Bacteriocins of Non-aureus StaphylococciIsolated from Bovine Milk

Domonique A. Carson, Herman W. Barkema, Sohail Naushad, Jeroen De BuckDepartment of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB,Canada

ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolatedfrom the bovine udder, potentially protect the udder against infection by majormastitis pathogens due to bacteriocin production. In this study, we determined theinhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bo-vine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a hu-man methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. Wedetermined the presence of bacteriocin clusters in NAS whole genomes using ge-nome mining tools, BLAST, and comparison of genomes of closely related inhibitingand noninhibiting isolates and determined the genetic organization of any identifiedbacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S.chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. war-neri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, fromS. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, alsoinhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc,and class IId) in 95 whole genomes from 16 species were identified. A total of 25novel bacteriocin precursors were described. In conclusion, NAS from bovine mam-mary glands are a source of potential bacteriocins, with �21% being possible pro-ducers, representing potential for future characterization and prospective clinical ap-plications.

IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costsCanadian dairy producers $400 million/year and is the leading cause of antibioticuse on dairy farms. With increasing antibiotic resistance and regulations regardinguse, there is impetus to explore bacteriocins (bacterially produced antimicrobial pep-tides) for treatment and prevention of bacterial infections. We examined the abilityof 441 NAS bacteria from Canadian bovine milk samples to inhibit growth of S. au-reus in the laboratory. Overall, 9% inhibited growth of S. aureus and 58% of thosealso inhibited MRSA. In NAS whole-genome sequences, we identified �21% of NASas having bacteriocin genes. Our study represents a foundation to further exploreNAS bacteriocins for clinical use.

KEYWORDS Staphylococcus, Staphylococcus aureus, bacteriocins, cattle, coagulase-negative staphylococci, mastitis

Non-aureus staphylococci (NAS), a heterogeneous group of approximately 50 spe-cies that can be considered teat skin opportunists and minor pathogens, are the

bacteria most commonly isolated from the bovine udder (1–4). Several studies reportedthat NAS may confer protection against intramammary infection (IMI) by major mastitispathogens (5, 6). In a challenge study, 53% of Staphylococcus chromogenes-infectedquarters were protected from S. aureus challenge (6). In another study, S. chromogenesisolates inhibited in vitro growth of all tested Staphylococcus aureus, Streptococcusdysgalactiae, and Streptococcus uberis isolates but none of the Gram-negative isolates

Received 9 May 2017 Accepted 23 June 2017

Accepted manuscript posted online 30June 2017

Citation Carson DA, Barkema HW, Naushad S,De Buck J. 2017. Bacteriocins of non-aureusstaphylococci isolated from bovine milk. ApplEnviron Microbiol 83:e01015-17. https://doi.org/10.1128/AEM.01015-17.

Editor Harold L. Drake, University of Bayreuth

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Jeroen De Buck,jdebuck@ucalgary.ca.

EVOLUTIONARY AND GENOMIC MICROBIOLOGY

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(5). Similarly, NAS strains from Brazilian bovine mastitis cases, including Staphylococcusepidermidis, Staphylococcus simulans, Staphylococcus saprophyticus, Staphylococcushominis, and Staphylococcus arlettae, inhibited growth of Corynebacterium fimi (7). Theisolated antimicrobial substances were considered to be bacteriocins due to sensitivityto proteolytic enzymes (7). Recently, Braem et al. identified NAS strains (from 6 species)that inhibited S. aureus, S. uberis, and S. dysgalactiae, and an inhibitory substance (froman inhibiting S. chromogenes) was isolated and identified to be a nukacin-like bacteri-ocin (8).

Bacteriocins are ribosomally synthesized and posttranslationally modified peptides(RiPPs) produced by Gram-positive bacteria that mainly inhibit growth of similarbacterial species or occasionally a broad spectrum of bacteria (9). As regulationssurrounding antibiotic usage get stricter, bacteriocins represent potential alternativesto antibiotics (10). Currently, nisin, produced by Lactococcus lactis, is available for usein the dairy industry in the form of Wipe Out, with germicidal activity against S. aureusand S. agalactiae (11). Lacticin 3147, another bacteriocin produced by L. lactis, wastested for use in a teat sealant and reduced the number of S. aureus organismsrecovered from the bovine mammary gland 18 h postchallenge compared to infusionwith teat sealant alone (12).

Traditionally, identification of novel bacteriocins used culture-based approachesthat involved screening numerous isolates for antimicrobial activity, followed bylengthy biochemical characterization. However, due to growing accessibility of genomesequence data, in silico screening (genome mining) is a promising approach to identifynovel biosynthetic gene clusters. Although bacteriocin precursor genes are often smalland lack homology, making in silico identification challenging, bacteriocin-associatedgenes present on the same operon are highly conserved. Therefore, the associatedmodification genes are used in classification schemes, with 2 classes: class I, posttrans-lationally modified bacteriocins, including lanthipeptides, sactipeptides, and lassopeptides; and class II, nonmodified or cyclic peptides (9). By screening genomes forbacteriocin-associated genes, new lanthipeptides (13, 14) and new class IIa bacteriocingene clusters (15) have been identified. In addition, BLAST-based approaches havebeen used to identify bacteriocins in cyanobacteria (16) and to identify lanthipeptideclusters by using a transport gene for screening (17). Current software-based ap-proaches (e.g., BAGEL3 and antiSMASH) combine direct mining for the structural genewith indirect mining for bacteriocin-associated genes. Using this approach, novelbacteriocins were identified in ruminal bacteria (18), anaerobic bacteria (19), lactic acidbacteria (20), and human gut microbiota (21). A large in silico screen apparently has notbeen done on NAS whole genomes, which could be the foundation for future inves-tigations into alternatives for antimicrobials in the dairy industry.

The first objective was to determine the inhibitory capability of 441 bovine NASisolates from 26 species against a bovine S. aureus isolate and a human methicillin-resistant S. aureus (MRSA) isolate. The second objective was to identify and describe theorganization of bacteriocin biosynthetic gene clusters in the corresponding 441 whole-genome sequences.

