and contributed intellectual

content. Both manuscripts were reproduced in their entirety as chapters in this dissertation.

Manuscript I) Domonique Carson, Herman W Barkema, Sohail Naushad, and Jeroen De Buck.

Bacteriocins of non-aureus staphylococci isolated from bovine milk. Accepted for publication in

Applied and Environmental Microbiology, 83 (17).

Manuscript II) Domonique Carson, Sohail Naushad, Herman W Barkema, and Jeroen De Buck.

Identifiying putative bacteriocin immunity genes in S. aureus whole genomes.

1

Chapter One: General Introduction

1.1 Mastitis in the Canadian dairy industry

Mastitis, inflammation of the mammary gland, is predominantly caused by microbial

infection in the mammary gland and costs Canadian dairy producers an estimated $400 million

per year due to direct losses in milk yield and treatment costs, and indirect losses, such as

discarded milk during treatment and future milk yield reduction (Rollin, Dhuyvetter, and

Overton 2015; Bradley 2002). Mastitis causing pathogens can be divided into major and minor

pathogens, as well as into environmental or contagious, depending on their transmission patterns

(Olde Riekerink et al. 2008). The most common contagious major pathogens are Staphylococcus

aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Streptococcus agalactiae,

whereas as Escherichia coli, and Klebsiella spp. are the most frequently isolated environmental

bacteria causing mastitis. Intramammary infection (IMI) of these pathogens can result in clinical

mastitis (CM), characterized by decrease in milk production and quality or by udder

abnormalities. Cows can also have subclinical mastitis (SCM), presenting with inflammation of

the udder determined by an increase in somatic cell count (SCC). Staphylococcus aureus and E.

coli are the most frequently isolated bacteria from CM on dairy farms in developed countries.

Staphylococcus aureus often leads to persistent IMIs that can influence culling decisions (Olde

Riekerink et al. 2006; Bradley 2002).

Traditionally, research and control programs have focused on decreasing the prevalence

of mastitis caused by major contagious pathogens, and producers and herd veterinarians have

been quite successful in this regard. Improvements have been made in hygiene, housing

2

management, milking management, and with timely treatment of infections has led to a

distribution shift of mastitis causing pathogens (Piepers et al. 2007). Non-aureus staphylococci

(NAS), generally considered minor mastitis pathogens, are the most commonly isolated bacteria

from the udder (Piepers et al. 2007; Pitkälä et al. 2004; Sampimon et al. 2009).

Even with the significant improvement being made on farm, mastitis is still a very

important disease for both the producer and consumer as it affects milk production, milk quality,

animal welfare, and can have public health concerns. Mastitis is the leading cause of antibiotic

usage for lactating dairy cows, for both the treatment and prevention of the disease (Saini et al.

2012; Oliveira and Ruegg 2014). In addition to treating any apparent CM in the lactating herd,

blanket dry cow therapy contributes heavily to the amount of overall of antibiotic use on farm

and high levels of antibiotic use are associated with higher levels of antibiotic resistant bacteria.

Consequently, consumer demands are driving the industry towards less antibiotic use, as evident

by the ever-increasing national production level of organic milk (Canadian dairy information

center, http://www.dairyinfo.gc.ca/pdf/org-bio_can_e.pdf). Although, the organic farm

regulations may result in producers being less likely to treat sick animals with antibiotics as

treated cows may be prohibited from returning to the milking herd, which may lead to welfare

implications (Barkema et al. 2015). Thus, in addition to judicious use of antibiotics, the need for

safe and effective alternatives is undeniable.

1.2 Non-aureus staphylococci

Staphylococci belong to the phylum Firmicutes, order Bacillales, class Bacilli, family

Staphylococcaceae, genus Staphylococcus. They are a group of Gram-positive and catalase-

3

positive bacteria that are round and form grape like structures. The heterogeneous genus

comprises over 50 species that are teat skin opportunists and generally considered minor udder

pathogens (White et al. 1989). NAS were originally differentiated from S. aureus based on the

ability of S. aureus to coagulate plasma (coagulase-positive), and therefore were named

coagulase-negative staphylococci (CNS). Originally, it was thought that only S. aureus was

pathogenic. At this point, several members of the group (e.g. S. agnetis) are now identified as

coagulase-variable (Dos Santos et al. 2016); it is, therefore, more appropriate that the group be

termed non-aureus staphylococci in order to encompass both the coagulase-negative and

coagulase-variable members.

Twenty-five NAS species have been isolated from cows, with S. chromogenes being the

most frequently isolated species from both milk and skin (Vanderhaeghen et al. 2015). While

NAS can be the cause of clinical mastitis, infection commonly results in subclinical mastitis,

raising the milk SCC of the infected quarter (Taponen et al. 2006). Therefore, a high prevalence

of NAS IMI can contribute to an increase in bulk milk SCC (BMSCC) in herds with a low

BMSCC (Schukken et al. 2009). Consequently, controlling NAS could allow producers to

further lower BMSCC. On the other hand, it has been reported that NAS IMI can result in

increased milk production when compared with uninfected cows (Schukken et al. 2009).

Interestingly, several studies have found that NAS have a protective effect against IMI by

major mastitis pathogens (De Vliegher et al. 2004; Matthews, Harmon, and Smith 1990). The

first challenge study using S. chromogenes reported a protective effect when 53% of S.

chromogenes-infected quarters were protected from a S. aureus challenge (Matthews, Harmon,

and Smith 1990). Later, De Vliegher et al. (2004) reported that two of 10 S. chromogenes

isolates were able to inhibit the in vitro growth of all S. aureus, S. dysgalactiae, and S. uberis,

4

but none of the tested E. coli isolates. On the other hand, field studies have reported no protective

effects from staphylococcal infections against environmental pathogens (Hogan et al. 1988) and

that NAS IMI was neither a risk factor or a protective against mastitis caused by S. aureus or S.

uberis (Zadoks et al. 2001). The inconsistent results regarding if NAS are protective or not

against IMI from major pathogens are likely due to undifferentiated NAS species and genotypes,

which may have different pathogenicity and effects in the udder.

When looking at the effects of NAS on udder health at a species level, certain species

have been shown to inhibit the growth of mastitis pathogens in vitro. NAS strains from Brazilian

bovine mastitis cases, including Staphylococcus epidermidis, Staphylococcus simulans,

Staphylococcus saprophyticus , Staphylococcus hominis , and Staphylococcus arlettae, inhibited

growth of indicator species Corynebacterium fimi (Nascimento et al. 2005). The isolated

antimicrobial substances were considered to be bacteriocins due to sensitivity to proteolytic

enzymes (Nascimento et al. 2005). Recently, Braem et al. (2014) identified NAS strains (from 6

species) that inhibited S. aureus, S. uberis, and S. dysgalactiae and an inhibitory substance (from

an inhibiting S. chromogenes) was isolated and identified to be a nukacin-like bacteriocin

(Braem et al. 2014). Thus, it is possible that the variation that was seen when examining NAS

inhibition was due to different species and isolates being able to produce bacteriocins, which are

antimicrobial peptides.

1.3 Bacteriocins

Bacteriocins are ribosomally synthesized and (generally) post-translationally modified

peptides (RiPPs) that are produced by bacteria to kill other bacteria, thus bacteriocin production

5

gives the producing strain an advantage in certain ecological niches, allowing them to compete

for common resources (Parada et al. 2007). The bacteriocins generally exert their bacteriocidal

activity against a narrow spectrum of bacteria. The first antimicrobial peptide compounds

identified, termed colicins, were produced by E. coli and now, bacteriocins have been found in

all major lineages of bacteria (Riley and Wertz 2002). The lactic acid bacteria (LAB) are

abundant producers of bacteriocins and LAB have been used for centuries to ferment foods

(Riley and Wertz 2002). Bacteriocins have the potential to be used as antimicrobial peptides in

the food and health industries. Nisin, a bacteriocin produced by Lactococcus lactis, was

discovered in 1947 (Mattick, Hirsch, and Berridge 1947) and is now the most widely used

bacteriocin.

Bacteriocins produced by Gram-positive bacteria are usually smaller than 8kDa and they are

usually pore forming. The producer contains specific proteins, which confer host immunity

against the bacteriocin (Cotter, Ross, and Hill 2013). Bacteriocins are generally classified into

two main groups, the modified peptides and the unmodified peptides (Cotter, Ross, and Hill

2013).

1.4 Classification of bacteriocins

A variety of classification systems for bacteriocins exist, based on chemical structure,

molecular mass, enzymatic susceptibility, mode of action, genetic mechanisms, thermo stability,

producing strains, spectrum of activity, or presence of post translationally modified residues

(Klaenhammer 1993). This assortment of classification schemes can result in bacteriocins being

6

assigned to multiple different classes (if they were discovered by different groups at the same

time), according to the classification scheme each group choses.

Historically, bacteriocins were first grouped in four main classes (Klaenhammer 1993).

Class I were the lantibiotics (subdivided into two groups), class II were the non-modified

peptides (subdivided into three groups), class III were the large and heat labile bacteriocins, and

class IV were the bacteriocins proposed to form large complexes with macromolecules

(Klaenhammer 1993). In 2005, Cotter, Hill, and Ross (2005) proposed a classification system

that recognized just two classes of bacteriocins: class I, the post translationally modified

lantibiotics and class II, the non-modified bacteriocins. The class II bacteriocins were broken

into four categories: IIa, small heat stable peptides, IIb, the two component bacteriocins, IIc, the

circular bacteriocins, and IId, the other class II bacteriocins, including sec-dependent and

leaderless bacteriocins (Cotter, Hill, and Ross 2005). Subsequently, Heng and Tagg (2006)

suggested that the class IIc bacteriocins, the circular bacteriocins, should be grouped as their own

class, class IV bacteriocins, and that the large bacteriolysins (which are non-bacteriocin lytic

proteins that were not included in the Cotter classification scheme) be classified as class III

bacteriocins.

In 2013, Arnison et al. (2013) proposed universal nomenclature for RiPPs, which has

been generally accepted (Cotter, Ross, and Hill 2013). This scheme includes all modified

bacteriocins grouped as class I, and the unmodified or circular bacteriocins grouped as subclasses

of class II bacteriocins (Arnison et al. 2013). Interestingly, a study in 2010 classified 107

bacteriocins according to amino acid structure, resulting in 12 different bacteriocin groups, each

representing a distinct branch on the phylogenetic tree and containing a conserved motif (Zouhir

et al. 2010). This study represented a unique classification scheme, although one that has not

7

been adapted by the bacteriocin researchers community. In 2016, Alvarez-Sieiro et al. (2016)

proposed a scheme modified from Arnison et al. (2013), where circular bacteriocins are included

in class I modified bacteriocins and the bacteriolysins are included as class III bacteriocins. In

this thesis, to remain consistent with the proposed universal nomenclature, we will follow the

two-class classification scheme recommended by Arnison et al. (2013).

1.4.1 Lanthipeptides

Traditionally termed lantibiotics (lanthionine-containing antibiotic), lanthipeptides are

characterized by the presence of several uncommon amino acids, including meso-lanthionine and

3-methyllanthionine, that are a result of post-translational modifications (Bierbaum et al. 1996).

The name lanthipeptides was changed to encompass the non-antibiotic peptides (type C and D

lanthipeptides) from the same biosynthetic origin (Goto et al. 2010), although the majority of

lanthipeptides are either type A or type B, which exert antimicrobial activity (Bierbaum and Sahl

2009). Lanthipeptides are the most common RiPPs in available genomes, with more than 95

lanthipeptides from Gram-positive bacteria having been isolated and described (Dischinger, Basi

Chipalu, and Bierbaum 2014). Genome mining has identified many more potential compounds

waiting to be characterized (Knerr and van der Donk 2012), many of which are closely related

and likely have common peptide ancestors. The most well known lanthipeptide, nisin, which was

isolated from L. lactis in 1947 (Mattick, Hirsch, and Berridge 1947), has been used in the food

preservation industry for over 50 years.

Lanthipeptides are the bacteriocins most frequently isolated from Staphylococcus species,

thus, there are many well-characterized “Staphylococcin” examples. The first to be discovered

and described were pep5 (Sahl et al. 1985) and epidermin (Allgaier et al. 1986) from S.

8

epidermidis and gallidermin from S. gallinarum (Schnell et al. 1989). Multiple additional type-A

lanthipeptides have since been identified. Staphylococcus cohnii produces staphyloccocin T,

which has an identical sequence to gallidermin and is active against a broad spectrum of Gram-

positive bacteria (Furmanek et al. 1999). Staphylococcus hyicus produces hyicin 3862 (Fagundes

et al. 2011), a bacteriocin likely related to epidermin and Bsa (a bacteriocin produced by S.

aureus) (Daly et al. 2010) and represents the first bacteriocin to be identified in S. hyicus. The

genetic organization of hyicin 3862 was recently elucidated and showed 91% identity with Bsa

but was found to have a broader spectrum of activity than Bsa (Fagundes et al. 2017). Along

with the bacteriocin pep5, S. epidermidis produces other lanthipeptides that are closely related to

pep5 including epilancin K7 (van de Kamp et al. 1995), epicidin 280 (Heidrich et al. 1998), and

epilancin 15X (Ekkelenkamp et al. 2005). Staphylococcus warneri produces a type-AII

lanthipeptide, nukacin ISK-1 (Sashihara et al. 2000), which is a member of the lacticin 481 group

(Bierbaum and Sahl 2009). Nukacin-like bacteriocins have also been identified in S. simulans

(Ceotto et al. 2010), S. hominis (Wilaipun et al. 2008), and S. chromogenes (Braem et al. 2014).

Lastly, S. aureus produces a two-component lanthipeptide, C55 (Maduwe, Sahl, and Tagg 1999).

1.4.1.1 Genetic Organization

Genes related to lanthipeptide synthesis are generically named with the locus symbol lan,

where each characterized bacteriocin has its individual naming system, for example nis for nisin.

All lanthipeptides have a precursor peptide, LanA, which contains both a leader peptide and a

core peptide, of which the former is cleaved off to yield the active peptide. The leader peptide is

thought to play roles in posttranslational modification, immunity, and export (Arnison et al.

2013). The mature lanthipeptide results from posttranslational modifications to the core peptide

9

by one or more enzymes, also encoded on the operon. There is a small subclass of lanthipeptides

that require two LanA peptides, A1 and A2, for complete antimicrobial activity. These low

identity peptides each require their own posttranslational modifications by separate modification

enzymes to be able to work synergistically together outside of the cell (Bierbaum and Sahl

2009).

The modification enzymes, which are responsible for creating thioether cross-links (by

dehydrating the serine and threonine residues followed by addition of a thiol of a cysteine

residue) and for cleaving off the leader peptide either during or before export, are the basis for

dividing lanthipeptides into their groups (Knerr and van der Donk 2012). Type A lanthipeptides

contain lanB and lanC genes, where the dehydration of the serine and threonine residues to

dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues in the pre-peptide are completed by a

dehydratase, LanB, and the thioether crosslinks are formed on the dehydrated amino acids by a

cyclase, generically called LanC. For type B lanthipeptides, a bifuncational enzyme, LanM,

carries out the dehydration and cyclisation. The C terminal of the LanM synthetase shares

homology with the LanC cyclases of the type A lanthipeptides (Blin et al. 2014; Asaduzzaman

and Sonomoto 2009).

Lanthipetides also contain LanP serine proteases, which cleave the leader peptide from

the core peptide, and a LanT, which is responsible for exporting the mature peptide. Immunity

genes are also located on the operon to provide the producer with self-immunity. In

lanthipeptides, immunity is conveyed with lanI, or the lanFE(G) immunity cluster. The LanI

protein is thought to function by interception or target shielding (Stein et al. 2005) or by

sequestering the bacteriocin on the bacterial cell wall membrane, as is the case with NisI from

nisin. In contrast, the LanFEG proteins work by removing the peptide via the dedicated ABC

10

transporter (Stein et al. 2005). Additional genes found on the gene clusters are lanR and lanK

which are both involved in lanthipeptide regulation by a quorum sensing system (Chatterjee et al.

2005). Presence of the active lanthipeptide leads to a signalling cascade initiated by the LanK

histidine kinase, followed by activation of the LanR response regulator to activate biosynthesis

of the bacteriocin (Dischinger, Basi Chipalu, and Bierbaum 2014).

1.4.1.2 Mode of Action

Type A lanthipeptides, like nisin, which are linear positively charged peptides, have dual

modes of action where they inhibit cell wall biosynthesis and form pores that mainly act upon

Gram-positive bacteria (Bierbaum and Sahl 2009). In the specific case of nisin, which has been

studied extensively, the cell wall precursor lipid II is the target and upon binding to lipid II the

nisin inhibits peptidoglycan synthesis. Additionally, upon binding, nisin is able to insert itself

into the membrane to form stable pores consisting of eight nisin and four lipid II molecules

(Hasper, de Kruijff, and Breukink 2004). This dual mode of action makes nisin very potent and

makes resistance harder to acquire (Bastos, Coelho, and Santos 2015). Other lanthipeptides, like

epidermin and gallidermin that are too short to span the lipid bilayer to form pores, also bind to

lipid II, yet are still able to kill target bacteria, indicating that they have other lipid II mediated

mechanisms (Bastos, Coelho, and Santos 2015). The proposed mode of action is that the

bacteriocin sequesters the lipid II away from its functional location, thus blocking cell wall

synthesis (Hasper et al. 2006). Targets have yet to be identified for pep5 and epilancin K7

(Draper et al. 2015). Type AII lanthipeptides (lacticin 481 and nukacin ISK-1 groups) consist of

an N-terminal linear region and a C-terminal globular region (Chatterjee et al. 2005), and it is

thought that their structures may prevent them from forming pores (Islam, Nagao, et al. 2012).

11

For certain bacteriocins in this group the positive lysine residues in the linear region are essential

for binding to the cell membrane, although not all members of this group have positively charged

residues (Islam, Nagao, et al. 2012), indicating additional areas of the bacteriocin that facilitate

antimicrobial activity. One member of this group, lacticin 481 binds lipid II leading to the

inhibition of the transglycosylation step, therefore stopping cell wall synthesis (Knerr et al.

2012). Nukacin ISK-1 contains a conserved region similar to the lipid II binding region of

mersacidin (a type B lanthipeptide), suggesting a similar mode of action. Preliminary studies

seem to indicate that nukacin ISK-1 indeed has a bacteriostatic mode of action caused by binding

to lipid II with its conserved A ring region to inhibit cell wall synthesis (Islam, Nishie, et al.

2012). This has been recently confirmed along with the illumination of other important residues

in the peptide (Elsayed et al. 2017). Therefore, type AII lanthipeptides are not pore formers but

interact with lipid II on the cell wall surface and inhibit cell wall biosynthesis, resulting in

termination of cell growth. Lacticin 3147, a two peptide lanthipeptide, has a proposed three step

mechanism where the a-peptide binds to lipid II inducing a conformational change in the a-

peptide which the b-peptide recognizes and binds to, resulting in insertion into the membrane,

therefore forming a pore (Srinivas et al. 2012). The type B lanthipeptides (e.g. mersacidin) are

globular peptides with no charge, or are negatively charged (Islam, Nagao, et al. 2012). These

lanthipeptides act by inhibiting cell wall biosynthesis and are also not pore formers. Mersacidin

binds to lipid II and interferes with the transglycosylation step of peptidoglycan synthesis (Brötz

et al. 1997). Mersacidin, as the name indicates, is active against methicillin-resistant S. aureus

(MRSA) (Kruszewska et al. 2004) and is also able to inhibit the growth of vancomycin-resistant

Enterococcus faecium strains even though mersacidin and vancomycin have the same target.

12

This is because mersacidin binds to a different site on the lipid II (Brötz et al. 1997; van Heel,

Montalban-Lopez, and Kuipers 2011).

1.4.2 Sactipeptides

Sactipeptides, formerly known as sactibiotics, are a small class of RIPPs that are

characterized by the presence of at least one thioether bond between the cysteine sulphur and the

a-carbon of an acceptor amino acid. They are all relatively hydrophobic with a 3D hairpin like

structure (Arnison et al. 2013). Sactipeptides originally had only been characterized in Bacillus

species, though recent genome approaches have identified them in Clostridium species (Murphy

et al. 2011; Haft, Basu, and Mitchell 2010). Subtilosin-A, produced by Bacillus subtilis 168, is

the best characterized sactipeptide (Babasaki et al. 1985) and has been reported to have

antimicrobial activity against both Gram-positive and Gram-negative pathogens (Shelburne et al.

2007). One member of the sactipeptide family, Thuricin CD, is effective against Clostridium

difficile, yet it has a narrow spectrum of activity thus not impacting the host commensal flora

(Rea et al. 2010). A sactipeptide was recently identified in S. hyicus, named hyicin 4244 (Duarte

et al. 2017), making it the first sactipeptide characterized from Staphylococcus.


The biosynthetic gene clusters of sactipeptides typically include a precursor peptide,

along with one radical SAM enzyme per precursor, a putative protease, and two potential export

and immunity proteins (Fluhe and Marahiel 2013). The nomenclature for these clusters is based

off the thuricin CD gene cluster (Rea et al. 2010), where “A” is the precursor peptide, “C” and

“D” refer to the radical-SAM protein(s), “F” and “G” indicate the ABC-transporters, and “P” is

13

used to identify any yet unidentified proteases in the cluster (Arnison et al. 2013). The SAM

enzyme is responsible for the sulphur to a-carbon crosslinks, formed from linking the sulphur of

a cysteine residue to the a-carbon of an acceptor amino acid (Fluhe and Marahiel 2013), and has

been used as the target for genome mining for novel sactipeptides (Murphy et al. 2011).


The precise modes of action of the characterized sactipeptides are not well understood,

although they seem to be able to interact with and disrupt target cell walls (Thennarasu et al.

2005; Wang et al. 2014). It has been demonstrated that thurincin H does not cause cell membrane

permeability or cell wall lysis, although it does decrease cell viability (Wang et al. 2014).

Subtilosin A seems to have variable modes of action, depending on the target organism. In a

study looking at subtilosin A’s effect on Gardnerella vaginalis, the bacteriocin resulted in an

immediate depletion of the cells pH and triggered an efflux of ATP, suggesting that subtilosin A

forms pores, leading to cell death (Sutyak Noll, Sinko, and Chikindas 2011). When investigating

subtilosin A’s effects on Listeria monocytogenes, there was no efflux of ATP and only minor

effects on pH and transmembrane potential, which the authors suggest indicates subtilosin A

interacts with the cell membrane, causing intracellular damage leading to cell death (Kuijk, Noll,

and Chikindas 2012). This mode of action was previously suggested, as evidence showed

subtilosin A adopts a partially buried orientation in the lipid bilayer, inducing conformational

changes and leading to membrane permeability (Thennarasu et al. 2005).

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1.4.3 Lasso Peptides

Lasso peptides get their name from their structure, which resembles a lariat with its

threaded configuration. Based on their structure and experimental reports, these peptides are

highly resistant to proteases and denaturing agents, which make them a topic of much interest

(Arnison et al. 2013), although not all lasso peptides seem to follow this pattern (Hegemann et al.

