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

Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012-2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.Peer reviewe

Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogen, Enterococcus faecium

Enterococcus faecium is a gut commensal of the gastro-digestive tract, but also known as nosocomial pathogen among hospitalized patients. Population genetics based on whole-genome sequencing has revealed that E. faecium strains from hospitalized patients form a distinct Glade, designated Glade A1, and that plasmids are major contributors to the emergence of nosocomial E. faecium. Here we further explored the adaptive evolution of E faecium using a genome-wide co-evolution study (GWES) to identify co-evolving single-nucleotide polymorphisms (SNPs). We identified three genomic regions harbouring large numbers of SNPs in tight linkage that are not proximal to each other based on the completely assembled chromosome of the Glade A1 reference hospital isolate AUS0004. Close examination of these regions revealed that they are located at the borders of four different types of large-scale genomic rearrangements, insertion sites of two different genomic islands and an IS30-like transposon. In non-Glade A1 isolates, these regions are adjacent to each other and they lack the insertions of the genomic islands and IS30-like transposon. Additionally, among the Glade A1 isolates there is one group of pet isolates lacking the genomic rearrangement and insertion of the genomic islands, suggesting a distinct evolutionary trajectory. In silico analysis of the biological functions of the genes encoded in three regions revealed a common link to a stress response. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. In conclusion, to our knowledge, this is the first study using GWES to identify genomic rearrangements, suggesting that there is considerable untapped potential to unravel hidden evolutionary signals from population genomic data.Peer reviewe

Global Spread of Vancomycin-resistant Enterococcus faecium from Distinct Nosocomial Genetic Complex

Vancomycin-resistant enterococci (VRE) have caused hospital outbreaks worldwide, and the vancomycin-resistance gene (vanA) has crossed genus boundaries to methicillin-resistant Staphylococcus aureus. Spread of VRE, therefore, represents an immediate threat for patient care and creates a reservoir of mobile resistance genes for other, more virulent pathogens. Evolutionary genetics, population structure, and geographic distribution of 411 VRE and vancomycin-susceptible Enterococcus faecium isolates, recovered from human and nonhuman sources and community and hospital reservoirs in 5 continents, identified a genetic lineage of E. faecium (complex-17) that has spread globally. This lineage is characterized by 1) ampicillin resistance, 2) a pathogenicity island, and 3) an association with hospital outbreaks. Complex-17 is an example of cumulative evolutionary processes that improved the relative fitness of bacteria in hospital environments. Preventing further spread of this epidemic E. faecium subpopulation is critical, and efforts should focus on the early disclosure of ampicillin-resistant complex-17 strains

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

Abstract Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012–2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance

mlplasmids : a user-friendly tool to predict plasmid- and chromosome-derived sequences for single species

Assembly of bacterial short-read whole-genome sequencing data frequently results in hundreds of contigs for which the origin, plasmid or chromosome, is unclear. Complete genomes resolved by long-read sequencing can be used to generate and label short-read contigs. These were used to train several popular machine learning methods to classify the origin of contigs from Enterococcus faecium, Klebsiella pneumoniae and Escherichia colt using pentamer frequencies. We selected support-vector machine (SVM) models as the best classifier for all three bacterial species (Fl-score E. faecium=0.92, F1-score K. pneumoniae=0.90, F1-score E. coli=0.76), which outperformed other existing plasmid prediction tools using a benchmarking set of isolates. We demonstrated the scalability of our models by accurately predicting the plasmidome of a large collection of 1644 E. faecium isolates and illustrate its applicability by predicting the location of antibiotic-resistance genes in all three species. The SVM classifiers are publicly available as an R package and graphical-user interface called 'mlplasmids'. We anticipate that this tool may significantly facilitate research on the dissemination of plasmids encoding antibiotic resistance and/or contributing to host adaptation.Peer reviewe

Restricted Gene Flow among Hospital Subpopulations of Enterococcus faecium

Peer reviewe

Identification of a Novel Genomic Island Associated with vanD-Type Vancomycin Resistance in Six Dutch Vancomycin-Resistant Enterococcus faecium Isolates

Genomic comparison of the first six Dutch vanD-type vancomycin-resistant Enterococcus faecium (VRE) isolates with four vanD gene clusters from other enterococcal species and anaerobic gut commensals revealed that the vanD gene cluster was located on a genomic island of variable size. Phylogenetic inferences revealed that the Dutch VRE isolates were genetically not closely related and that genetic variation of the vanD-containing genomic island was not species specific, suggesting that this island is transferred horizontally between enterococci and anaerobic gut commensals.Peer reviewe

Whole Genome Sequence Analysis of the First Vancomycin-Resistant Enterococcus faecium Isolates from a Libyan Hospital in Tripoli

The purpose of the study was to investigate the molecular characteristics and genetic relatedness of the first reported cases of vancomycin-resistant enterococci (VRE) from the Tripoli Medical Center, Libya. In total, 43 VRE isolates were obtained from various clinical sites throughout the years 2013-2014, including 40 vanA-type and 2 vanB-type vancomycin-resistant Enterococcus faecium isolates and 1 vanC1-type Enterococcus gallinarum. Of the 42 E. faecium, 19 isolates were subjected to whole genome sequencing. Core genome multilocus sequence typing (cgMLST) analysis revealed three sequence clusters (SCs) of clonally related isolates, which were linked to different hospital wards. The first two VRE isolates, isolated early 2013 from patients in the medical intensive care unit, were grouped in SC1 (MLST [ST] 78, vanB) and differed in only 3 of 1423 cgMLST alleles. The SC2 (n = 16, special care baby unit, neonatal intensive care unit, pediatric surgery ward, and oncology ward) and SC3 (n = 1, antenatal ward) were all ST80 vanA-VRE, but the single SC3 isolate differed in 233 alleles compared with SC2. Within SC2, isolates differed in 1-23 alleles. Comparison with a larger database of E. faecium strains indicated that all isolates clustered within the previously defined hospital clade A1. A combination of Resfinder and mlplasmid analysis identified the presence of resistance genes on different plasmid predicted genetic elements among different SCs. In conclusion, this study documents the first isolates causing outbreaks with VRE in the Libyan health care system. Further surveillance efforts using molecular typing methods to monitor spread of multidrug-resistant bacteria in the Libyan health care system are urgently needed

Detecting co-selection through excess linkage disequilibrium in bacterial genomes

Population genomics has revolutionized our ability to study bacterial evolution by enabling data-driven discovery of the genetic architecture of trait variation. Genome-wide association studies (GWAS) have more recently become accompanied by genome-wide epistasis and co-selection (GWES) analysis, which offers a phenotype-free approach to generating hypotheses about selective processes that simultaneously impact multiple loci across the genome. However, existing GWES methods only consider associations between distant pairs of loci within the genome due to the strong impact of linkage-disequilibrium (LD) over short distances. Based on the general functional organisation of genomes it is nevertheless expected that majority of co-selection and epistasis will act within relatively short genomic proximity, on co-variation occurring within genes and their promoter regions, and within operons. Here, we introduce LDWeaver, which enables an exhaustive GWES across both short- and long-range LD, to disentangle likely neutral co-variation from selection. We demonstrate the ability of LDWeaver to efficiently generate hypotheses about co-selection using large genomic surveys of multiple major human bacterial pathogen species and validate several findings using functional annotation and phenotypic measurements. Our approach will facilitate the study of bacterial evolution in the light of rapidly expanding population genomic data