Genomic surveillance of vancomycin-resistantEnterococcus faeciumreveals spread of a linear plasmid conferring a nutrient utilization advantage

Abstract

Healthcare-associated outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are a worldwide problem with increasing prevalence. The genomic plasticity of this hospital-adapted pathogen contributes to its efficient spread despite infection control measures. Here, we aimed to identify the genomic and phenotypic determinants of healthcare-associated transmission of VREfm. We assessed the VREfm transmission networks at the tertiary-care University Hospital of Zurich (USZ) between October 2014 and February 2018 and investigated microevolutionary dynamics of this pathogen. We performed whole-genome sequencing for the 69 VREfm isolates collected during this timeframe and assessed the population structure and variability of the vancomycin resistance transposon. Phylogenomic analysis allowed us to reconstruct transmission networks and to unveil external or indirect transmission networks, not detectable by traditional surveillance. Notably, it unveiled a persistent clone, sampled 31 times over a 29-month period. Exploring the evolutionary dynamics of this clone and characterizing the phenotypic consequences revealed the spread of a variant with decreased daptomycin susceptibility and the acquired ability to utilize N acetyl galactosamine (GalNAc), one of the primary constituents of the human gut mucins. This nutrient utilization advantage was conferred by a novel plasmid, termed pELF_USZ, which exhibited a linear topology. This plasmid, which was harbored by two distinct clones, was transferable by conjugation. Overall, this work provides an example of the potential of the integration of epidemiological, functional genomic and evolutionary perspectives to understand adaptation strategies contributing to the successful spread of VREfm.

Linear plasmids in Klebsiella and other Enterobacteriaceae

“…Hundreds of genomes of each of these clones are present in the NCBI database and the vast majority do not harbour linear plasmid sequences, suggesting that the linear-plasmid-positive variants are rare, and likely result from recent horizontal transfer but this has not resulted in clonal expansion during which the plasmid has been stably maintained. This is in contrast to the recent report in Enterococcus faecium where the linear plasmid pELF_USZ was stably maintained in a host lineage during >2 years of clonal spread in a hospital [ 4 ].…”

“…This is in contrast to the recent report in Enterococcus faecium where the linear plasmid pELF_USZ was stably maintained in a host lineage during >2 years of clonal spread in a hospital [4].…”

“…The vast majority of plasmid molecules exist in a circular conformation; however, linear plasmids have been found in several bacterial species, with the first description in Streptomyces in 1979 [ 1 ], and later in Borrelia [ 2 ] (where they are universally present) and Rhodococcus [ 3 ]. A study of clinical Enterococcus faecium isolates recently reported the existence of a 143 kbp linear plasmid carrying a N -acetylgalactosamine (GalNAc) utilization operon that could be transferred between strains via conjugation [ 4 ]. Linear plasmids appear to be exceedingly rare within Enterobacteriaceae , with the first, pBSSB1 (27 kbp), described in 2007 from Salmonella enterica Typhi isolated in Indonesia [ 5 ].…”

“…For replicons that are linear, there is a requirement to stabilize the terminal ends to ensure stability and appropriate replication, which in eukaryotes is achieved through the use of telomeres. In contrast, bacterial linear plasmids can either (i) create hairpin loops, as in Borrelia [ 9 ] and Enterococcus [ 4 ], or (ii) bind telomere-associated proteins to each end of the molecule with the assistance of terminal inverted repeats (TIRs), as in Streptomyces [ 10 ]. The Salmonella linear plasmid pBSSB1 was found to carry 1230 bp TIRs with covalently bound proteins on the end, similar to Streptomyces ; however, these had no homology to any previously identified TIRs [ 5 ].…”

Genomic analysis of 600 vancomycin-resistant Enterococcus faecium reveals a high prevalence of ST80 and spread of similar vanA regions via IS1216E and plasmid transfer in diverse genetic lineages in Ireland

“…To investigate whether the large transferable vanA -encoding contigs identified in isolates SJ11 and BSI_SJ40 by hybrid assembly were linear plasmids, the sequences of the contigs were compared with the corresponding sequences of linear plasmids pELF1 (143 316 bp), pELF2 (108 102 bp) and pELF_USZ (101 837 bp) recently described in VREfm isolates from Japan 30 , 31 and Switzerland, respectively. 32 Comparing the vanA contigs from SJ11 (116 749 bp) and BSI_SJ40 (131 341 bp) revealed a clear homology ( Figure S6 ). Each plasmid and contig harboured genes encoding putative replication initiation proteins and proteins with a putative role in DNA partitioning and transfer.…”