2015). Actinobacteria, and occasionally Proteobacteria, most commonly produce lasso peptides

(Arnison et al. 2013), although putative gene clusters have been identified in genomes from other

phyla (Hegemann et al. 2015). As of 2015, 38 lasso peptides had been discovered, largely due to

genome mining (Hegemann et al. 2015). A few of these lasso peptides have a narrow spectrum

of antimicrobial activity (Iwatsuki et al. 2006; Knappe et al. 2008; Salomón and Farías 1992).

Lasso peptides require only two posttranslational modifications, cleavage of the leader

peptide and formation of a disulphide bridge(s). The presence or absence of disulphide bridges in

the structure determine if the peptide belongs to class one, two, or three (Maksimov and Link

2014), where class I lasso peptides contain two disulphide bridges, class II contain none, and

class III contain just one. To the best of our knowledge, no lasso peptides have been identified in

Staphylococcus.


The recently adapted universal nomenclature for gene organization of lasso peptides

follows an “ABCD” structure (Arnison et al. 2013). Studies report that the ABC genes are

necessary for lasso peptide production (Maksimov and Link 2014), where A is the structural

gene, B encodes for the ATP dependent protease, and C encodes for the enzyme responsible for

isopeptide bond formation (Pan and Link 2011). Some clusters contain an ABC transporter,

15

encoded by the D gene, which transports the peptide out of the cell and is also responsible for

producer immunity (Pan and Link 2011). For clusters where the D gene is absent, the peptide is

still excreted from the cell and there is no host cell death, indicating the producer may use an

existing ABC transporter or that the peptide is able to diffuse out of the cell membrane (Knappe

et al. 2008). Newer clusters have been reported that contain highly conserved genes adjacent to

the ABC genes, such as isopeptidases (Hegemann et al. 2015), which may be indicative of the

evolutionary nature of the lasso peptide as clusters with these genes branch together in clades

(Maksimov and Link 2013).


In general, lasso peptides are enzyme inhibitors or receptor antagonists, with a narrow

spectrum of activity against closely related bacteria (Arnison et al. 2013). The most well

characterized lasso peptide, microcin J25 produced by E. coli AY25, exerts its antimicrobial

effects by entering the target cell via the iron siderophore receptor (Destoumieux-Garzón et al.

2005) and inhibiting RNA polymerase (Delgado et al. 2001). Likewise, capistruin, produced by

Burkholderia thailandensis E264, was experimentally shown to have the same mode of action as

microcin J25 and the authors propose that all structurally similar lasso peptides potentially have

the same target (Kuznedelov et al. 2011). Lassomycin is a protease inhibitor that specifically

inhibits Mycobacterium tuberculosis (Gavrish et al. 2014).

1.4.4 Class IIa Bacteriocins

Class IIa bacteriocins refer to pediocin-like bacteriocins with a broad inhibitory spectrum

including potent anti-listerial activity (Kjos et al. 2011). These bacteriocins contain an N

16

terminal consensus sequence (YGNGVxCxxxxCxVxWxxA, where x is any amino acid) (Cotter,

Hill, and Ross 2005). These bacteriocins normally contain two distinct regions separated by a

flexible hinge (Kjos et al. 2011). Pediocin PA-1, produced by Pediococcus acidilactici UL5, is

the model bacteriocin for this group.

1.4.4.1 Genetic organization

The pediocin PA-1 gene cluster contains four genes, ABCD, where A is the structural

gene, B encodes the immunity protein, C is the ABC transporter and D encodes for an accessory

protein (Alvarez-Sieiro et al. 2016).


Class IIa bacteriocins form pores in target cells, and unlike other class II bacteriocins,

these mechanisms have been elucidated. The target for these bacteriocins is the proteins of the

sugar transporter mannose phosphotransferase system (Man-PTS) (Kjos et al. 2011). However, it

is unknown if the bacteriocins use the Man-PTS the same way lanthipeptides use lipid II as a

docking molecule or if the bacteriocin interacts with the Man-PTS gate causing it to permanently

open, for which the latter model is more likely (Kjos et al. 2011)

1.4.5 Class IIb Bacteriocins

Class IIb bacteriocins require the presence of two distinct peptides that work

synergistically to provide maximum antimicrobial activity. Lactococcin G, produced by L. lactis

(Nissen-Meyer et al. 1992), was the first class IIb bacteriocin to be isolated and is subsequently

the best characterized at this point. As of 2015, this class contained 15 additional bacteriocins

17

(Kjos et al. 2014). All peptides in this class require two different peptides (located on the same

operon) each produced in equal amounts to obtain peak antimicrobial activity (Kjos et al. 2014).


These bacteriocins require at least five different genes, on one or two operons (Alvarez-

Sieiro et al. 2016). Generally, there are two structural genes, an ABC transporter, an immunity

protein gene and a gene encoding an accessory protein.


Lactococcin G, produced by L. lactis, was initially reported to interact with a receptor on

the cell membrane of the target bacteria to induce cell leakage (Rogne et al. 2008). Recently, it

was determined the target is likely bacA, a membrane protein involved in peptidoglycan

synthesis (Kjos et al. 2014), which was the first time a target has been identified for class IIb

bacteriocins.

1.4.6 Class IIc Bacteriocins

Class IIc bacteriocins are circular bacteriocins, characterized by an amide bond between

the N and C termini (Maqueda et al. 2008) and as of 2011 have only been identified in Gram-

positive bacteria (van Belkum, Martin-Visscher, and Vederas 2011). Their head to tail

cyclization attribute to their reported resistance to proteases and pH and heat treatment (van

Belkum, Martin-Visscher, and Vederas 2011). The first and most well characterized circular

bacteriocin is Enterocin AS-48, isolated from Enterococcus (Martínez-Bueno et al. 1994; Samyn

et al. 1994). At least nine bacteriocins from this group have been isolated, purified, and

18

characterized (Arnison et al. 2013). The first report of a circular bacteriocin in Staphylococcus is

aureocyclicin 4185, produced by S. aureus (Potter, Ceotto, Coelho, Guimaraes, et al. 2014).


The genetic organization for many circular bacteriocins has been well-described (van

Belkum, Martin-Visscher, and Vederas 2011), although a universal nomenclature has not been

adopted for this group yet, except for the use of A for the precursor peptide and the

recommendation to use B for the putative membrane protein (Arnison et al. 2013). The enterocin

AS-48 gene cluster contains ten genes, termed A, B, C, C1, D, D1, E, F, G, and H, which function

as production, modification, transport, and immunity genes (Maqueda et al. 2008).


In general, circular bacteriocins exert their antimicrobial activity by targeting cell

membranes and forming pores (Arnison et al. 2013). Enterocin AS-48 interacts with the cell

membrane, inserting itself in a voltage-independent manner causing loss of membrane potential

and cell death (Maqueda et al. 2008). Carnocyclin A, on the other hand, is able to form pores in a

voltage-dependent manner (Gong et al. 2009).

1.4.7 Class IId Bacteriocins

Class IId bacteriocins are a heterogenous group of bacteriocins that are single linear

peptides. The most well characterized bacteriocin from this group is lactococcin 972, produced

by L. lactis subsp. lactis IPLA 972 (Martı́nez et al. 1999).

19


Along with the 91aa structural gene, LclA, the complete lactococcin 972 cluster contains

a transporter, LclB, and an immunity protein (Martı́nez et al. 1999). The genetic structure of

lactococcin A is similar, with a structural gene, an immunity gene, an ABC transporter, and an

accessory gene (Stoddard et al. 1992).


Latococcin 972 blocks the incorporation of lipid II of closely related bacteria.

Lactococcin A, like Class IIa bacteriocins, targets the Man-PTS proteins, although unlike the

Class IIa bacteriocins, it is very specific and targets only the lactococcal Man-PTS system

(Alvarez-Sieiro et al. 2016).

1.5 Immunity genes and cross immunity

Bacteriocin producers encode for specific immunity proteins to provide protection from

the lethal activities of their products. The peptide structural and modification/transport genes are

present on the same operon as the immunity genes, meaning that the producing strain is sensitive

to the bacteriocin product when in a non-producing state (Eijsink 1998).

Lanthipeptides have the most complex immunity proteins, termed LanI and LanFEG.

These two proteins are thought to function independently and have different mechanisms of

protection (Stein et al. 2005). LanFEG have been experimentally determined to be involved in

exporting bacteriocin out of the cell and fall into the ABC-2 subfamily of drug resistance

exporters (Stein et al. 2005). On the other hand, LanI appears to sequester lanthipeptides on the

20

surface, thus preventing pore formation. One interesting note from a study done examining the

lanthipeptide subtilin and its immunity proteins was that SpaI interacts specifically with subtilin,

and not with the structurally similar nisin (Stein et al. 2005). Similarly, NisI only confers

protection against nisin, and not against subtilin (Stein et al. 2003). This indicates that cross

immunity due to bacteriocin cluster immunity proteins can be rare, even between closely related

lanthipeptides. For certain bacteriocins, like nisin, lanI and lanFEG are both needed for optimal

immunity, however certain bacteriocin clusters only contain one self protection mechanism, for

example the pep5 cluster contains only lanI whereas the epidermin cluster only contains the

lanFEG transporter (Stein et al. 2003).

There are additional genes related to immunity that have been identified, although not

well described. Abi proteins, or CAAX immunity proteins, are putative membrane bound

metalloproteases and have been shown to be involved with self-immunity for Class IIb

bacteriocins (Kjos et al. 2011). They can show extensive cross immunity, which could mean that

the immunity proteins give immunity by a common, shared mechanism (Kjos et al. 2010). These

mechanisms could be degradation of the bacteriocin, or by modifying the bacteriocins receptor

(Kjos et al. 2010). Class IIa bacteriocins, as well as lactococcin A and B, which target the Man-

PTS system studies have shown that their immunity proteins bind and lock the bacteriocin onto

the receptor target to prevent pore formation (Diep et al. 2007).

1.6 Bacteriocin discovery and purification

Traditionally, discovery of bacteriocins starts with a large screen of bacterial isolates to

assess their inhibitory capability, and thus potential bacteriocin production, against indicator

21

bacteria in vitro. A commonly used method of detection is the ‘spot on lawn’ assay (Fleming,

Etchells, and Costilow 1975), where drops of the producer broth are spotted onto agar and

incubated overnight to allow colonies to develop. The top of the agar is overlayed with soft agar

(0.5% agar) inoculated with the indictor species, and incubated overnight, followed by

examination and measuring of the zones of inhibition around the producer colonies (Fleming,

Etchells, and Costilow 1975). De Vliegher et al. (2004) utilized a cross-streaking method, where

the potential producer was inoculated as a center streak down a blood agar plate and incubated

overnight. The indicator species was spread over the entirety of the agar after flipping the agar

over, with the producer center streak ending up on the bottom of the plate. The zones of

inhibition were measured after incubation for 24hr, perpendicular to the center streak (De

Vliegher et al. 2004). Well diffusion assays are also used to assess inhibition (Schillinger and

Lücke 1989). For this assay, agar plates are overlayed with soft agar inoculated with the

indicator species, followed by the drilling of wells into the agar once set. Cell free supernatant of

the potential producer is added to each well, and following overnight incubation, zones of

inhibition are assessed (Schillinger and Lücke 1989). Identified potential producers are then

grown up in conditions conducive for bacteriocin production. Staphylococcus aureus isolates

were shown to have peak bacteriocin production during the late-log or early stationary growth

phase of cultures grown in brain heart infusion (BHI) medium at 37°C (Nascimento et al. 2004).

Bacteriocin production during the late-log or early stationary phase can be 4.6 to 7.5 fold higher

than production in the early exponential growth phase (Sedgley, Clewell, and Flannagan 2009).

The bacteriocin is extracellularly secreted into the medium during growth, normally in small

quantities, so a large amount of medium is recommended to be able to isolate enough quantity of

bacteriocin. Optimal isolation and purification protocols depend on the bacteriocin, as they have

22

different properties that can be taken advantage of for separation from the culture medium.

Therefore, there is not one technique that is suitable for all classes of bacteriocin (Kaškonienė et

al. 2017). Common isolation techniques take advantage of their charge and hydrophobic natures

(Parada et al. 2007). Ammonium sulphate precipitation is the most commonly used method to

reduce the volume and concentrate the bacteriocin from the producer medium (Kaškonienė et al.

2017), although chloroform extractions have proven to be less expensive, less time consuming,

and result in a higher bacteriocin yield (Burianek and Yousef 2000). Upon obtaining an active

partially purified bacteriocin (at this point there are still proteins and peptides from the growth

medium present), Reverse-Phase High Pressure Liquid Chromatography (RP-HPLC) is most

commonly performed to purify the bacteriocin (Kaškonienė et al. 2017). Other techniques, such

as ultrafiltration or filter assisted size exclusion protein fractionation, can be used for purification

(Kaškonienė et al. 2017). Upon obtaining a purified bacteriocin, SDS-PAGE can be used to

determine an approximate molar mass, and the bacteriocin can be sent to sequencing to obtain

the amino acid sequence, or it can be detected using matrix-assisted laser desorption ionisation

time-of-flight mass spectrometry (MALDI-TOF MS) (Zhu et al. 2016). This lab-based

bacteriocin discovery approach is time consuming, as well as labour intensive, requiring

qualified technicians to use the equipment needed (Kaškonienė et al. 2017). There also is the risk

of “re-discovery” of a bacteriocin, as was the case with a bacteriocin discovered in an S.

simulans isolate, initially named simulancin 3299, although after purification and identification it

was discovered to be identical to known bacteriocin nukacin ISK-1 (Ceotto et al. 2010). There is

also a risk of missing identification of some bacteriocins that are not expressed under normal

laboratory conditions.

23

1.7 In silico screening

In silico screening, or genome mining, is an approach that is being used more frequently

for the discovery of new bacteriocins. This methodology is able to examine whole genome

sequences of bacteria and identify bacteriocin biosynthetic gene clusters, independent of

laboratory phenotype analysis.

One of the initial genome screenings for bacteriocins was done using the lanM protein

(from type B lanthipeptides) as the driver sequence. Because the associated genes are well

conserved in specific classes of bacteriocins, this approach could yield identification of novel

type B lanthipeptides. The study resulted in 89 LanM homologues identified, 61 of which were

in bacteria not associated with lanthipeptide production. For identified genomes, with both a

LanM homologue and available whole genomes, BAGEL was used to further analyze the

genome for potential bacteriocin clusters. One bacteriocin containing isolate that was able to

inhibit pathogens was selected for further laboratory analysis and bacteriocin characterization as

a proof of concept. Consequently, lichenicidin was isolated and characterized, providing

testimony that in silico screening is a valuable tool that can result in the identification of novel

bacteriocins (Begley et al. 2009).

LanT, a lanthipeptide associated transporter, was used as the driver sequence to identify

novel lanthipeptides in available sequences on NCBI, taking the top 72 hits for further analysis

(Singh and Sareen 2014). This approach led to the identification of 54 strains containing LanT

homologues, strains that were not previously associated with lanthipeptide production. Overall,

the study identified 8 novel two-component lanthipeptides for further characterization (Singh and

Sareen 2014). Likewise, the radical SAM enzyme present in sactipeptide clusters was used as the

24

driver sequence in genome mining for novel sactipeptides, yielding putative sactipeptides in

phyla not typically associated with bacteriocin production (Murphy et al. 2011). Using a similar

associated gene homology approach with lasso peptides, Burkholderia thailandensis E264 was

identified to contain putative lasso peptide associated genes. Capistriun, a novel lasso peptide

was subsequently isolated (Knappe et al. 2008).

A unique precursor-centric genome mining approach was created to search for small

areas of conserved regions in the structural gene of lasso peptides (Maksimov, Pelczer, and Link

2012). Using this approach, out of 3000 prokaryotic genomes mined, 78 were identified to be

putative producers. To validate this approach, one putative producer was selected and the lasso

peptide was expressed in E. coli, leading to the production of a novel lasso peptide, astexin-1

(Maksimov, Pelczer, and Link 2012). In total, genome mining has resulted in the considerable

increase in identified members of the lasso peptide family, inflating to 38 members as of 2015

(Hegemann et al. 2015).

Currently, approaches have been automated that combine direct mining for the structural

gene along with indirect mining for associated genes in order to comprehensively search the

genomes. BAGEL3 is one such available software, which mines for bacteriocins in single or

multiple DNA sequences such as (un)finished genomes, scaffold files but also meta-genomics

data (van Heel et al. 2013). BAGEL3 is also a source for databases of structural genes in each

class of bacteriocin, although these databases were last updated in 2013. antiSMASH (antibiotics

and Secondary Metabolite Analysis SHell) is another tool for genome mining that identifies all

secondary metabolite biosynthetic gene clusters, not just bacteriocins (Weber et al. 2015).

Novel bacteriocins were recently identified in anaerobic bacteria using a combination of

antiSMASH, and BAGEL and bactibase databases (Letzel, Pidot, and Hertweck 2014). Out of

25

221 anaerobe genomes from 18 different phyla, they identified 25% of genomes (from 8

different phyla) were able to encode for bacteriocins. This study determined 43 out of 81

identified clusters were novel and described 23 clusters that had not been identified in anaerobes

before, although had similarities to previously identified bacteriocins from other phyla (Letzel,

Pidot, and Hertweck 2014). In another study, 34 genomes from 34 different species of LAB that

have not been identified as bacteriocin producers were mined through BAGEL3, resulting in the

identification of 20 of the species containing bacteriocin gene clusters (Singh et al. 2015).

Azevedo et al. (2015) screened 224 ruminal bacteria strains and 5 ruminal archaea to determine

the distribution and diversity of ruminal bacteria. This study identified 46 bacteriocin gene

clusters in 33 strains of bacteria. Whole and partial genome sequences were uploaded into

Bagel3 and antiSMASH software for the detection of bacteriocin gene clusters. Before that study

only 9 bacteriocins had been fully or partially characterized from ruminal bacteria (Azevedo et

al. 2015). In another study of substantial size, 382 genome isolates from the gastrointestinal tract,

available as a subset of the Human Microbiome Project, were mined for bacteriocin clusters

using BAGEL3 (Walsh et al. 2015). In total, 74 clusters from 59 isolates were detected, and the

majority of the species containing isolates were from species not previously associated with

bacteriocin production (Walsh et al. 2015). It is apparent that genome mining is an incredibly

useful tool to identify bacteriocin gene clusters.

Although, caution has to be taken to draw conclusions from genome mining, as precursor

peptides may be modified in different ways than anticipated from examining the modification

genes and may result in a mature peptide belonging to a different class than once thought

(Arnison et al. 2013). Additionally, proximity on a contig may not mean there is a

target/substrate relationship. Additionally, in silico screening may not ultimately result in

26

identification and purification of bacteriocin, though there have been successes (Begley et al.

2009; Dischinger et al. 2009; Knappe et al. 2008). There is also the potential to miss completely

novel clusters, as neither the precursor genes nor associated genes are known or lack sufficient

homology to be identified.

It is nevertheless a good starting point to identify isolates for future characterization in

the laboratory and to identify the distribution of bacteriocin associated gene clusters in large

groups of related bacteria, or bacteria from unique environmental niches. In general, due to

extensive genome mining projects, bacteriocins are now known to be more prevalent and present

in more phyla of bacteria than what was once thought (Arnison et al. 2013).

1.8 Applications of bacteriocins

Nisin is currently the most widely studied and used bacteriocin. In 1969, the Joint Food

and Agriculture Organization/World Health Organization approved nisin for use as a food

additive (Shin et al. 2016). In 1988, nisin was given a generally regarded as safe (GRAS) status

for use in cheeses by the Food and Drug Administration in the United States (Cotter, Hill, and

Ross 2005). Presently, it is licensed in over 50 countries as a food preservative (Shin et al. 2016).

The two main identified areas of research for bacteriocin utilization are in the food preservation

industry and in the medical and veterinary fields (Pieterse and Todorov 2010). This thesis will

focus on the potential applications of bacteriocins in the medical and veterinary fields.

Bacteriocins have been identified as attractive alternatives to antibiotics (Cotter, Ross,

and Hill 2013). Although it is generally accepted that bacteriocins produced by Gram-positive

bacteria possess less potential to be used in a clinical setting for treatment of Gram-negative

27

pathogens because these bacteriocins normally only have activity against other Gram-positive

pathogens. However, nisin and epidermin have both demonstrated activity against Gram-

negative pathogens in vitro (Kuwano et al. 2005; Lacroix et al. 2001). Subtilosin A also has

antimicrobial effects against both Gram-positive and Gram-negative pathogens (Shelburne et al.

2007), so there is potential for other bacteriocins to be identified to be useful against Gram-

negative pathogens. Additionally, applying heat stress increased the effectiveness of subtilosin A

against Gram-negative bacteria (Shelburne et al. 2007), which was similar to what was reported

for nisin and Gram-negative bacteria (Boziaris and Adams 2001).

The prospective applications in the human medical field range from topical treatments of

skin infections to treatments of ulcers (Pieterse and Todorov 2010). One reason that bacteriocins

could be so useful is that medicine could exploit their narrow spectrum of activity. While there

are many bacteriocins that have broad spectrums of activity, which is appealing while dealing

with an infection of unknown etiology, there are also many bacteriocins with a narrow spectrum

of activity. These bacteriocins are of value because they can target a specific pathogen while

leaving the commensal microbiota untouched. An example of this is Thuricin CD, a sactipeptide

produced by Bacillus thuringiensis DPC 6431, which has promising results as a therapeutic

against C. difficile infections, while showing no significant adverse effects on the normal colon

microbiota (Rea et al. 2010). Another promising study showed that nisin exerted anti-biofilm

effects against saliva derived biofilms without causing cytotoxic effects to the human oral cells

(Shin et al. 2015). Yet another benefit to using narrow spectrum bacteriocins in place of

antibiotics is that antibiotics could be used less frequently, thus reducing the selection pressure

for resistance, and therefore maintaining the usefulness of that antibiotic for future need (Riley

and Wertz 2002).

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Nevertheless, there are some complications that must be addressed to put these

bacteriocins to clinical use. Bacteria that are able to encapsulate themselves, such as Klebsiella

species, are resistant to subtilosin A by way of preventing the bacteriocin by getting to the cell

wall surface, either by exclusion or by binding on the capsule surface (Shelburne et al. 2007).

With nisin for example, it has low solubility and low activity at high pH, and it has a tendency to

interact with blood components (Dischinger, Basi Chipalu, and Bierbaum 2014). Additionally,

because lanthipeptides lack stability against intestinal enzymes, optimal methods of delivery

would have to be investigated. Studies have suggested the use of a pill (Arthur, Cavera, and

Chikindas 2014) to encapsulate and protect the bacteriocin from the proteolytic enzymes in the

stomach. Another proposed mechanism to avoid degradation by digestive enzymes is to colonize

the gastrointestinal tract with a strain that produce the bacteriocin. In a study assessing L.

monocytogenes inhibition by a bacteriocin produced by Lactobacillus salivarius UCC118, mice

were fed either a control, a bacteriocin producing strain, or a bacteriocin knockout strain then

orally infected with luciferase-tagged Listeria (Corr et al. 2007). Thirty minutes post-challenge,

there was no fluorescence in the bacteriocin-positive infected mice, indicating anti-listerial

activity from the probiotic strain (Corr et al. 2007).

