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

Anna C Shore,David C Coleman,Andrea T Feßler,Orla M Brennan,Gráinne I Brennan,Brian O'Connell,Stefan Schwarz,Alexandros Lazaris,Peter M Kinnevey

doi:10.1128/aac.02949-15

Abstract

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.

Highlights

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections
The ST22-MRSA-IV clone is a pandemic nosocomial MRSA clone, and previous studies have revealed the ability of this clone to adapt to the introduction of different antimicrobial agents into the health care environment [38]
We report another step in the evolution of this MRSA clone, with the first report of the transferable multidrug resistance gene cfr in two independent ST22-MRSA-IV isolates

Summary

Introduction

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. The cfr gene was first reported in a bovine Staphylococcus sciuri isolate in 1997 and was subsequently found in many different staphylococcal species, including methicillin-susceptible S. aureus (MSSA), MRSA, and coagulase-negative and coagulase-variable (Staphylococcus hyicus) staphylococci, as well as in Bacillus, Enterococcus, Streptococcus, Macrococcus, Jeotgalicoccus, Proteus, and Escherichia species [10,11,12]. It has been detected in isolates from humans, livestock, meat products, and the environment and has been identified on a variety of plasmids, chromosomal locations have been reported [10, 13]. Specific insertion sequences (ISs) have been shown to play a role in cfr mobility and integration into different plasmid types, and cfr is often colocated with other resistance determinants, allowing for the coselection of cfr [10]

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