Dataset Information

The use of high-throughput sequencing to investigate an outbreak of glycopeptide-resistant Enterococcus faecium with a novel quinupristin-dalfopristin resistance mechanism.

ABSTRACT: High-throughput sequencing (HTS) has successfully identified novel resistance genes in enterococci and determined clonal relatedness in outbreak analysis. We report the use of HTS to investigate two concurrent outbreaks of glycopeptide-resistant Enterococcus faecium (GRE) with an uncharacterised resistance mechanism to quinupristin-dalfopristin (QD). Seven QD-resistant and five QD-susceptible GRE isolates from a two-centre outbreak were studied. HTS was performed to identify genes or predicted proteins that were associated with the QD-resistant phenotype. MLST and SNP typing on HTS data was used to determine clonal relatedness. Comparative genomic analysis confirmed this GRE outbreak involved two distinct clones (ST80 and ST192). HTS confirmed the absence of known QD resistance genes, suggesting a novel mechanism was conferring resistance. Genomic analysis identified two significant genetic determinants with explanatory power for the high level of QD resistance in the ST80 QD-resistant clone: an additional 56aa leader sequence at the N-terminus of the lsaE gene and a transposon containing seven genes encoding proteins with possible drug or drug-target modification activities. However, HTS was unable to conclusively determine the QD resistance mechanism and did not reveal any genetic basis for QD resistance in the ST192 clone. This study highlights the usefulness of HTS in deciphering the degree of relatedness in two concurrent GRE outbreaks. Although HTS was able to reveal some genetic candidates for uncharacterised QD resistance, this study demonstrates the limitations of HTS as a tool for identifying putative determinants of resistance to QD.

SUBMITTER: Shaw TD

PROVIDER: S-EPMC5916979 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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The use of high-throughput sequencing to investigate an outbreak of glycopeptide-resistant Enterococcus faecium with a novel quinupristin-dalfopristin resistance mechanism.

Shaw Timothy D TD Fairley D J DJ Schneiders T T Pathiraja M M Hill R L R RLR Werner G G Elborn J S JS McMullan R R

European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 20180224 5

High-throughput sequencing (HTS) has successfully identified novel resistance genes in enterococci and determined clonal relatedness in outbreak analysis. We report the use of HTS to investigate two concurrent outbreaks of glycopeptide-resistant Enterococcus faecium (GRE) with an uncharacterised resistance mechanism to quinupristin-dalfopristin (QD). Seven QD-resistant and five QD-susceptible GRE isolates from a two-centre outbreak were studied. HTS was performed to identify genes or predicted p ...[more]

PMID: 29478197

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Project description:To investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) sequence type 78 (ST78) in a large tertiary Australian hospital. A collection of 63 VREfm ST78 isolates, identified during a routine genomic surveillance program, were subjected to genomic epidemiological analysis based on whole-genome sequencing (WGS) data. The population structure was reconstructed using phylogenetic analysis, and a collection of publicly available VREfm ST78 genomes were used to provide global context. Core genome single nucleotide polymorphism (SNP) distances and available clinical metadata were used to characterize outbreak clusters and reconstruct transmission events. In silico genotyping confirmed that all study isolates were vanB-type VREfm carrying virulence characteristics of the hospital-associated E. faecium. Phylogenetic analysis identified two distinct phylogenetic clades, only one of which was responsible for a hospital outbreak. Four outbreak subtypes could be defined with examples of recent transmissions. Inference on transmission trees suggested complex transmission routes with unknown environmental reservoirs mediating the outbreak. WGS-based cluster analysis with publicly available genomes identified closely related Australian ST78 and ST203 isolates, highlighting the capacity for WGS to resolve complex clonal relationships between the VREfm lineages. Whole genome-based analysis has provided a high-resolution description of an outbreak of vanB-type VREfm ST78 in a Queensland hospital. Combined routine genomic surveillance and epidemiological analysis have facilitated better understanding of the local epidemiology of this endemic strain, providing valuable insight for better targeted control of VREfm. IMPORTANCE Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of health care-associated infections (HAIs) globally. In Australia, the spread of hospital-adapted VREfm is largely driven by a single clonal group (clonal complex [CC]), CC17, to which the lineage ST78 belongs. While implementing a genomic surveillance program in Queensland, we observed increased incidence of ST78 colonizations and infections among patients. Here, we demonstrate the use of real-time genomic surveillance as a tool to support and enhance infection control (IC) practices. Our results show that real-time whole-genome sequencing (WGS) can efficiently disrupt outbreaks by identifying transmission routes that in turn can be targeted using resource-limited interventions. Additionally, we demonstrate that by placing local outbreaks in a global context, high-risk clones can be identified and targeted prior to them becoming established within clinical environments. Finally, the persistence of these organism within the hospital highlights the need for routine genomic surveillance as a management tool to control VRE transmission.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

The use of high-throughput sequencing to investigate an outbreak of glycopeptide-resistant Enterococcus faecium with a novel quinupristin-dalfopristin resistance mechanism.

Publications

The use of high-throughput sequencing to investigate an outbreak of glycopeptide-resistant Enterococcus faecium with a novel quinupristin-dalfopristin resistance mechanism.

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