RESULTSPhenotypic testing. 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) inhibited growth of the bovine clinical mastitis S. aureusisolate (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 MRSAisolate.

Effect of proteinase K on inhibition. Out of 21 inhibitors that were potentialproducers of bacteriocins, five isolates inhibited growth of S. aureus in the well diffusionassay after chloroform extraction. Inhibition by all 5 isolates was eliminated with theaddition of proteinase K.

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TABLE 1 Bacteriocin gene clusters identified in bovine non-aureus staphylococcal genomes and inhibitory phenotypes tested againstS. aureus and MRSA

Species (n)No. ofisolatesa Group ID

In vitro inhibition(n) No. of bacteriocin gene clusters

S. aureus MRSAb

Class I Class II

TotalLanthipeptide SactipeptideLassopeptide a b c d

S. agnetis (13) 13 SAG � NT 0 0 0 0 0 0 0 0S. arlettae (15) 15 SAR � NT 0 0 0 0 0 0 0 0S. auricularis (2) 2 SAU � NT 0 0 0 0 0 0 0 0

S. capitis (22) 5 SCAP1 � NT 1 0 0 0 0 0 0 15 SCAP2 � � 1 0 0 0 0 0 0 11 SCAP3 � NT 1 1 0 0 0 0 0 21 SCAP4 � � 0 1 0 0 0 0 0 11 SCAP4 � � 1 1 0 0 0 0 0 27 SCAP5 � (7) � (5) 0 0 0 0 0 0 0 02 SCAP6 � NT 0 0 0 0 0 0 0 0

S. caprae (1) 1 SCAR � NT 0 0 0 0 0 0 0 0

S. chromogenes (82) 2 SCH1 � � 1 0 0 0 0 0 0 11 SCH2 � � 0 0 0 0 0 0 0 079 SCH3 � NT 0 0 0 0 0 0 0 0

S. cohnii (24) 2 SCO1 � NT 1 0 0 0 0 0 0 122 SCO2 � NT 0 0 0 0 0 0 0 0

S. devriesei (8) 8 SDE � NT 0 0 0 0 0 0 0 0

S. epidermidis (26) 2* SEP1 � � 1 0 0 0 0 0 0 124 SEP2 � NT 0 0 0 0 0 0 0 0

S. equorum (17) 2 SEQ1 � NT 0 0 0 0 0 0 1 13 SEQ2 � NT 0 0 0 1 0 0 0 11 SEQ3 � NT 1 0 0 0 0 0 0 111 SEQ4 � NT 0 0 0 0 0 0 0 0

S. fleurettii (2) 2 SFL1 � NT 0 0 1 0 0 0 0 1

S. gallinarum (21) 14 SGA1 � NT 0 0 0 0 0 1 0 12 SGA2 � NT 1 0 0 0 0 1 0 25 SGA3 � NT 0 0 0 0 0 0 0 0

S. haemolyticus (29) 1 SHA1 � NT 0 0 0 0 0 1 0 128 SHA2 � NT 0 0 0 0 0 0 0 0

S. hominis (11) 11 SHO � NT 0 0 0 0 0 0 0 0

S. hyicus (3) 1 SHY1 � NT 0 0 0 0 0 0 1 12 SHY2 � NT 0 0 0 0 0 0 0 0

S. kloosii (1) 1 SKL � NT 0 0 0 0 0 0 0 0S. nepalensis (2) 2 SNE � NT 0 0 0 0 0 0 0 0

S. pasteuri (6) 1 SPA1 � � 0 0 0 0 0 0 0 05 SPA2 � NT 0 0 0 0 0 0 0 0

S. saprophyticus (16) 1 SSA1 � NT 0 0 0 1 0 0 0 11 SSA2 � � 0 0 0 0 0 0 0 014 SSA2 � NT 0 0 0 0 0 0 0 0

S. sciuri (30) 3 SSC1 � NT 0 0 0 0 0 0 1 11 SSC2 � � 1 0 0 0 0 0 0 11 SSC3 � � 0 0 0 0 0 0 1 11 SSC4 � � 0 0 1 0 0 0 0 11 SSC4 � NT 0 0 1 0 0 0 0 1

(Continued on next page)

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Screening of genomes for bacteriocin clusters. In the 441 NAS genomes, 184putative bacteriocin gene clusters belonging to 143 isolates were identified. A total of105 clusters from 95 isolates belonging to 16 species were determined to be viableclusters, whereas the others were eliminated due to the absence of either a structuralgene or essential bacteriocin-associated genes (Table 1). Overall, 21.5% of NAS poten-tially produced bacteriocins. Ten of the 441 genomes encoded 2 clusters of differentclasses/types, whereas the remaining 85 potential producers contained 1 cluster. Of the40 inhibitors, 21 were putative producers, whereas no viable bacteriocin gene clusterswere identified in 19 inhibitors.

Class II bacteriocins were most frequently identified, with 69 clusters in 68 isolatesfrom 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 potentialproducers were also inhibitors. For class I bacteriocins, lanthipeptides were the mostfrequently 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, andS. vitulinus (Fig. 1). Fifteen of the 29 potential lanthipeptide producers were alsoinhibitors. Three sactipeptide clusters were identified from 3 S. capitis isolates, 2 ofwhich were inhibitors. Lastly, 4 lasso peptide clusters were identified in 2 noninhibitingS. fleurettii genomes, a noninhibiting S. sciuri genome, and an inhibiting S. sciurigenome. Putative bacteriocin-associated genes were distributed throughout the phy-logeny of NAS (22) (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 putativebacteriocin gene clusters. There was no obvious clustering based on phylogeny or classof bacteriocin.

