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Effect of carbapenems on the transcriptional expression of the oprD, oprM and oprN genes in Pseudomonas aeruginosa

^1–3,5Mikrobiyoloji ve Klinik Mikrobiyoloji AD¹, Tibbi Biyoloji AD², Biyofizik AD³ and Enfeksiyon Hastaliklari & Klinik Mikrobiyoloji AD⁵, KOU Tip Fakultesi, Sopali Ciftligi, 41900 Derince Kocaeli, Turkey ⁴IU, Deneysel Tip Arastirma Enstitusu, Istanbul, Turkey

Correspondence Haluk Vahaboglu vahabo{at}hotmail.com

Received April 7, 2004
Accepted May 27, 2004

The effects of imipenem and meropenem on the transcriptional expression of resistance-related genes oprD, oprM and oprN in Pseudomonas aeruginosa 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 nalB mutant, overexpressing the MexAB–OprM pump, and the derivative PT149 is a nfxC-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 oprD was relatively stable against carbapenem antibiotics. oprM was induced significantly by imipenem in only one strain and oprN 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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