In terms of applications for bacteriocins in the dairy industry, bacteriocins have been

studied for use in preventing mastitis, although to date, only nisin and lacticin 3147 have been

explored extensively for use. Nisin was first observed to considerably reduce the amount of S.

aureus and E. coli recovered from the bovine teat skin after 1 minute of exposure to the nisin

preparation when compared with conventional iodine and chlorohexidine treatments (Sears et al.

1992). Additionally, the product posed little risk of skin irritation (Sears et al. 1992). Immucell

Corporation developed two nisin based products, Wipe-Out® and Mast Out® for use in the

29

industry, although they do not appear to be available anymore (Pieterse and Todorov 2010).

Though, according to Immucell Corp’s website, they are in the construction stage of a new

pharmaceutical facility for the production of nisin for use in a product to treat SCM

(http://immucell.com/products/purified_nisin/). Lacticin 3147 has been investigated to replace

antibiotics in dry cow therapy by use in teat sealants. Initial studies indicated lacticin 3147

inhibited mastitis pathogen S. dysgalatiae in vitro and resulted in no irritation upon inoculation

into the teat (Ryan et al. 1998). In an experimental trial on 68 uninfected quarters lacticin 3147

was tested on its ability to inhibit a challenge of S. dyscalactiae (issued 3 days post bacteriocin

infusion) for 8 days (Ryan et al. 1999). The results indicated that only 6% of the quarters

inoculated with both teat sealant and lacticin 3147 contracted clinical mastitis or shed S.

dysgalactiae in the 8 days post challenge, compared to 66% of the control quarters (Ryan et al.

1999). In a subsequent study, the usefulness of lacticin 3147 against S. aureus was assessed

(Twomey et al. 2000). In this study, one quarter of each lactating cow was infused with lacticin

3147 and teat sealant, where the other two quarters were left as untreated controls. Two hours

after inoculation, the quarters were challenged with S. aureus and 18 h later the recovery count

of S. aureus was assessed. The quarters with lacticin 3147 and teat sealant showed less S. aureus

recovery (Twomey et al. 2000). Lacticin 3147 was also assessed for mastitis prevention in the

lactating herd in the use of a teat dip (Klostermann et al. 2010). Here, teats were coated with S.

aureus, S. dysgalactiae, or S. uberis and then dipped with a teat dip containing lacticin 3147. The

dip was able to eliminate 80% of the S. aureus, 97% of S. dysgalactiae and 90% of S. uberis

after a 10-minute contact time (Klostermann et al. 2010).

Many other bacteriocins have demonstrated activity against mastitis causing pathogens.

Five characterized bacteriocins from B. thuringiensis were tested for inhibition against 50 bovine

30

subclinical mastitis S. aureus isolates, and all of the S. aureus isolates were sensitive to at least

one of the bacteriocins (Barboza-Corona et al. 2009). More recently, a study reported that yet

unidentified bacteriocins from B. thuringiensis were able to inhibit 60% of pathogens isolated

from dairy goat mastitis cases in vitro, many of which were multi-drug resistant isolates

(Gutiérrez-Chávez et al. 2016). Nukacin 3299, isolated from S. simulans, showed inhibitory

activity against Staphylococcus strains from mastitis origins (Ceotto et al. 2010). Staphylococcus

aureus (165 strains) and S. agalactiae (74 strains) from bovine mastitis were assessed for

susceptibility to three bacteriocins produced by S. aureus and four bacteriocins produced by S.

epidermidis. Epidermin (an S. epidermidis bacteriocin) showed the most widespread inhibition of

mastitis pathogens (>85%), followed by using a combination of S. aureus bacteriocins (Coelho et

al. 2007).

The delivery of these inhibiting bacteriocins will need to be studied more, although it

appears that teat dips, wipes, and teat sealants containing the bacteriocin are the most promising.

The benefits of combining bacteriocins with teat sealants mean that the bacteriocin is localized in

the teat, allowing for antimicrobial activity against pathogens that manage to enter the teat canal

after dry off. These potential products could allow dairy farmers to reduce their antibiotic use by

implementing selective dry cow therapy, only treating cows with infected quarters with

antibiotics and teat sealants at dry off, and treating uninfected cows with the teat sealant plus

bacteriocin product. Studies longer than 8 days will need to be done in order to ensure the cow is

provided with protection for the entire duration of the dry period. Teat wipes and dips could

provide effective control against Gram-positive mastitis in the lactating herd, potentially limiting

antibiotic use by decreasing the cases of CM. Thus, there is great potential for bacteriocins to be

31

used for treatment and control of mastitis. Future studies need to be done on the best ways to use

these bacteriocins, as well as on the best delivery system.

1.9 Bacteriocin resistance

An important consideration when moving forward with investigating bacteriocins for

potential use in health and food industries is to assess the risk of target bacteria to develop

resistance (Cotter, Ross, and Hill 2013). As with antibiotic resistance, the specific mechanisms

of resistance can be classified into two groups, acquired resistance (developed by a formerly

susceptible strain) and innate resistance (intrinsic). Only a few bacteriocins have been studied

with respect to the development of resistance: nisin, lacticin 3147, pediocin-like bacteriocins,

and lysostaphin (Bastos, Coelho, and Santos 2015). Little resistance to nisin has been reported

among food spoilage bacteria in the field; therefore, the evidence of bacteriocin resistance is

limited to laboratory experiments and lanthipeptide resistance has indeed been induced in

laboratory settings (Draper et al. 2015). The mechanisms which target bacteria confer resistance

to lanthipeptides are often innate and provide protection against cationic antimicrobial peptides

in general, not specifically lanthipeptides (Draper et al. 2015).

One very specific mechanism of innate resistance is through immune mimicry, which

occurs when non-lanthipeptide producing strains are immune to lanthipeptides as a result of

possessing immunity gene functional homologues (Draper et al. 2009). The expression of these

orphan immunity clusters confers protection against the associated bacteriocin (Draper et al.

2015). Additionally, some bacteria may encode for enzymes, which specifically degrade

bacteriocins. Nisinase, which degrades nisin, is an example and confers protection against nisin

32

and subtilin (Bastos, Coelho, and Santos 2015). Nisin resistance protein NSR protease is another

anti-nisin enzyme that has been identified in non-producer L. lactis strains (Sun et al. 2009).

Transcriptome analysis revealed 92 genes directly or indirectly involved in the

acquisition of nisin resistance in L. lactis (Kramer et al. 2006), indicating that resistance can

potentially be incredibly complex. The authors suggest four ways that L. lactis was able to

decrease susceptibility to nisin. The first and main mechanism is the prevention of nisin from

interacting with the cell membrane, and therefore lipid II. It likely does this by increasing the

thickness of the cell wall, becoming more densely packed, and by becoming less negatively

charged, effectively repelling nisin. Secondly, expression of genes changes in order to change the

local pH at the outside of the cytoplasmic membrane, which may either sequester the nisin or

promote its degradation. Thirdly, the phospholipids can become less saturated and more loosely

packed by changing expression of genes that are involved in elongation and saturation of said

phospholipids. This may hamper nisins ability to insert itself and form pores. Lastly, ABC

transporters may be able to remove nisin from the cytoplasmic membrane, keeping it from

binding to lipid II (Kramer et al. 2006). It is notable that the structure of lipid II does not change

as a mechanism of resistance, as where nisin binds is synthesized early and necessary for

function (Kramer et al. 2006).

In terms of acquired resistance, the frequency of spontaneous mutations that result in

nisin resistance can vary from <10-9 to 10-2 in L. monocytogenes and from 10-8 to 10-2 in other

Gram-positive organisms (Bastos, Coelho, and Santos 2015). Nisin resistance seems to have

little effect on bacterial fitness, and the resistance phenotype appears to be stable in the

population (Bastos, Coelho, and Santos 2015). For lacticin 3147, the frequency of resistance in

L. lactis appears to be low, with resistant isolates only able to resist low levels of the bacteriocin

33

after one round of pressure. Although, higher levels of resistance to lacticin 3147 can be selected

for and the resistance phenotype also appears to be stable (Bastos, Coelho, and Santos 2015).

Moderate to high levels of resistance have been reported in L. monocytogenes and E.

faecalis for Class IIa bacteriocins, where the resistant strains can decrease expression of the

Man-PTS system, which is the bacteriocins target (Guinane et al. 2006). Additionally, sensitivity

of cells to Class IIa bacteriocin depends on the sequence of the Man-PTS proteins, as only a

specific clade in the Man-PTS proteins’ phylogeny is susceptible (Kjos, Nes, and Diep 2009).

The authors’ approach of examining receptor sequences (in this case the Man-PTS protein) in

relation to susceptibility can potentially be used for all bacteriocins once a target receptor is

determined in order to quantify the inhibitory ability of the bacteriocin on a large group of target

organisms.

Because of the resistance observed in in vitro studies, it is not unlikely that bacteriocin

resistance will arise with the application of bacteriocins in clinical settings (Bastos, Coelho, and

Santos 2015). Although, a study examining the risk of bacteriocin E-760 resistance developing in

Campylobacter in a chicken model only found low levels of resistance (Hoang, Stern, and Lin

2011). Resistance risk may depend on the bacteriocins mode of action; specifically, bacteriocins

with dual modes of action may be less likely to induce resistance. Therefore, the lack of apparent

resistance towards nisin may be due to its multiple modes of action with a number of distinct

targets (Shin et al. 2015). More studies need to be done on the specific risk of resistance

developing in food model systems. Once resistance mechanisms are determined, ways can be

created to mitigate the resistance in clinical applications. For example, An ABC pump confers

resistance of Campylobacter to E-760, therefore it is postulated that use of the bacteriocin with

efflux pump inhibitors may increase the susceptibility of Campylobacter to E-760 for use in

34

production systems (Hoang, Stern, and Lin 2011). More studies need to be done on the risk of

resistance development in clinical settings for Staphylococci bacteriocins. To do this,

information needs to be obtained on specific bacteriocins, their structure, modes of action, and

spectrums of activity. Identification of bacteriocins is the first step in this process. With the NAS

being such ubiquitous members of the udder microbiome, paired with evidence of NAS

inhibition on major mastitis pathogens due to bacteriocin production, large-scale investigations

of bacteriocin production in NAS has merit.

1.10 Thesis outline

The overall aim of this thesis research was to assess the species-specific inhibitory

capability of bovine NAS, to identify bacteriocin genes potentially relating to inhibition, and in

an attempt to understand the mechanisms of susceptibility, identify genes related to susceptibility

in a large group of bovine S. aureus to an S. chromogenes bacteriocin. The study consisted of

two parts with distinct (albeit complementary) aims and hypotheses.

1.10.1 Bacteriocins of non-aureus staphylococci isolated from bovine milk

Aim: To determine the inhibitory capability of 441 bovine NAS isolates from 26 different

species against a bovine S. aureus and a human methicillin-resistant S. aureus (MRSA). The

second aim was to identify and describe the organization of bacteriocin biosynthetic gene

clusters in the corresponding 441 whole genome sequences.

Hypothesis: Phenotypic inhibition will be widespread across isolates and genotype will be

able to predict phenotypic inhibition in most cases.

35

The research to address this aim and hypothesis is described in Chapter 2.

1.10.2 Identifying putative bacteriocin resistance genes in Staphylococcus aureus whole genomes

Aim: To determine the sensitivity of 139 bovine S. aureus isolates to a bacteriocin

producing S. chromogenes isolate and to identify and describe putative bacteriocin resistance

genes in S. aureus whole genome sequences.

Hypothesis: The presence of specific bacteriocin resistance genes in S. aureus isolates will

correspond to the in vitro phenotype.

The research to address this aim and hypothesis is described in Chapter 3.

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Chapter Two: Bacteriocins of non-aureus staphylococci isolated from bovine milk

2.1 Abstract

Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine

udder, potentially protect the udder against infection by major mastitis pathogens due to

bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS

isolates (comprising 25 species) against bovine S. aureus. Furthermore, inhibiting isolates were

tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking

method. We determined the presence of bacteriocin clusters in NAS whole genomes using

genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and non-

inhibiting isolates and determined the genetic organization of any identified bacteriocin

biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S.

epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus)

inhibited growth of S. aureus in vitro; 23 of which, from S. capitis, S. chromogenes, S.

epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. 105 putative

bacteriocin gene clusters in 95 whole genomes from 16 species were identified. Twenty-five

novel bacteriocin precursors were described. Additionally, 7 NAS species were identified with

clusters for the first time and sactipeptides and lasso peptides, which have never been reported in

Staphylococcus species, were identified. In conclusion, NAS from bovine mammary glands are a

source of potential bacteriocins, with >21% being possible producers, representing potential for

future characterization and clinical applications.

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Importance

Mastitis (particularly infections caused by Staphylococcus aureus) cost Canadian dairy

producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With

increasing antibiotic resistance and regulations regarding use, there is impetus to explore

bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of

bacterial infections. We examined the ability of 441 non-aureus staphylococci (NAS) bacteria

from Canadian bovine milk samples to inhibit growth of S. aureus in the laboratory. Overall, 9%

inhibited growth of S. aureus and 58% of those also inhibited methicillin-resistant S. aureus

(MRSA). In NAS whole-genome sequences, we identified 21% of NAS as having bacteriocin

genes. Our study provides the first comprehensive genomic report of NAS bacteriocins from

bovine milk and represents the foundation to further explore these antimicrobial peptides for

clinical use.

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2.2 Introduction

Non-aureus staphylococci (NAS), a heterogeneous group of approximately 50 species that

can be considered teat skin opportunists and minor pathogens, are the bacteria most commonly

isolated from the bovine udder (Piepers et al. 2007; Pitkälä et al. 2004; Sampimon et al. 2009;

White et al. 1989). Several studies reported that NAS may confer protection against

intramammary infection (IMI) by major mastitis pathogens (De Vliegher et al. 2004; Matthews,

Harmon, and Smith 1990). In a challenge study, 53% of Staphylococcus chromogenes-infected

quarters were protected from S. aureus challenge (Matthews, Harmon, and Smith 1990). In

another study, S. chromogenes isolates inhibited in vitro growth of all tested Staphylococcus

aureus, Streptococcus dysgalactiae, and Streptococcus uberis isolates, but none of the Gram-

negative isolates (De Vliegher et al. 2004). Similarly, NAS strains from Brazilian bovine mastitis

cases, including Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus

saprophyticus , Staphylococcus hominis , and Staphylococcus arlettae, inhibited growth of

Corynebacterium fimi (Nascimento et al. 2005). The isolated antimicrobial substances were

considered to be bacteriocins due to sensitivity to proteolytic enzymes (Nascimento et al. 2005).

Recently, Braem et al. (2014) identified NAS strains (from 6 species) that inhibited S. aureus, S.

uberis, and S. dysgalactiae and an inhibitory substance (from an inhibiting S. chromogenes) was

isolated and identified to be a nukacin-like bacteriocin.

Bacteriocins are ribosomally synthesized and post-translationally modified peptides

(RiPPs) produced by Gram-positive bacteria that mainly inhibit growth of similar bacterial

species, or occasionally a broad spectrum of bacteria (Cotter, Hill, and Ross 2005). As

regulations surrounding antibiotic usage get stricter, bacteriocins represent potential alternatives

39

to antibiotics (Cotter, Ross, and Hill 2013). Nisin, produced by Lactococcus lactis, was available

for use in the dairy industry in the form of Wipe Out®, with germicidal activity against S. aureus

and S. agalactiae (Sears et al. 1992). Lacticin 3147, another bacteriocin produced by L. lactis,

was tested for use in a teat sealant and reduced the number of S. aureus recovered from the

bovine mammary gland 18 h post challenge when compared to infusion with teat sealant alone

(Crispie et al. 2005).

Traditionally, identification of novel bacteriocins used culture-based approaches that

involved screening numerous isolates for antimicrobial activity, followed by lengthy biochemical

characterization. However, due to growing accessibility of genome sequence data, in silico

screening (genome mining) is a promising approach to identify novel biosynthetic gene clusters.

Although bacteriocin precursor genes are often small and lack homology, making in silico

identification challenging, bacteriocin-associated genes present on the same operon are highly

conserved. Therefore, the associated modification genes are used in classification schemes, with

2 classes: Class 1, post-translationally modified bacteriocins, including lanthipeptides,

sactipeptides, and lasso peptides; and Class 2, non-modified or cyclic peptides (Cotter, Hill, and

Ross 2005). By screening genomes for bacteriocin-associated genes, new lanthipeptides (Marsh

et al. 2010; Begley et al. 2009) and new Class IIa bacteriocin gene clusters (Kjos et al. 2011)

have been identified. In addition, BLAST-based approaches have been used to identify

bacteriocins in cyanobacteria (Wang, Fewer, and Sivonen 2011) and to identify lanthipeptide

clusters by using a transport gene for screening (Singh and Sareen 2014). Current software-based

approaches (e.g. BAGEL3 and antiSMASH) combine direct mining for the structural gene with

indirect mining for bacteriocin-associated genes. Using this approach, novel bacteriocins were

identified in ruminal bacteria (Azevedo et al. 2015), anaerobic bacteria (Letzel, Pidot, and

40

Hertweck 2014), lactic acid bacteria (Singh et al. 2015), and human gut microbiota (Walsh et al.

2015). A large in silico screen has apparently not been done on NAS whole genomes, which

could be the foundation for future investigations into alternatives for antimicrobials in the dairy

industry.

The first objective was to determine the inhibitory capability of 441 bovine NAS isolates

from 25 species against a bovine S. aureus and a human methicillin-resistant S. aureus (MRSA).

The second objective was to identify and describe the organization of bacteriocin biosynthetic

gene clusters in the corresponding 441 whole genome sequences.

2.3 Materials and methods

2.3.1 Isolates

NAS isolates and a S. aureus isolate from a clinical case of bovine mastitis were collected

in the National Cohort of Dairy Farms (NCDF) conducted across Canada during 2007 and 2008,

as described by Reyher et al. (2011). The Canadian Bovine Mastitis and Milk Quality Research

Network (CBMQRN) at the University of Montreal stored the samples before sending them to

the University of Calgary. Overall, 441 NAS isolates were selected from the stock of 5507

isolates over the 25 NAS species identified previously (Naushad et al. 2016; Condas et al. 2017

). These isolates originated from 87 herds across Canada from Nova Scotia, Prince Edward

Island, New Brunswick (representing Atlantic Canada), Québec and Ontario (representing

Central Canada), and Alberta (representing Western Canada) (Reyher et al. 2011). Isolates

included 68 NAS isolates from clinical mastitis cases, 26 multi-drug resistant (MDR) isolates,

maximum 1 isolate per cow of any uncommon species (defined as <20 unique isolates at cow

41

level), and maximum 1 randomly selected isolate per cow for all other species until 441 were

chosen (Naushad et al. 2016). The multi-drug resistant MRSA clinical strain H176 from human

origin was obtained from Dr. K. Zhang’s laboratory at the University of Calgary.

2.3.2 Phenotypic testing

All 441 NAS isolates were tested for antimicrobial activity against a S. aureus isolate

(derived from clinical mastitis). Only the NAS isolates that inhibited this S. aureus were tested

against the MRSA. Testing was done using a cross-streaking method, modified from a previous

report (De Vliegher et al. 2004). Each isolate was plated on 5% defimbrinated sheep blood agar

plates (BD Diagnostics, Mississauga, ON, Canada) and incubated overnight at 37°C. A single

colony was diluted in PBS to a McFarland 0.5 standard and was used to inoculate a centre streak

(5 mm) on a 5% sheep blood agar plate and subsequently incubated at 37°C for 24 h. On day 2,

the agar was loosened from the plate with sterile metal tweezers and flipped onto the lid of the

plate so the NAS centre streak was face down. Then, 100 µL of a 10-3 dilution in PBS of a

McFarland 0.5 standard of a single colony from an overnight culture on 5% sheep blood agar of

S. aureus was spread over the entire agar surface and incubated at 37° for 24 h. On day 3, plates

were examined for bacterial growth and any inhibition (total or partial) of pathogen growth was

recorded. All experiments included a negative control (PBS was used to make the center streak

on day 1).

2.3.3 Effect of proteinase K on inhibition

Strains that were both inhibitors of S. aureus and potential bacteriocin producers were

tested to confirm the peptide nature of the inhibitory product using proteinase K (20 mg ml -1;

42

Sigma Aldrich) and an agar well diffusion assay. Concentrated cell free supernatant was obtained

from BHI cultures of the 21 inhibiting and potentially producing NAS isolates by performing a

modified version of a chloroform extraction described previously (Burianek and Yousef 2000).

Briefly, 40ml of BHI broth was inoculated with 0.1% of an overnight culture of NAS and

incubated at 37° for approximately 20hr. Cells were removed by centrifugation at 4500g at 4° for

15 min. 20ml of chloroform (Sigma-Aldrich) was mixed with the cell free supernatant of each

NAS and stirred vigorously for 20 min, followed by centrifugation at 4500 g at 4° for 15 min.

The cloudy interfacial precipitate was collected and dried overnight. The dried product was

dissolved in 1ml phosphate buffer solution (PBS) using a magnetic spinner at 4° for 24h.

Proteinase K was added to each concentrated cell free supernatant. Samples with and

without proteinase K were incubated at 37° for 1 hr. Residual inhibition was tested using an agar

well diffusion assay (Schillinger and Lücke 1989).

2.3.4 Whole genome sequencing, assembly, and annotation

Sequencing, assembly, and annotation for NAS and the clinical mastitis S. aureus isolate

were performed as described (Naushad et al. 2016). Briefly, genomic DNA was extracted with

DNeasy Blood and Tissue Kit (Qiagen, Toronto, ON, Canada), according to the corresponding

protocol for Gram-positive bacteria. Sequencing of these samples was performed using the

Illumina MiSeq platform (Illumina, San Diego, CA, USA); DNA libraries for sequencing were

prepared using a Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). All

sequencing steps, including cluster generation, paired-end sequencing (2 × 250 bp), and primary

data analysis for quality control, were performed on the instrument. Genome assembly was

automated using the Snakemake workflow engine (Köster and Rahmann 2012). Raw read pairs

43

were screened for adapters and quality trimmed using Cutadapt 1.8.3 (Martin 2011) as

implemented in Trim Galore! 0.4.0 (with default parameters). Genomes were assembled using

Spades 3.6.0 (Nurk et al. 2013) using the built-in error correction and default parameters. To

assess coverage, reads were mapped back to the assembled genome using BWA 0.7.12-r1039 (Li

and Durbin 2009). Contigs > 200 bp were annotated with Prokka 1.11 (Seemann 2014) using the

provided Staphylococcus database. Assembly quality was evaluated with Quast 3.0 (Gurevich et

al. 2013). Contigs, as well as the annotated protein sequences, were used for custom blast

searches using SequenceServer (Priyam et al. 2015). Data were previously submitted to NCBI

under BioProject ID PRJNA342349.