Class I bacteriocins. (i) Lanthipeptide clusters. Twenty-nine lanthipeptide geneclusters were detected in NAS genomes (Table 1). Fifteen clusters were classified as type

TABLE 1 (Continued)

Species (n)No. ofisolatesa Group ID

In vitro inhibition(n) No. of bacteriocin gene clusters

S. aureus MRSAb

Class I Class II

TotalLanthipeptide SactipeptideLassopeptide a b c d

6 SSC5 � (6) � 0 0 0 0 0 0 0 017 SSC6 � NT 0 0 0 0 0 0 0 0

S. simulans (42) 19 SSI1 � NT 0 0 0 0 0 0 1 11 SSI2 � NT 1 0 0 0 0 0 1 21* SSI3 � � 1 0 0 0 0 0 1 21* SSI4 � � 0 0 0 0 0 1 1 23 SSI5 � � 1 0 0 0 0 0 1 22 SSI6 � � 0 0 0 0 0 0 1 11 SSI7 � � 0 0 0 0 0 0 0 014 SSI8 � NT 0 0 0 0 0 0 0 0

S. succinus (15) 1 SSU1 � NT 1 0 0 0 0 0 0 114 SSU2 � NT 0 0 0 0 0 0 0 0

S. vitulinus (6) 1 SVI1 � NT 1 0 0 0 0 0 0 15 SVI2 � NT 0 0 0 0 0 0 0 0

S. warneri (19) 5 SWA1 � NT 0 0 0 0 0 0 1 11 SWA2 � � 0 0 0 0 0 0 0 013 SWA3 � NT 0 0 0 0 0 0 0 0

S. xylosus (28) 8 SXY1 � NT 0 0 0 0 0 0 1 11* SXY2 � � 0 0 0 0 0 1 0 119 SXY3 � NT 0 0 0 0 0 0 0 0

aAn asterisk indicates inhibitory activity was achieved with the chloroform-extracted product and inhibition was suppressed with the addition of proteinase K.bNT, not tested.

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FIG 1 Distribution of bacteriocin biosynthetic gene clusters in species of non-aureus staphylococci isolated from milk of Canadian dairycows, displayed on the phylogenetic tree from Naushad et al. (22). Bacteriocin types are indicated by the following abbreviations: L,lanthipeptide; S, sactipeptide; Ls, lasso peptide; II, class II double glycine leader peptides; C, circular bacteriocins; Lt, lactococcin-like.

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1 (Fig. 2); 12 of those clusters, all from S. capitis, had identical LanA structural peptides(Fig. 2). Six of the 12 potential S. capitis producers also were inhibitors. The 44-amino-acid (aa) precursor shared 59% identity with nisin (a Lactococcus lactis bacteriocin) andcontained the conserved domain pfam02052. Two of the remaining type 1 clusterscame from 2 inhibiting isolates of S. epidermidis, containing an identical 52-aa precursorpeptide, including the epidermin-conserved domain (TIGR03731), and shared 96%identity with epidermin (GenBank accession number P08136) (Fig. 2). The cluster in S.epidermidis 1778 additionally contained the LanFEG immunity system and a LanTprotein (Fig. 2). These 2 isolates were both the only inhibitors and only potentialproducers identified in S. epidermidis, suggesting that the bacteriocin was responsible

FIG 2 Biosynthetic gene clusters and LanA alignments of type 1 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 boldface and identical precursors in different clusterorganizations indicated by an asterisk. (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). �, the modal value of that column is shared by more than one residue.

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for in vitro inhibition (Fig. 3). The final type 1 lanthipeptide cluster identified wasdetected in noninhibitor S. equorum 1644. This cluster harbored a 57-aa precursor thatcontained the type 1 lanthipeptide conserved domain pfam08130 and shared 53%identity with Pep5 and 48% identity with epicidin 280 (Fig. 2). This precursor was notidentified in any of our other 440 isolates. Four of the 5 clusters contained a LanRregulator, whereas 3 of the 5 contained a LanP protease.

Fourteen of the 29 lanthipeptide clusters contained the type 2 LanM modificationsystem. Ten of those clusters contained single LanM proteins (Fig. 4). Of those 10clusters, 2 inhibitors from S. chromogenes contained 61-aa LanA1 and 82-aa A2 precursors,which contained the mersacidin conserved domain (pfam16934); the A1 precursor shared20% identity with the Cyl-L component of cytolysin produced by Enterococcus faecalis, and

FIG 3 Phylogenetic tree of Staphylococcus epidermidis isolates from bovine milk indicating growth inhibition against Staphylococcusaureus and genomically identified bacteriocin clusters. The percentage of trees in which the associated isolates clustered together isshown next to the branches, with branch lengths measured in numbers of substitutions per site. Phenotypically inhibiting isolates aresurrounded with a box. L, lanthipeptide bacteriocin gene cluster.

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the A2 precursor shared 26% identity with the Cyl-S component of cytolysin (Fig. 4),whereas A1 and A2 shared 40% identity with each other (Fig. 4). The S. chromogenes1348 cluster additionally contained a LanT transporter (Fig. 4). Staphylococcus cohnii 5contained a cluster with a 39-aa hypothetical protein that shared 19% identity withLanA from mersacidin, although no bacteriocin-associated conserved domains wereidentified during BLASTp analysis. Staphylococcus cohnii 1067 also had no identifiedstructural peptide, although a 41-aa hypothetical protein adjacent to LanM shared 21%identity with the S. cohnii 5 potential LanA and 25% with LanA from S. simulans 1336

FIG 4 Biosynthetic gene clusters and LanA alignments of type 2 lanthipeptides with a single LanM identified in non-aureus staphylococci isolated from milkof Canadian dairy cows. (A) Biosynthetic gene clusters of type 2 lanthipeptides, with inhibiting NAS isolates in boldface and identical precursors in various clusterorganizations within the same species indicated by an asterisk. (B) Multiple-sequence alignments of LanA genes identified in type 2 lanthipeptide clusters andknown 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).

