Bacteriocins of bovine non-aureus staphylococci

Bacteriocins of bovine non-aureus staphylococciGraduate Studies The Vault: Electronic Theses and Dissertations
2017
Carson, Domonique
thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25092
http://hdl.handle.net/11023/4124
University of Calgary graduate students retain copyright ownership and moral rights for their
thesis. You may use this material in any way that is permitted by the Copyright Act or through
licensing that has been assigned to the document. For uses that are not allowable under
copyright legislation or licensing, you are required to seek permission.
Downloaded from PRISM: https://prism.ucalgary.ca
by
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES
CALGARY, ALBERTA
SEPTEMBER, 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
iii
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.
v
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
vi
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.
vii
Dedication
and to my dogs
viii
List of Symbols, Abbreviations and Nomenclature ................................................................ xiv
Preface .......................................................................................................................................... xv
1.1 Mastitis in the Canadian dairy industry ........................................................................... 1
1.2 Non-aureus staphylococci ................................................................................................... 2
1.4.1 Lanthipeptides ................................................................................................................ 7
1.4.2 Sactipeptides ................................................................................................................ 12
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
genomes ................................................................................................................................ 35
Chapter Two: Bacteriocins of non-aureus staphylococci isolated from bovine milk ........... 36
2.1 Abstract .............................................................................................................................. 36
2.2 Introduction ....................................................................................................................... 38
2.3.1 Isolates ......................................................................................................................... 40
2.3.6 BLAST ......................................................................................................................... 44
2.4 Results ................................................................................................................................ 45
2.5 Discussion .......................................................................................................................... 51
2.6 Conclusions ........................................................................................................................ 58
whole genomes ............................................................................................................................. 79
3.1 Abstract .............................................................................................................................. 79
3.2 Introduction ....................................................................................................................... 81
3.3.1 Isolates ......................................................................................................................... 83
3.3.5 Screening of genomes for immunity related genes ...................................................... 86
x
3.4.3 Screening of genomes for putative resistance related genes ........................................ 88
3.5 Discussion .......................................................................................................................... 90
3.6 Conclusions ........................................................................................................................ 95
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
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
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
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).
14
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 (Martnez et al. 1999).
19
Along with the 91aa structural gene, LclA, the complete lactococcin 972 cluster contains
a transporter, LclB, and an immunity protein (Martnez 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).
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 (Kaškonien 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 (Kaškonien 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 (Kaškonien et al. 2017). Other techniques, such
as ultrafiltration or filter assisted size exclusion protein fractionation, can be used for purification
(Kaškonien 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 (Kaškonien 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.
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).
28
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.
36
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.
37
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-gen

Bacteriocins of bovine non-aureus staphylococci

Documents

Transcript of Bacteriocins of bovine non-aureus staphylococci

Staphylococci Fred Tenover

Resistance of Staphylococcal and Streptococcal Clinical ... Sabbah.pdf · negative Staphylococci (CONS) was 76.9%, which was higher than that among S. aureus (64.7%). With respect

Altamash classification & staphylococci

S. aureus Biofilms. Staphylococcus aureus ALL STAPHYLOCOCCI are:ALL STAPHYLOCOCCI are: 1. Gram-positive 1. Gram-positive 2. Cocci 2. Cocci 3. Clusters.

University of Groningen The Staphylococcus aureus ...111 Chapter 6 Partially overlapping substrate specificities of sortases A and C of staphylococci M.J.J.B. Sibbald #, X.M. Yang

ANTIBIOTIC SUSCEPTIBILITY PROFILE OF METHICILLIN RESISTANT Staphylococci aureus IN POULTRY FARM, IN ZARIA, NIGERIA. BY Onaolapo J. A., Igwe J. C and Bala.

Aerobic Gram- Positive Cocci Negative Streptococci See Streptococci identification chart Positive Staphylococci See Staphylococci Identification chart.

Emerging Applications of Bacteriocins as Antimicrobials ...

Thermo Scientific Microbiology Products Food Testing Microbiology.pdf · ISO 6888 Horizontal methods for the enumeration of coagulase-positive Staphylococci (Staphylococcus aureus

1 Staphylococci

Methicillin Resistant Staphylococcus · PDF filethe mecA or mecC genes. ... Methicillin Resistant Staphylococcus aureus S. aureus S. aureus S. aureus S. aureus. Staphylococcus aureus.

01: ' # '8& *#0 & 934 Vascular Surgery Principles and Practice 4. Bacteriology Staphylococci ( Staphylococcus aureus and coagulase negative staphylococci) account for more than 75%

Staphylococci Ppt

Isolation & Identification of Staphylococci. Staphylococci Characteristics Staphylococci are often found in the human nasal cavity (and on other mucous.

Staphylococci 09 10 Med

Staphylococci - Columbia University · • Microbiology of staphylococci • Epidemiology of S. aureus infections • Pathogenesis of S. aureus infections – Invasive disease –

‘Ikvo-Year Assessment of the Pathogen Frequency and ... · patient’s endogenous flora. Staphylococcus aureus, coag- ulase-negative staphylococci (CONS), and Enterococcus spp are

209 Staphylococci

Staphylococci and streptococci

Journal of Microbiology, Biotechnology and Food Sciences · enterobacteria and total coliforms counts), Baird-Parker agar (for Staphylococci+micrococci and Staphylococcus aureus counts)