2.3.5 Screening of genomes for bacteriocin clusters

Identification of biosynthetic gene clusters related to secondary metabolite production and

analysis of sequences of interest was done using antiSMASH 3 (Weber et al. 2015). Each gene in

identified clusters was further examined using the BlastP web server on NCBI

(http://www.ncbi.nlm.nih.gov/BLAST), and the presence of conserved domains from the

Conserved Domain Database (Marchler-Bauer et al. 2017) was noted in each coding region and

compared to previously identified known conserved domains in bacteriocin-associated genes

(Azevedo et al. 2015). Putative gene clusters were classified according to Cotter et al. (Cotter,

Ross, and Hill 2013). Additionally, any identified structural genes in the NAS genomes were

used to BLAST back against our NAS genomes, to potentially identify any clusters not detected

by antiSMASH.

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2.3.6 BLAST

The 441 NAS whole genomes were assessed by BLAST for any bacteriocin structural

genes contained in Classes I, II, and III databases from BAGEL

(http://bagel.molgenrug.nl/index.php/bacteriocin-database). Any genomic regions with identified

bacteriocin-associated genes after the BLAST search were visualized using Geneious version

8.1.6 (Kearse et al. 2012) to determine if the bacteriocin gene cluster was complete by assessing

if the structural gene and known essential associated genes were present using the BlastP web

server on NCBI (http://www.ncbi.nlm.nih.gov/BLAST).

2.3.7 Genome comparison

To further identify potential bacteriocin-associated genes, genomes of the inhibiting NAS

were compared to closely related genomes of non-inhibiting NAS of the same species, according

to the phylogenetic trees of each species. For this purpose, the phylogenetic trees for each

species of NAS were constructed using methods as described (Naushad et al. 2016). Briefly,

trees were rooted using Macrococcus caseolyticus and created based on the core genome of the

individual NAS species. The core set were identified using the UCLUST algorithm (Edgar 2010)

and protein families with at least 30% sequence identity and 50% sequence length were

considered core. However, protein families present in ≥ 95% of the input genomes were

considered core and protein families containing potential paralogous sequences (duplicated

sequence in same genome) were excluded. Each protein family was individually aligned using

MAFFT 7 (Katoh and Standley 2013). Aligned amino acid positions which contained gaps in

more than 50% of genomes, were excluded from further analysis. Remaining amino acid

positions were concatenated to create a combined dataset. A maximum-likelihood tree based on

45

this alignment was constructed using FastTree 2.1 (Price, Dehal, and Arkin 2010) using the

Whelan and Goldman substitution model (Whelan and Goldman 2001).

Comparisons were done by identifying shared genes, present in both closely related

inhibiting and non-inhibiting isolates using Spine, a web based application that identifies

common sequences in the input genomes (Ozer, Allen, and Hauser 2014). Sequences unique to

inhibiting isolates were then determined using AGEnt, by subtracting the output of shared

sequences acquired from Spine from the genome of an inhibiting isolate (Ozer, Allen, and

Hauser 2014). Sequences unique to inhibiting isolates were then visualized using Geneious

version 8.1.6 (Kearse et al. 2012) and genes and conserved domains were determined using the

BLASTn web server on NCBI (http://www.ncbi.nlm.nih.gov/BLAST) and Conserved Domain

Database (Marchler-Bauer et al. 2017) to establish any additional bacteriocin-associated

sequences.

2.3.8 Precursor gene alignments

Protein alignments of precursor peptides were generated using MUSCLE (Edgar 2004).

Sequence alignments were viewed and edited with Jalview alignment editor (Waterhouse et al.

2009).

2.4 Results


Out of 441 NAS isolates, 40 isolates (9.1%) from 9 species (S. capitis, S. chromogenes, S.

epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus)

46

inhibited growth of the bovine clinical mastitis S. aureus isolate (Table 1). Of the 40 inhibiting

isolates, 23 (57.5%) from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and

S. xylosus also inhibited growth of the MRSA isolate.

2.4.2 Effect of proteinase K on inhibition

Out of 21 inhibitors that were potential producers, four isolates showed inhibition in the

well diffusion assay, and for all of which inhibition was eliminated with the addition of

proteinase K.


In the 441 NAS genomes, 184 putative bacteriocin gene clusters belonging to 143 isolates

were identified. A total of 105 clusters from 95 isolates belonging to 16 species were determined

to be viable clusters, whereas the others were eliminated due to absence of either a structural

gene or essential bacteriocin-associated genes (Table 1). Overall, 21.5% of NAS potentially

produced bacteriocins. Ten of the 441 genomes encoded 2 clusters of different classes/types,

whereas the remaining 85 potential producers contained 1 cluster. Of the 40 inhibitors, 21 were

putative producers, whereas no viable bacteriocin gene clusters were identified in 19 inhibitors.

Class II bacteriocins were most frequently identified, with 69 clusters in 68 isolates from S.

equorum, S. gallinarum, S. haemolyticus, S. hyicus, S. saprophyticus, S. sciuri, S. simulans, S.

succinus, S. warneri, and S. xylosus (Fig 1). Nine of the Class II producers were also inhibitors.

For Class I bacteriocins, lanthipeptides were the most frequently identified type, with 29 clusters

in 29 isolates from S. capitis, S. chromogenes, S. cohnii, S. epidermidis, S. equorum, S.

gallinarum, S. sciuri, S. simulans, S. succinus, and S. vitulinus (Fig 1). Fifteen of the 29

47

lanthipeptide producers were also inhibitors. Three sactipeptide clusters were identified from 3 S.

capitis isolates, 2 of which were inhibitors. Lastly, 4 lasso peptide clusters were identified in 2

non-inhibiting S. fleuretti genomes, a non-inhibiting S. sciuri genome, and an inhibiting S. sciuri

genome. Putative bacteriocin-associated genes were distributed throughout the phylogeny of

NAS (Naushad et al. 2016) (Fig 1), although no isolates from S. agnetis (n=13), S. arlettae

(n=15), S. auricularis (n=2), S. caprae (n=1), S. devriesei (n=8), S. hominis (n=11), S. kloosii

(n=1), S. nepalensis (n=2), and S. pasteuri (n=6) contained putative bacteriocin gene clusters.

There was no obvious clustering based on phylogeny or class of bacteriocin.

2.4.3.1 Class I bacteriocins

Lanthipeptide clusters. Twenty-nine lanthipeptide gene clusters were detected in NAS genomes

(Table 1). Fifteen clusters were classified as type 1 (Fig 2); 12 of those clusters, all from S.

capitis, had an identical LanA structural peptide (Fig 2). Six of the 12 S. capitis producers were

also inhibitors. The 44 aa precursor had 59% identity with nisin (a Lactococcus lactis

bacteriocin) and contained the conserved domain, pfam02052. Two of the remaining type 1

clusters came from 2 inhibiting isolates of S. epidermidis, containing an identical 52 aa precursor

peptide, including the epidermin-conserved domain (TIGR03731) and had 96% identity with

epidermin (accession number P08136; Fig 2). The cluster in S. epidermidis 1778 additionally

contained the LanFEG immunity system and a LanT protein (Fig 2). These 2 isolates were both

the only inhibitors and only producers identified in S. epidermidis, suggesting that the

bacteriocin was responsible for in vitro inhibition (Fig 3). The final type 1 lanthipeptide cluster

identified was detected in non-inhibitor S. equorum 1644. This cluster harboured a 57 aa

precursor that contained the type 1 lanthipeptide conserved domain pfam08130 and had 53%

48

identity with Pep5 and 48% identity with epicidin 280 (Fig 2). This precursor was not identified

in any of our other 441 isolates. Four of the 5 clusters contained a LanR regulator, whereas 3 of

the 5 contained a LanP protease.

Fourteen of the 29 lanthipeptide clusters contained the type 2 LanM modification system.

Ten of those clusters contained single LanM proteins (Fig 4). Of those 10 clusters, 2 inhibitors

from S. chromogenes contained 61 aa LanA1 and 82 aa A2 precursors, which contained the

mersacidin conserved domain (pfam16934); the A1 precursor had 20% identity with the Cyl-L

component of cytolysin produced by Enterococcus faecalis and the A2 precursor had 26%

identity with the Cyl-S component of cytolysin (Fig 3), whereas A1 and A2 had 40% identity

with each other (Fig 4). The S. chromogenes 1348 cluster additionally contained a LanT

transporter (Fig 4). Staphylococcus cohnii 5 contained a cluster with a 39 aa hypothetical protein

that had 19% identity to LanA from mersacidin, although no bacteriocin-associated conserved

domains were identified during BLASTp analysis. Staphylococcus cohnii 1067 also had no

identified structural peptide, although a 41 aa hypothetical protein adjacent to the LanM had 21%

identity with the S. cohnii 5 potential LanA and 25% with LanA from S. simulans 1336 identified

in this study. Staphylococcus simulans 1336, a non-inhibitor, contained a 2-component

lanthipeptide, where the 68 aa A1 had 56% identity with the Cyl-L component of cytolysin and

the 68aa A2 had 57% identity with Cyl-L. Furthermore, A1 and A2 have 80% identity with one

another and contained the 2-component Enterococcus faecalis cytolysin (EFC) conserved

domain (pfam16934). The next 3 lanthipeptide clusters were from 3 inhibiting S. simulans

isolates and all contained identical clusters with LanA peptides (conserved domain pfam04604)

that were identical to NukA, the structural peptide for Nukacin ISK-1(accession number

Q9KWM4). Another cluster, in S. simulans 3061, contained a 105 aa hypothetical protein which

49

may function as a LanA (Fig 3) and had 15% identity with a potential LanA in S. cohnii 5.

Lastly, S. vitulinus 730 contained 2 LanA peptides, which had 53% identity with each other.

Furthermore, A1 had 35% identity with Cyl-L and A2 had 32% identity with both Cyl-S and

mersacidin. Three clusters contained an NADPH-dependent FMN reductase (not normally

associated with lanthipeptide clusters).

The remaining 4 type lanthipeptide clusters were part of a different category of type 2

lanthipeptides that contain 2 structural peptides, plus dual lanM proteins, where each lanM is

responsible for modifying a distinct precursor peptide (Fig 5). Staphylococcus succinus 6028

contained a 73 aa A1 precursor and a 68 aa A2 precursor. Staphylococcus succinus 6028A1 had

34% identity with Lacticin 3147A1, whereas the A2 precursor had 32% identity with Lacticin

3147A2. In inhibitor S. sciuri 225, the 63 aa A1 precursor had 28% identity to Lacticin 3147A1

and the 68 aa A2 precursor had 36% identity with Lacticin 3147A2 and 40% identity with

Lichenicidin A2. Lastly, S. gallinarum 2094 and 1388 contained identical novel two-peptide type

2 lanthipeptide biosynthetic clusters with 83aa A1 precursors (conserved domain pfam14867)

that had 34% identity with Lichenicidin A1 and 68aa A2 precursors that had 43% identity with

Lichenicidin A2. The 2 precursors, A1 and A2, had 21.9% identity with each other. Three of the

4 clusters contained a lanP protease.

Sactipeptide clusters. A total of 26 potential Subtilosin-A like clusters were identified, although

23 were excluded due to absence of the critical AlbF gene (Azevedo et al. 2015), leaving 3 S.

capitis isolates that contained viable clusters (Table 1). In S. capitis 1319 (non-inhibitor), 2487

(inhibitor), and 3379 (inhibitor), the structural peptides were all identical and had 63% identity

with the Subtilosin-A precursor, SboA (accession number O07623; Fig 6).

50

Lasso Peptide clusters. Three non-inhibiting NAS isolates, 2 S. fleuretti and 1 S. sciuri, along

with an inhibiting S. sciuri, contained identical clusters encoding for a lasso peptide (Fig 7). The

40 aa structural peptide, ‘A’, had no identified conserved domains, but had 31% identity with a

previously characterized lasso peptide lariatin, produced by Rhodococcus sp. K01-B0171

(Iwatsuki et al. 2006).

2.4.3.2 Class II bacteriocins

Double-glycine leader. Three S. equorum and 1 S. saprophyticus isolate (all non-inhibitors)

encoded for bacteriocin clusters that contained 2 precursor peptides that were annotated as

bacteriocin class II with double-glycine leader peptides (Fig 8). Although the precursor peptides

in all 4 clusters were identical, additional associated proteins varied. The S. saprophyticus and 2

of the S. sciuri clusters contained a SecA protein, possibly related to secretion.

Class IIc circular bacteriocin clusters. Nineteen isolates, 2 of which were inhibitors (across

various clades of NAS phylogeny; Fig 1) contained circular bacteriocin gene clusters encoding 4

distinct bacteriocins (Fig 9). Of the 19 identified clusters, 16 were in S. gallinarum isolates, and

all 16-precursors were identical, although the clusters contained varying bacteriocin-associated

genes (Fig 9). The S. gallinarum precursor showed 25% identity with both gassericin and

enterocin AS-48 (Fig 9). All but 2 of the clusters contained a signal peptidase. Staphylococcus

haemolyticus 109 contained a 97 aa structural peptide with 25% identity with both circularin and

enterocin AS-48. The cluster identified in inhibiting S. simulans 1355 contained a structural

peptide (conserved domain TIGR03651) with 23% identity with enterocin AS-48. The final

51

cluster, identified in inhibiting S. xylosus 4938 contained a precursor with 32% identity with

circularin A.

Class IId clusters. Forty-seven NAS genomes contained complete lactococcin-like clusters (Fig

10). Of these 47 producers, 8 were inhibitors. Twenty-seven lactococcin-like clusters were

identified in 27 S. simulans isolates. Eleven isolates contained a 94 aa structural precursor,

whereas the remaining 15 contained a 105 aa structural precursor, with 88% identity with each

other (Fig 10).

Fourteen isolates contained clusters with the same organization of genes, although the

precursor gene (conserved domain pfam09683) varied slightly among isolates (Fig 10). All 8 S.

xylosus isolates in this group contained an identical 93 aa structural gene with 62% identify with

the structural gene identified in the 2 S. equorum clusters. The S. hyicus cluster’s structural

peptide had 52% identity with the S. xylosus’s structural peptide. Lastly, 3 S. sciuri isolates

structural peptides had 43% identity with the S. xylosus precursor.

An additional S. sciuri isolate, with an identical structural peptide to the other S. sciuri

isolates mentioned above, was identified, although it lacked the ABC transporter in the cluster.

The remaining 5 clusters were identified in S. warneri. The 95 aa putative precursor had 60%

with the 94 aa precursor from S. simulans (Fig 10).

2.5 Discussion

In this study 95 isolates (22%) of 441 NAS encoded for 105 bacteriocin biosynthetic gene

clusters, making them potential bacteriocin producers. Bacteriocin gene clusters were detected,

52

apparently for the first time, in S. equorum, S. haemolyticus, S. succinus, S. capitis, S. cohnii, S.

vitulinus, and S. fleuretti, although some species had demonstrated antimicrobial activity in

previous studies (Braem et al. 2014). According to the phylogenetic tree, bacteriocin gene

clusters are spread throughout the NAS phylogeny, although lasso peptides were only present in

2 species from the same clade and sactipeptides were only identified in S. capitis. In contrast,

only 40 (9%) of 441 NAS were inhibitors when antimicrobial activity was tested in vitro against

a bovine clinical mastitis S. aureus isolate. We identified a higher percentage of NAS with

antimicrobial activity than a previous study that reported only 6.4% of NAS from bovine mastitis

cases were inhibitory (Nascimento et al. 2005). The NAS isolated from the teat apex skin of

dairy cows may have potential for greater bacteriocin production, as 13% of those isolates had

antimicrobial properties in one study(Braem et al. 2014).

Our study identified 29 lanthipeptide clusters from 29 NAS isolates. Both Class I and II

lanthipeptides were identified in NAS genomes. Lanthipeptides are characterized by the presence

of several uncommon amino acids, including meso-lanthionine and 3-methyl-lanthionine, the

result of post-translational modifications (Bierbaum et al. 1996). For Class I lanthipeptides,

dehydration of the serine and threonine residues to dehydroalanine (Dha) and dehydrobutyrine

(Dhb) residues in the pre-peptide, LanA, are completed by a dehydratase (LanB). Thereafter,

thioether crosslinks are formed on the dehydrated amino acids by a cyclase (generically termed

LanC), whereas each specific bacteriocin has its individual naming system, for example nisB and

nisC for nisin. More than 50 lanthipeptides from Gram-positive bacteria have been isolated and

described (Asaduzzaman and Sonomoto 2009), with genome mining identifying many more

potential compounds (Knerr and van der Donk 2012). Previously uncommon genes, e.g. FMN

reductase and N-acetyltransferases, were identified in some clusters in our study, which have

53

been reported in multiple lanthipeptide clusters identified by Singh and Sareen (2014). As we

learn more about post-translational modifications, these genes could help identify novel clusters

in additional genomes. Class I lanthipeptides were identified in S. capitis, S. epidermidis, and S.

equorum. The S. epidermidis strains harbouring the bacteriocin biosynthetic gene cluster also

inhibited S. aureus. The structural gene had 96% similarity with epidermin, the most frequently

produced lanthipeptide by NAS (Bastos et al. 2009) and reported to inhibit human MRSA

(Nascimento et al. 2006) and S. aureus isolated from bovine mastitis (Coelho et al. 2007). Only 6

of 12 S. capitis lanthipeptide producers were inhibitors; therefore, if it is in fact the bacteriocin

identified that is responsible for the inhibition then the remaining 6 non-inhibitors could be

producers of a yet unidentified inhibitory substance, or perhaps the bacteriocin cluster is not

being expressed in the 6 non-inhibitors. This study apparently represents the first time

lanthipeptides have been identified in S. capitis. The S. equorum producer was also a non-

inhibitor, but harbours a gene that likely encodes for a novel bacteriocin (similar to pep5 and

epidicin 280). Class I lanthipeptides pre-peptides typically have a FNLD conserved region

approximately at positions -15 to -20 and a proline at -2 from the cleavage site, which have roles

in modifications of the peptide (Lubelski et al. 2008). Only the LanA identified in the S.

epidermidis strains had the FNLD conserved region and the proline at -2. The LanA identified in

S. capitis had a FDLD motif with the proline at -2, similar to gallidermin and lanthipeptides

recently identified in ruminal bacteria (Azevedo et al. 2015). However, the S. equorum LanA had

a FDLE motif, similar to pep5 and epicidin 280, which had the closest identity when aligned.

One of the most common lanthipeptides identified in NAS to date has been nukacin-like

bacteriocin. In our study, nukacin was discovered in 3 inhibiting S. simulans isolates. The LanA

precursor was identical to Nukacin ISK-1 produced by S. warneri (Sashihara et al. 2000),

54

Nukacin 3299 produced from S. simulans (Ceotto et al. 2010), and Nukacin L217 produced by S.

chromogenes (Braem et al. 2014). That both Nukacin 3299 and Nukacin L217 producer strains

were also isolated from bovine milk and the potential nukacin producer in our study was able to

inhibit S. aureus suggests a role for this bacteriocin in NAS colonization and pathogen inhibition

in the udder environment.

Class II lanthipeptides are characterized by a bifuncational enzyme, LanM, responsible for

both dehydration and cyclisation, whereas the C terminal shares homology with LanC cyclases

of the Class I lanthipeptides (Asaduzzaman and Sonomoto 2009). Ten of 14 clusters identified

contained a single LanM, with 4 containing 2 LanA precursors. The 2 LanA precursors in each

cluster had a high sequence identity (80% for the S. simulans 1336 cluster), suggesting that the

same single LanM enzyme could modify both precursors. For dual precursors with low sequence

identity (e.g. in lichenicidin and haloduracin), there are multiple lanM enzymes to modify each

unique precursor (Dischinger et al. 2009) (McClerren et al. 2006). These represent a distinct

group of type II lanthipeptides, of which 3 were identified in our study. Herein, A1 and A2

precursors had 26, 35, and 22% identity with one another for clusters in S. succinus, S. sciuri,

and S. gallinarum, respectively. However, only the S. sciuri isolate was an inhibitor. Unlike class

II lanthipeptides produced by ruminal bacteria (Azevedo et al. 2015), eight type II clusters in this

study harboured the LanP protease.

Sactipeptides are a group of class I bacteriocins that contain a sulphur to α-carbon linkage,

catalyzed by a recombinant S-adenosylmethionine (rSAM) protein (Fluhe and Marahiel 2013;

Arnison et al. 2013). They were originally only isolated from Bacillus species, although genome

mining has now identified putative gene clusters in the genera Clostridium, Blautia, Kandleria,

Lachnobacterium, Peptostreptococcus, Roseburia, and Ruminococcus (Azevedo et al. 2015;

55

Walsh et al. 2015; Murphy et al. 2011). Hyicin 4244, produced by S. hyicus, was recently

characterized and represents the first sactipeptide in Staphylococcus (Duarte et al. 2017). One

sactipeptide cluster was identified in 3 isolates in our study, although the bacteriocin-associated

genes varied slightly between clusters. Our analysis identified a histidine kinase (HK) and

response regulator (RR) in each cluster, indicating that bacteriocins may be subjected to a 2-

component regulatory system, previously only reported in sactipeptides clusters from ruminal

bacteria (Azevedo et al. 2015). Two of the 3 clusters containing isolates were also inhibitors,

although the S. capitis 3379 isolate additionally contained a lanthipeptide, which could be

responsible for inhibition. The inhibiting S. capitis 2784 did not contain any additional clusters to

our knowledge, although, subtilosin-A, which this novel bacteriocin is most related to,

moderately inhibited S. aureus in vitro (Shelburne et al. 2007). Thus, the non-inhibiting S. capitis

producer was potentially not expressing its bacteriocin gene or the inhibition seen in the other

two isolates was due to another product.

Lasso peptides are an emerging group of RiPPs that do not undergo extensive

modification, although they are folded so that the C terminal is threaded through a ring formed

by a single isopeptide bond, yielding their signature lariat-like form (Maksimov and Link 2014).

To the best of our knowledge, no lasso peptides have been identified in Staphylococcus species

(Hegemann et al. 2015). In this study, an identical, novel cluster was identified in 3 non-

inhibiting isolates, 2 from S. fleuretti and 1 from S. sciuri, along with 1 inhibiting S. sciuri. The

identified clusters contained all 4 of the essential enzymes (‘ABCD’) for peptide production

(Letzel, Pidot, and Hertweck 2014), a transglutaminase-like protein that is likely the ‘B’ gene

which acts as a protease to cleave the leader sequence, a protein with an asparagine synthase

conserved domain, which is likely the ‘C’ gene responsible for isopeptide bond formation, and 2

56

units of an ABC-type dipeptide/oligopeptide/nickel transport system functioning as the ‘D’ gene,

as well as a 53 aa putative protein.