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identified in this study. Staphylococcus simulans 1336, a noninhibitor, contained a2-component lanthipeptide, where the 68-aa A1 shared 56% identity with the Cyl-Lcomponent of cytolysin and the 68-aa A2 shared 57% identity with Cyl-L. Furthermore,A1 and A2 have 80% identity with one another and contained the 2-componentEnterococcus faecalis cytolysin (EFC) conserved domain (pfam16934). The next 3lanthipeptide clusters were from 3 inhibiting S. simulans isolates, and all containedidentical clusters with LanA peptides (conserved domain pfam04604) that wereidentical to NukA, the structural peptide for nukacin ISK-1 (GenBank accessionnumber Q9KWM4). Another cluster, in S. simulans 3061, contained a 105-aa hypothet-ical protein which may function as a LanA (Fig. 4) and shared 15% identity with apotential LanA in S. cohnii 5. Lastly, S. vitulinus 730 contained 2 LanA peptides, whichshared 53% identity with each other. Furthermore, A1 shared 35% identity with Cyl-Land A2 shared 32% identity with both Cyl-S and mersacidin. Three clusters containedan NADPH-dependent flavin mononucleotide (FMN) reductase (not normally associatedwith lanthipeptide clusters).

The remaining 4 lanthipeptide clusters were part of a different category of type2 lanthipeptides that contain 2 structural peptides, plus dual LanM proteins, whereeach LanM protein is responsible for modifying a distinct precursor peptide (Fig. 5).Staphylococcus succinus 6028 contained a 73-aa A1 precursor and a 68-aa A2precursor. Staphylococcus succinus 6028A1 shared 34% identity with lacticin 3147A1,whereas the A2 precursor shared 32% identity with lacticin 3147A2. In inhibitor S. sciuri225, the 63-aa A1 precursor shared 28% identity with lacticin 3147A1, and the 68-aa A2precursor shared 36% identity with lacticin 3147A2 and 40% identity with lichenicidinA2. Lastly, S. gallinarum 2094 and 1388 contained identical novel two-peptide type 2lanthipeptide biosynthetic clusters with 83-aa A1 precursors (conserved domainpfam14867) that shared 34% identity with lichenicidin A1 and 68-aa A2 precursors thatshared 43% identity with lichenicidin A2. The 2 precursors, A1 and A2, shared 21.9%identity with each other. Three of the 4 clusters contained a LanP protease.

(ii) Sactipeptide clusters. A total of 26 potential subtilosin A-like clusters wereidentified, although 23 were excluded due to the absence of the critical AlbF gene (18),leaving 3 S. capitis isolates that contained viable clusters (Table 1). In S. capitis 1319(noninhibitor), 2487 (inhibitor), and 3379 (inhibitor), the structural peptides all wereidentical and shared 63% identity with the subtilosin A precursor, SboA (accessionnumber O07623) (Fig. 6).

(iii) Lasso peptide clusters. Three noninhibiting NAS isolates, 2 S. fleurettii isolatesand 1 S. sciuri isolate, along with an inhibiting S. sciuri isolate, contained identicalclusters encoding a lasso peptide (Fig. 7). The 40-aa structural peptide, termed A,shared no identified conserved domains but shared 31% identity with a previouslycharacterized lasso peptide, lariatin, produced by Rhodococcus sp. strain K01-B0171(23).

Class II bacteriocins. (i) Double-glycine leader. Three S. equorum isolates and 1 S.

saprophyticus isolate (all noninhibitors) encoded bacteriocin clusters that contained 2precursor peptides that were annotated as bacteriocin class II with double-glycineleader peptides (Fig. 8). Although the precursor peptides in all 4 clusters were identical,additional associated proteins varied. The S. saprophyticus isolate and 2 of the S. sciuriclusters contained a SecA protein, possibly related to secretion.

(ii) Class IIc circular bacteriocin clusters. Nineteen isolates, 2 of which wereinhibitors (across various clades of NAS phylogeny; Fig. 1), contained circular bacteri-ocin 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 theclusters contained various bacteriocin-associated genes (Fig. 9). The S. gallinarumprecursor shared 25% identity with both gassericin and enterocin AS-48 (Fig. 9). All but2 of the clusters contained a signal peptidase. Staphylococcus haemolyticus 109 con-tained a 97-aa structural peptide sharing 25% identity with both circularin and ente-rocin AS-48. The cluster identified in inhibiting S. simulans 1355 contained a structural

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peptide (conserved domain TIGR03651) with 23% identity with enterocin AS-48. Thefinal cluster, identified in inhibiting S. xylosus 4938, contained a precursor with 32%identity with circularin A.

(iii) Class IId clusters. Forty-seven NAS genomes contained complete lactococcin-like clusters (Fig. 10). Of these 47 potential producers, 8 were inhibitors. Twenty-sevenlactococcin-like clusters were identified in 27 S. simulans isolates. Eleven isolatescontained a 94-aa structural precursor, whereas the remaining 15 contained a 105-aastructural precursor, with 88% identity with each other (Fig. 10).

Fourteen isolates contained clusters with the same organization of genes, althoughthe precursor gene (conserved domain pfam09683) varied slightly among isolates(Fig. 10). All 8 S. xylosus isolates in this group contained identical 93-aa structural genes

FIG 5 Biosynthetic gene clusters and LanA alignments of type 2 lanthipeptides with dual LanM enzymes identified in non-aureus staphylococci isolated frommilk of Canadian dairy cows. (A) Biosynthetic gene clusters of identified dual precursor type 2 lanthipeptides, with inhibiting NAS isolates in boldface. (B-1)Multiple-sequence alignments of LanA1 genes identified in type 2 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 genesidentified in type 2 lanthipeptide clusters and known bacteriocins lacticin 3147 A2 (accession number O87237), lichenicidin A2 (accession number P86476), andstaphylococcin C55 A2 (accession number Q9S4D2).

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with 62% identity with the structural gene identified in the 2 S. equorum clusters. TheS. hyicus cluster’s structural peptide shared 52% identity with the S. xylosus cluster’sstructural peptide. Lastly, 3 S. sciuri isolate structural peptides shared 43% identity withthe S. xylosus precursor.

An additional S. sciuri isolate with a structural peptide identical to that of theother S. sciuri isolates mentioned above was identified, although it lacked the ABCtransporter in the cluster. The remaining 5 clusters were identified in S. warneri. The95-aa putative precursor shared 60% identity with the 94-aa precursor from S.simulans (Fig. 10).

