Molecular characterization of linezolid-resistant CoNS isolates in Japan.
OBJECTIVE
Linezolid has been reported to remain active against 98% of staphylococci with resistance identified in 0.05% of Staphylococcus aureus and 1.4% of CoNS. The objective of this study was to characterize the linezolid-resistance mechanisms in the linezolid-resistant CoNS strains isolated in Japan.


METHODS
Staphylococcus capitis strains exhibiting linezolid MICs >8 mg/L isolated from inpatients between 2012 and 2014 were screened for cfr and mutations in 23S rRNA, L3 and L4 by PCR/sequencing. Isolates were also examined for mutations in the rlmN gene.


RESULTS
S. capitis had six 23S rRNA alleles. Five S. capitis isolates displayed linezolid MICs of 8, 16 and 32 mg/L. G2576U mutations were detected in three, four or five copies of 23S rRNA in all isolates. In two isolates exhibiting the highest linezolid MIC (32 mg/L) there was a large deletion in a single copy of 23S rRNA. Repeated 10 bp sequences were found in both 16S and 23S rRNAs, suggesting deletion by recombination between the repeats. One isolate had the mutation Ala-142→Thr in the ribosomal protein L3. All linezolid-resistant isolates also demonstrated mutations in the gene encoding RlmN methyltransferase, leading to Thr-62→Met and Gly-148→Ser.


CONCLUSIONS
Multiple mechanisms appeared to be responsible for the elevated linezolid resistance in S. capitis isolates: a G2576U mutation in different numbers of copies of 23S rRNA, loss of a single copy of 23S rRNA and a mutation in the ribosomal protein L3, suggesting the accumulation of independent mutational events.
Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Akiko
A
Takaya
Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Asahi
A
Kimura
Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan.
Yoshiharu
Y
Sato
Division of Control and Treatment of Infectious Diseases, Chiba University Hospital, Chiba 260-8677, Japan.
Naruhiko
N
Ishiwada
Division of Laboratory Medicine, Chiba University Hospital, Chiba 260-8677, Japan.
Masaharu
M
Watanabe
Department of Bacteriology II, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan.
Mari
M
Matsui
Department of Bacteriology II, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan.
Keigo
K
Shibayama
Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan tomoko-y@faculty.chiba-u.jp.
Tomoko
T
Yamamoto
D000081:Acetamides / Q000494:pharmacology*; D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D003030:Coagulase / Q000172:deficiency; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006801:Humans; D007564:Japan; D000069349:Linezolid; D008826:Microbial Sensitivity Tests; D009154:Mutation; D023303:Oxazolidinones / Q000494:pharmacology*; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D013210:Staphylococcus / Q000187:drug effects* / Q000302:isolation & purification
D000081:Acetamides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003030:Coagulase; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D023303:Oxazolidinones; D012338:RNA, Ribosomal, 23S; C000716547:RPL3 protein, human; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D000069349:Linezolid
S. capitis; ribosomal mutations; rlmN