Although most identified putative bacteriocin clusters in this study were class II, there are

only 12 unique precursors in those clusters, compared to the 15 unique lanthipeptide precursors

identified. To date, the majority of Class II bacteriocins in Staphylococcus species have been

identified in S. aureus (Bastos et al. 2009). The first circular bacteriocin in Staphylococcus

species, discovered in 2014, was aureocyclicin 4185 from S. aureus 4185 (Potter, Ceotto,

Coelho, Guimarães, et al. 2014). Herein, we identified putative novel circular bacteriocin gene

clusters from S. gallinarum, S. haemolyticus, S, simulans, and S. xylosus. These bacteriocin

precursors had limited similarity with previously characterized circular bacteriocins, but all

contained the conserved domain associated with the circularin A/uberolysin family. Although the

S. xylosus producer was not the only producer in the species (lactococcin-like clusters were also

identified in other isolates), it was the only inhibitor in the species, suggesting that this

bacteriocin is responsible for activity against S. aureus, although further investigation after

isolation and purification of the peptide is needed. The S. simulans isolate was also an inhibitor

in vitro, but producers from S. gallinarum and S. haemolyticus were non-inhibitors, suggesting

these bacteriocins either have a different spectrum of activity or were not activated.

Class IId bacteriocins are described as linear non-pediocin-like. Lactococcin 972 belongs

to this group and is produced by Lactococcus lactis subsp. lactis IPLA 972 (Martı́nez et al.

1999). The gene cluster, along with the LclA precursor, encodes for a transporter, LclB, and an

immunity gene (Letzel, Pidot, and Hertweck 2014). The 7 class IId lactococcin-like precursors

all had varying degrees of similarity with one another, but all contained the lactococcin 972

conserved domain. Generally, lactococcin-like bacteriocins have a narrow spectrum of activity

57

against Lactococcus species, due to the nature of their binding to receptors (Kjos, Nes, and Diep

2009), indicating it was unlikely that these producers would be inhibitory against S. aureus.

Nonetheless, 8 of 47 lactococcin-like producers were inhibitors. Of these, 8 inhibitors, 4 also

contained a lanthipeptide cluster, whereas 1 also contained a circular bacteriocin cluster, which

could have been the bacteriocins responsible for S. aureus inhibition. Remaining inhibitors could

harbour an unidentified novel bacteriocin responsible for inhibition, or could be producing a non-

bacteriocin inhibitory substance. All of these novel bacteriocins will need further assessments for

spectrum of activity and biochemical characterization to complete identification.

When comparing phenotype and genotype, 95 NAS contained bacteriocin biosynthetic

gene clusters, whereas only 40 of the NAS had inhibition towards S. aureus. This could be due to

several factors, including bacteriocins produced were not effective against inhibiting S. aureus or

the condition tested in vitro did not lead to sufficient levels of bacteriocin production

(Nascimento et al. 2004). This highlights a substantial benefit of genome mining, as variability

of in vitro inhibition testing is negated, enabling clusters that may be silent or repressed in vitro

to be identified. Of the 40 inhibitors, 21 were identified as potential producers and the peptide

nature of the inhibitory product was verified in four of these isolates by elimination of the

inhibition the addition of with proteinase K. Further investigation into the remaining isolates

should be carried out to optimize the extraction conditions and confirm the proteinaceous nature

of the inhibitory compound. For the remaining 19 inhibiting isolates bacteriocin gene clusters

were not detected, perhaps due to inhibition from production of other inhibiting substances, e.g.

low molecular weight antibiotics, lytic enzymes, or metabolic by-products (Leroy and De Vuyst

2004). It could also be due to the nature of the detection software, as identification of clusters

using antiSMASH is based on similarity to previously described genes, with potential to miss

58

completely novel clusters. However, as knowledge increases regarding bacteriocin-associated

genes and structural precursors, detection methods will improve and more bacteriocins will be

described, allowing for even greater detection. In order to conduct the most comprehensive

analysis of bacteriocins currently available, our analysis methods were ordered and combined to

maximize detection. By first using antiSMASH and BLAST searches using the BAGEL

databases, we identified the bulk of the genomes containing bacteriocin gene clusters. We then

used any precursor genes in clusters identified by antiSMASH for further BLAST searches in our

whole genomes, which led to identification of additional lasso peptide clusters not detected by

antiSMASH. Lastly, using our phenotypic results and comparing genomes of inhibitors to

closely related non-inhibitors in the same species and using BLAST searches to analyze unique

sequences in the inhibitor led to identification of bacteriocin-associated genes in 6 of 19

inhibitors not initially identified as producers. However, no complete clusters were identified in

these isolates, due to the absence of peptide precursor genes in close proximity upon

visualization in Geneious.

2.6 Conclusions

In conclusion, all clusters identified, excluding the nukacin identified in S. simulans and

the epidermin variant identified in S. epidermidis, were novel bacteriocin clusters, having less

than 70% identity with previously described bacteriocins. The combination of genome mining

tools, such as antiSMASH along with BLAST searches, makes discovery of novel bacteriocins

quicker and more comprehensive than conventional approaches. The identified putative

producers should be further studied to characterize the bacteriocins described here in order to

59

elucidate structures, modes of action, and spectrum of activity. The NAS isolated from mammary

origin are a rich source of bacteriocins, with >21% being potential producers, thereby

representing a promising source for future research and potential clinical application.

60

Table 2-1 Bacteriocin gene clusters identified in bovine non-aureus staphylococci genomes and inhibitory phenotypes tested

against Staphylococcus aureus and MRSA.

Species No. isolates Group ID

In vitro inhibition No. bacteriocin gene clusters

S. aureus MRSA1 Class I

Class II Total

Lanthi-peptide

Sacti-peptide

Lasso Peptide

IIa IIb IIc IId

S. agnetis (n=13) 13 SAG - 0 0 0 0 0 0 0 0 S. arlettae (n=15) 15 SAR - 0 0 0 0 0 0 0 0 S. auricularis (n=2) 2 SAU - 0 0 0 0 0 0 0 0 S. capitis (n=22) 5 SCAP1 - 1 0 0 0 0 0 0 1

5 SCAP2 + + 1 0 0 0 0 0 0 1 1 SCAP3 - 1 1 0 0 0 0 0 2 1 SCAP4 + + 0 1 0 0 0 0 0 1 1 SCAP4 + - 1 1 0 0 0 0 0 2 7 SCAP5 + (7) + (5) 0 0 0 0 0 0 0 0 2 SCAP6 - 0 0 0 0 0 0 0 0

S. caprae (n=1) 1 SCAR - 0 0 0 0 0 0 0 0 S. chromogenes (n=82) 2 SCH1 + + 1 0 0 0 0 0 0 1

1 SCH2 + + 0 0 0 0 0 0 0 0 79 SCH3 - 0 0 0 0 0 0 0 0

S. cohnii (n=24) 2 SCO1 - 1 0 0 0 0 0 0 1 22 SCO2 - 0 0 0 0 0 0 0 0

S. devriesei (n=8) 8 SDE - 0 0 0 0 0 0 0 0 S. epidermidis (n=26) 2* SEP1 + + 1 0 0 0 0 0 0 1

24 SEP2 - 0 0 0 0 0 0 0 0 S. equorum (n=17) 2 SEQ1 - 0 0 0 0 0 0 1 1

3 SEQ2 - 0 0 0 1 0 0 0 1 1 SEQ3 - 1 0 0 0 0 0 0 1

11 SEQ4 - 0 0 0 0 0 0 0 0 S. fleurettii (n=2) 2 SFL1 - 0 0 1 0 0 0 0 1 S. gallinarum (n=21) 14 SGA1 - 0 0 0 0 0 1 0 1

2 SGA2 - 1 0 0 0 0 1 0 2 5 SGA3 - 0 0 0 0 0 0 0 0

61

S. haemolyticus (n=29) 1 SHA1 - 0 0 0 0 0 1 0 1 28 SHA2 - 0 0 0 0 0 0 0 0

S. hominis (n=11) 11 SHO - 0 0 0 0 0 0 0 0

S. hyicus (n=3) 1 SHY1 - 0 0 0 0 0 0 1 1 2 SHY2 - 0 0 0 0 0 0 0 0

S. kloosii (n=1) 1 SKL - 0 0 0 0 0 0 0 0 S. nepalensis (n=2) 2 SNE - 0 0 0 0 0 0 0 0 S. pasteuri (n=6) 1 SPA1 + + 0 0 0 0 0 0 0 0

5 SPA2 - 0 0 0 0 0 0 0 0 S. saprophyticus (n=16) 1 SSA1 - 0 0 0 1 0 0 0 1

1 SSA2 + - 0 0 0 0 0 0 0 0 14 SSA2 - 0 0 0 0 0 0 0 0

S. sciuri (n=30) 3 SSC1 - 0 0 0 0 0 0 1 1 1 SSC2 + - 1 0 0 0 0 0 0 1 1 SSC3 + - 0 0 0 0 0 0 1 1 1 SSC4 + - 0 0 1 0 0 0 0 1 1 SSC4 - 0 0 1 0 0 0 0 1 6 SSC5 + (6) - 0 0 0 0 0 0 0 0

17 SSC6 - 0 0 0 0 0 0 0 0 S. simulans (n=42) 19 SSI1 - 0 0 0 0 0 0 1 1

1 SSI2 - 1 0 0 0 0 0 1 2 1* SSI3 + - 1 0 0 0 0 0 1 2 1* SSI4 + - 0 0 0 0 0 1 1 2 3 SSI5 + + 1 0 0 0 0 0 1 2 2 SSI6 + + 0 0 0 0 0 0 1 1 1 SSI7 + - 0 0 0 0 0 0 0 0

14 SSI8 - 0 0 0 0 0 0 0 0 S. succinus (n=15) 1 SSU1 - 1 0 0 0 0 0 0 1

14 SSU2 - 0 0 0 0 0 0 0 0 S. vitulinus (n=6) 1 SVI1 - 1 0 0 0 0 0 0 1

5 SVI2 - 0 0 0 0 0 0 0 0 S. warneri (n=19) 5 SWA1 - 0 0 0 0 0 0 1 1

1 SWA2 + - 0 0 0 0 0 0 0 0 13 SWA3 - 0 0 0 0 0 0 0 0

S. xylosus (n=28) 8 SXY1 - 0 0 0 0 0 0 1 1 1* SXY2 + + 0 0 0 0 0 1 0 1 19 SXY3 - 0 0 0 0 0 0 0 0

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Grey shading: not tested; 1MRSA = methicillin-resistant Staphylococcus aureus; aAn asterisk in the No. isolates column indicates

inhibitory activity was achieved with the chloroform-extracted product and inhibition was suppressed with the addition of proteinase

K.

63

Figure 2-1 Distribution of bacteriocin biosynthetic gene clusters in species of non-aureus

staphylococci isolated from milk of Canadian dairy cows displayed on the phylogenetic tree

from Naushad et al (2017).

Bacteriocin types are indicated according to the following; L, lanthipeptide; S, sactipeptide; Ls,

lasso peptide; II, Class II double glycine leader peptides; C, circular bacteriocins; Lt,

lactococcin-like.

Macrococcus caseolyticus ATCC 13548 Macrococcus caseolyticus JCSC5402

Staphylococcus sciuri (n = 29)

Staphylococcus fleuretti (n = 2) Staphylococcus vitulinus (n = 6)

Staphylococcus chromogenes (n = 83)

Staphylococcus hyicus (n = 3)

Staphylococcus agnetis (n = 13)

Staphylococcus simulans (n = 42)

Staphylococcus aureus (n = 9)

Staphylococcus hominis (n = 11)

Staphylococcus devriesei (n = 8)

Staphylococcus haemolyticus (n= 29)

Staphylococcus pasteuri (n = 6)

Staphylococcus warneri (n = 19)

Staphylococcus epidermidis (n = 26)

Staphylococcus caprae SCR4023 4023

Staphylococcus capitis (n = 22)

Staphylococcus auricularis (n = 2) Staphylococcus kloosii SKL4696 4696

Staphylococcus arlettae (n = 15)

Staphylococcus gallinarum (n = 21)

Staphylococcus succinus (n = 15)

Staphylococcus nepalensis (n = 2)

Staphylococcus cohnii (n = 24)

Staphylococcus equorum (n = 17)

Staphylococcus saprophyticus (n = 16)

Staphylococcus xylosus (n = 28)

100

100

100

10099

100

100

100

10030

100

100

100

100

100

100

100

100

100

100

100

100

100

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86

82

100

100

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100

75

100

99

100

100

100

100

100

100

100

0.5

L

L

L

L

L

L

L

L

L

Ls

Ls

S

Lt

Lt

Lt

Lt

II

II

C

C

C

C

64

A.

B.

Figure 2-2 Biosynthetic gene clusters and LanA alignments of type A lanthipeptides

identified in non-aureus staphylococci isolated from milk of Canadian dairy cows.

(A) Biosynthetic gene clusters of type 1 lanthipeptides with inhibiting non-aureus staphylococci

isolates in bold and identical structural precursor genes in different cluster organizations

10 20 30 40 50GalliderminEpiderminPep5EpilancinNisinEpicidinS._capitisS._epidermidis_1778/3868S._equorum_1644

ME A V K E K N E L F D L D V K V N A K E S N D S G A E P R I A S K F L C T P G C A K T G S F N S Y C C - - - - - - - -ME A V K E K N D L F N L D V K V N A K E S N D S G A E P R I A S K F I C T P G C A K T G S F N S Y C C - - - - - - - -MK N N K N L F D L E I K K E T S Q N T D E L E P Q T A G P A I R A S V K Q C Q K T L K A T R L F T V S C K G K N G C KMN N S L F D L N L N K G V E T Q K S D L S P Q S A S V L K T S I K V S K K Y C K G V T L T C G C N I T G G K - - - - -MS T K D F N L D L V S V S K K D S G A S P R I T S I S L C T P G C K T G A L MG C NMK T A T C H C S I H V S K - - -ME N K K D L F D L E I K K D NME N N N E L E A Q S L G P A I K A T R Q V C P K A T R F V T V S C K K S D C Q - - - -MK V V K E K K E L F D L D V K V N A R DMN N S E S G P P N T S L I WC T D G C A K R - - - - - - - - - - - - - - - -ME A V K E K N D L F N L D V K V N A K E S N D S G A E P R V A S K F L C T P G C A K T G S F N S Y C C - - - - - - - -MK S V D T E S L F D L E I K K D V Q K R D G E L E A Q S L G P A I R A S V K Q C K K T G R L I T I G C G E E N K - - -

Conservation

Quality

Consensus

* 4 6 3 3 4 3 2 4 8 3 2 2 2 3 6 3 2 6 3 2 3 2 4 4 4 5 3 4 3 5 4 3 4 2 2 4 3 1 5 5 5 4 5 1 0 0 0 0 0 1 0 - - - - - - - -

ME + V K E K N D L F + L D V K V N A K E S N + S G A E P + + A S K F L C T P G C A K T G + F N S Y C C G + + + K G C K

65

indicated by *. (B) Multiple sequence alignments of LanA genes identified in type 1

lanthipeptide clusters and known bacteriocins nisin (accession number P13068), gallidermin

(accession number P21838), epidermin (accession number P08136), epilancin K7 (accession

number Q57312), pep5 (accession number P19578), and epicidin 280 (accession number

O54220).

66

Figure 2-3 Phylogenetic tree of Staphylococcus epidermidis isolates from bovine milk

indicating growth inhibition against Staphylococcus aureus and genomically identified

bacteriocin clusters.

L L

67

The percentage of trees in which the associated isolates clustered together is shown next to the

branches, with branch lengths measured in numbers of substitutions per site. Phenotypically

inhibiting isolates are surrounded with a box. Bacteriocin gene clusters are identified by the

following; L, lanthipeptide.

68

A.

B.

Figure 2-4 Biosynthetic gene clusters and LanA alignments of type B lanthipeptides with a

single LanM identified in non-aureus staphylococci isolated from milk of Canadian dairy

cows.

10 20 30 40 50 60 70 80 90 100 110CinnamycinCylL-LCylL-SMersacidinNukacin_ISK-1S._chromogenes_803/1348AS._chromogenes_803/1348BS._cohnii_1067S._cohnii_5S._simulans_1336AS._simulans_1336BS._simulans_1390/1346/1897S._simulans_3061S._vitulinus_730AS._vitulinus_730B

- - - - - - - - - - - - - - - - - - - - - - - - - - - MT A S I L Q Q S V V D A D F R A A L L E N P A A F - - - - G A S A A A L P T P V E A Q D Q - - A S L D F WT K D I A A T E A F A C R Q S C S - - F G P F T F V C D G N T K- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ME N L S V V P S F E E L S V E E ME A I Q G - - S G D V Q A E T T P V C A V A - - - - - - A T A A A S S A A C GWV G G G I F T G V T V V V S L K H C - - - - -- - - - - - - - - - - - - - - - - - - - ML N K E N Q E N Y Y S N K L E L V G P S F E E L S L E E ME A I Q G - - S G D V Q A E T T P A C - - - - - - - - - - - - - - - - - - - - - F T I G L G V G - - A L F S A K F C - - - - -- - - - - - - - - - - - - - MS Q E A I I R S WK D P F S R E N S T Q N P A G N P F S E L K E A QMD K L V G - - A G DME A A C T F T L P G G - - - - - - - - - - - - - - - - - - - - G G - - - - - - V C T L T S E C I C - - -- - - - - - - - - - - - - - - - - - - - - - - - - - ME N S K VMK D I E V A N L L E E V Q E D E L N E V L G - - - A K K K S G V I P T V S H D - - - C HMN S - - - - - - - - - - - - - - - - - - - - F Q F V F T C C S - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - MS E K A F T E E I V G K S L H S L T Q E E MH S F Y G D F D D N A E V R A S P V A S A V V K E T V K Q G A K A S S A K C A G V I S L S G I G G - A A S S A N D C L G - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - MK F N V E E I V G K S I Q T L S E E D L K D F Y N - Q N D D V E V R A T P L T T A P - - - - - - - - - - - - - - - - V S F L A S Y - - - - - L A S E T I D C G S D K -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MD E I L K L Q K L N Q A N H T S E DMN S L I D Y R T G R I F T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - WS S L S N H C - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ME D V L E L Q K L A S D E - - - - - - - - A D V S E K G Y T P T T V T T - - - V G - - - - - - - - - - - - - - - - - - - - - - - L S T I S N G C - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - MF N K E L K E V V P S F D S L T T E E ME A L I G - - E G D I Q A E T T P A C A A A - - - - - - - - - A A S S G A C A A G A G G F I V G A T A I F S V K Q C - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - MF N K E L K E V V P S F D S L T T E E ME A L I G - - E G D I Q A E T T P V C A A A - - - - - - - - - A A S S G T C A S F I T G A A S G - - V A F S I K E C - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - ME N S K VMK D I E V A N L L E E V Q E D E L N E V L G - - - A K K K S G V I P T V S H D - - - C HMN S - - - - - - - - - - - - - - - - - - - - F Q F V F T C C S - - - -

MK Q K I Q L L F S P A I I F L L I Y V I S F T S S Y I T K S Y F I F ML F V Y I F L T I F I H E L G H Y I L - - A R I K K G I L I KM I V G P - - - F Q F S R D A Y I S S N K E WA Y V G G L T - - - I I Y F L N D E K I MY L- - - - - - - - - - - - - - - - - - - - - - - - - - - - - ML N E T N N L V G L S F T E L N Q E E MD F I S G - A E G T V E P Q A T P T I A S P - - - F T P Y A V E I S I G A T V S A V S G - - - - - - L V S Y T K D C L - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MK N E N I I G K S F ME L E Q E E MD F I S G - A E G T V E P Q A T P T I S S A - - - V C V R V S L G V S A A G A S F V V S Y - - - - - V A S A T S D C D - - - -

Conservation

Quality

Consensus

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 0 0 0 0 0 2 2 2 2 1 2 6 1 3 3 1 3 2 7 0 0 0 1 0 0 - - 0 3 2 3 2 2 0 0 0 0 1 0 1 0 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - 5 5 3 2 4 2 1 5 - - - - -

MK Q K I Q L L F S P A I I + + + + + + I + + + + + ME NMK N + + L + E V G P S F E E L T E E E ME A + I G + A E G D V E A E + T P T C A A A + K E + HMN S A A A S S A A C A S F + G G Y G + G + T V A + S T K D C + G + + +

69

(A) Biosynthetic gene clusters of type B lanthipeptides with inhibiting NAS isolates in bold and

identical precursors in various cluster organizations within the same species indicated by *. (B)

Multiple sequence alignments of LanA genes identified in type B lanthipeptide clusters and

known bacteriocins cinnamycin (accession number P29827), cytolysin-L (accession number

H7C7B0), cytolysin-S (accession number H7C7B5), mersacidin (accession number P43683), and

nukacin ISK-1 (accession number Q9KWM4).

70

A.

B-1.

B-2.

Figure 2-5 Biosynthetic gene clusters and LanA alignments of type B lanthipeptides with

dual lanM enzymes identified in non-aureus staphylococci isolated from milk of Canadian

dairy cows.