DISCUSSION

In this study, 95 isolates (22%) of 441 NAS encoded 105 bacteriocin biosyntheticgene clusters, making them potential bacteriocin producers. Bacteriocin gene clusterswere detected in 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. According to the phylogenetic tree,bacteriocin gene clusters are spread throughout the NAS phylogeny, although lassopeptides were only present in 2 species from the same clade and sactipeptides wereonly identified in S. capitis. In contrast, only 40 (9%) of 441 NAS were inhibitors whenantimicrobial activity was tested in vitro against a bovine clinical mastitis S. aureusisolate. We identified a higher percentage of NAS with antimicrobial activity than a

FIG 6 Biosynthetic gene clusters and alignments of precursor peptides from sactipeptides identified in non-aureus staphylococci isolated from milk of Canadiandairy cows. (A) Biosynthetic gene clusters of identified sactipeptides, with inhibiting non-aureus staphylococcal isolates indicated using boldface and identicalprecursors indicated by an asterisk. (B) Sequence alignment of the identified sactipeptide precursor and known sactipeptide subtilosin A (accession numberO07623).

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previous study that reported only 6.4% of NAS from bovine mastitis cases wereinhibitory (7). The NAS isolated from the teat apex skin of dairy cows may havepotential for greater bacteriocin production, as 13% of those isolates had antimicrobialproperties in one study (8).

Our study identified 29 lanthipeptide clusters from 29 NAS isolates. Both type 1 and2 lanthipeptides were identified in NAS genomes. Lanthipeptides are characterized bythe presence of several uncommon amino acids, including meso-lanthionine and

FIG 7 Biosynthetic gene clusters and alignments of precursor peptides from the lasso peptide identified in non-aureus staphylococciisolated from milk of Canadian dairy cows. (A) Biosynthetic gene cluster of the identified lasso peptide. (B) Sequence alignments of theidentified lasso peptide precursor and known lasso peptides lariatin (UniProtKB accession number H7C8I3) and microcin J25 (accessionnumber Q9X2V7).

FIG 8 Biosynthetic gene clusters of class II double glycine leader peptide bacteriocins identified in non-aureus staphylococci isolated from milk of Canadiandairy cows. Identical precursors are identified by an asterisk.

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3-methyl-lanthionine, the result of posttranslational modifications (24). For type 1lanthipeptides, dehydration of the serine and threonine residues to dehydroalanine(Dha) and dehydrobutyrine (Dhb) residues in the prepeptide, LanA, is completed by adehydratase (LanB). Thereafter, thioether cross-links are formed on the dehydratedamino acids by a cyclase (generically termed LanC), whereas each specific bacteriocinhas its individual naming system, for example, nisB and nisC for nisin. More than 50lanthipeptides from Gram-positive bacteria have been isolated and described (25), withgenome mining identifying many more potential compounds (26). Previously uncom-

FIG 9 Biosynthetic gene clusters and precursor alignments of class IIc circular bacteriocins identified in non-aureus staphylococci isolated from milk of Canadiandairy cows. (A) Biosynthetic gene clusters of identified class IIc bacteriocins, with inhibiting NAS isolates indicated in boldface and identical precursors in S.gallinarum indicated by asterisks. (B) Alignment of precursor peptides from identified class IIc bacteriocins and known bacteriocins carnocyclin A (accessionnumber B2MVM5), circularin A (accession number Q5L226 [Bactibase accession number BAC164]), enterocin AS-48 (accession number Q47765), gassericin(accession number O24790), and uberolysin (accession number A5H1G9).

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mon genes, e.g., those encoding FMN reductase and N-acetyltransferases, were iden-tified in some clusters in our study, which have been reported in multiple lanthipeptideclusters identified by M. Singh and D. Sareen (17). As we learn more about posttrans-lational modifications, these genes could help identify novel clusters in additionalgenomes. Type 1 lanthipeptides were identified in S. capitis, S. epidermidis, and S.equorum. The S. epidermidis strains harboring the bacteriocin biosynthetic gene clusteralso inhibited S. aureus. The structural gene had 96% similarity to the gene encodingepidermin, the lanthipeptide most frequently produced by NAS (27) and reported toinhibit human MRSA (28) and S. aureus isolated from bovine mastitis (29). Only 6 of 12S. capitis potential lanthipeptide producers were inhibitors; therefore, if it is in fact thebacteriocin identified that is responsible for the inhibition, perhaps the bacteriocincluster is not being expressed in the 6 noninhibitors. The S. equorum potential producerwas also a noninhibitor but harbors a gene that likely encodes a novel bacteriocin

FIG 10 Biosynthetic gene clusters and precursor alignments of class IId lactococcin-like bacteriocins identified in non-aureus staphylococci isolated frommilk of Canadian dairy cows. (A) Biosynthetic gene clusters of identified class IId bacteriocins, with inhibiting NAS isolates indicated in boldface, identicalprecursors in S. equorum indicated by a superscript letter A, and identical precursors in S. sciuri indicated by asterisks. (B) Alignments of precursorpeptides from identified class II bacteriocins and known bacteriocins lactococcin 972 (accession number O86283) and lactococcin A (accession numberP0A313).

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(similar to pep5 and epidicin 280). Type 1 lanthipeptide prepeptides typically have anFNLD conserved region at approximately positions �15 to �20 and a proline at �2from the cleavage site and have roles in modifications of the peptide (30). Only theLanA identified in the S. epidermidis strains had the FNLD conserved region and theproline at �2. The LanA identified in S. capitis had an FDLD motif with the proline at�2, similar to gallidermin and lanthipeptides recently identified in ruminal bacteria (18).However, the S. equorum LanA had an 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 beennukacin-like bacteriocin. In our study, nukacin was discovered in 3 inhibiting S. simulansisolates. The LanA precursor was identical to nukacin ISK-1 produced by S. warneri (31),nukacin 3299 produced by S. simulans (32), and nukacin L217 produced by S. chromo-genes (8). The findings that both nukacin 3299 and nukacin L217 producer strains werealso isolated from bovine milk and the potential nukacin producer in our study was ableto inhibit S. aureus suggest a role for this bacteriocin in NAS colonization and pathogeninhibition in the udder environment.

