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.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) cause a wide range of hospital infections. Ireland has had one of the highest invasive VREfm infection rates in Europe over the last decade, yet little is known about Irish VREfm. OBJECTIVES: To investigate the population structure of Irish VREfm, explore diversity by analysing the vanA transposon region and compare Irish, Danish and global isolates. METHODS: E. faecium (n = 648) from five Irish hospitals were investigated, including VREfm [547 rectal screening and 53 bloodstream infection (BSI)] isolates and 48 vancomycin-susceptible (VSEfm) BSI isolates recovered between June 2017 and December 2019. WGS and core-genome MLST (cgMLST) were used to assess population structure. Genetic environments surrounding vanA were resolved by hybrid assembly of short-read (Illumina) and long-read (Oxford Nanopore Technologies) sequences. RESULTS: All isolates belonged to hospital-adapted clade A1 and the majority (435/648) belonged to MLST ST80. The population structure was highly polyclonal; cgMLST segregated 603/648 isolates into 51 clusters containing mixtures of screening and BSI isolates, isolates from different hospitals, and VREfm and VSEfm. Isolates within clusters were closely related (mean average ≤16 allelic differences). The majority (96.5%) of VREfm harboured highly similar vanA regions located on circular or linear plasmids with multiple IS1216E insertions, variable organization of vanA operon genes and 78.6% harboured a truncated tnpA transposase. Comparison of 648 Irish isolates with 846 global E. faecium from 30 countries using cgMLST revealed little overlap. CONCLUSIONS: Irish VREfm are polyclonal, yet harbour a characteristic plasmid-located vanA region with multiple IS1216E insertions that may facilitate spread.

Linezolid resistance in Enterococcus faecium and Enterococcus faecalis from hospitalized patients in Ireland: high prevalence of the MDR genes optrA and poxtA in isolates with diverse genetic backgrounds.

OBJECTIVES: To investigate the prevalence of the optrA, poxtA and cfr linezolid resistance genes in linezolid-resistant enterococci from Irish hospitals and to characterize associated plasmids. METHODS: One hundred and fifty-four linezolid-resistant isolates recovered in 14 hospitals between June 2016 and August 2019 were screened for resistance genes by PCR. All isolates harbouring resistance genes, and 20 without, underwent Illumina MiSeq WGS. Isolate relatedness was assessed using enterococcal whole-genome MLST. MinION sequencing (Oxford Nanopore) and hybrid assembly were used to resolve genetic environments/plasmids surrounding resistance genes. RESULTS: optrA and/or poxtA were identified in 35/154 (22.7%) isolates, the highest prevalence reported to date. Fifteen isolates with diverse STs harboured optrA only; one Enterococcus faecium isolate harboured optrA (chromosome) and poxtA (plasmid). Seven Enterococcus faecalis and one E. faecium harboured optrA on a 36 331 bp plasmid with 100% identity to the previously described optrA-encoding conjugative plasmid pE349. Variations around optrA were also observed, with optrA located on plasmids in five isolates and within the chromosome in three isolates. Nine E. faecium and 10 E. faecalis harboured poxtA, flanked by IS1216E, within an identical 4001 bp region on plasmids exhibiting 72.9%-100% sequence coverage to a 21 849 bp conjugative plasmid. E. faecalis isolates belonged to ST480, whereas E. faecium isolates belonged to diverse STs. Of the remaining 119 linezolid-resistant isolates without linezolid resistance genes, 20 investigated representatives all harboured the G2576T 23S RNA gene mutation associated with linezolid resistance. CONCLUSIONS: This high prevalence of optrA and poxtA in diverse enterococcal lineages in Irish hospitals indicates significant selective pressure(s) for maintenance.

First description of arginine catabolic mobile element (ACME) type VI harboring the kdp operon only in Staphylococcus epidermidis using short and long read whole genome sequencing: Further evidence of ACME diversity.

The arginine catabolic mobile element (ACME) was first described in methicillin-resistant Staphylococcus aureus and is considered to enhance transmission, persistence and survival. Subsequently ACMEs were shown to be more prevalent in the coagulase-negative Staphylococcus epidermidis. Previously, ACME types were distinguished by characteristic combinations of the arc and opp3 operons [I (arc+, opp3+), II (arc+, opp3-) and III (arc-, opp3+)] encoding an arginine deaminase pathway and oligopeptide permease transporter, respectively. Recently two novel ACME types harboring the potassium transporter-encoding operon kdp were described in oral S. epidermidis isolates [IV (arc+, opp3-, kdp+), and V (arc+, opp3+, kdp+)]. This study investigated two independent oral S. epidermidis isolates that yielded amplimers with kdp-directed primers only when subjected to ACME typing PCRs. Hybrid assemblies based on Illumina MiSeq short-read and Oxford Nanopore MinION long-read whole genome sequences revealed that both isolates harbored a sixth, novel ACME type (VI) integrated into orfX. Both ACME VIs lacked the arc and opp3 operons, harbored the kdp operon adjacent to other commonly ACME-associated genes including speG, hsd, sdr, and rep, but the structural organization of the adjacent regions were distinct. These ACMEs were flanked by different direct repeat sequences and the ACME VI-positive isolates belonged to unrelated genetic clusters. Overall these findings are indicative of independent evolution. The identification of ACME type VI further illustrates the diversity of ACME elements in S. epidermidis. The presence of ACMEs harboring kdp may confer a selective advantage on oral S. epidermidis in a potassium-rich environment such as found in dental plaque.