State-Wide Genomic and Epidemiological Analyses of Vancomycin-Resistant Enterococcus faecium in Tasmania's Public Hospitals

Abstract

From 2015 onwards, the number of vancomycin-resistant Enterococcus faecium (VREfm) isolates increased in Tasmania. Previously, we examined the transmission of VREfm at the Royal Hobart Hospital (RHH). In this study, we performed a state-wide analysis of VREfm from Tasmania's four public acute hospitals. Whole-genome analysis was performed on 331 isolates collected from screening and clinical specimens of VREfm. In silico multi-locus sequence typing (MLST) was used to determine the relative abundance of broad sequence types (ST) across the state. Core genome MLST (cgMLST) was then applied to identify potential clades within the ST groupings followed by single-nucleotide polymorphic (SNP) analysis. This work revealed that differences in VREfm profiles are evident between the state's two largest hospitals with the dominant vanA types being ST80 at the RHH and ST1421 at Launceston General Hospital (LGH). A higher number of VREfm cases were recorded at LGH (n = 54 clinical, n = 122 colonization) compared to the RHH (n = 14 clinical, n = 67 colonization) during the same time period, 2014-2016. Eleven of the clinical isolates from LGH were vanA and belonged to ST1421 (n = 8), ST1489 (n = 1), ST233 (n = 1), and ST80 (n = 1) whereas none of the clinical isolates from the RHH were vanA. For the recently described ST1421, cgMLST established the presence of individual clusters within this sequence type that were common to more than one hospital and that included isolates with a low amount of SNP variance (≤16 SNPs). A spatio-temporal analysis revealed that VREfm vanA ST1421 was first detected at the RHH in 2014 and an isolate belonging to the same cgMLST cluster was later collected at LGH in 2016. Inclusion of isolates from two smaller hospitals, the North West Regional Hospital (NRH) and the Mersey Community Hospital (MCH) found that ST1421 was present in both of these institutions in 2017. These findings illustrate the spread of a recently described sequence type of VREfm, ST1421, to multiple hospitals in an Australian state within a relatively short time span.

Keywords: Enterococcus faecium; multi-locus sequence typing; single nucleotide polymorphism; vancomycin; whole genome sequencing.

FIGURE 1

Multi-Locus Sequence Types (MLST) of vancomycin-resistant Enterococcus faecium (VREfm) isolates collected at Tasmania’s four acute public hospitals: (A) Royal Hobart Hospital (RHH) and (B) Launceston General Hospital (LGH) from 2014 to 2016; (C) North West Regional Hospital (NRH) and (D) Mersey Community Hospital (MCH). The MLST (and vancomycin-resistance locus) of the VREfm isolates were determined in silico from whole genome sequence data. Boxed numbers above histograms represent the number of new clinical VREfm isolates collected within each sequence type.

FIGURE 2

Core genome Multi-Locus Sequence Type (cgMLST) analysis of vancomycin-resistant Enterococcus faecium (VREfm) isolates from the four public hospitals in Tasmania (RHH, Royal Hobart Hospital; LGH, Launceston General Hospital; NRH, North West Regional Hospital; MCH, Mersey Community Hospital). The isolates were analyzed using cgMLST in Ridom SeqSphere+ and are visualized in a minimum spanning tree. Isolates with zero allele differences between them grouped together in clusters with isolates per cluster shown in the circles. The number of different alleles between clusters and unique isolates is shown on the connecting lines (not to scale).

FIGURE 3

cgMLST of ST1421 vancomycin-resistant Enterococcus faecium (VREfm) isolates (n = 50). VREfm isolates belonging to sequence type ST1421 were analyzed by cgMLST in Ridom SeqSphere+ and are visualized in a minimum spanning tree. The isolates further differentiated into three clusters and one unique isolate (grouped into circles). The number of different alleles between clusters and the unique isolate is shown on the connecting lines (not to scale).

FIGURE 4

SNP-based analysis of vancomycin-resistant Enterococcus faecium (VREfm) isolates. (A) A SNP-based maximum-likelihood (PhyML) phylogenetic tree was generated from the VREfm isolates collected from the four public hospitals in Tasmania (RHH, Royal Hobart Hospital; LGH, Launceston General Hospital; NRH, North West Regional Hospital; MCH, Mersey Community Hospital) that were whole genome sequenced (n = 331) with reference genome Enterococcus faecium DO (TX16_NC-017960) to root the tree. Multi-locus sequence types (MLST) and vancomycin (van)-resistance loci are indicated. The clade of ST1421 isolates is highlighted in red. (B) Clades from the PhyML phylogenetic tree of VREfm isolates belonging to multi-locus sequence type ST1421 (n = 50) show concordance with the clusters determined by core-genome MLST (cgMLST) with Cluster 3 sub-dividing further into Clusters 3A and 3B.