Type 2 lanthipeptides are characterized by a bifunctional enzyme, LanM, responsiblefor both dehydration and cyclization, whereas the C terminus shares homology withLanC cyclases of the type 1 lanthipeptides (25). Ten of 14 clusters identified containeda single LanM, with 4 containing 2 LanA precursors. The 2 LanA precursors in eachcluster had a high sequence identity (80% for the S. simulans 1336 cluster), suggestingthat the same single LanM enzyme could modify both precursors. For dual precursorswith low sequence identity (e.g., in lichenicidin and haloduracin), there are multipleLanM enzymes to modify each unique precursor (33, 34). These represent a distinctgroup of type 2 lanthipeptides, of which 3 were identified in our study. Here, A1 andA2 precursors shared 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 wasan inhibitor. Unlike type 2 lanthipeptides produced by ruminal bacteria (18), eight type2 clusters in this study harbored the LanP protease.

Sactipeptides are a group of class I bacteriocins that contain a sulfur-to-�-carbonlinkage, catalyzed by a recombinant S-adenosylmethionine (rSAM) protein (35, 36).They were originally only isolated from Bacillus species, although genome mining hasnow identified putative gene clusters in the genera Clostridium, Blautia, Kandleria,Lachnobacterium, Peptostreptococcus, Roseburia, and Ruminococcus (18, 21, 37). Onesactipeptide cluster was identified in 3 isolates in our study, although the bacteriocin-associated genes varied slightly between clusters. Our analysis identified a histidinekinase (HK) and response regulator (RR) in each cluster, indicating that bacteriocins aresubjected to a 2-component regulatory system, previously only reported in sactipep-tide clusters from ruminal bacteria (18). Two of the 3 cluster-containing isolates werealso inhibitors, although the S. capitis 3379 isolate additionally contained a lanthipep-tide that could be responsible for inhibition. The inhibiting S. capitis 2784 did not containany additional clusters to our knowledge, although subtilosin A, which this novel bacteri-ocin is most related to, moderately inhibited S. aureus in vitro (38). Thus, the noninhibitingS. capitis potential producer may not have been expressing its bacteriocin gene, or theinhibition by the other two isolates was due to another product.

Lasso peptides are an emerging group of RiPPs that do not undergo extensivemodification, although they are folded so that the C terminus is threaded through aring formed by a single isopeptide bond, yielding their signature lariat-like form (39). Tothe best of our knowledge, no lasso peptides have been identified in Staphylococcusspecies (40). In this study, an identical, novel cluster was identified in 3 noninhibitingisolates, 2 from S. fleurettii and 1 from S. sciuri, along with 1 inhibiting S. sciuri. Theidentified clusters contained all 4 of the essential enzymes (ABCD) for peptide produc-tion (19), a transglutaminase-like protein that is likely encoded by the B gene and actsas a protease to cleave the leader sequence, a protein with an asparagine synthaseconserved domain, which is likely encoded by the C gene responsible for isopeptide

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bond formation, and 2 units of an ABC-type dipeptide/oligopeptide/nickel transportsystem functioning as the D gene product, 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 uniquelanthipeptide precursors identified. To date, the majority of class II bacteriocins inStaphylococcus species have been identified in S. aureus (27). The first circular bacteri-ocin in Staphylococcus species, discovered in 2014, was aureocyclicin 4185 from S.aureus 4185 (41). Here, we identified putative novel circular bacteriocin gene clustersfrom S. gallinarum, S. haemolyticus, S. simulans, and S. xylosus. These bacteriocinprecursors had limited similarity to previously characterized circular bacteriocins, but allcontained the conserved domain associated with the circularin A/uberolysin family.Although the S. xylosus potential producer was not the only producer in the species(lactococcin-like clusters were also identified in other isolates), it was the only inhibitorin the species, suggesting that this bacteriocin is responsible for activity against S.aureus, although further investigation after isolation and purification of the peptide isneeded. The S. simulans isolate was also an inhibitor in vitro, but potential producersfrom S. gallinarum and S. haemolyticus were noninhibitors, suggesting these bacterio-cins either have a different spectrum of activity or were not activated.

Class IId bacteriocins are described as linear non-pediocin-like. Lactococcin 972belongs to this group and is produced by Lactococcus lactis subsp. lactis IPLA 972 (42).The gene cluster, along with the LclA precursor, encodes a transporter, LclB, and animmunity gene product (19). The 7 class IId lactococcin-like precursors all had variousdegrees of similarity with one another, but all contained the lactococcin 972 conserveddomain. Generally, lactococcin-like bacteriocins have a narrow spectrum of activityagainst Lactococcus species due to the nature of their binding to receptors (43),indicating it was unlikely that these potential producers would be inhibitory against S.aureus. Nonetheless, 8 of 47 lactococcin-like potential producers were inhibitors. Ofthese 8 inhibitors, 4 also contained a lanthipeptide cluster, whereas 1 also contained acircular bacteriocin cluster, which could have been the bacteriocins responsible for S.aureus inhibition. Remaining inhibitors could harbor an unidentified novel bacteriocinresponsible for inhibition or could be producing a nonbacteriocin inhibitory substance.All of these novel bacteriocins will need further assessments for spectrum of activityand biochemical characterization to complete identification.

