Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk

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 26 species) against bovine Staphylococcus 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 noninhibiting 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 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 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 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications.IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs 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 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 MRSA. In NAS whole-genome sequences, we identified >21% of NAS as having bacteriocin genes. Our study represents a foundation to further explore NAS bacteriocins for clinical use.

Keywords: Staphylococcus; Staphylococcus aureus; bacteriocins; cattle; coagulase-negative staphylococci; mastitis.

FIG 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. (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.

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 cluster organizations 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.

FIG 3

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

FIG 4

Biosynthetic gene clusters and LanA alignments of type 2 lanthipeptides with a single LanM identified in non-aureus staphylococci isolated from milk of Canadian dairy cows. (A) Biosynthetic gene clusters of type 2 lanthipeptides, with inhibiting NAS isolates in boldface and identical precursors in various cluster organizations within the same species indicated by an asterisk. (B) Multiple-sequence alignments of LanA genes identified in type 2 lanthipeptide clusters and known bacteriocins cinnamycin (accession number P29827), cytolysin-L (accession number H7C7B0), cytolysin-S (accession number H7C7B5), mersacidin (accession number P43683), and nukacin ISK-1 (accession number Q9KWM4).

FIG 5

Biosynthetic gene clusters and LanA alignments of type 2 lanthipeptides with dual LanM enzymes identified in non-aureus staphylococci isolated from milk 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 genes identified in type 2 lanthipeptide clusters and known bacteriocins lacticin 3147 A2 (accession number O87237), lichenicidin A2 (accession number P86476), and staphylococcin C55 A2 (accession number Q9S4D2).

FIG 6

Biosynthetic gene clusters and alignments of precursor peptides from sactipeptides identified in non-aureus staphylococci isolated from milk of Canadian dairy cows. (A) Biosynthetic gene clusters of identified sactipeptides, with inhibiting non-aureus staphylococcal isolates indicated using boldface and identical precursors indicated by an asterisk. (B) Sequence alignment of the identified sactipeptide precursor and known sactipeptide subtilosin A (accession number O07623).

FIG 7

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

FIG 8

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

FIG 9

Biosynthetic gene clusters and precursor alignments of class IIc circular bacteriocins identified in non-aureus staphylococci isolated from milk of Canadian dairy 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 (accession number 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).

FIG 10

Biosynthetic gene clusters and precursor alignments of class IId lactococcin-like bacteriocins identified in non-aureus staphylococci isolated from milk of Canadian dairy cows. (A) Biosynthetic gene clusters of identified class IId bacteriocins, with inhibiting NAS isolates indicated in boldface, identical precursors in S. equorum indicated by a superscript letter A, and identical precursors in S. sciuri indicated by asterisks. (B) Alignments of precursor peptides from identified class II bacteriocins and known bacteriocins lactococcin 972 (accession number O86283) and lactococcin A (accession number P0A313).