FIGURE 5

Phylogenetic analysis of vancomycin-resistant Enterococcus faecium (VREfm) isolates from ST1421 Cluster 1. (A) A SNP-based maximum-likelihood (PhyML) phylogenetic tree. (B) Matrix of pairwise comparison of SNP differences between isolates expressed as: (i) Total number of SNP differences; (ii) Number of SNPs inside homologous recombination regions, and (iii) Number of SNPs outside of homologous recombination regions. The previously described recombination-filtered SNP threshold of ≤16 SNPs for VREfm has been used as a guide for identifying clonally related or non-unique isolates. (C) Overview of recombination-filtered SNPs between isolates. The numbers of different SNPs between the isolates are shown on the solid black connecting lines. SNP differences above the threshold of 16 SNPs are shown as blue dotted lines. (D) Spatio-temporal location of patients in Cluster 1 who tested positive for VREfm at the Royal Hobart Hospital and Launceston General Hospital. The movement of patients following admission to the Royal Hobart Hospital through to date of discharge are indicated with respect to time (x-axis) and hospital ward location (y-axis). Each line color represents an individual patient. Patient 14S_RHH008 was admitted to RHH on September 30, 2014 and was screened for VREfm on October 1, 2014 from which a positive test was reported.

FIGURE 6

Phylogenetic analysis of vancomycin-resistant Enterococcus faecium (VREfm) isolates from ST1421 Cluster 2. (A) A SNP-based maximum-likelihood (PhyML) phylogenetic tree. (B) Matrix of pairwise comparison of SNP differences between isolates expressed as: (i) Total number of SNP differences; (ii) Number of SNPs inside homologous recombination regions, and (iii) Number of SNPs outside of homologous recombination regions. The previously described recombination-filtered SNP threshold of ≤16 SNPs for VREfm has been used as a guide for identifying clonally related or non-unique isolates. (C) Overview of recombination-filtered SNPs between isolates. The numbers of different SNPs between the isolates are shown on the solid black connecting lines. SNP differences above the threshold of 16 SNPs are shown as blue dotted lines. (D) Spatio-temporal location of patients in Cluster 2 who tested positive for VREfm at the Launceston General Hospital. The movement of patients following admission to hospital through to date of discharge are indicated with respect to time (x-axis) and hospital ward location (y-axis). Each line color represents an individual patient. As illustrated, patient 16S_LGH063 had admissions to both the Royal Hobart Hospital and Launceston General Hospital but was confirmed VREfm positive at the latter hospital.

FIGURE 7

Phylogenetic analysis of vancomycin-resistant Enterococcus faecium (VREfm) isolates from ST1421 Cluster 3A. The previously described recombination-filtered SNP threshold of ≤16 SNPs for VREfm has been used as a guide for identifying clonally related or non-unique isolates. (A) Overview of recombination-filtered SNPs between isolates. The numbers of different SNPs between the isolates are shown on the solid black connecting lines. SNP differences above the threshold of 16 SNPs are shown as blue dotted lines. (B) Spatio-temporal location of patients in Cluster 3A who tested positive for VREfm. The movement of patients following admission to hospital through to date of discharge are indicated with respect to time (x-axis) and hospital ward location (y-axis). Each line color represents an individual patient. As illustrated, a number of patients had multiple admissions to more than one hospital over the time course.

FIGURE 8

Phylogenetic analysis of vancomycin-resistant Enterococcus faecium (VREfm) isolates from ST1421 Cluster 3B. The previously described recombination-filtered SNP threshold of ≤16 SNPs for VREfm has been used as a guide for identifying clonally related or non-unique isolates. (A) Overview of recombination-filtered SNPs between isolates. The numbers of different SNPs between the isolates are shown on the solid black connecting lines. SNP differences above the threshold of 16 SNPs are shown as blue dotted lines. (B) Spatio-temporal location of patients in Cluster 3B who tested positive for VREfm. The movement of patients following admission to hospital through to date of discharge are indicated with respect to time (x-axis) and hospital ward location (y-axis). Each line color represents an individual patient. As illustrated, a number of patients had multiple admissions to more than one hospital over the time course.

FIGURE 9

Dissemination of the vanA ST1421 vancomycin-resistant Enterococcus faecium (VREfm) in Tasmania’s public hospital system. The first reported case of ST1421 VREfm in Tasmania occurred at the Royal Hobart Hospital in 2014. Correlation of genomic data with epidemiological data revealed a network in which recently identified sequence type of VREfm (ST1421) emerged in each of the state’s other public hospitals.