Comprehensive analysis of Enterococcus spp. from two European healthy infant cohorts shows stable genomic traits including antimicrobial resistance (AMR)

Abstract

Enterococcus spp. some of which are pathogenic, are common gut microbiota members, including also infants. Infants may be more susceptible to Enterococcus due to their developing gut ecosystems. It is unclear whether antibiotic resistance genes (ARGs) and certain genomic traits in enterococci are restricted to the human subpopulation or more widespread. Furthermore, the correlation between these traits and geographic variation is poorly understood. Therefore, we sequenced 100 strains isolated from full-term healthy infants' fecal samples from two geographically distant European cohorts (MAMI in Spain and LucKi from the Netherlands) to explore the diversity of Enterococcus spp. within the infant's gut microbiome and assess cohort-specific traits such as ARGs. Most isolates were E. faecalis and E. gallinarum, with a total of 11 species identified. We found a rich reservoir of ARGs, plasmids, prophages and virulence factors in the infant strains, with minimal cohort-specific differences in resistome profiles. In addition, Epx, a pore-forming toxin associated with pathogenicity, was found in E. hirae strains. While metabolic profiles were similar across cohorts, E. faecalis strains harbored more virulence genes and prophages compared to other species. An analysis of public Enterococcus genomes revealed that multi-drug resistant (MDR) strains exist without any significant geographic or temporal pattern. Phenotypic resistance analysis indicated that 28% of MAMI strains were gentamicin resistant, compared to 5% of the strains from the LucKi cohort, though LucKi isolates were also resistant to other antibiotics. We also selected ten E. faecalis isolates with varying virulence gene repertoires for phenotypic virulence testing in Caenorhabditis elegans and found them killing at various rates, however no clear pattern emerged in correlation with any specific genetic determinant. Overall, our results suggest that Enterococcus spp. including ARGs, are highly mobile across Europe and beyond. Their adaptability likely facilitates long-distance dissemination, with strains being acquired early in life from community environments.

Keywords: AMR; Enterococcus; genomics; healthy infants; phylogenomics; resistome.

Figure 1.

Left, cladogram (rooted at midpoint) using mash distances, based on distances between any two genomes of Enterococcus, using C. perfringens as outgroup. The tree is color coded for the species. Next, columns indicate birth mode – vaginal (light green), C-emergency (dark green) and C-elective (black); the cohort genomes for MAMI (black), LucKi (red) and reference genomes (yellow); the number of total and intact prophages (darker color, more prophages). The matrix to the right shows, presence (color) or absence (no color) for antimicrobial resistance genes, according to CARD (pink); plasmids, according to PlasmidFinder (purple); and virulence genes according to VFDB (brown).

Figure 2.

Phenotypic antibiotics resistance profiles of enterococci from the two cohorts. All strains were sensitive to linezolid. (a) Number of resistant isolates within each species, broken down into antibiotics (non-susceptible species against each antibiotics are not shown); black asterisk to indicate MAMI and red asterisk for LucKi; for gentamicin – its MAMI (pink) and LucKi (checkered pink). (b) Comparison of resistant strains concerning cohorts for tested antibiotics. Black; MAMI and Red; LucKi.

Figure 3.

Pangenome of 61 genomes of E. faecalis (showing gene presence/absence) revealing 5,640 gene clusters of 3402 accessory and 2238 core genes in our isolates plus the type strain (E. faecalis DSM 20478, GCF_000392875.1). The blue heatmap with dendogram displayed above the pangenome presents the average nucleotide identity (ANI); below this are layers representing number of gene clusters, singletons, redundancy, GC content and total length. Outer rings represent core genes, total genes in gene cluster, combined, functional and geometric homogeneity index and functional annotations (CAZyme, COG20, KOfam and KEGG). Functional homogeneity indicates how conserved aligned amino acid residues across genes are. Geometric homogeneity compares the positions of gaps in the aligned residues without considering specific amino acids.

Figure 4.

Heatmap indicating presence of functional modules along with their completeness concerning each biochemical pathway in the E. faecalis metabolism (excerpt, full heatmap in supplementary Figure S1). On top, the cohort is indicated: black, MAMI; red, LucKi, while the yellow labeled genome indicates the reference genome of E. faecalis. The class of the pathway is also indicated by shades of teal: lighter teal refers to ‘pathway modules’ (functional units of gene sets in metabolic pathways, including molecular complexes), while darker teal refers to ‘signature modules’ (functional units of gene sets that characterize phenotypic features). In the heatmap itself, darker shades indicate complete or near complete pathways, while lighter shades indicate a low percent pathway completeness. Thus, only a few genes are shared concerning a specific pathway found in the KEGG database (KO).

Figure 5.

The bar plot illustrates the enriched functions identified by anvi-compute-functional-enrichment program using four different sources: cog20_function, KEGG_Module, KEGG_Britte, and KOfam. Each bar represents a specific function (terms of molecular function, cellular component and biological process) with the bar length indicating the enrichment score and the significance denoted by the color-coded p-value; <0.05 was considered to be significant.

Figure 6.

Global Enterococcus diversity and distribution (a) midpoint rooted tree of 687 Enterococcus genomes (587 public genomes and 100 genomes from this study). A total of 15 species are found in public genomes from over 30 countries, with the first isolate from 1900 until 2023. Ten highly prevalent plasmids (DOp1, pNb2354p1, pRE25, pRUM, pB82, pE1p13, pAmalpha1, pAD1, p200B, and pQY003) were selected and a presence/absence matrix is shown as inner circle next to cohorts (black, presence; pink, absence); the number of total and intact prophages (darker color, more prophages; next bar is of continents followed by dates of collection. (b) Resistance of all public Enterococcus from panel a sorted for prevalence. (c) Host distribution among different Enterococcus species (public and infants) with highest number of hosts occupied by E. faecalis and E. faecium. (d) A world map shows the prevalence of 587 public strains in each region.