title: Amino Acid Substitutions of CrrB Responsible for Resistance to Colistin through CrrC in Klebsiella pneumoniae.
abstract: Colistin is a last-resort antibiotic for treatment of carbapenem-resistant Klebsiella pneumoniae A recent study indicated that missense mutations in the CrrB protein contribute to colistin resistance. In our previous study, mechanisms of colistin resistance were defined in 17 of 26 colistin-resistant K. pneumoniae clinical isolates. Of the remaining nine strains, eight were highly resistant to colistin. In the present study, crrAB sequences were determined for these eight strains. Six separate amino acid substitutions in CrrB (Q10L, Y31H, W140R, N141I, P151S, and S195N) were detected. Site-directed mutagenesis was used to generate crrB loci harboring individual missense mutations; introduction of the mutated genes into a susceptible strain, A4528, resulted in 64- to 1,024-fold increases in colistin MICs. These crrB mutants showed increased accumulation of H239_3062, H239_3059, pmrA, pmrC, and pmrH transcripts by quantitative reverse transcription (qRT)-PCR. Deletion of H239_3062 (but not that of H239_3059) in the A4528 crrB(N141I) strain attenuated resistance to colistin, and H239_3062 was accordingly named crrC Similarly, accumulation of pmrA, pmrC, and pmrH transcripts induced by crrB(N141I) was significantly attenuated upon deletion of crrC Complementation of crrC restored resistance to colistin and accumulation of pmrA, pmrC, and pmrH transcripts in a crrB(N141I) ΔcrrC strain. In conclusion, novel individual CrrB amino acid substitutions (Y31H, W140R, N141I, P151S, and S195N) were shown to be responsible for colistin resistance. We hypothesize that CrrB mutations induce CrrC expression, thereby inducing elevated expression of the pmrHFIJKLM operon and pmrC (an effect mediated via the PmrAB two-component system) and yielding increased colistin resistance.
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Yi-Hsiang
authors.initials: YH
authors.lastname: Cheng
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Tzu-Lung
authors.initials: TL
authors.lastname: Lin
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan School of Medicine, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Yi-Tsung
authors.initials: YT
authors.lastname: Lin
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan wangjt@ntu.edu.tw.
authors.forename: Jin-Town
authors.initials: JT
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003091:Colistin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009876:Operon; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C510752:PmrC protein, E coli; D012333:RNA, Messenger; C081902:pmrA protein, Bacteria; D003091:Colistin