Cloning, sequencing, and characterization of the SdeAB multidrug efflux pump of Serratia marcescens.
Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.
Kumar
Ayush
A
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Manitoba, Canada.
Worobec
Elizabeth A
EA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D003001:Cloning, Molecular; D024901:Drug Resistance, Multiple, Bacterial; D004996:Ethidium / Q000494:pharmacology; D024841:Fluoroquinolones / Q000494:pharmacology; D006586:Hexanes / Q000494:pharmacology; D008969:Molecular Sequence Data; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012706:Serratia marcescens / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D024841:Fluoroquinolones; D006586:Hexanes; D017494:Proton Pumps; C026385:n-hexane; D012967:Sodium Dodecyl Sulfate; D002701:Chloramphenicol; D004996:Ethidium