1887

Abstract

Constantly expressed genes are used as internal controls in relative quantification studies. Suitable internal controls for such studies have not yet been defined for . In this study, the genes , , , , and of were compared in terms of expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistance phenotypes were studied. Stability of expression among the housekeeping genes was assessed on the basis of correlation coefficients, with the best-correlated pair accepted as being the most stable one. Eventually, and formed the most stable pair ( = 0.958; < 0.001). Next, in four ciprofloxacin-selected -like mutants, levels of , and mRNA were compared with those of their wild-type counterparts. The comparison was made after correcting the raw values by the geometric mean of the internal control genes and . The level of mRNA was significantly up-regulated, while the gene was down-regulated (although this difference was statistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of -type mutants; this experiment therefore lends further support to the use of and genes simultaneously as internal controls for such studies.

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2003-05-01
2020-10-24
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References

  1. Gygi S. P, Rochon Y, Franza B. R, Aebersold R. 1999; Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730
    [Google Scholar]
  2. Jacobs C, Joris B, Jamin M. 7 other authors 1995; AmpD, essential for both beta-lactamase regulation and cell wall recycling, is a novel cytosolic N -acetylmuramyl-l-alanine amidase. Mol Microbiol 15:553–559 [CrossRef]
    [Google Scholar]
  3. Johnson M. R, Wang K, Smith J. B, Heslin M. J, Diasio R. B. 2000; Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem 278:175–184 [CrossRef]
    [Google Scholar]
  4. Kerr J. R, Moore J. E, Curran M. D, Graham R, Webb C. H, Lowry K. G, Murphy P. G, Wilson T. S, Ferguson W. P. 1995; Investigation of a nosocomial outbreak of Pseudomonas aeruginosa pneumonia in an intensive care unit by random amplification of polymorphic DNA assay. J Hosp Infect 30:125–131 [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. Kutchma A. J, Hoang T. T, Schweizer H. P. 1999; Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A : ACP transacylase (FabD). J Bacteriol 181:5498–5504
    [Google Scholar]
  7. Livermore D. M. 1992; Interplay of impermeability and chromosomal beta-lactamase activity in imipenem-resistant Pseudomonas aeruginosa . Antimicrob Agents Chemother 36:2046–2048 [CrossRef]
    [Google Scholar]
  8. 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]
  9. 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]
  10. Morita Y, Komori Y, Mima T, Kuroda T, Mizushima T, Tsuchiya T. 2001; Construction of a series of mutants lacking all of the four major mex operons for multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1: MexCD-OprJ is an inducible pump. FEMS Microbiol Lett 202:139–143 [CrossRef]
    [Google Scholar]
  11. 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]
  12. Okamoto K, Gotoh N, Nishino T. 2001; Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay. Antimicrob Agents Chemother 45:1964–1971 [CrossRef]
    [Google Scholar]
  13. Poole K. 2001; Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms. J Mol Microbiol Biotechnol 3:255–264
    [Google Scholar]
  14. Renders N, Romling Y, Verbrugh H, van Belkum A. 1996; Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments. J Clin Microbiol 34:3190–3195
    [Google Scholar]
  15. Schnider U, Keel C, Blumer C, Troxler J, Defago G, Haas D. 1995; Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities. J Bacteriol 177:5387–5392
    [Google Scholar]
  16. Sumita Y, Fukasawa M. 1996; Meropenem resistance in Pseudomonas aeruginosa . Chemotherapy 42:47–56
    [Google Scholar]
  17. 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]
  18. Vahaboglu H, Ozturk R, Akbal H, Saribas S, Tansel O, Coskunkan F. 1998; Practical approach for detection and identification of OXA-10-derived ceftazidime-hydrolyzing extended-spectrum beta-lactamases. J Clin Microbiol 36:827–829
    [Google Scholar]
  19. Vandecasteele S. J, Peetermans W. E, Merckx R, Van Eldere J. 2001; Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J Bacteriol 183:7094–7101 [CrossRef]
    [Google Scholar]
  20. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002; Accurate normalization of real- time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034.1-0034.11 http://genomebiology.com/2002/3/7/research/0034
    [Google Scholar]
  21. Wang T, Brown M. J. 1999; mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal Biochem 269:198–201 [CrossRef]
    [Google Scholar]
  22. Wei Y, Lee J. M, Richmond C, Blattner F. R, Rafalski J. A, LaRossa R. A. 2001; High-density microarray-mediated gene expression profiling of Escherichia coli . J Bacteriol 183:545–556 [CrossRef]
    [Google Scholar]
  23. Yucesoy M, Yulug N, Kocagoz S, Unal S, Cetin S, Calangu S. 2000; Antimicrobial resistance of gram-negative isolates from intensive care units in Turkey: comparison to previous three years. J Chemother 12:294–298 [CrossRef]
    [Google Scholar]
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