Comprehensive analysis of bacteriocins produced by clinical enterococcal isolates and their antibacterial activity against Enterococci including VRE

Abstract

It is well-known that Enterococcus species produce bacteriocins that have antibacterial activity. However, a comprehensive analysis of the bacteriocin distribution among Enterococcus strains has not been conducted. In this study, we identified the bacteriocin genes from 80 Enterococcus faecalis and 38 Enterococcus faecium strains and investigated their antibacterial activity. In the 80 E. faecalis strains, the cytolysin gene (61.3%), enterolysin A gene (27.5%) and BacL₁ gene (45.0%) were identified. In the 38 E. faecium strains, the enterocin A gene (97.4%), enterocin B gene (2.6%), enterocin NKR-5-3B gene (21.0%), bacteriocin T8 gene (36.8%) and BacAS9 gene (23.7%) were identified. The antibacterial activity of all strains was tested against E. faecalis and E. faecium. The strains positive for the cytolysin, enterolysin A, BacL₁, bacteriocin T8 or BacAS9 genes presented variable antibacterial activity. Several bacteriocin-positive strains showed antibacterial activity against other enterococcal species, but not against Staphylococcus or Escherichia coli. In addition, the enterolysin A-positive strain showed antibacterial activity against vancomycin-resistant E. faecium, and the bacteriocin T8- or BacAS9-positive strains showed activity against vancomycin-resistant E. faecalis and E. faecium. Our findings suggest that E. faecium and E. faecalis strains that carry different bacteriocin genes may affect the composition of the surrounding bacterial community.

Fig. 1

Alignments of the cytolysin-encoding genes of the 3 E. faecalis strains and amino acid alignments of the three cytolysin types. (a) The cytolysin gene cluster (cytL-L1 and cytL-S1, cyt-LS-fusion) and its neighboring region were extracted from 3 strains (JARB-HU0762, HU0680, HU0828) and compared. The grayscale indicates the percent sequence identity. (b) Amino acid alignment of three types of cytolysin, namely, CytL-La + CytL-Sa, CytL-Lb + CytL-Sb and cytolysin-LS-fusion.

Fig. 2

Evaluation of the antibacterial activity of E. faecalis and E. faecium strains harboring bacteriocin genes via a soft-agar overlay assay. A soft-agar overlay assay was performed to evaluate the antibacterial activity against E. faecalis JARB-HU0683 and E. faecium JARB-HU0748. Overnight cultures of enterococcal strains (2.0 µl) were spotted onto TSA plates and incubated for 24 h at 37 °C. The indicator strain was added to 3.0 mL of prewarmed soft TSA agar medium, and the mixed medium was poured onto agar plates containing the previously spotted colonies and incubated aerobically overnight at 37 °C. The diameter of the growth-inhibitory zone surrounding the bacteriocin-producing bacteria was measured.

Fig. 3

Antibacterial activity of the E. faecalis and E. faecium strains that carry bacteriocin genes against E. faecalis and E. faecium. A soft-agar overlay assay was performed using E. faecalis JARB-HU0683 (a, c) and E. faecium JARB-HU0748 (b, d). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (one-way ANOVA for comparisons between groups by Dunnett’s post hoc multiple comparison test).

Fig. 4

Expression of bacteriocin genes and antibacterial activity of Enterococcus strains with or without bacteriocin genes against several bacterial species. Antibacterial activity and bacteriocin gene expression of representative enterococcal strains. RNA extraction and quantitative PCR were performed on representative isolates of each bacteriocin type to observe the correlation between the zones of inhibition and gene expression. * JARB-HU notation is abbreviated to HU.

Fig. 5

Phylogenetic tree analysis of E. faecalis and E. faecium. Phylogenetic tree analysis of E. faecalis (a) and E. faecium (b) strains was performed, and the relationships among the phylogenetic tree results, MLST types and bacteriocin genes were analyzed.

Fig. 6

Antibacterial activity of bacteriocin-producing strains against VRE. A soft-agar overlay assay was performed using 3 E. faecalis strains, including 2 vancomycin-resistant strains, and 7 E. faecium strains, including 6 vancomycin-resistant strains. VS: vancomycin susceptible, VR: vancomycin resistant, EFs: E. faecalis, EFm: E. faecium. VSEFs1: JARB-HU0683 (no bacteriocin gene), VSEFm1: JARB-HU0748 (no bacteriocin gene), VREFs1, 2: vanA, cytL-L, cytL-S and bacL₁ positive, VREFm1-4: vanA, enterocin A and bacteriocin T8 positive, VREFm5, 6: vanB, enterocin A and bacteriocin T8 positive.

Fig. 7

Amino acid alignment of BacAS9 and similar bacteriocins. Amino acids alignment of 4 bacteriocins is shown. Gray background shows the consensus region among bacteriocins.