When comparing phenotype and genotype, 95 NAS contained bacteriocin biosyn-thetic gene clusters, whereas only 40 of the NAS had inhibition toward S. aureus. Thiscould be due to several factors, including that bacteriocins produced were not effectiveagainst inhibiting S. aureus or the condition tested in vitro did not lead to sufficientlevels of bacteriocin production (44). This highlights a substantial benefit of genomemining, as variability of in vitro inhibition testing is negated, enabling clusters that maybe silent or repressed in vitro to be identified. Of the 40 inhibitors, 21 were identifiedas potential producers, and the peptide nature of the inhibitory product was confirmedin five of these isolates by elimination of the inhibition with the addition of proteinaseK. Further investigation into the remaining isolates should be carried out to optimizethe extraction conditions to obtain inhibition in the well diffusion assay and to confirmthe proteinaceous nature of the inhibitory compound. Bacteriocin gene clusters werenot detected for the remaining 19 inhibiting isolates, perhaps due to inhibition fromproduction of other inhibiting substances, e.g., low-molecular-weight antibiotics, lyticenzymes, or metabolic by-products (45). It could also be due to the nature of thedetection software, as identification of clusters using antiSMASH is based on similarityto previously described genes, with potential to miss completely novel clusters. How-ever, as knowledge increases regarding bacteriocin-associated genes and structuralprecursors, detection methods will improve and more bacteriocins will be described,allowing for even greater detection. In order to conduct the most comprehensiveanalysis of bacteriocins currently available, our analysis methods were ordered andcombined to maximize detection. By first using antiSMASH and BLAST searches usingthe BAGEL databases, we identified the bulk of the genomes containing bacteriocin

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gene clusters. We then used any precursor genes in clusters identified by antiSMASH forfurther BLAST searches in our whole genomes, which led to identification of additionallasso peptide clusters not detected by antiSMASH. Lastly, using our phenotypic results,comparing genomes of inhibitors to closely related noninhibitors in the samespecies, and using BLAST searches to analyze unique sequences in the inhibitor ledto identification of bacteriocin-associated genes in 6 of 19 inhibitors not initiallyidentified as producers. However, no complete clusters were identified in theseisolates due to the absence of peptide precursor genes in close proximity uponvisualization in Geneious.

In conclusion, all clusters identified, excluding the nukacin identified in S. simulansand the epidermin variant identified in S. epidermidis, were novel bacteriocin clusters,having less than 70% identity with previously described bacteriocins. The combinationof genome mining tools, such as antiSMASH along with BLAST searches, makesdiscovery of novel bacteriocins quicker and more comprehensive than conventionalapproaches. The identified putative producers should be further studied to characterizethe bacteriocins described here in order to elucidate structures, modes of action, andspectrum of activity. The NAS isolated from mammary origin are a rich source ofbacteriocins, with �21% being potential producers, thereby representing a promisingsource for future research and potential clinical applications.

MATERIALS AND METHODSIsolates. NAS isolates and an S. aureus isolate from a clinical case of bovine mastitis were collected

by the National Cohort of Dairy Farms (NCDF), conducted across Canada during 2007 and 2008, asdescribed by Reyher et al. (46). The Canadian Bovine Mastitis and Milk Quality Research Network(CBMQRN) at the University of Montreal stored the samples before sending them to the Universityof Calgary. Overall, 441 NAS isolates were selected from the stock of 5,507 isolates of the 25 NASspecies identified previously (22, 47). These isolates originated from 87 herds across Canada fromNova Scotia, Prince Edward Island, New Brunswick (representing Atlantic Canada), Québec andOntario (representing Central Canada), and Alberta (representing Western Canada) (46). Isolatesincluded 68 NAS isolates from clinical mastitis cases, 26 multidrug-resistant (MDR) isolates, amaximum of 1 isolate per cow of any uncommon species (defined as �20 unique isolates at cowlevel), and a maximum of 1 randomly selected isolate per cow for all other species until 441 werechosen (22). The multidrug-resistant MRSA clinical strain H176 was obtained from K. Zhang’slaboratory at the University of Calgary.

Phenotypic testing. All 441 NAS isolates were tested for antimicrobial activity against an S. aureusisolate (derived from clinical mastitis). Only the NAS isolates that inhibited this S. aureus strain weretested against the MRSA strain. Testing was done using a cross-streaking method, modified from aprevious report (5). Each isolate was plated on 5% defibrinated sheep blood agar plates (BD Diagnostics,Mississauga, ON, Canada) and incubated overnight at 37°C. A single colony was diluted in phosphate-buffered saline (PBS) to a McFarland 0.5 standard and was used to inoculate a center streak (5 mm) ona 5% sheep blood agar plate and subsequently incubated at 37°C for 24 h. On day 2, the agar wasloosened from the plate with sterile metal tweezers and flipped onto the lid of the plate so the NAScenter streak was face down. A 100-�l volume of a 10�3 dilution in PBS of a McFarland 0.5 standard ofa single colony from an overnight culture on 5% sheep blood agar of S. aureus then was spread over theentire agar surface and incubated at 37°C 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 anegative control (PBS was used to make the center streak on day 1).

Effect of proteinase K on inhibition. Strains that were both inhibitors of S. aureus and potentialbacteriocin producers were tested to confirm the peptide nature of the inhibitory product usingproteinase K (20 mg ml�1; Sigma-Aldrich) and an agar well diffusion assay (48). Concentrated cell-freesupernatant was obtained from brain heart infusion (BHI) cultures of the 21 inhibiting and potentiallyproducing NAS isolates by performing a modified version of chloroform extraction as described previ-ously (49). Briefly, 40 ml of BHI broth was inoculated with 0.1% of an overnight culture of NAS andincubated at 37°C for approximately 20 h. Cells were removed by centrifugation at 4,500 � g at 4°C for15 min. Twenty milliliters of chloroform (Sigma-Aldrich) was mixed with the cell-free supernatant of eachNAS and stirred vigorously for 20 min, followed by centrifugation at 4,500 � g at 4°C for 15 min. Thecloudy interfacial precipitate was collected and dried overnight. The dried product was dissolved in 1 mlPBS using a magnetic spinner at 4°C for 24 h. Proteinase K was added to a sample of each concentratedcell-free supernatant, and samples with and without proteinase K were incubated at 37°C for 1 h. Residualinhibition was tested using an agar well diffusion assay (48) using the same bovine S. aureus isolate asused in the cross-streaking test.

