1887

Abstract

is a prominent opportunistic nosocomial pathogen resistant to several classes of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is active efflux. Our group previously identified SdeAB, a resistance-nodulation-cell division (RND) efflux pump complex, and a TolC-like outer-membrane protein (HasF), which together mediate energy-dependent fluoroquinolone efflux. In addition, a regulatory protein-encoding gene in the upstream region of was identified () and found to be 40 % homologous to MarA, an transcriptional regulator. To provide conclusive evidence as to the role of these components in , , and deletion mutants were constructed. Suicide vectors were created and introduced via triparental mating into UOC-67 (wild-type) and, for and , T-861 (clinical isolate). We have analysed these genetically altered strains using minimal inhibitory concentration (MIC) assays for a wide range of compounds (fluoroquinolones, SDS, novobiocin, ethidium bromide and chloramphenicol). Intracellular accumulation of a variety of fluoroquinolones was measured fluorospectroscopically. The , and knockout strains were consistently more susceptible to antibiotics than the parent strains, with the / double knockout strain showing the highest susceptibility. A marked increase in fluoroquinolone (ciprofloxacin) accumulation was observed for strains deficient in either the or genes when compared to the parental strains, with the highest ciprofloxacin accumulation observed for the / double knockout. Antibiotic accumulation assays for the knockout mutant strains performed in the presence of carbonyl cyanide -chlorophenylhydrazone (CCCP), a proton-motive-force inhibitor, demonstrated that SdeAB-mediated efflux is proton-motive-force dependent. Due to the comparable susceptibility of the and the individual knockouts, we conclude that HasF is the sole outer-membrane component of the SdeAB pump. In addition, MIC data for -deficient and overexpressing strains confirm that SdeR is an activator of and acts to enhance the overall multidrug resistance of .

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2019-10-13
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References

  1. Alekshun, M. N. & Levy, S. B. ( 1997; ). Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother 41, 2067–2075.
    [Google Scholar]
  2. Aucken, H. M. & Pitt, T. L. ( 1998; ). Antibiotic resistance and putative virulence factors of Serratia marcescens with respect to O and K serotypes. J Med Microbiol 47, 1105–1113.[CrossRef]
    [Google Scholar]
  3. Barbosa, T. M. & Levy, S. B. ( 2000; ). Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA. J Bacteriol 182, 3467–3474.[CrossRef]
    [Google Scholar]
  4. Buckley, A. M., Webber, M. A., Cooles, S., Randall, L. P., La Ragione, R. M., Woodward, M. J. & Piddock, L. J. V. ( 2006; ). The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis. Cell Microbiol 8, 847–856.[CrossRef]
    [Google Scholar]
  5. Chen, J., Kuroda, T., Huda, M. N., Mizushima, T. & Tsuchiya, T. ( 2003; ). An RND-type multidrug efflux pump SdeXY from Serratia marcescens. J Antimicrob Chemother 52, 176–179.[CrossRef]
    [Google Scholar]
  6. Chollet, R., Chevalier, J., Bollet, C., Pages, J. M. & Davin-Regli, A. ( 2004; ). RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 48, 2518–2523.[CrossRef]
    [Google Scholar]
  7. CLSI ( 2006; ). Performance Standards for Antimicrobial Susceptibility Testing. Supplement M100–S16. Wayne, PA: Clinical and Laboratory Standards Institute.
  8. Finan, T. M., Kunkel, B., De Vos, G. F. & Signer, E. R. ( 1986; ). Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167, 66–72.
    [Google Scholar]
  9. Fujimaki, K., Fujii, T., Aoyama, H., Sato, K.-I., Inoue, Y., Inoue, M. & Mitsuhashi, S. ( 1989; ). Quinolone resistance in clinical isolates of Serratia marcescens. Antimicrob Agents Chemother 33, 785–787.[CrossRef]
    [Google Scholar]
  10. Hachler, H., Cohen, S. P. & Levy, S. B. ( 1991; ). marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli. J Bacteriol 173, 5532–5538.
    [Google Scholar]
  11. Kaniga, K., Delor, I. & Cornelis, G. R. ( 1991; ). A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene 109, 137–141.[CrossRef]
    [Google Scholar]
  12. Kumar, A. ( 2004; ). Characterization of RND efflux pumps of Serratia marcescens. PhD thesis, Department of Microbiology, University of Manitoba.
  13. Kumar, A. & Worobec, E. A. ( 2005a; ). Cloning, sequencing and characterization of the SdeAB multidrug efflux pump of Serratia marcescens. Antimicrob Agents Chemother 49, 1495–1501.[CrossRef]
    [Google Scholar]
  14. Kumar, A. & Worobec, E. A. ( 2005b; ). HasF, a TolC-homolog of Serratia marcescens, is involved in energy-dependent efflux. Can J Microbiol 51, 497–500.[CrossRef]
    [Google Scholar]
  15. Lambert, H. P. & O'Grady, F. W. ( 1992; ). Antibiotics and Chemotherapy. Edinburgh: Churchill Livingstone.
  16. Li, X.-Z. & Nikaido, H. ( 2004; ). Efflux-mediated drug resistance in bacteria. Drugs 64, 159–204.[CrossRef]
    [Google Scholar]
  17. Ma, D., Cook, D. N., Alberti, M., Pon, N. G., Nikaido, H. & Hearst, J. E. ( 1995; ). Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol 16, 45–55.[CrossRef]
    [Google Scholar]
  18. Maxwell, A. & Lawson, D. M. ( 2003; ). The ATP-binding site of type II topoisomerases as a target for antibacterial drugs. Curr Top Med Chem 3, 283–303.[CrossRef]
    [Google Scholar]
  19. Mortimer, P. G. & Piddock, L. J. ( 1991; ). A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. J Antimicrob Chemother 28, 639–653.[CrossRef]
    [Google Scholar]
  20. Nagakubo, S., Nishino, K., Hirata, T. & Yamaguchi, A. ( 2002; ). The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system, MdtABC. J Bacteriol 184, 4161–4167.[CrossRef]
    [Google Scholar]
  21. Pearson, J. P., Pesci, E. C. & Iglewski, B. H. ( 1997; ). Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J Bacteriol 179, 5756–5757.
    [Google Scholar]
  22. Posadas, D. M., Martin, F. A., Sabio y Garcia, J. V., Spera, J. M., Delpino, M. V., Baldi, P., Campos, E., Cravero, S. L. & Zorreguieta, A. ( 2007; ). The TolC homologue of Brucella suis is involved in resistance to antimicrobial compounds and virulence. Infect Immun 75, 379–389.[CrossRef]
    [Google Scholar]
  23. Pumbwe, L. & Piddock, J. V. ( 2002; ). Identification and molecular characterization of CmeB, a Campylobacter jejuni multidrug efflux pump. FEMS Microbiol Lett 206, 185–189.[CrossRef]
    [Google Scholar]
  24. Ruzin, A., Keeney, D. & Bradford, P. A. ( 2007; ). AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus–Acinetobacter baumannii complex. J Antimicrob Chemother 59, 1001–1004.[CrossRef]
    [Google Scholar]
  25. van Amsterdam, K., Bart, A. & van der Ende, A. ( 2005; ). A Helicobacter pylori TolC efflux pump confers resistance to metronidazole. Antimicrob Agents Chemother 49, 1477–1482.[CrossRef]
    [Google Scholar]
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vol. 154, part 2, pp. 454 - 461

Details of some of the strains and plasmids given in the original paper, and of the construction and complementation of -, - and -deficient strains, were missing or incorrect. Details are provided in the accompanying [ PDF] (76 kb).



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