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 .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/012427-0
2008-02-01
2020-02-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/2/454.html?itemId=/content/journal/micro/10.1099/mic.0.2007/012427-0&mimeType=html&fmt=ahah

References

  1. Alekshun M. N., Levy S. B.. 1997; Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob Agents Chemother41:2067–2075
    [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 Microbiol47:1105–1113
    [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 Bacteriol182:3467–3474
    [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 Microbiol8:847–856
    [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 Chemother52:176–179
    [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 Chemother48:2518–2523
    [Google Scholar]
  7. CLSI 2006; Performance Standards for Antimicrobial Susceptibility Testing . Supplement M100–S16 Wayne, PA: Clinical and Laboratory Standards Institute;
    [Google Scholar]
  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 Bacteriol167:66–72
    [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 Chemother33:785–787
    [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 Bacteriol173:5532–5538
    [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 . Gene109:137–141
    [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 Chemother49:1495–1501
    [Google Scholar]
  14. Kumar A., Worobec E. A.. 2005b; HasF, a TolC-homolog of Serratia marcescens , is involved in energy-dependent efflux. Can J Microbiol51:497–500
    [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. Drugs64:159–204
    [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 Microbiol16:45–55
    [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 Chem3:283–303
    [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 Chemother28:639–653
    [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 Bacteriol184:4161–4167
    [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 Bacteriol179:5756–5757
    [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 Immun75:379–389
    [Google Scholar]
  23. Pumbwe L., Piddock J. V.. 2002; Identification and molecular characterization of CmeB, a Campylobacter jejuni multidrug efflux pump. FEMS Microbiol Lett206:185–189
    [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 Chemother59:1001–1004
    [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 Chemother49:1477–1482
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/012427-0
Loading
/content/journal/micro/10.1099/mic.0.2007/012427-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error