Diverse Bacteriocins Produced by Strains From the Human Milk Microbiota

Abstract

Microbial colonization of the infant gut is a convoluted process dependent on numerous contributing factors, including age, mode of delivery and diet among others that has lifelong implication for human health. Breast milk also contains a microbiome which acts as a source of colonizing bacteria for the infant. Here, we demonstrate that human milk harbors a wide diversity of bacteriocin-producing strains with the potential to compete among the developing gut microbiota of the infant. We screened 37 human milk samples and found isolates with antimicrobial activity and distinct cross-immunity profiles. From these isolates, we detected 73 putative gene clusters for bacteriocins of all known sub-classes, including 16 novel prepeptides. More specifically, we detected two novel lantibiotics, four sactibiotics and three class IIa bacteriocins with an unusual modification of the pediocin box that is composed of YDNGI instead of the highly conserved motif YGNGV. Moreover, we identified a novel class IIb bacteriocin, four novel class IIc and two class IId bacteriocins. In conclusion, human milk contains a variety of bacteriocin-producing strains which may provide them a competitive advantage in the colonization of the infant gut and suggests that the milk microbiota is a source of antimicrobial potential.

Keywords: antibiotic resistance; antimicrobials; bacteriocins; genome mining; human microbiota; human milk; lantibiotics; sactibiotics.

FIGURE 1

Representative zones of inhibition from a selection of the 29 antimicrobial producers that demonstrated the strongest inhibition against S. aureus APC 3759 and M. luteus DSM 1790 against a selection of the 29 indicator strains.

FIGURE 2

Flow chart of bacteriocin screening performed for this study. PBGC stands for Potential Bacteriocin Gene Cluster.

FIGURE 3

Multiple Sequence Alignments (MSA) of lantibiotic prepeptides and biosynthetic gene clusters. (A) Class IA MSA and predicted biosynthetic gene cluster. Sep stands for S. epidermidis. The same prepeptide was identified in S. epidermidis APC 3775 and APC 3810. (B) Class IB MSA and predicted biosynthetic gene clusters. Shom and Efaec stand for S. hominis and E. faecalis respectively.

FIGURE 4

Multiple Sequence Alignments (MSA) of sactibiotic prepeptides and biosynthetic gene clusters. (A) MSA of the identified sactibiotic prepeptides and the known sactibiotic hyicin. Sep stands for S. epidermidis. The same prepeptide was identified in S. epidermidis APC 3785, APC 3796, APC 3797, and APC 3883. The same applied for the prepeptide identified in S. epidermidis APC 3775 and APC 3882. (B) Biosynthetic gene clusters of identified sactibiotics.

FIGURE 5

Multiple Sequence Alignments (MSA) of class IIa bacteriocin prepeptides and biosynthetic gene clusters. (A) MSA of the identified class IIa prepeptides and known bacteriocins Enterocin P (Uniprot accession number O30434) and Enterocin A (Uniprot accession number C8C4N1). Ef stands for E. faecium. The same prepeptide was detected in E. faecium APC 3826 and APC 3828. The same applied for E. faecium APC 3827, APC 3832, APC 3836, and APC 3837. Moreover, an identical prepeptide was found in the genomes of E. faecium APC 3830, APC 3833, APC 3835, and APC 3880. (B) Biosynthetic gene clusters of identified class IIa bacteriocins. Class IIa-related proteins are surrounded by a solix box while class IIc-related proteins are encompassed in a dashed box.

FIGURE 6

Multiple Sequence Alignments (MSA) of class IIb bacteriocin prepeptides and biosynthetic gene clusters. (A) MSA of the identified class IIb α prepeptides and known bacteriocins Enterocin 1071 (Uniprot accession number for α: Q9L9U6) and Lactococcin G (Uniprot accession number for α: C5IHS6). (B) MSA of the identified class IIb β prepeptides and known bacteriocins Enterocin 1071 (Uniprot accession number for β: Q9L9U5) and Lactococcin G (Uniprot accession number for β: C5IHS7). Efaec stands for E. faecalis and Sep corresponds to S. epidermidis. The same prepeptides were identified in S. epidermidis APC 3779, APC 3801, and APC 3802. (C) Biosynthetic gene clusters of identified class IIb bacteriocins.

FIGURE 7

Multiple Sequence Alignments (MSA) of class IIc bacteriocin prepeptides and biosynthetic gene clusters. (A) MSA of the identified class IIc prepeptides and known bacteriocin EnterocinNKR-5-3B (Uniprot accession number A0A0P0YL94). Sep corresponds to S. epidermidis, Sa stands for S. aureus and Ef for E. faecium. The same prepeptide was identified in S. epidermidis APC 3782, APC 3804, and APC 3805. The same prepeptide was detected in S. aureus APC 3813, APC 3814, APC 3815, APC 3818, APC 3819, APC 3820, APC 3822, APC 3884, APC 3886, and APC 3887. Also, identical prepeptide was found in the genomes of E. faecium APC 3826, APC 3827, APC 3828, APC 3830, APC 3831, APC 3832, APC 3833, APC 3835, APC 3836, APC 3837, and APC 3880. (B) Biosynthetic gene clusters of identified class IIc bacteriocins. Class IIc-related proteins are surrounded by a dashed box while class IIa-related proteins are encompassed in a solid box.

FIGURE 8

Multiple Sequence Alignments (MSA) of class IId bacteriocin prepeptides and biosynthetic gene clusters. (A) MSA of the identified class IId prepeptides and known bacteriocin Lactococcin 972 (Uniprot accession number O86283). Sep corresponds to S. epidermidis and Sa stands for S. aureus. The same prepeptide was identified in S. epidermidis APC 3775, APC 3782, APC 3804, APC 3805, APC 3806, and APC 3810. The same prepeptides were detected in S. aureus APC 3774, APC 3809, APC 3812, APC 3813, APC 3816, APC 3820, and APC 3823. (B) Biosynthetic gene clusters of identified class IId bacteriocins.