10 20 30 40 50 60 70 80Lacticin_3174A1Lichenicidin_A1Staphylococci_C55A1*S._gallinarum_2094/1388A1S._sciuri_225A1S._succinus_6028A1

- - - - - - - - - - - - - MN K N E I E T Q - - - - P V T WL E E V S D Q N F - - - - - - - - - - - D E D V F G A C S T N T F S L S D YWG N N G AWC T L T H E C MAWC KMS K K E M I L S WK N P MY R T E S S Y H - - - P A G N I L K E L Q E E E Q H S I A G - - - - - - G T I T L S T C A I - - - - L S K P L G N N G Y L C T V T K E C MP S C N- - - - - - - - - MK S S F L E K D I E E Q - - - - - V T WF E E V S E Q E F - - - - - - - - - - - D D D I F G A C S T N T F S L S D YWG N K G NWC T A T H E C MS WC K- MR K N L E - - - R N P Y L R N E N Q E S V D L P L D N P I R E L K E D E L L K L N G A K S R F K P G A E I A V S T I G C Y A G S V Y L G N D G F MC T T T V E C Q N Q C K- - - - - - - - - - - - - MN K Q E I E L F E - - S A G N F A K E L E N G S L E N I F G G D S - - - Q P R E F T S D N D G K Y - K S I T W - - E C S I C P - T H T C F G - C -- - - MS D F F K Q R K N L T S N T A E K L - - - - D E A I L E E V E D Q S S MG G F - - - - - - - N T WN L T P T S T V G I A A S V G L G N K G K V C T Y T V E C V N N C S

Conservation

Quality

Consensus

- - - - - - - - - - - - - 7 5 4 6 6 5 6 2 2 - - - - 0 3 7 4 6 7 * 9 4 7 4 6 4 - - - - - - - - - - - 3 3 1 4 8 7 5 5 7 4 1 1 1 - 4 * 3 4 7 3 2 5 9 3 6 * 7 1 * 4 6 * 5 6 1 * 1

M+ + K + + + + + + + N P M+ + N E I E E Q + D L P A + N + L E E + + E Q E + + + I F G + + S R F K D T D E F + A C S T + T + + L S + Y + G N + G + WC T + T H E C MNWC K

10 20 30 40 50 60 70 80Lacticin_3174A2Lichenicidin_A2Staphylococci_C55A2S._gallinarum_2094/1388A2S._sciuri_225A2S._succinus_6028A2

- - MK E K NMK K - N D T I E L Q L G K Y L E D DM I E L A E G D E S H G G T T - P - - - - - - - A T P A I S I L S A Y - - - I S T N T C P T T K C T - - - R A C - - -- - MK N S A A R E A F K G A N H P A GMV S E E E L K A L V G G N D V N P E T T - P - - - A T T S S WT C I T A G V T - - - - V S A S L C P T T K C T - - - S R C - - -Q S DMK N E L G K F L E E N E L E L G K F S E S DML E I T D - D E V Y A A G T - P - - - - L A L L G G A A T G V I G Y - - - I S N Q T C P T T A C T - - - R A C - - -- - MK K E D Y K G - - - - - - - - I G L V D E K E L K KMA G A G E V T P R T T I P C G A A V I T A G G V I G G G V K Y T L Q D S A N S C P T G Q C T - - - S K - - - -- - MT N - - - - - - - - - - - - - I Q K V T I K E L E N I S K G S A S S P R V T - P - - - - T T T V V P A S L A V - - - - - - - - - - - C P T T K C A S V V K A C P G K- - MS K D Q E K Q L N E I - - - - T G L I S E D E L E E S L L - E D T F G G T H - P - - - T L S Y I G G A L G G V S A V T A V T A Q S P C P T S A C S - - - K S C - - -

Conservation

Quality

Consensus

- - 5 4 5 1 0 0 0 0 - - - - - - - - 6 6 6 6 3 5 4 9 9 4 3 5 5 2 - 4 4 6 3 7 3 7 6 - * - - - - 1 0 0 5 5 6 8 5 5 7 7 0 1 - - - - 0 2 0 1 0 * * * 7 4 * 7 - - - 5 4 2 - - -

Q S MK K + + + K K + N E + + E L + + G K V S E + E L + E + A G G D E V + P + T T I P C G A A + T T A G G A I + G V + A Y T + + I S A + T C P T T K C T S V V + A C P G K

71

(A) Biosynthetic gene clusters of identified dual precursor type B lanthipeptides with inhibiting

NAS isolates in bold. (B-1) Multiple sequence alignments of LanA1 genes identified in type B

lanthipeptide clusters and known bacteriocins lacticin 3147 A1 (accession number O87236),

lichenicidin A1 (accession number P86475), and staphylococcin C55 A1 (accession number

Q9S4D3). (B-2) Multiple sequence alignments of LanA2 genes identified in type 2 lanthipeptide

clusters and known bacteriocins lacticin 3147 A2 (accession number O87237), lichenicidin A2

(accession number P86476), and staphylococcin C55 A2 (accession number Q9S4D2).

72

A.

B.

Figure 2-6 Biosynthetic gene clusters and alignments of precursor peptides from

sactipeptides identified in non-aureus staphylococci isolated from milk of Canadian dairy

cows.

(A) Biosynthetic gene clusters of identified sactipeptides with inhibiting non-aureus

staphylococci isolates indicated using bold and identical precursors indicated by *. (B) Sequence

alignment of the identified sactipeptide precursor and known sactipeptide subtilosin A (accession

number O07623).

10 20 30 40S._capitisSubtilosin_A

ME Q G VMV S N K G C S A C A V G A A C L A D G P I P D F E V A G I T G T F GMA SMK K A V I V E N K G C A T C S I G A A C L V D G P I P D F E I A G A T G L F G L WG

Conservation

Quality

Consensus

* 7 7 + * 9 * 6 * * * * 8 8 * 8 9 * * * * * 8 * * * * * * * * 9 * * 7 * * 7 * * 9 6 8

M+ + + V + V + N K G C + + C + + G A A C L + D G P I P D F E + A G + T G + F G + + +

73

A.

B.

Figure 2-7 Biosynthetic gene clusters and alignments of precursor peptides from the lasso

peptide identified in non-aureus staphylococci isolated from milk of Canadian dairy cows.

(A) Biosynthetic gene cluster of the identified lasso peptide. Identical precursors indicated with

an * (B) Sequence alignments of the identified lasso peptide precursor and known lasso peptides

lariatin (accession number H7C8I3) and microcin J25 (accession number Q9X2V7).

10 20 30 40 50LariatinMicrocin_J25S._fleuretti/S._sciuri

- - - - - - - - - - MT S Q P S K K T Y N A P S L V Q R G K F A R - - T T A G S Q L V Y R E WV G H S N V I K P G PM I K H F H F N K L S S G K K N N V P S P A K G V I Q I K K S A S Q L T K G G A G H V P E Y F V G I G T P I S F Y G- - - - - - - - - - - - - - - MK K T Y T S P S L V L L G N A K D L I K G S H S S R I P E N A V G I S - - - F Y K G

Conservation

Quality

Consensus

- - - - - - - - - - - - - - - 6 6 5 7 6 7 8 4 8 9 9 7 5 5 6 5 5 5 - - 7 5 8 4 8 6 4 9 5 8 5 6 * * 5 8 - - - 3 5 4 7

M I K H F H F N K L + + + + + + K K T Y + A P S L V Q + G K + A + + + T + + G S + + V P E + + V G I S + + I + + + G

74

Figure 2-8 Biosynthetic gene clusters of Class II double glycine leader peptide bacteriocins

identified in non-aureus staphylococci isolated from milk of Canadian dairy cows.

Identical precursors are identified by *.

75

A.

B.

Figure 2-9 Biosynthetic gene clusters and precursor alignments of Class IIc circular

bacteriocins identified in non-aureus staphylococci isolated from milk of Canadian dairy

cows.

10 20 30 40 50 60 70 80 90 100 110Carnocyclin_ACircularin_AEnterocin_AS-48GassericinUberolysinS._gallinarumS._haemolyticus_109S._simulans_1355

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - ML Y E L V A - - - - - - - Y G I - - A Q G T A E K V V S L I N A G L T V G S I I S I L G G V - - - - - T V G L S G V F T A V K A - - - - - - - - - - - A I A K Q G I K K A I Q L- - - - - - - - - - - - - - - - - - - - - - - - - - - MS L L A L V A G T - - - - - - L G V - - S Q S I A T T V V S I V L T G - - - S T L I S I I L G I T - - - - A I L S G G V D A I L E I GWS A F V A T V K K I V A E R G K A A A I AW- - - - MV K E N K F S K I F I L MA L S F L G L A L F S A S L Q F L P I A HMA K E F G I - - P A A V A G T V L N V V E A G GWV T T I V S I L T - - - - - - - A V G S G G L S L L A A A G R E S I K A Y L K K E I K K K G K R A V I AW

MV T K Y G R N L G L N K V E L F A I - - - WA V L V V A L L L T T A N I YW I A D Q F G I H L A T G T A R K L L D AMA S G A S L G T A F A A I L G V T L P AWA L A A A G A L G A T A A - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - MD I L L E L A G Y - - - - - - T G I - - A S G T A K K V V D A I D K G A A A F V I I S I I S - - - - - - - T V I S A G A L G A V S A S A D F I I L T V K N Y I S R N L K A Q A V I W

MF L R L K N S K E E I K I AMF I T I S S L A V L L I G P L F N A A A A - - - - - - MG L - - N A A T A T S L Y Y A L N F V GWG A T V A S I V A S F - - - - - G L A S I G A Y T I WA A - - - - - - - - - - - - V K R MA L R S F I KW- - - - MN A T K T I N S I I F L T A L V AMS S I A F V L F S T V S P N - - L V S ML S I - - D S G T A L V I V N R V L D T V N L A T I V A S V G T L T - - - - G A G A I G V G L L Q T A KW - - - - - - - - - L A L K F G K K R A Q AW- - - - - MS K V Q D S N V S F L L T L S A S L L V V T V L MF T L P N A P F I A Q Q L G L - - S T A A S L G L A Q A L K T V G N V A T A L T I I G T F T - - - - G V G T I G S S L A A V I - - - - - - - - - L Q K V K K E G A K K A A A F

Conservation

Quality

Consensus

- - - - - - - - - - - - - - - - - - - - - - - - - - - 1 1 5 4 4 4 5 4 6 0 - - - - - - 6 8 9 - - 4 6 8 6 8 2 3 9 5 4 3 8 2 2 7 1 0 1 5 6 7 6 7 5 9 5 - - - - - - - 8 7 7 5 5 * 5 2 6 7 4 2 7 - - - - - - - - - - - - 1 0 2 0 1 1 0 0 1 0 0 1

M+ + + M+ + + K + + + K I + F L + + L S A L A + L + + + L L L T L A + + + + I A + Q L G I H L A + G T A + K V V + A V + + G G + V A T I I S I I G G + T L P AW+ V G S I G + + L A + A A GW+ + I + A T V K K + + K K + G K K + A I AW

76

(A) Biosynthetic gene clusters of identified Class IIc bacteriocins with inhibiting NAS isolates

indicated in bold and identical precursors in S. gallinarum are indicated by *. (B) Alignment of

precursor peptides from identified class IIc bacteriocins and known bacteriocins carnocyclin A

(accession number B2MVM5), and circularin A (accession number BAC164), enterocin AS-48

(accession number Q47765), gassericin (accession number O24790), and uberolysin (accession

number A5H1G9).

77

A.

B.

Figure 2-10 Biosynthetic gene clusters and precursor alignments of Class IId lactococcin-

like bacteriocins identified in non-aureus staphylococci isolated from milk of Canadian

dairy cows.

(A) Biosynthetic gene clusters of identified Class IId bacteriocins with inhibiting NAS isolates

indicated in bold and identical precursors in S. equorum indicated by A and identical precursors

in S. sciuri indicated by *. (B) Alignments of precursor peptides from identified Class II

10 20 30 40 50 60 70Lactococcin_972Lactococcin_AS._equorumS._hyicusS._scuriS._simulans_AS._simulans_BS._warneriS._xylosus

MK T K S L V L A L S A V T L F S A G G I V - - - - - - A Q A E G T WQ H G Y G V S S A Y S N Y H H G S K T H S A T V V N N L T E K A S F Y Y N F W -MK N Q L N F N I V S D E E L S E A N G G K L T F I Q S T A A G D L Y Y N T N T H K Y V Y Q Q T Q N A F G A A A N T I V N GWMG G A A G G F G L H HMK K T I I S V L L Y L A I V L G S G T I A N A A T I - Y A Q G G I WN Y G V G S K Y VWS Y Y S N N Y K A H G S T A V G K H S S - - - - - - - - - -MK K K F MT C C I A G T I L L G I A H T A D A T T V - D V G G G KWS H G V G S K Y VWS H Y S H N S R N H G A T A I G K - WT G N K A Y Y S L H -MK K F F I T L L L T I I I A S G A G I V K A Y Q V - - N V D G G T WN Y G V S N K Y VWS N Y Y H G K K A H Y T T V Q G R I WR V N K S Y Y G F Y -MK K Y V A R T I I I A T L L L GMG T T T I A N A Y E WA E G G KWS H G I G S T Y VWS Y Y T H N S Y G H D S T A I G KWWG - N Q A Y Y R V Y -MK K Y V A R T I I I A T L L L GMG T T T I A N A Y E WA E G G KWS H G I G S T Y VWS Y Y T H N S Y G H D S T A I G K L G G V T K H I I E Y I RMK N K V L G L S L Y L S L A L G I S T V T Y A T A Y E Y A H G G T W I H G V G S K Y VWS Y Y Y H S S K G H G S T A I G K F WG I N K A Y Y N V Y -MK K T I I S I L F T G I V V F G S G T V A K A V T I - Y A E G G L WN Y G V G S S Y VWS Y Y N H N S K A H G S T A I G K YWD - N Q T Y Y K V Y -

Conservation

Quality

Consensus

* * 6 4 4 5 2 6 5 7 4 3 3 4 7 4 4 5 7 3 6 4 0 1 0 1 0 - 3 5 2 4 5 5 + 4 5 8 5 7 2 5 6 8 + 8 5 8 4 5 7 3 3 7 4 3 7 * 7 6 7 4 1 5 3 - 1 0 0 1 1 0 1 1 -

MK K K V I + L + L + A T + L L G A G T + + I A + + Y E Y A E G G + W+ H G V G S K Y VWS Y Y + H N S K A H G S T A I G K + WG + N K A Y Y + V Y +

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bacteriocins and known bacteriocins Lactococcin 972 (accession number O86283) and Lactoccin

A (accession number P0A313).

79

Chapter Three: Identifying putative bacteriocin resistance genes in Staphylococcus aureus whole genomes

3.1 Abstract

Mastitis is the most costly disease in dairy cattle and is the largest contributor to antibiotic

use on farm. Staphylococcus aureus is the most detrimental mastitis causing pathogens. In this

study, we determined the susceptibility of 139 bovine S. aureus isolates to a S. chromogenes

isolate that was previously identified to inhibit the growth of S. aureus and to encode a

lanthipeptide bacteriocin. We also determined the presence of bacteriocin gene clusters in the S.

aureus whole genomes to examine the association with susceptibility to the growth inhibition

caused by the S. chromogenes isolate. At the pan genome level, we also identified genes

potentially related to susceptibility and resistance by comparing the presence of putative

immunity related genes or genes encoding hypothetical proteins between susceptible and

resistant S. aureus phenotypes. The distributions of additional putative bacteriocin resistance

genes, which were obtained through the NCBI database and through examining accessory

genomes of resistant isolates, were also determined for each susceptibility group. Forty-nine

(35%) S. aureus isolates were susceptible to the S. chromogenes, whereas 90 (65%) isolates were

resistant in the in vitro inhibition assay. Seventy-four putative bacteriocin gene clusters in 73 S.

aureus whole genomes were identified. The presence of having a bacteriocin cluster was

associated with an isolate being susceptible to the S. chromogenes. The pan genome of S. aureus

isolates contained 6559 genes. Genes that were present in over 90% of all isolates, as well as

genes contained in less than 40% of isolates, were disregarded. Thence, genes were selected for

further analysis on the basis of them either encoding for hypothetical proteins or for proteins that

80

are known to be potentially involved in bacteriocin resistance. This resulted in 273 genes to be

assessed for the distribution between the susceptible and resistant groups. Seventy-seven genes

had a positive association with an isolate being resistant to the S. chromogenes, while 76 genes

were associated with the S. aureus isolate being susceptible. In conclusion, susceptibility seems

to be linked to a large number of genes, the majority of which are annotated as hypothetical

proteins and will need further assessment to determine their role in S. aureus susceptibility.

Key-words: Staphylococcus aureus, non-aureus staphylococci, coagulase-negative

staphylococci, bacteriocin

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3.2 Introduction

Staphylococcus aureus is most harmful mastitis pathogen on dairy farms, causing clinical

mastitis (CM) cases, increased somatic cell count (SCC), and large economic losses due to

decreased milk yield and increased culling. Antibiotic therapy is frequently inadequate against

chronic intramammary infections (IMI) caused by S. aureus and with increased consumer

interest and stricter antibiotic use policies on the horizon due to the mounting antibiotic

resistance there is a need to investigate alternatives to antibiotic use for the treatment of bacterial

infections on farm. Conceivable alternatives are bacteriocins, which have been shown to inhibit

mastitis pathogens in vitro (Braem et al. 2014; Brito, Somkuti, and Renye 2011; Ceotto‐Vigoder

et al. 2016; Chaimanee et al. 2009; Coelho et al. 2007; Kim et al. 2010; De Vliegher et al. 2004;

Leon-Galvan et al. 2015; Nascimento et al. 2005) and have been investigated for use in a teat dip

(Klostermann et al. 2010) and teat sealants (Crispie et al. 2004).

Bacteriocins are ribosomally synthesized and post-translationally modified peptides

(RiPPs) that generally exert bacteriocidal action across a small spectrum of closely related

bacteria (Cotter, Ross, and Hill 2013). Bacteriocins from Gram-positive bacteria are classified

into two main groups, the lanthipeptides, containing post translationally modified peptides, and

class II bacteriocins, containing the circular and the non-modified peptides (Cotter, Ross, and

Hill 2013). Bacteriocins have been garnering attention as potential alternatives to antibiotics due

to their ability to inhibit the growth of drug resistant bacteria (Nascimento et al. 2006;

Kruszewska et al. 2004) and to not cause significant effects on host natural microbiota (Rea et al.

2010). Bacteriocin producers encode specific proteins in the bacteriocin biosynthetic gene cluster

to provide self-protection. In lanthipeptides, immunity is conveyed with proteins designated as

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LanI, or the LanFE(G) immunity clusters. LanI is thought to function by interception, target

shielding (Stein et al. 2005) or by sequestering the bacteriocin on the bacterial cell wall

membrane, as is the case with NisI from nisin (Stein et al. 2003). In contrast, the lanFEG cluster

works by removing the peptide via a dedicated ABC transporter (Stein et al. 2003). Immunity for

class IIa and IIc bacteriocins is obtained when the immunity protein binds directly to the

bacteriocin receptor, inhibiting pore formation (Diep, D 2007). A variety of other families of

genes have been identified as potential immunity proteins, although with no known mechanisms

such as Abi genes, which encode CAAX metalloproteases (Kjos et al. 2010).

Cross immunity between closely related lanthipeptides was initially thought to be rare

(Draper et al. 2009); the nisin immunity proteins, NisI and NisFEG, conferred no protection to

subtilin, a closely related lanthipeptide (Stein et al. 2003), and vice versa (Stein et al. 2005), but

cross-immunity has been reported (Draper et al. 2009). Immunity to the two-peptide

lanthipeptide lacticin 3147 has been demonstrated in isolates that encode the closely related

bacteriocin staphylococcin C55, as well as in pathogens that contain functional immunity

homologues (Draper et al. 2009). The fact that pathogens can contain orphan immunity

homologues has implications for clinical use, as a natural reservoir of resistant organisms

containing immunity homologues could be selected for under conditions of antimicrobial

pressure and render the bacteriocin therapeutic unusable. There is also the potential to transfer

these genes to other organisms, including mastitis pathogens. Although there are many suspected

immunity genes, an investigation into the occurrence of these genes in a large group of S. aureus

isolates has not been explored to date. This information could potentially be extrapolated to

predict the usefulness of a specific bacteriocin as a preventative or therapeutic against S. aureus

infections.

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The first objective of this study was to determine the susceptibility of 139 bovine S.

aureus isolates against a previously identified lanthipeptide producing bovine S. chromogenes in

vitro (Carson et al. 2017). The second objective was to identify any bacteriocin biosynthetic gene

clusters using genome-mining software in the corresponding 139 S. aureus whole genomes in an

attempt to explain the resistance phenotype observed in vitro. The third objective was to identify

putative bacteriocin resistance genes that are associated with resistant isolates by determining the

distribution of genes of interest from the pan genome in both the susceptible and resistant groups

and examining the associations.

3.3 Materials and methods

3.3.1 Isolates

Staphylococcus aureus and NAS isolates were collected in the National Cohort of Dairy

Farms (NCDF) study conducted across Canada during 2007 and 2008, as described by Reyher et

al. (2011). The Canadian Bovine Mastitis and Milk Quality Research Network (CBMQRN) at

the University of Montreal stored the samples before sending them to the University of Calgary.

A total of 146 S. aureus isolates were selected. The isolates originated from 125 lactating cows

from 52 herds across Canada from Nova Scotia, Prince Edward Island, New Brunswick

(representing Atlantic Canada), Québec and Ontario (representing Central Canada), and Alberta

(representing Western Canada) (Reyher et al. 2011). Two random selections for isolates were

made, one for the clinical cases (51 out of 387 isolates) and one for IMI (95 out of 2915 isolates).

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An S. chromogenes isolate, which was previously identified to inhibit S. aureus in vitro

and encode a lanthipeptide bacteriocin gene cluster (Carson et al. 2017) was tested for inhibition

against 139 S. aureus isolates. Testing was done using a modified cross-streaking method (De

Vliegher et al. 2004). The S. chromogenes isolate was plated on 5% defimbrinated sheep blood

agar plates (BD Diagnostics, Mississauga, ON, Canada) and incubated overnight at 37°C. A

single colony was diluted in PBS to a McFarland 0.5 standard and was used to inoculate a centre

streak (5 mm) on a 5% sheep blood agar plate and subsequently incubated at 37°C for 24 h. On

day 2, the agar was loosened from the plate with sterile metal tweezers and flipped onto the lid of

the plate so the NAS centre streak was face down. 100µL of a 10-3 dilution in PBS of a

McFarland 0.5 standard of a single colony from an overnight culture on 5% sheep blood agar of

each S. aureus isolate was spread over the entire agar surface and incubated at 37° for 24 h. On

day 3, plates were examined for bacterial growth and any inhibition (total or partial) of the

pathogen growth was recorded. All experiments included a negative control whereby only PBS

was used to make the center streak on day 1.

3.3.3 Whole genome sequencing, assembly, and annotation

Sequencing, assembly, and annotation for NAS and S. aureus isolates were performed as

described previously (Naushad et al. 2016). Briefly, genomic DNA was extracted with DNeasy

Blood and Tissue Kit (Qiagen, Toronto, ON, Canada), according to the corresponding protocol

for Gram-positive bacteria. Sequencing of these samples was performed using the Illumina

MiSeq platform (Illumina, San Diego, CA, USA); DNA libraries for sequencing were prepared

using a Nextera XT DNA library preparation kit (Illumina, San Diego, CA, US). All sequencing

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steps, including cluster generation, paired-end sequencing (2 × 250 bp), and primary data

analysis for quality control, were performed on the instrument. Genome assembly was automated

using the Snakemake workflow engine (Köster and Rahmann 2012). Raw read pairs were

screened for adapters and quality trimmed using cutadapt 1.8.3 (Martin 2011) as implemented in

Trim Galore! 0.4.0 with default parameters. Genomes were assembled using Spades 3.6.0 (Nurk

et al. 2013) using the built-in error correction and default parameters. To assess coverage, reads

were mapped back to the assembled genome using BWA 0.7.12-r1039 (Li and Durbin 2009).

Contigs larger than 200 bp were annotated with Prokka 1.11 (Seemann 2014) using the provided

Staphylococcus database. Assembly quality was evaluated with Quast 3.0 (Gurevich et al. 2013).

Contigs, as well as the annotated protein sequences, were used for custom blast searches using

SequenceServer (Priyam et al. 2015). The data for NAS were previously submitted to NCBI

under BioProject ID PRJNA342349. The process to submit S. aureus data has been initiated.

A phylogenetic tree for the S. aureus was constructed using methods as described

previously (Naushad et al. 2016). Briefly, a genome-based phylogenetic tree of 136 S. aureus

isolates was constructed using the published pipeline PhyloPhlAn (Segata et al. 2013). The

PhyloPhlAn approach is based on the use of 400 ubiquitous and phylogenetically informative

proteins. Orthologs of these proteins in S. aureus genomes were detected using USEARCH

v5.2.32 (Edgar 2010). Multiple sequence alignments of these proteins were generated using

MUSCLE v3.8.31 (Edgar 2004) and a Maximum-Likelihood tree was constructed and formatted

using MEGA 6.0 (Tamura et al. 2013).