Whole-genome sequencing, assembly, and annotation. Sequencing, assembly, and annotation forNAS and the clinical mastitis S. aureus isolate were performed as described previously (22). Briefly,genomic DNA was extracted with a DNeasy blood and tissue kit (Qiagen, Toronto, ON, Canada) accordingto the corresponding protocol for Gram-positive bacteria. Sequencing of these samples was performed

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using the Illumina MiSeq platform (Illumina, San Diego, CA, USA); DNA libraries for sequencing wereprepared using a Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). All sequencingsteps, including cluster generation, paired-end sequencing (2 by 250 bp), and primary data analysis forquality control, were performed on the instrument. Genome assembly was automated using theSnakemake workflow engine (50). Raw read pairs were screened for adapters and quality trimmed usingCutadapt 1.8.3 (51) as implemented in Trim Galore! 0.4.0 (with default parameters). Genomes wereassembled using Spades 3.6.0 (52) using the built-in error correction and default parameters. To assesscoverage, reads were mapped back to the assembled genome using BWA 0.7.12-r1039 (53). Contigs of�200 bp were annotated with Prokka 1.11 (54) using the provided Staphylococcus database. Assemblyquality was evaluated with Quast 3.0 (55). Contigs, as well as the annotated protein sequences, were usedfor custom BLAST searches using SequenceServer (56).

Screening of genomes for bacteriocin clusters. Identification of biosynthetic gene clusters relatedto secondary metabolite production and analysis of sequences of interest was done using antiSMASH 3(57). 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 ConservedDomain Database (58) was noted in each coding region and compared to previously identified knownconserved domains in bacteriocin-associated genes (18). Putative gene clusters were classified accordingto Cotter et al. (10). Additionally, any identified structural genes in the NAS genomes were used forfurther BLAST searches against our NAS genomes to potentially identify any clusters not detected byantiSMASH.

BLAST. The 441 NAS whole genomes were assessed by BLAST for any bacteriocin structural genescontained in class 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 searchwere visualized using Geneious version 8.1.6 (59) to determine if the bacteriocin gene cluster wascomplete by assessing if the structural gene and known essential associated genes were present usingthe BLASTP web server on NCBI (http://www.ncbi.nlm.nih.gov/BLAST).

Genome comparison. To further identify potential bacteriocin-associated genes, genomes of theinhibiting NAS were compared to closely related genomes of noninhibiting NAS of the same speciesaccording to the phylogenetic trees of each species. For this purpose, the phylogenetic trees for eachspecies of NAS were constructed using methods described previously (22). Briefly, trees were rootedusing Macrococcus caseolyticus and created based on the core genome of the individual NAS species. Thecore set was identified using the UCLUST algorithm (60), and protein families with at least 30% sequenceidentity and 50% sequence length were considered core. However, protein families present in �95% ofthe input genomes were considered core, and protein families containing potential paralogous se-quences (duplicated sequence in the same genome) were excluded. Each protein family was individuallyaligned using MAFFT 7 (61). Aligned amino acid positions which contained gaps in more than 50% ofgenomes were excluded from further analysis. Remaining amino acid positions were concatenated tocreate a combined data set. A maximum-likelihood tree based on this alignment was constructed usingFastTree 2.1 (62) using the Whelan and Goldman substitution model (63).

Comparisons were done by identifying shared genes, present in both closely related inhibiting andnoninhibiting isolates, using Spine, a Web-based application that identifies common sequences in theinput genomes (64). Sequences unique to inhibiting isolates were then determined using AGEnt bysubtracting the output of shared sequences acquired from Spine from the genome of an inhibitingisolate (64). Sequences unique to inhibiting isolates were then visualized using Geneious version 8.1.6(59), 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 (58) to establish any additionalbacteriocin-associated sequences.

Precursor gene alignments. Protein alignments of precursor peptides were generated usingMUSCLE (65). Sequence alignments were viewed and edited with Jalview alignment editor (66).

Accession number(s). Data were previously submitted to NCBI under BioProject numberPRJNA342349.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01015-17.

SUPPLEMENTAL FILE 1, PDF file, 1.9 MB.

ACKNOWLEDGMENTSWe thank all dairy producers, animal health technicians, and Canadian Bovine

Mastitis and Milk Quality Research Network (CBMQRN) regional coordinators (Trevor DeVries, University of Guelph, Canada; Jean-Philippe Roy and Luc Des Côteaux, Universityof Montreal, Canada; Kristen Reyher, University of Prince Edward Island, Canada; andHerman Barkema, University of Calgary, Canada) who participated in data collection.The bacterial isolates were provided by the CBMQRN. We also thank Matthew Worken-tine for his assistance with bioinformatics and Aaron Lucko for his work in thelaboratory. Finally, we thank John Kastelic for editing the manuscript.

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This work was partially funded through the NSERC Industrial Research Chair inInfectious Diseases of Dairy Cattle. This project was also part of the Canadian BovineMastitis and Milk Quality Research Network (CBMQRN) program, funded by DairyFarmers of Canada and Agriculture and Agri-Food Canada through the Dairy ResearchCluster 2 Program. The CBMQRN pathogen and data collections were financed by theNatural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada),Alberta Milk (Edmonton, AB, Canada), Dairy Farmers of New Brunswick (Sussex, NewBrunswick, Canada), Dairy Farmers of Nova Scotia (Lower Truro, NS, Canada), DairyFarmers of Ontario (Mississauga, ON, Canada), Dairy Farmers of Prince Edward Island(Charlottetown, PE, Canada), Novalait, Inc. (Quebec City, QC, Canada), Dairy Farmers ofCanada (Ottawa, ON, Canada), Canadian Dairy Network (Guelph, ON, Canada), Agricul-ture and Agri-Food Canada (Ottawa, ON, Canada), Public Health Agency of Canada(Ottawa, ON, Canada), Technology PEI, Inc. (Charlottetown, PE, Canada), Université deMontréal (Montréal, QC, Canada), and University of Prince Edward Island (Charlotte-town, PE, Canada) through the CBMQRN (Saint-Hyacinthe, QC, Canada). D.A.C. and S.N.were supported by an NSERC-CREATE in milk quality scholarship.

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