Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium

Abstract

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium. Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faecium. IMPORTANCE Enterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Conditionally essential genes for survival during starvation inEnterococcus faeciumE745

“…Abricate (https://github.com/tseemann/abricate, version 0.8) was used to identify presence of the 18 E. faecium E745 genes that had the largest role in survival in PBS, as determined by Tn-seq, against the draft assemblies from 1,644 E. faecium genomes that represent the global diversity of the species E. faecium [58]. We considered a minimum identity and coverage of 95% and 80%, respectively, to consider a gene as present in a particular draft assembly.…”

“…We considered a minimum identity and coverage of 95% and 80%, respectively, to consider a gene as present in a particular draft assembly. To visualize the presence and absence of these genes in the context of the phylogeny of E. faecium , we used the neighbour-joining tree described in [58], based on a RAxML tree-based of 955 E. faecium core genes, and used the R package ggtree (version 1.14.6) to plot the presence of the different E745 genes.…”

Mode and dynamics of vanA-type vancomycin-resistance dissemination in Dutch hospitals

“…Recombination events and estimation of sequence clusters using BratNextGen and hierBAPS were performed as previously described (21). PopPUNK (version 2.0.1) was run specifying the flag '--easy-run' with a minimum k-mer size of 13 (flag --min-k) and creating the files required to generate a microreact project (flag --microreact) (27).…”

Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogenEnterococcus faecium

“…Previous whole genome sequencing (WGS) studies identified a split in the E. faecium population into two lineages, including a hospital-associated clade (clade A) and a communityrelated clade (clade B) (5,6). Later, clade A was subdivided into clade A1 representing the majority of hospital associated isolates and clade A2 for animal-related isolates (7), although two studies which included a larger collection of animal-related isolates suggested that these isolates clustered in polyphyletic groups and not in a distinct clade A (8,9). In a recent study also including 55 isolates from Latin America, phylogenomics suggests that the animal isolates represent multiple lineages that diverged prior to the emergence of the clinical subclades in clade A (10).…”

“…Recently, we determined the plasmid content (plasmidome) of 1,644 E. faecium isolates from different sources, countries and years using short-and long-read whole genome sequencing technologies in combination with machine-learning classifiers (9,11). This analysis revealed that the hospital-associated isolates carried a larger number of plasmid sequences compared to isolates from other sources and different configurations of plasmidome populations in the hospital environment.…”