Genomic insights into antibiotic-resistance and virulence genes of Enterococcus faecium strains from the gut of Apis mellifera

Abstract

Enterococcus faecium is a lactic acid bacterium that confers beneficial health effects in humans. However, lately, a number of E. faecium strains have been linked to the spread of nosocomial infections in the hospital environment. Therefore, any potential commercial usage of E. faecium isolates should be preceded by an assessment of infection risk. In the current study, the genomes of two novel E. faecium strains Am1 (larval isolate) and Bee9 (adult bee isolate) isolated from the gut of Apis mellifera L. (honeybee) were sequenced to allow evaluation of their safety. In particular, their genomes were screened for antibiotic-resistance and virulence genes. In addition, their potential to spread resistance in the environment was evaluated. The analysis revealed that Am1 and Bee9 possess 2832 and 2844 protein-encoding genes, respectively. In each case, the genome size was 2.7 Mb with a G+C content of 37.9 mol%. Comparative analysis with probiotic, non-pathogenic and pathogenic enterococci revealed that there are variations between the two bee E. faecium isolates and pathogenic genomes. They were, however, closely linked to the probiotic comparison strains. Phenotypically, the Am1 and Bee9 strains were susceptible to most antibiotics tested, but showed intermediate sensitivity towards erythromycin, linezolid and trimethoprim/sulfamethoxazole. Notably, no genes associated with antibiotic resistance in clinical isolates (e.g. vancomycin resistance: vanA, vanB, vanS, vanX and vanY) were present. In addition, the insertion sequences (IS16, ISEfa11 and ISEfa5), acting as molecular pathogenicity markers in clinically relevant E. faecium strains, were also absent. Moreover, the analysis revealed the absence of three key pathogenicity-associated genes (acm, sgrA, ecbA) in the Am1 and Bee9 strains that are found in the prominent clinical isolates DO, V1836, Aus0004 and Aus0085. Overall, the findings of this investigation suggest that the E. faecium isolates from the bee gut have not suffered any recent clinically relevant antibiotic exposure. It also suggests that E. faecium Am1 and Bee9 are safe potential probiotic strains, because they lack the phenotypic and genetic features associated with strains eliciting nosocomial infections.

Keywords: Apis mellifera; Enterococcus faecium; antibiotic resistance; comparative genomics; lactic acid bacteria (LAB); virulence.

Fig. 1.

Growth of E. faecium Am1 (a) and Bee9 (c) on the surface of blood agar after 24 h incubation at 37 °C. (b) and (d) demonstrate SEM examinations of Am1 and Bee9 cells, respectively.

Fig. 2.

Subsystem categories and features distribution of the E. faecium genomes based on the RASTk annotation server. For each E. faecium strain, the number of genes identified in each category is displayed. The colour scale represents the number of genes found in each category; the deeper the colour, the more genes were detected in that category.

Fig. 3.

Phylogenetic analyses of E. faecium Am1 and Bee9 with closely related homologues inferred using the maximum-likelihood method (a) and Bayesian inference (b). The evolutionary analyses were carried out in phylogeny.fr (available at https://www.phylogeny.fr/), with tree building using PhyML and MrBayes programs, respectively. The evolutionary trees were graphically displayed using the FigTree program (available at http://tree.bio.ed.ac.uk/software/figtree/).

Fig. 4.

blast Atlas overview of E. faecium (Am1 and Bee9) and selected genomes from the NCBI database based on blastn. The outermost circles demonstrate the two E. faecium , Am1 (red) and Bee9 (brown), and the genomes of strains used in comparison: SM21 (orange), T110 (dark green), FS86 (light green), NRRL B-2354 (purple), Aus0085 (blue), Aus0004 (red violet), V1836 (light purple). Strain E. faecium DO (navy blue) was used as a reference genome. The innermost circles represent the G+C content (black), G+C skew curve (violet) and Clusters of Orthologous Genes (COG) categories. The genome sequences were analysed using the GView server using both alignment length and percent identity cut-off values of 80 %.

Fig. 5.

Heat map representation of virulence genes/factors and antibiotic-resistance genes identified in E. faecium Am1 and Bee9 using the VFanalyzer platform and the k-mer-based ARG detection method available through PATRIC, respectively. The dark and light blue colours represent present and absent genes, respectively.

Fig. 6.

Analysis of the GIs of E. faecium Am1 (a) and Bee9 (b) predicted by IslandViewer web server. The predicted GIs are shown in different colours within the circular image based on the tools used: sigi-hmm, which predicts GIs based on a hidden Markov model (orange); IslandPath-DIMOB, which predicts GIs based on features associated with GIs (blue); and an integration of three methods (red).

Fig. 7.

Putative bacteriocin-related gene clusters using the BAGEL4 web server. (a, b) Predicted enterocin P (EntP) and enterolysin A (EnlA) in E. faecium Am1 genome; (c, d) Predicted EntP and EnlA in E. faecium Bee9 genome.

Fig. 8.

Examination of some LAB characteristics. Histograms in (a) and (b) show the hydrophobicity and autoaggregation percentage achieved by E. faecium Am1 and Bee9. The histogram shows the percentages obtained by L. plantarum strain 10CH and L. plantarum laboratory isolate (+ve C). The results of the antibiotic disc diffusion method for Am1 and Bee9 are shown in (c) and (d), respectively. The (e), (f), (g) and (h) plates demonstrate the antagonistic activity of Am1 and Bee9 neutralized CFS against S. aureus (ATCC 25923), Escherichia coli (ATCC 8739), P. aeruginosa (ATCC 27853) and B. subtilis (ATCC 6633), respectively.