1887

Abstract

The effects of imipenem and meropenem on the transcriptional expression of resistance-related genes , and in were studied by quantitative real-time PCR. Four strains were examined: the type strain PT5 (PAO1), its derivatives M7 and PT149, and a clinical isolate, PaKT3. The derivative M7 is a mutant, overexpressing the MexAB–OprM pump, and the derivative PT149 is a C-type mutant, overexpressing the MexEF–OprN pump while it is down-regulated for the OprD protein. After 18 h incubation in broth, the cultures were divided into three portions. Two were supplemented with antibiotics and the other was left antibiotic-free as the control. After a further 45 min incubation, total RNA was isolated from the strains by guanidine denaturation and acid-phenol/chloroform extraction. DNA-free total RNAs were immediately reverse-transcribed by MMuLV reverse transcriptase. Concentrations of mRNAs obtained by quantitative PCR were expressed relative to uninduced portions of the strains. The results showed that was relatively stable against carbapenem antibiotics. was induced significantly by imipenem in only one strain and was induced by imipenem in most of the strains. The responses at the mRNA level found here were unexpected and suggested a chaotic, unpredictable regulatory mechanism.

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2004-09-01
2024-03-28
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References

  1. Aires J. R., Pechere J. C., van Delden C., Kohler T. 2002; Amino acid residues essential for function of the MexF efflux pump protein of Pseudomonas aeruginosa . Antimicrob Agents Chemother 46:2169–2173 [CrossRef]
    [Google Scholar]
  2. Evans K., Passador L., Srikumar R., Tsang E., Nezezon J., Poole K. 1998; Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa . J Bacteriol 180:5443–5447
    [Google Scholar]
  3. Huang H., Hancock R. E. 1996; The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa . J Bacteriol 178:3085–3090
    [Google Scholar]
  4. Kohler T., Michea-Hamzehpour M., Henze U., Gotoh N., Curty L. K., Pechere J. C. 1997; Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa . Mol Microbiol 23:345–354 [CrossRef]
    [Google Scholar]
  5. Kohler T., van Delden C., Curty L. K., Hamzehpour M. M., Pechere J. C. 2001; Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa . J Bacteriol 183:5213–5222 [CrossRef]
    [Google Scholar]
  6. Maseda H., Yoneyama H., Nakae T. 2000; Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa . Antimicrob Agents Chemother 44:658–664 [CrossRef]
    [Google Scholar]
  7. Masuda N., Sakagawa E., Ohya S., Gotoh N., Tsujimoto H., Nishino T. 2000; Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa . Antimicrob Agents Chemother 44:3322–3327 [CrossRef]
    [Google Scholar]
  8. Ochs M. M., McCusker M. P., Bains M., Hancock R. E. 1999; Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids. Antimicrob Agents Chemother 43:1085–1090
    [Google Scholar]
  9. Perez F. J., Gimeno C., Navarro D., Garcia-de-Lomas J. 1996; Meropenem permeation through the outer membrane of Pseudomonas aeruginosa can involve pathways other than the OprD porin channel. Chemotherapy 42:210–214 [CrossRef]
    [Google Scholar]
  10. Pfaffl M. W., Horgan G. W., Dempfle L. 2002; Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36. e36: [CrossRef]
    [Google Scholar]
  11. Poole K. 2000; Efflux-mediated resistance to fluoroquinolones in gram-negative bacteria. Antimicrob Agents Chemother 44:2233–2241 [CrossRef]
    [Google Scholar]
  12. Poole K. 2001; Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. J Mol Microbiol Biotechnol 3:255–264
    [Google Scholar]
  13. Savli H., Karadenizli A., Kolayli F., Gundes S., Ozbek U., Vahaboglu H. 2003; Expression stability of six housekeeping genes : a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 52:403–408 [CrossRef]
    [Google Scholar]
  14. Trias J., Dufresne J., Levesque R. C., Nikaido H. 1989; Decreased outer membrane permeability in imipenem-resistant mutants of Pseudomonas aeruginosa . Antimicrob Agents Chemother 33:1202–1206 [CrossRef]
    [Google Scholar]
  15. Wylie J. L., Worobec E. A. 1995; The OprB porin plays a central role in carbohydrate uptake in Pseudomonas aeruginosa . J Bacteriol 177:3021–3026
    [Google Scholar]
  16. Yoneyama H., Nakae T. 1993; Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa . Antimicrob Agents Chemother 37:2385–2390 [CrossRef]
    [Google Scholar]
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