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Identification of biosynthetic gene clusters related to secondary metabolite production and

analysis of sequences of interest were done using antiSMASH 3 (Weber et al. 2015). Putative

gene clusters were classified according to Cotter et al. (Cotter, Ross, and Hill 2013).

3.3.5 Screening of genomes for immunity related genes

3.3.5.1 Pan genome

The pan genome of all S. aureus isolates was computed using BPGA (Bacterial Pan

Genome Analysis tool) (Chaudhari et al., 2016). Genes were discarded that were present in over

90% of isolates, as they were likely a part of the core genome and would not have enough

differences between the resistant and resistant groups for differentiation. Genes were also

discarded that were present in less than 40% of genomes, as there likely would not be enough

representation in each group to determine differences. Genes were then further selected for

analysis based on annotation. Only genes that would reasonably be considered to be involved in

immunity, such as those encoding any ABC transporters, proteases, lipoproteins, hypothetical

proteins, bacteriocin precursors, and any genes annotated as bacteriocin immunity proteins, were

selected. The presence of these genes in susceptible and resistant groups was determined using

an in-house python BLAST script. Two x two tables were created to examine the association

between an isolate being susceptible or resistant and having a specific gene or not having that

specific gene. The significance of that association was examined using Fisher’s exact test

(Fisher’s exact test of independence). Data were analyzed using Microsoft Excel, with P < 0.05

considered statistically significant.

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3.3.5.2 BLAST

Two groups of 10 genomes, each containing 5 randomly selected susceptible and 5

resistant S. aureus isolates, were compared using Spine, a web based application that identifies

both common and accessory sequences in the input genomes (Ozer, Allen, and Hauser 2014).

Sequences identified in the accessory genome of the resistant isolate group were explored for

genes potentially related to immunity based on their annotations. A list of genes was compiled

and the distribution of putative bacteriocin resistance genes was determined in all S. aureus

genomes in both the resistant and susceptible groups by conducting BLASTp searches against a

local S. aureus BLAST database, using command line BLAST+ 2.5.0 (Camacho et al. 2009).

Additionally, an immunity gene database based on literature was compiled to BLAST on

the local S. aureus database to determine the distribution of genes in resistant and susceptible

genomes. Data were analyzed as stated above.

3.4 Results

3.4.1 Phenotypic test

Out of 139 S. aureus isolates, 49 (35%) had growth inhibition from the S. chromogenes

(Table 1) and were deemed susceptible. The remaining 90 isolates were not susceptible and

therefore were deemed resistant.


Seventy-four putative bacteriocin gene clusters belonging to 73 isolates were identified

(Table 1). Overall, 53% of S. aureus potentially produces bacteriocins. Of the 90 resistant S.

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aureus, 36 (40%) contained putative clusters. Of the 49 susceptible S. aureus, 37 (76%)

contained putative clusters. Lanthipeptides were the main bacteriocin class identified in the

genomes (73 clusters contained lanthipeptides). A class IId lactococcin-like bacteriocin cluster

was also identified. Two distinct lanthipeptide gallidermin-like precursors were identified, which

had 79% identity with one another (Fig 3-1). The precursors, although annotated as gallidermin

precursors, had only 55 and 53% identity with gallidermin (Fig 3-1).

3.4.3 Screening of genomes for putative resistance related genes

The pan genome of the S. aureus isolates consisted of 6559 genes. 847 genes were present

in less than 90% of all isolates but greater than or equal to 40%. Out of this, 273 were selected

based on their potential for being involved in resistance, or were hypothetical proteins. Based on

the p-values of the fisher’s exact test, 156 had significant associations to be potentially related to

susceptibility (124 of which encoded for hypothetical proteins, with no known function).

Of the 156 genes with significant associations, 73 were associated with an isolate being

resistant. An association with a resistant isolate was defined as having a significant p-value from

the fisher’s exact test and by the gene being present in over 50% of the resistant isolates and in

less than 50% of the susceptible isolates. The resulting list consisted of genes encoding three

ABC superfamily ATP binding cassette transporters, a cytosol aminopeptidase, 61 hypothetical

proteins, a lipoprotein, three oligopeptide ABC superfamily ATP binding cassette transporters, a

putative ABC-type transporter, a putative staphylococcal lipoprotein, a putative staphylococcal

tandem lipoprotein, and a S1B family serine protease SplE (Table 3-2).

Seventy-three genes with significant associations were present in greater than 50% of

susceptible isolates and less than 50% of resistant isolates and were thus attributed to an isolate

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being susceptible. This list contained six ABC superfamily ATP binding cassette transporters,

an ABC-2 family transporter protein, 56 hypothetical proteins, two gallidermin precursors, a

nisin biosynthesis NisC protein, four oligopeptide ABC superfamily ATP binding cassette

transporters, a putative ABC-type transporter, a serine protease SplE_1, and a thermophilic

serine protease precursor (Table 3-3).

Putative bacteriocin resistance genes identified in the accessory genomes of resistant

isolates using the SPINE approach yielded 10 potential genes of interest, three ABC superfamily

ATP binding cassette transporters, an antimicrobial peptide ABC transporter ATPase, a CAAX

amino terminal protease self-immunity, a gallidermin precursor, a macrolide export

ATO_binding/permease protein, two oligopeptide ABC superfamily ATP binding cassette

transporter, and an P-ATPase superfamily P-type ATPase potassium (K+) transporter subunit A.

One oligopeptide ABC superfamily ATP binding cassette transporter was associated with an

isolate being resistant, with 62% of the resistant isolates encoding for that protein, while only

27% of the susceptible isolates did (Table 3-2). Additionally, two ATP transporters were

associated with isolates being non-susceptible, with 54% and 50% presence in resistant isolates,

compared to 22% for both in susceptible isolates (Table 3-2). Presence of the gallidermin

precursor was associated with an isolate being susceptible (Table 3-3).

From the BLAST searches based on literature, no additional genes were identified that

were associated with an isolate being resistant. However, two self-immunity proteins present on

the epidermin biosynthetic gene cluster, EpiE and EpiF, were associated with an isolate being

susceptible (Table 3-3).

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3.5 Discussion

In this study, 90 isolates (65%) of 139 S. aureus were not susceptible to growth inhibition

by an S. chromogenes isolate that harbours genes encoding a lanthipeptide. A total of 74

bacteriocin biosynthetic gene clusters were identified with antiSMASH and of the 90 resistant

genomes, 36 (40%) were identified to contain 37 clusters, while of the 49 (76%) susceptible

isolates contained 37 putative bacteriocin clusters. Genomes of the susceptible and resistant S.

aureus were examined and 73 genes were identified that were associated with an isolate being

resistant to the S. chromogenes.

Only two classes of bacteriocins were identified in the S. aureus genomes, a lanthipeptide

and a class IId lactococcin-like bacteriocin gene cluster. The presence of a bacteriocin gene

cluster was associated with an isolate being susceptible to the S. chromogenes bacteriocin. The

lanthipeptide clusters had two distinct precursors that were frequently identified in the same

cluster. They were annotated as gallidermin precursors, although they only showed 55 and 53%

identity with gallidermin. In the phylogenetic tree created with other lanthipeptide precursors, it

appears the identified precursors and gallidermin share a common ancestor but the S. aureus

precursors are probable novel lanthipeptides. Further characterization will need to be done to

conclude firstly, if they are viable clusters followed by determining their spectrum of activity,

structures, and modes of action. From the BLAST data looking at the distribution of potential

immunity related proteins in susceptible and resistant isolates, it indicates that the gallidermin

precursor (as it was annotated) is associated with an isolate being susceptible to the S.

chromogenes bacteriocin producing strain. One conceivable mechanism for this is that the

lanthipeptide cluster the S. aureus contains can provide cross immunity to the S. chromogenes

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bacteriocin, but because resistance is dependent on expression of immunity proteins (Eijsink et

al. 1998), the S. aureus likely was not producing bacteriocin at the time when it was streaked on

the plate, therefore the immunity proteins on the lanthipeptide operon may not have been

produced, thus leaving the S. aureus susceptible to the S. chromogenes. Although, this would

mean that by containing this gallidermin precursor, and thus a lanthipeptide operon, other

bacteriocin immunity genes are excluded, or that the operon affects other bacteriocin immunity

genes, leaving it susceptible.

One method of bacteriocin resistance is immune mimicry, where the target organism

contains an orphan immunity homologue, which normally confers self-immunity to the

bacteriocin producer (Draper et al. 2009). Therefore, investigating the target genomes for

immunity homologues would be a straightforward way to predict susceptibility to a specific

bacteriocin. Unfortunately, the bacteriocin biosynthetic gene cluster of the producing S.

chromogenes did not contain any recognizable immunity proteins or protein clusters (Carson et

al. 2017) to use as a driver sequence to BLAST in the S. aureus genomes. It is possible that the S.

chromogenes contains immunity genes elsewhere on the genome to provide self-protection,

although further investigation into that isolate would need to be conducted for confirmation.

Investigating the gene differences between susceptible and resistant isolates by creating a

pan genome and describing the distribution in each group, as well as by conducting BLAST

searches from accessory genomes of resistant isolates and from literature, we identified 76 genes

where its presence in a genome is associate with that isolate being resistant to a S. chromogenes

isolate. The majority of these genes encoded for hypothetical proteins, meaning that no known

function has been described. Therefore, there is a need for characterization of these hypothetical

proteins to determine their specific functions and how they are potentially involved in immunity

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to the S. chromogenes isolate. Fifty-six hypothetical proteins were associated with the isolate

containing them to be susceptible to the S. chromogenes isolate. Characterization and

investigation into the roles of these proteins should also be assessed to have a better

understanding of how these proteins contribute to the susceptibility phenotype observed in vitro.

Homology to known genes can be assessed, as well as presence of conserved domains to predict

functions of the hypothetical proteins. Many different strategies can be utilized simultaneously

for resistance to a bacteriocin, as proven with nisin resistance (Kramer et al. 2006), so it is no

surprise that there are so many potential proteins associated with susceptibility. Although, the

high number of hypothetical proteins that seem to be involved in susceptibility is quite large and

it seems unlikely that they are all involved in immunity. The methodology used may result in a

high level of false discovery.

The LanI immunity proteins in lanthipeptide clusters are lipoproteins, which confer the

producer self-immunity by, in the case of nisin, sequestering nisin and thus rendering it inactive

(Stein et al. 2003). Only the structures for NisI (Hacker et al. 2015) and SpaI (for subtilin)

(Christ et al. 2012) have been elucidated. Even though nisin and subtilin are sequentially and

structurally similar, the two immunity lipoproteins do not show cross-resistance for the two

bacteriocins (Hacker et al. 2015). Therefore, the lipoproteins that confer immunity to the

producer need to be quite specific for that bacteriocin, although, it is plausible that other

lipoproteins present in non-producing strains may be able to provide protection against certain

bacteriocins. We identified three lipoproteins that are associated with an isolate being resistant to

the S. chromogenes strain. The next step would be to do multiple sequence alignments with

known lipoproteins involved in immunity to assess any relatedness and also to compare the

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structures of each lipoprotein to assess for any similar regions that could be indicative of being

related to immunity.

An S1B family serine protease was found to be associated with an isolate being not

susceptible to the S. chromogenes. This family of protease is typically involved in intracellular

protein turnover (Di Cera 2009). These proteases are also found associated with bacteriocin gene

clusters for cleaving the leader peptide off of the core peptide before exporting from the cell. In

terms of bacteriocin resistance, it in feasible that the protease is able to degrade the bacteriocin

before it has a chance to interact with the cell membrane. Studies to indicate where the protease

localizes in the cell could provide some evidence of potential interactions with bacteriocins, as

the nisin resistance protein (NSR), which is able to proteolytically inactivate nisin, localizes on

the cell membrane where it interacts with nisin to provide resistance to the non-nisin producing

strain (Sun et al. 2009). Nisin resistance protein genes (nsr) have been identified across many

species, including Staphylococcus epidermidis, in an operon that potentially provides nisin

immunity (Draper et al. 2015), although it was not identified in any S. aureus genomes in this

study. A serine protease SplE_1 and thermophilic serine protease precursors were both identified

in the pan genome of the S. aureus isolates and were determined to be associated with an isolate

being susceptible to inhibition from the S. chromogenes. The thermophilic serine protease

precursor was identified with the lanthipeptide clusters in antiSMASH (data not shown),

therefore, the possible mechanisms for how it is related to an isolates sensitivity to the S.

chromogenes is likely the same one mentioned previously for the gallidermin precursors.

ATP transporters are also known to be involved in bacteriocin immunity, as they make up

the LanFEG components of lanthipeptide immunity clusters (Stein et al. 2003). They are also

involved in nisin resistance for non-nisin producing strains, by way of removing nisin from the

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cytoplasmic membrane to prevent it from binding to lipid II to form pores (Kramer et al.

2006). After hypothetical proteins, the ABC transporters were the most identified proteins to be

associated with susceptibility to the S. chromogenes isolate. Although, they were involved in

both isolates being resistant and isolates being susceptible. We could postulate that the proteins

associated with susceptible isolates are likely the FEG components of the lanthipeptide clusters

identified with antiSMASH and it would make sense that they showed the same association to

the susceptible isolates that the gallidermin precursor did.

Studies have demonstrated Abi proteins, which are metalloproteases also called CAAX

proteases, are involved in bacteriocin immunity (Kjos et al. 2010). In this study, one gene that

was annotated as a CAAX amino terminal protease self-immunity proteins were identified in the

S. aureus pan genome and assessed for differences in proportions in the susceptible and resistant

groups, although no associations were significant. Potentially, this protein is not involved in

resistance to the specific S. chromogenes strain tested, but could provide resistance for the S.

aureus strains to a different bacteriocin. There is also potential for the Abi proteins to belong to a

bacteriocin biosynthetic gene cluster (Kjos et al. 2010), and therefore provide the producer with

self-immunity, although no clusters were identified in antiSMASH that contained these proteins.

Future analysis visualizing the areas of the S. aureus genome containing these Abi proteins

should be conducted to rule out the presence of a novel bacteriocin that antiSMASH overlooked.

Further directions could involve transcriptome analyses to determine how the levels of

expression of certain genes change between a susceptible and resistant isolate, as was done with

nisin (Kramer et al. 2006). There is also the potential to focus on characterizing the bacteriocin

produced by S. chromogenes in vitro. Once a mode of action is determined, assessing

mechanisms of resistance and therefore predicting susceptibility to the bacteriocin will be more

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straightforward. Identifying a target for the bacteriocin can lead to sequence analyses of the

receptors in the S. aureus genomes to potentially link differences in sequences to susceptibility,

as was done with the man PTS system and Class IIa bacteriocins (Kjos, Nes, and Diep 2009).

Additionally, the whole pan genome could be analysed for protein associations between

susceptibility groups, as there are possibilities for proteins to be involved in susceptibility that

are not necessarily recognizable to be classified as putative bacteriocin resistance genes.

3.6 Conclusions

In conclusion, 73 genes were identified that were associated with S. aureus isolates being

resistant to a bacteriocin producing S. chromogenes strain. The majority of identified genes were

coding for hypothetical proteins, but ABC transporters, lipoproteins, and a serine protease were

also detected. The identified putative bacteriocin resistance genes should be further studied to

elucidate their potential role in bacteriocin immunity.

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Table 3-1 Bacteriocin gene clusters identified in bovine Staphylococcus aureus and in

vitro susceptibility to a lanthipeptide-producing S. chromogenes.

In vitro susceptibility

No. isolates

Group ID

No. bacteriocin gene clusters

Class I

Class II Total

Lanthi-peptide

Sacti-peptide

Lasso Peptide

IIa IIb IIc IId

Susceptible (n=49)

37 AUR1 1 0 0 0 0 0 0 1

12 AUR2 0 0 0 0 0 0 0 0

Resistant (n=90)

35 AUR3 1 0 0 0 0 0 0 1

1 AUR4 1 0 0 0 0 0 1 2

54 AUR4 0 0 0 0 0 0 0 0

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Figure 3-1 Maximum Likelihood phylogenetic tree of Staphylococcus aureus isolates with

isolates resistant to a bacteriocin producing S. chromogenes indicated with a black dot.

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Table 3-2 The number and percentage of S. aureus isolates containing a putative

bacteriocin resistance gene associated with an isolate being resistant to a lanthipeptide

encoding S. chromogenes.

Protein

Isolates encoding for protein

Fisher’s exact p-value Susceptible (n=49) Resistant (n=90)

# of isolates % # of isolates %

ABC superfamily ATP binding cassette transporter, ABC/membrane protein

14 29 49 54 0.0025

12 24 51 57 0.00011

14 29 49 54 0.0025

Cytosol aminopeptidase 25 49 70 79 0.00061 Hypothetical protein 18 37 62 69 9.44x10-5 Hypothetical protein 13 27 55 61 4.48 x10-5

Oligopeptide ABC superfamily ATP binding cassette transporter

13 25 50 56 7.89 x10-5 *

15 31 50 56 0.0027

14 29 45 50 0.0082

15 31 56 62 0.00020

Lipoprotein 13 25 47 53 0.0027 Putative ABC type transporter 13 25 52 58 0.00020

ABC transporter ATP-binding protein 11 22 49 54 0.00031 *

Putative staphylococcal lipoprotein 14 27 55 62 0.00011

Putative staphylococcal tandem lipoprotein 13 25 48 54 0.0014

ABC superfamily ATP binding cassette transporter 11 22 45 50 0.0019 *

S1B family serine protease SplE 13 25 51 57 0.00038

Note: all hypothetical proteins were not included in this table, just a sample.

*gene was identified in accessory genome analysis using SPINE.

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Table 3-3 The number of and percentage of S. aureus isolates containing genes associated

with an isolate being susceptible to a lanthipeptide encoding S. chromogenes.

Protein

Isolates encoding for protein

Fisher’s exact p-value Susceptible (n=49) Resistant (n=90)

# of isolates % # of isolates %

ABC superfamily ATP binding cassette transporter, ABC protein

34 67 37 42 0.0051

32 63 29 33 0.00073

32 63 31 35 0.0016

40 78 45 50 0.0012 38 75 39 44 0.00072 30 59 30 34 0.0047

ABC-2 family transporter 28 55 32 36 0.034 Epidermin biosynthetic protein EpiF 30 59 30 34 0.0022 *

Epidermin biosynthetic protein EpiF 34 67 30 34 7.32 x10-5 *

Hypothetical protein 40 78 44 49 0.0011 Hypothetical protein 32 63 27 30 0.00033

Gallidermin precursor 35 69 34 38 0.00078

34 67 33 37 0.00087

Nisin biosynthetic protein NisC 31 61 26 29 0.00034

Oligopeptide ABC superfamily ATP binding cassette transporter

34 67 58 35 0.00039

32 63 56 37 0.0047

30 59 56 37 0.014

35 69 56 37 0.00042 Putative ABC-type transporter 38 75 39 44 0.00072

Serine protease splE_1 35 69 33 37 0.00042 Thermophilic serine protease precursor 34 67 34 38 0.0015

Note: all hypothetical proteins were not included in this table, just a sample

*gene was identified in genomes from the literature based BLAST search

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A.

B.

Figure 3-2 LanA alignments and phylogenetic tree of type A lanthipeptides identified in S.

aureus isolated from milk of Canadian dairy cows.

10 20 30 40 50 60 70

NisinS._aureus_aS._aureus_bS._capitisEpiderminS._epidermidis_1778/3868GalliderminEpilancin_K7S._equorum_1644Pep5Epicidin_280

- - - - - MS TKD FN L D L - VSV - - SKKDSGASPR I TS I S L - - C TPGC - K T - - - - - - - - - - GA L MGCNMK TA TCHCS I HVSK- - - - - - MENV L D L DVQVKAKND TSDSAGDER I TS F I G - - C TPGCGK T - - - - - - - - - - GS F - - - - - - - - NS FCC - - - - -- - - - - - MEKV L D L DVQVKGNNN TNDSAGDER I TSH F L - - CS FGCGK T - - - - - - - - - - GS F - - - - - - - - NS FCC - - - - -MKVVKEKKE L FD L DVKVNAR - DMNNSESGPPN TS L I W- - C TDGCAKR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -MEAVKEKND L FN L DVKVNAK - ESNDSGAEPR I ASK F I - - C TPGCAK T - - - - - - - - - - GS F - - - - - - - - NSYCC - - - - -MEAVKEKND L FN L DVKVNAK - ESNDSGAEPRVASK F L - - C TPGCAK T - - - - - - - - - - GS F - - - - - - - - NSYCC - - - - -MEAVKEKNE L FD L DVKVNAK - ESNDSGAEPR I ASK F L - - C TPGCAK T - - - - - - - - - - GS F - - - - - - - - NSYCC - - - - -- - - - - MNNS L FD L N L NKGV - - E TQKSD L SPQSAS - V L K TS I KVSKKY - - - - - - - - - - CKG - - - - - V T L TCGCN I TGGK- MKSVD TES L FD L E I KKDV - - QKRDGE L EAQS L GPA I RASVKQCKK T - - - - - - - - - - GR L - - - - - - - - I T I GCGEENK- - - MKNNKN L FD L E I KKE T - - SQN TDE L EPQTAGPA I RASVKQCQK T L KA TR L F TVSCKG - - - - - - - - KNGCK - - - - -- - - MENKKD L FD L E I KKDN - - MENNNE L EAQS L GPA I KA TRQVCPKA - - - - - - - - - - TR F - - V TVSCKKSDCQ - - - - -

Conservation

Quality

Consensus

- - - - - 0 4 5 4 4 8 8 * 7 9 1 5 3 6 - - 4 3 5 5 5 4 5 4 3 5 5 6 8 0 4 5 - - 7 3 2 5 7 0 * 3 - - - - - - - - - - 2 0 1 - - - - - - - - 0 1 0 2 0 - - - - -

MEAVKEKND L FD L DVKVNAKNE +NDS + +EPR I AS + F L +AC TPGCAK T L KA TR L F TVSGS FMG+ + + + T +NSYCC I + + +K

65.13 Pep5

169.88 Nisin

49.39 S._aureus_a

53.61 S._aureus_b

64.06

14.00Epidermin

14.00S._epidermidis_1778/3868

5.61

15.89 Gallidermin

56.94

13.26

145.74 S._capitis

33.99

27.90

141.57 Epilancin_K7

25.16

127.18 S._equorum_1644

21.32

116.73 Epicidin_280

65.13

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(A) Multiple sequence alignments of LanA identified in type A lanthipeptide clusters and

known bacteriocins nisin (accession number P13068), gallidermin (accession number P21838),

epidermin (accession number P08136), epilancin K7 (accession number Q57312), pep5

(accession number P19578), epicidin 280 (accession number O54220), and lanthipeptide

precursors identified in Chapter 2. (B) Neighbour joining tree of LanA.

