Date Published: June 13, 2019
Publisher: Public Library of Science
Author(s): Kimon Lemonidis, Talal S. Salih, Stephanie J. Dancer, Iain S. Hunter, Nicholas P. Tucker, Yung-Fu Chang.
Multi-locus sequencing typing (MLST) is widely used to monitor the phylogeny of microbial outbreaks. However, several strains of vancomycin-resistant Enterococcus faecium (VREfm) with a missing MLST locus (pstS) have recently emerged in Australia, with a few cases also reported in England. Here, we identified similarly distinct strains circulating in two neighbouring hospitals in Scotland. Whole genome sequencing of five VREfm strains isolated from these hospitals identified four pstS-null strains in both hospitals, while the fifth was multi-locus sequence type (ST) 262, which is the first documented in the UK. All five Scottish isolates had an insertion in the tetM gene, which is associated with increased susceptibility to tetracyclines, providing no other tetracycline-resistant gene is present. Such an insertion, which encompasses a dfrG gene and two currently uncharacterised genes, was additionally identified in all tested vanA-type pstS-null VREfm strains (5 English and 68 Australian). Phylogenetic comparison with other VREfm genomes indicates that the four pstS-null Scottish isolates sequenced in this study are more closely related to pstS-null strains from Australia rather than the English pstS-null isolates. Given how rapidly such pstS-null strains have expanded in Australia, the emergence of this clone in Scotland raises concerns for a potential outbreak.
Vancomycin-resistant Enterococcus spp. (VRE) was first identified about three decades ago and has now become a major nosocomial pathogen. It typically infects immunocompromised patients and can cause endocarditis, bloodstream, urinary tract, and skin and skin structure infections . VRE infections are generally more serious than those caused by vancomycin-susceptible enterococci, and are associated with higher mortality rates . Among all VRE species, vancomycin-resistant Enterococcus faecium (VREfm) is responsible for the majority of hospital infections. VREfm has been recently listed as a high priority pathogen for research and development of new antibiotics by the World Health Organisation . Various measures have been implemented to monitor the spread of VREfm infections, including multi-locus sequence typing (MLST); this relies on characterising the allelic profile of seven “house-keeping” genes, located in the E. faecium chromosome . Although useful, this MLST scheme is of limited resolution to accurately capture the clonal type of isolated E. faecium strains [5–7]. Furthermore, VREfm isolates lacking the pstS MLST-gene locus have recently emerged in both England  and Australia .
VRE strains were isolated from five patients in two Scottish hospitals, between January to October 2017. These patients developed complications following either biliary or colonic surgery, and had been treated with various combinations of penicillin, amoxicillin, flucloxacillin, Tazocin, gentamicin, metronidazole and vancomycin during their hospital stay. The VRE strains were cultured on horse blood agar with single colonies transferred to Mueller Hinton broth (Oxoid) liquid. These were subsequently placed on Mueller Hinton agar. Single colony cultures of these were subsequently used for DNA isolation. Genomic DNA was extracted using an Isolate II genomic DNA kit (Bioline), using the manufacturer’s instructions for difficult to lyse Gram-positive bacteria. DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies). Enriched ISPs were loaded into ion v2 BC 316 chips (2 genomes per chip) and sequenced on an Ion PGM system (Life Technologies). Low quality reads (quality score threshold: 0.05) were trimmed using CLC genomics Workbench (Qiagen, version 9.5.2), and resulting reads were assembled using SPAdes (St. Petersburg genome assembler, version 3.9). Contigs having less than 1000 bp sequences were discarded. The remaining contigs were reordered on Mauve (version 20150226) using the complete E. faecium Aus0004 genome  as reference, and resulting genome sequences were submitted to GenBank under the accessions PJZU00000000 (VREF001), PJZT00000000 (VREF002), PJZS00000000 (VREF003), PJZR00000000 (VREF004) and PJZQ00000000 (VREF005). Antibiotic resistant genes in VREF001-5 genomes were predicted using the Resistance Gene Identifier tool within the comprehensive antibiotic resistance database (CARD) (https://card.mcmaster.ca/analyze/rgi). This tool utilizes algorithms for identification of perfect, strict or loose matches against resistant genes of the comprehensive antibiotic resistance database, based on homology and SNP models . Sequence reads of pstS-null genomes from England and Australia were obtained from European Nucleotide Archive, and reads were assembled using CLC genomics Workbench; contigs having less than 200 bp were discarded. In silico MLST analysis was performed using the PubMLST website (http://pubmlst.org/) . Alignments of genomes were done using the REALPHY (Reference sequence Alignment based Phylogeny builder) online tool (version 1.12) , with E. faecium Aus0004 , Aus0085  as references, using default options (read length: 20, seed length: 22, polymorphism threshold: 0.95) and merging of reference alignments. All input genome sequences were assembled contigs in FASTA format and the two different reference genome alignments were combined to produce the resulting phylogenetic tree. Minimum inhibitory concentrations (MICs; μg/ml) for vancomycin, streptomycin, spectinomycin, tetracycline, oxytetracycline, doxycycline, minocycline and rifampicin were calculated using the microdilution method in cation adjusted Mueller-Hinton broth media (BD Biosciences), whereas MICs (μg/ml) for all other antibiotics were obtained using VITEK 2 (bioMerieux).
The Scottish VRE strains sequenced (VREF001-5) had 2.9–3.0 Mb genomes with 37.6–37.7% GC content (Table 1). Analysis of average nucleotide identity and Tetra Correlation Search (TCS) against database genome sequences, using JSpeciesWS (version 3.0.12) , showed that all five VRE isolates were E. faecium. VREF001, VREF002, VREF004 and VREF005 were highly related with over 99.9% identity among aligned (>97%) sequences, whereas VREF003 displayed around 16% unique sequences compared with the other four genomes. In silico MLST analysis of VREF001-5 genomes identified VREF003 as sequence type (ST) 262, which has not been previously reported in the UK. The other four genomes had exact matches for six MLST alleles (atpA-9, ddl-1, gdh-1, purK-1, gyd-12, adk-1), but no match for pstS. This MLST profile has now been assigned as ST1424 in the E. faecium MLST database (https://pubmlst.org/efaecium/). However, despite the missing pstS in these four genomes, all VREfm strains sequenced in this study did have a pstS homologue within a pst operon (also referred to as pstS2), which is thought to be the actual pstS housekeeping gene in E. faecium [7,8].
MLST analysis is inferior to whole genome sequence analysis for outbreak investigations of VREfm , due to the high rate of recombination events occurring within the E. faecium chromosome that cannot be captured adequately by MLST analysis [27,28]. Furthermore, the emergence of non-typeable VREfm strains poses an extra obstacle in implementing the MLST scheme for VREfm phylogenetic analysis. Non-typeable VREfm strains have recently emerged and have been shown to be very rare in the UK. In a study encompassing whole genome sequencing of nearly 500 E. faecium healthcare-associated isolates from 2001–2011 in the UK and Ireland, there were only five cases of pstS-null VRE strains, all of which were isolated from a single English hospital, between 2004 and 2005 .The emergence of such strains was even more recent in Australia: the first two strains were isolated in 2013, but numbers increased rapidly to a total of 89 cases, by the end of 2015 [8,29,30] and to about 300 cases, by the end of 2016 [25,30,31].