In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment.
Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB(L82R) in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB(L82R) mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.
Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Antonio
A
Cannatelli
Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Vincenzo
V
Di Pilato
Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Tommaso
T
Giani
Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Fabio
F
Arena
Bacteriology Laboratory, St. Orsola University Hospital, Bologna, Italy.
Simone
S
Ambretti
Regional Laboratory for Microbiological Emergencies, Operative Unit of Clinical Microbiology, St. Orsola University Hospital, Bologna, Italy.
Paolo
P
Gaibani
Department of Medical Biotechnologies, University of Siena, Siena, Italy.
Marco Maria
MM
D'Andrea
Department of Medical Biotechnologies, University of Siena, Siena, Italy Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy Clinical Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy gianmaria.rossolini@unifi.it.
Gian Maria
GM
Rossolini
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002999:Clone Cells; D003091:Colistin / Q000378:metabolism / Q000494:pharmacology*; D054796:Drug Dosage Calculations; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007710:Klebsiella Infections / Q000188:drug therapy / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D008070:Lipopolysaccharides / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009876:Operon; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; C409039:PmrB protein, bacteria; D014157:Transcription Factors; C081902:pmrA protein, Bacteria; D001618:beta-Lactamases; C063912:carbapenemase; D003091:Colistin