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Chapter Four: Summarizing discussion

The foremost goal of this thesis was to gain knowledge on the ability of NAS to inhibit S.

aureus. NAS are the most frequently isolated bacteria from the bovine mammary gland, yet are

considered minor pathogens and there are conflicting reports in the literature about the effects of

NAS on udder health. Some studies report that NAS increase SCC (Schukken et al. 2009) and

reduce milk yield, whereas others report an increase in milk yield (Piepers et al. 2010) and a

protective effect against IMI by major pathogens (Matthews, Harmon, and Smith 1990; Braem et

al. 2014). The discrepancy in study results could be due to considering NAS as a group instead

of at the species level. The majority of reports of NAS inhibition seem to be due to S.

chromogenes (De Vliegher et al. 2004; Braem et al. 2014). The apparent protective effect seems

to be due, at least in part, to the production of bacteriocins, which are small antimicrobial

peptides, although a large-scale investigation on bacteriocin production by NAS had not been

done. Therefore, the overall goal of this thesis was to determine the inhibitory capability of NAS

against bovine S. aureus and to determine the distribution and describe any bacteriocin

biosynthetic gene clusters in 441 NAS whole genomes. Additionally, the thesis investigated the

susceptibility of 139 S. aureus isolates to a bacteriocin producing S. chromogenes strain and

aimed to identify any bacteriocin biosynthetic gene clusters and any potential genes related to

susceptibility in the S. aureus whole genomes.

4.1 NAS phenotypic testing

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The modified cross-streaking method was chosen for this portion of the experiment

due to the ease of assessing inhibition with this methodology, though there are some limitations.

The expression of certain bacteriocins may be dependent on the culture conditions (Espeche et

al. 2014), so different NAS strains that potentially produce different bacteriocins may not all

express that bacteriocin in the conditions that were used in this thesis. Additionally, there is

potential to miss inhibition by only using one indicator species, as different bacteriocins have

different spectrums of activity. The variation in concentration of bacteriocin being produced and

subsequently diffused through the agar is another limitation, which could lead to false negatives

when assessing inhibition. Inhibition can be a result of inhibiting substances other than

bacteriocins, e.g. low molecular weight antibiotics, lytic enzymes, or metabolic by-products

(Leroy and De Vuyst 2004). In order to confirm inhibition is due to a bacteriocin, the bacteriocin

can be isolated, purified, and subsequently tested in an inhibition assay. Although, an often

quicker and less expensive option is to assess cessation of inhibition with the addition of

proteolytic enzyme, which gives a good indication that inhibition is due to a substance that is

protein-like in nature. Though, the cross-streaking method we used for phenotypic assessment is

not well suitable for testing the potential bacteriocin producing strain with proteinase K;

therefore, the well-diffusion assay was employed to test this. Although, because bacteriocins are

produced in broth culture in such low concentrations, the cell-free supernatant of the producing

strain was not concentrated enough to elicit an inhibitory effect in the well diffusion assay.

Consequently, chloroform extractions were carried out on the 21 potential producer/inhibitors in

order to concentrate the bacteriocin from the cell-free supernatant to assess for protease

sensitivity. To this end, we only obtained five NAS chloroform extracted products that were able

to inhibit the S. aureus in the well diffusion assay. For each of the five that we were able to

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obtain inhibition for, we were able to eliminate inhibition with the addition of proteinase K to

the bacteriocin product, thus indicating that the inhibitory compound is protein-like in nature and

therefore, likely due to bacteriocins. For the remaining 16 isolates that inhibited S. aureus in the

cross streaking method yet did not inhibit in the well diffusion assay via chloroform extractions,

there could be a number of different reasons. The chloroform extraction technique may not have

been the optimal extraction procedure for all of the bacteriocins potentially being produced, as

the properties of the bacteriocins likely differ. It is thus possible that another extraction protocol

could result in a higher yield of bacteriocin from the cell free supernatant. Additionally, the

growing conditions (e.g. temperature) of the producer in broth could have affected the inhibition,

as chloroform extracted products of a nukacin-like bacteriocin were compared and products that

were initially incubated at 30°C had stronger inhibitory effects than those grown at 37°C (Braem

et al. 2014). As well, some bacteria may not produce similar amounts of bacteriocin when grown

in broth, compared to when grown on solid medium. A variety of temperatures while incubating

the producer in broth medium can be explored in the future to optimize bacteriocin extraction.

Another method of assessing proteinase K sensitivity could be done directly on the agar, to

eliminate the need to perform any extractions on cell free supernatant.

In conclusion, the cross-streaking method to initially assess phenotype was sufficient at

identifying 40 NAS that inhibited the growth of S. aureus in vitro. Using this method, combined

with the well diffusion assay to assess for proteolytic enzyme effects on inhibition was adequate

at determining the species-specific effects on NAS on S. aureus and was able to confirm the

protein-like nature of the inhibitory product for 5 NAS potential bacteriocin producers.

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4.2 Bacteriocin clusters in NAS

Various NAS species have been reported to produce bacteriocins. Staphylococcus

epidermidis produces pep5 (Sahl et al. 1985), epidermin (Allgaier et al. 1986), epilancin K7 (van

de Kamp et al. 1995), epicidin 280 (Heidrich et al. 1998), and epilancin 15X (Ekkelenkamp et al.

2005). Staphylococcus gallinarum produced gallidermin (Schnell et al. 1989). Staphylococcus

cohnii produces staphyloccocin T (Furmanek et al. 1999). Staphylococcus hyicus produces the

lanthipeptide hyicin 3862 (Fagundes et al. 2011) and the sactipeptide, hyicin 4244 (Duarte et al.

2017). Staphylococcus warneri produces a type-AII lanthipeptide, nukacin ISK-1 (Sashihara et

al. 2000). Nukacin-like bacteriocins have also been identified in S. simulans (Ceotto et al. 2010),

S. hominis (Wilaipun et al. 2008), and S. chromogenes (Braem et al. 2014). Lastly, S. aureus

produces Bsa (Daly et al. 2010), the circular bacteriocin aureocyclicin 4185 (Potter, Ceotto,

Coelho, Guimaraes, et al. 2014), and a two-component lanthipeptide, C55 (Maduwe, Sahl, and

Tagg 1999). In this study, we were able to identify 105 putative bacteriocin clusters in 95 NAS

isolates. We identified potential new additions to the pep5 and epidermin groups of

lanthipeptides, along with novel lanthipeptides not closely related to previously described

lanthipeptides. Staphylococcus epidermidis 1778 and 3868 had the strongest inhibition of any

isolate out of the 441 tested, and the identified lanthipeptide varied in one aa from epidermin.

Thus, it is likely a variant of epidermin, and its spectrum of activity should be explored further as

epidermin has shown promise for use in clinical settings (Fontana, de Bastos, and Brandelli

2006; Nascimento et al. 2006).

There are some limitations associated with in silico screening that need to be stated.

Genome mining does not provide any information about biological activity, as evident when

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comparing our phenotypic test with our genomic screen. We identified isolates with potential

clusters that were not active against S. aureus in vitro, although there is the possibility that the

isolate potentially produces a bacteriocin that is not active against S. aureus or the clusters

identified may not be functional in producing active bacteriocins. One of the initial problems

regarding identifying bacteriocins using software was that bacteriocin-finding tools only looked

for precursor genes in the genomes and as a result clusters were often missed due to the fact that

precursor genes are so small and are frequently overlooked in annotation (Wang, Fewer, and

Sivonen 2011). Current approaches combine mining for the structural gene with mining for the

homologous bacteriocin-associated genes present on the operon, which has increased the

identification of clusters. However, completely novel bacteriocin classes will likely be missed

because they lack homology to known biosynthetic genes and/or structural genes. In this study

however, as discussed in the discussion of Chapter 2, we utilized multiple tools for discovery, in

order to have the most comprehensive analysis of the available genomes. Lastly, although it is

possible to deduce which classes the bacteriocins we identified belong to, the placement into

classes based on bioinformatics alone are not always correct (Arnison et al. 2013), thus further

characterization in the laboratory is necessary for confirmation. Similarity in sequence to a

known precursor also does not guarantee that the two belong to the same class, additionally,

proximity between precursors and biosynthetic genes do not guarantee a target/substrate

relationship (Arnison et al. 2013). For these reasons, biochemical characterization in the

laboratory is necessary to completely characterize the discovered bacteriocins. That being said,

genome mining is a valuable resource to identify potential clusters to be characterized in vitro.

Henceforth, genome mining and laboratory analysis should be paired together in a cyclic

process to maximize bacteriocin discovery. Genome mining can identify isolates to pursue with

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laboratory analysis, leading to elucidating the bacteriocins biology, which will provide

further information to enhance genome-mining algorithms, leading to the identification of more

bacteriocins.

4.3 Staphylococcus aureus susceptibility

The S. chromogenes isolate for this experiment was chosen based on initial pilot

experiment, wherein the susceptibility of nine S. aureus isolates to the S. chromogenes was

variable (data not shown). The same cross-streaking method utilized in Chapter 2 was used for

this experiment. The limitations of this method discussed above (the differential expression of

bacteriocins in different environments or the spectrum of activity variation) do not apply here, as

we know that the S. chromogenes strain is able to inhibit S. aureus in vitro. We also know that

the S. chromogenes isolate is able to produce a protein-like inhibitory product (as evident from

the positive results of the proteinase K experiment in Chapter 2) that is likely a bacteriocin.

Therefore, the variability in inhibition that we see in the S. aureus is due to something the

specific S. aureus isolate inherently contains, making it resistant to the S. chromogenes.

4.4 Potential immunity genes

Utilizing access to the S. aureus whole genome sequences and determining phenotypic

data for inhibition, assessing putative bacteriocin resistance genes present in the S. aureus was

the next logical step. By comparing the distribution of genes of interest (ones that have been

previously established as being involved in immunity or annotated as hypothetical proteins)

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present in susceptible and resistant isolates, potential genes could be determined that

contribute to susceptibility. By using the pan genome to select genes for investigation, we were

able to determine 77 genes that were associated with an isolate not being susceptible to the S.

chromogenes and we also were able to determine 76 genes that were associated with an isolate

being susceptible to the S. chromogenes. The majority of these genes encoded for hypothetical

proteins, which will require functional determination to evaluate their role in susceptibility. One

future method that could potentially yield a smaller pool of genes would be to use a different

NAS strain for the phenotypic assessment, one that is less variable in inhibition. The large

amount of variation seen in the susceptibility of S. aureus to the S. chromogenes bacteriocin is no

doubt likely due to multiple resistance mechanisms, whereas with fewer resistant isolates to a

different NAS could potentially mean there are a smaller number of resistance mechanism at

work. Therefore, less variation may result in a better chance at determining what susceptible

strains are lacking that resistant strains contain. Nonetheless, these results show that S. aureus

isolates from the same ecological niche show variable susceptibility to the bacteriocin encoding

S. chromogenes strain and there are a large number of proteins in play that determine

susceptibilities.

4.5 Conclusions and future research

The current studies contribute towards understanding bacteriocin production among

Staphylococcus strains isolated from bovine milk. The results indicate that in silico screening for

bacteriocins is a promising and time effective method to identify potential novel bacteriocins.

This knowledge will be the foundation for future in vitro and in vivo investigations. The results

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also indicate that susceptibility in S. aureus to an S. chromogenes lanthipeptide can be varied

and there are many genes that are potentially associated with susceptibility.

Chapter one, a review of NAS inhibition and bacteriocins was provided. Bacteriocins

produced by select NAS are likely the reason for the inhibition on mastitis pathogens reported by

previous studies. Indeed, bacteriocins have been recently isolated and characterized from NAS

following in vitro inhibition studies (Braem et al. 2014; Ceotto et al. 2010).

Overall, 40 isolates from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S.

saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus inhibited growth of a bovine

clinical mastitis S. aureus isolate. Twenty-three of the 40 inhibiting isolates from S. capitis, S.

chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus also inhibited growth of the

MRSA isolate. Ninety-five isolates belonging to 16 species, S. capitis, S. chromogenes, S. cohnii,

S. epidermidis, S, equorum, S. fleurettii, S. gallinarum, S. haemolyticus, S, hyicus, S.

saprophyticus, S. sciuri, S. simulans, S. succinus, S. vitulinus, S. warneri, and S. xylosus, were

identified to contain putative bacteriocin biosynthetic gene clusters, and thus potentially produce

bacteriocins. Only 21 of the inhibiting NAS were also potential producers, as we were not able to

identify bacteriocin clusters in the remaining 19 genomes from inhibitors, although this is not to

definitively say that they do not contain them. It should be noted that although species

identification is important with NAS for a multitude of reasons, bacteriocin production appears

to be strain specific, and not species-related.

The susceptibility of a group of 139 S. aureus to a single S. chromogenes bacteriocin was

examined in Chapter 3, as well as the possible association between susceptibility and putative

bacteriocin resistance genes. Forty-nine S. aureus isolates were inhibited by the S. chromogenes,

while the remaining 90 isolates were not susceptible. After assessing the S. aureus pan genome,

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as well as creating a BLAST database for genes present in the resistant isolates accessory

genomes and using putative bacteriocin resistance genes based on the literature, 77 proteins were

found that were associated with the resistant S. aureus isolates. The majority of the identified

proteins were hypothetical proteins, which will require future studies for elucidating of their role

in susceptibility.

Described below are suggestions for future research mainly regarding the next steps to

take in the laboratory working towards potential future applications in the dairy industry. One of

the initial next steps could focus on optimizing the growing conditions and purification of

bacteriocins from potential NAS and S. aureus producers, as mentioned previously. Generally,

following a large-scale screen of isolates for inhibitory activity, isolation and characterization of

the bacteriocin from the isolate that exhibited the strongest inhibiting potential in vitro (Braem et

al. 2014) or from those isolates in which a bacteriocin has not been previously described (Ceotto

et al. 2010) is common practice. If we were to pursue the strongest inhibitors from this thesis, the

two inhibiting S. epidermidis isolates exhibited the strongest inhibition of the S. aureus. As for

pursuing bacteriocin isolation from species that have not been identified as having clusters, S.

equorum, S. haemolyticus, S. succinus, S. capitis, S. cohnii, S. vitulinus, and S. fleuretti all were

identified with having clusters in this study and none of which were previously reported to,

although some species had demonstrated antimicrobial activity in previous studies (Braem et al.

2014). An advantage of this study is the availability of the genomic data for presence of

bacteriocin biosynthetic gene clusters. This will eliminate the chance of “re-discovery” of

bacteriocins, as has happened previously with selecting isolates based on inhibition experiments

alone (Ceotto et al. 2010; Braem et al. 2014). With the exception of the nukacin bacteriocin

identified in 3 inhibiting S. chromogenes isolates, all the bacteriocins identified from the in silico

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screen of NAS and S. aureus are novel. The S. epidermidis isolates that were the strongest

inhibitors contained a bacteriocin cluster, likely encoding for an epidermin variant. Additionally,

because this study represents the first time lasso peptides have been identified in Staphylococcus

genomes the second time sactipeptides have been noted (Duarte et al. 2017), characterization of

those bacteriocins would be noteworthy.

Following purification of a bacteriocin, it can be characterized by determining structure,

size, class, stability, and spectrum of activity. A wide range of pathogens should be used for

spectrum of activity experiments in order to assess the potential usefulness because along with

the benefits of identifying bacteriocins active against mastitis pathogens, identifying bacteriocins

with inhibition against food pathogens, such as Listeria, could be beneficial and offer new

bacteriocins for use in the food preservation industry. Once the bacteriocin is characterized,

experiments can take place to determine the bacteriocins mode of action and cellular target(s).

Certainly, as mentioned previously, assessing the risk of resistance would have to be

thoroughly examined and deemed low, or at least manageable, for there to be any merit of

developing bacteriocins for applications in the industry. The resistance distribution should be

first assessed. The target receptor sequences, if known, can be taken from a wide range of target

organisms and a comparison of receptor sequence and isolate susceptibility can take place, much

like the experiment looking at the man-PTS sequence and Class IIa bacteriocins (Kjos, Nes, and

Diep 2009) to determine the range of isolate susceptibility. Additionally, transcriptome analyses

can be done in order to determine factors related to acquire resistance by determining which

genes up and down-regulate in resistant isolates (Kramer et al. 2006). The rate of acquiring

resistance in vitro should also be examined. Once mechanisms of resistance have been

documented, steps can be taken to decrease the amount of resistance when administering

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bacteriocins for clinical use. Some recommendations to avoid resistance include using

multiple bacteriocins with different modes of action (Cotter, Ross, and Hill 2013), which can

also increase spectrum of activity (Coelho et al. 2007). Using bacteriocins in conjunction with

antibiotics could also reduce the risk of resistance developing, as bacteriocins and antibiotics

have different modes of action. Activity can also be increased for certain bacteriocins when

combined with other antimicrobials or membrane-active substances (Cotter, Ross, and Hill

2013), which would reduce the selection pressure for resistance. Bioengineering the bacteriocins

is also a possibility for decreasing some resistance, examples of which include increasing the

bacteriocins resistance to proteases (Rink et al. 2010), or to enable them to bind to receptors

which carry gene modifications that made the target bacteria previously resistant (Cotter, Ross,

and Hill 2013).

Once novel bacteriocins have been fully described and risk of resistance has been

assessed, studies can continue to assess the bacteriocins potential for use in the dairy industry. As

indicated previously, the most likely use of NAS bacteriocins in the dairy industry would be in

teat wipes or teat sealants for the prevention of mastitis from Gram-positive pathogens and to

reduce the use of antibiotics. When the Dutch dairy industry stopped blanket dry cow therapy,

antibiotic usage decreased by 56% from 2007 to 2013 (Speksnijder et al. 2015). Although, with

the decrease in antibiotic use at dry off, the incidence rate of CM was 1.7 times higher in quarters

dried off without antibiotics compared to quarters dried off with antibiotics (Scherpenzeel et al.

2014). The pathogen responsible for these CM cases was S. uberis (Scherpenzeel et al. 2014),

and it should be noted that S. uberis is a Gram-positive pathogen that has been shown to be

inhibited by bacteriocins in vitro. Thus, the use of bacteriocins with a teat sealant at dry off could

potentially prevent these CM mastitis cases and mitigate the increase of CM observed. Using just

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teat sealant without any antibiotics could conceivably push any bacteria in the teat duct up

into the canal during application; having bacteriocin in the teat sealant therefore would reduce

new infections that could occur as a result of this mechanism of infection (Crispie et al. 2005).

Consequently, more studies need to be done on the persistence of bacteriocin in a teat sealant to

determine how long protection could be provided to the cow to be able to establish the usefulness

of bacteriocins in teat sealants at dry off.

There is also the possibility to use bacteriocins to treat mastitis cases in the lactating herd.

Occasionally, intramammary antibiotic treatment for mastitis is ineffective due to the formation

of biofilms in the teat canal, which antibiotics are not able to access (Pieterse and Todorov

2010). Lysostaphin, which is a bacteriolysin, or class III bacteriocin according to some

classification schemes (Heng and Tagg 2006) produced by S. simulans, shows antimicrobial

activity against staphylococci (Bastos, Coutinho, and Coelho 2010) and was recently

demonstrated to disrupt S. aureus biofilms and result in bacterial cell death (Ceotto‐Vigoder et

al. 2016). Nisin application also resulted in S. aureus cell death in pre-formed biofilms (Ceotto‐

Vigoder et al. 2016). The authors also suggest that using a combination of bacteriocins for

prevention and treatment of S. aureus biofilms will decrease the risk of developing resistance, as

the two bacteriocins have different modes of action (Ceotto‐Vigoder et al. 2016). A recent study

from Argentina identified an S. chromogenes isolated from a non-infected quarter that was able

to prevent the formation of biofilms, but had no antimicrobial activity (Isaac et al. 2017). The

bioactive component of the S. chromogenes cell free supernatant was inhibited with the addition

of proteinase K, indicating that it is protein-like in nature (Isaac et al. 2017), and therefore, the

anti-biofilm effects could be due to a bacteriocin. These results indicate that bacteriocins can

potentially be used to treat persistent S. aureus infections with a biofilm component, potentially

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resulting in less antibiotic use and less economic losses due to IMI. Another benefit of using

bacteriocins to treat lactating cows is the potential reduction of withdrawal times because the

digestive enzymes present in milk would degrade bacteriocins more quickly than antibiotics,

therefore reducing the cost association with lost milk after treatment (Pieterse and Todorov

2010). The persistence of the bacteriocin in the mammary gland would have to be assessed, as

well as its activity and stability at physiological pH.

Also, with mounting consumer interest in probiotic use, one could appreciate the use of

probiotics to battle mastitis and because NAS have shown to inhibit mastitis pathogens in the

udder, it raises the question if they could be used as such. However, it remains to be determined

if the producing strain would be able to produce enough bacteriocin in milk to be effective, and

what the effect the colonization of a bacteriocin producing NAS strain would have on the udder

and milk quality. Lactococcus lactis 19.3 produces a bacteriocin with a wide inhibitory spectrum,

including against L. monocytogenes and was able to grow and produce similar amounts of

bacteriocin in MRS medium, cow's milk and soya milk, respectively (Zamfir et al. 2016).

Another study infused live L. lactis 3147 (which produces lacticin 3147) into the udder, which

had previously showed killing effects of Gram-positive mastitis pathogens (Klostermann et al.

2008), and the authors concluded the inhibition likely came from stimulating the host’s

mammary immune system and the stimulation may be a result of the bacteriocin production

(Crispie et al. 2008). If NAS strains behaved the same in bovine milk, there is potential for

bacteriocin production in the milk of the udder and could explain the inhibition of mastitis

pathogens observed in the field (Matthews, Harmon, and Smith 1990; Piepers et al. 2010), and

why teats colonized by S. chromogenes produce milk with low SCC (De Vliegher et al. 2003).

Staphylococcus chromogenes is the most frequently isolated NAS from the mammary gland

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(Condas et al. 2017 ) and it has been identified as a producer of bacteriocins in the literature

(Braem et al. 2014; De Vliegher et al. 2004). Although, S. capitis, S. gallinarum, S. hyicus, S.

agnetis, and S. simulans are the most frequently isolated NAS species from quarters with high

SCC (Condas et al. 2017), so using isolates from those species has the potential to increase the

SCC which would not be desirable to the dairy producer. Although, the increase of SCC with

IMI from NAS is relatively small (Condas et al. 2017) and the strain selected for potential

probiotic use would have to be assessed for how it persists in the udder and how it specifically

impacts the udder health and milk quality. Because enzymes in the milk can potentially degrade

purified bacteriocins, administering bacteriocin-producing bacteria that can persist may be a

more effective approach for treatment or prevention of infection. Although, to be used in food

production the strain would have to have a GRAS (generally regarded as safe) status.

In conclusion, this study indicates that NAS are potentially prolific producers of

bacteriocins, some of which have the ability to inhibit S. aureus and MRSA in vitro. This study

also highlights the larger number of proteins that are potentially involved in bacteriocin

resistance in S. aureus. The substantial amount of information generated from the in silico

analyses warrants further investigation.

116

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Bacteriocins of bovine non-aureus staphylococci

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