Novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (VRE) from a hospital outbreak: co-location of cfr and optrA in VRE.

Linezolid is often the drug of last resort to treat infections caused by Gram-positive cocci. Linezolid resistance can be mutational (23S rRNA or L-protein) or, less commonly, acquired [predominantly cfr, conferring resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A compounds (PhLOPSA) or optrA, encoding oxazolidinone and phenicol resistance]. To investigate the clonality and genetic basis of linezolid resistance in 13 linezolid-resistant (LZDR) methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered during a 2013/14 outbreak in an ICU in an Irish hospital and an LZDR vancomycin-resistant Enterococcus faecium (VRE) isolate from an LZDR-MRSE-positive patient. All isolates underwent PhLOPSA susceptibility testing, 23S rRNA sequencing, DNA microarray profiling and WGS. All isolates exhibited the PhLOPSA phenotype. The VRE harboured cfr and optrA on a novel 73 kb plasmid (pEF12-0805) also encoding erm(A), erm(B), lnu(B), lnu(E), aphA3 and aadE. One MRSE (M13/0451, from the same patient as the VRE) harboured cfr on a novel 8.5 kb plasmid (pSEM13-0451). The remaining 12 MRSE lacked cfr but exhibited linezolid resistance-associated mutations and were closely related to (1-52 SNPs) but distinct from M13/0451 (202-223 SNPs). Using WGS, novel and distinct cfr and cfr/optrA plasmids were identified in an MRSE and VRE isolate, respectively, as well as a cfr-negative LZDR-MRSE ICU outbreak and a distinct cfr-positive LZDR-MRSE from the same ICU. To our knowledge, this is the first report of cfr and optrA on a single VRE plasmid. Ongoing surveillance of linezolid resistance is essential to maintain its therapeutic efficacy.

Lazaris A ，Coleman DC ，Kearns AM ，Pichon B ，Kinnevey PM ，Earls MR ，Boyle B ，O'Connell B ，Brennan GI ，Shore AC ... -  《-》

Hospital outbreak of linezolid-resistant and vancomycin-resistant ST80 Enterococcus faecium harbouring an optrA-encoding conjugative plasmid investigated by whole-genome sequencing.

Linezolid is an antibiotic used to treat infections caused by multi-drug-resistant Gram-positive bacteria. Linezolid resistance in enterococci has been reported with increasing frequency, with a recent rise in resistance encoded by optrA, poxtA or cfr. To investigate a hospital outbreak of linezolid- and vancomycin-resistant Enterococcus faecium (LVREfm) using whole-genome sequencing (WGS). Thirty-nine VREfm from patient screening (19 isolates, 17 patients) and environmental sites (20 isolates) recovered in October 2019 were investigated. Isolates were screened using polymerase chain reaction for optrA, poxtA and cfr, and underwent Illumina MiSeq WGS. Isolate relatedness was assessed using E. faecium core genome multi-locus sequence typing (cgMLST). One LVREfm underwent MinION long-read WGS (Oxford Nanopore Technologies) and hybrid assembly with MiSeq short-read sequences to resolve an optrA-encoding plasmid. Twenty isolates (51.3%) were LVREfm and optrA-positive, including the LVREfm from the index patient. A closely related cluster of 28 sequence type (ST) 80 isolates was identified by cgMLST, including all 20 LVREfm and eight linezolid-susceptible VREfm, with an average allelic difference of two (range 0-10), indicating an outbreak. Nineteen (95%) LVREfm harboured a 56,684-bp conjugative plasmid (pEfmO_03). The remaining LVREfm exhibited 44.1% sequence coverage to pEfmO_03. The presence of pEfmO_03 in LVREfm and the close relatedness of the outbreak cluster isolates indicated the spread of a single strain. The outbreak was terminated by enhanced infection prevention and control (IPC) and environmental cleaning measures, ceasing ward admissions and ward-dedicated staff. WGS was central in investigating an outbreak of ST80 LVREfm. The rapid implementation of enhanced IPC measures terminated the outbreak.

Egan SA ，Corcoran S ，McDermott H ，Fitzpatrick M ，Hoyne A ，McCormack O ，Cullen A ，Brennan GI ，O'Connell B ，Coleman DC ... -  《-》

First Report of cfr-Carrying Plasmids in the Pandemic Sequence Type 22 Methicillin-Resistant Staphylococcus aureus Staphylococcal Cassette Chromosome mec Type IV Clone.

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for ribosomal proteins; cfr, encoding phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) resistance; its homologue cfr(B); or optrA, conferring oxazolidinone and phenicol resistance. Linezolid resistance is rare in S. aureus, and cfr is even rarer. This study investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from patients in separate Irish hospitals. Isolates were subjected to cfr PCR, PhLOPSA susceptibility testing, 23S rRNA PCR and sequencing, DNA microarray profiling, spa typing, pulsed-field gel electrophoresis (PFGE), plasmid curing, and conjugative transfer. Whole-genome sequencing was used for single-nucleotide variant (SNV) analysis, multilocus sequence typing, L protein mutation identification, cfr plasmid sequence analysis, and optrA and cfr(B) detection. Isolates M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 23S rRNA alleles, compared to 1/5 in M13/0401. Both isolates were sequence type 22 MRSA staphylococcal cassette chromosome mec type IV (ST22-MRSA-IV)/spa type t032 isolates, harbored cfr, exhibited the PhLOPSA phenotype, and lacked optrA and cfr(B). They differed by five PFGE bands and 603 SNVs. Isolate M12/0145 harbored cfr and fexA on a 41-kb conjugative pSCFS3-type plasmid, whereas M13/0401 harbored cfr and lsa(B) on a novel 27-kb plasmid. This is the first report of cfr in the pandemic ST22-MRSA-IV clone. Different cfr plasmids and mutations associated with linezolid resistance in genotypically distinct ST22-MRSA-IV isolates highlight that prudent management of linezolid use is essential.

Shore AC ，Lazaris A ，Kinnevey PM ，Brennan OM ，Brennan GI ，O'Connell B ，Feßler AT ，Schwarz S ，Coleman DC ... -  《-》

Tedizolid susceptibility in linezolid- and vancomycin-resistant Enterococcus faecium isolates.

Klupp EM ，Both A ，Belmar Campos C ，Büttner H ，König C ，Christopeit M ，Christner M ，Aepfelbacher M ，Rohde H ... -  《-》

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.

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. 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. 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. This high prevalence of optrA and poxtA in diverse enterococcal lineages in Irish hospitals indicates significant selective pressure(s) for maintenance.

Egan SA ，Shore AC ，O'Connell B ，Brennan GI ，Coleman DC ... -  《-》