Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa

The gene mexR regulates negatively the expression of the MexA-MexB-OprM eff lux pump in Pseudomonas aeruginosa, and mutations in mexR cause a multiple antibiotic resistance phenotype. Five hundred and forty resistant clones of P. aeruginosa PAO503 were isolated after selection for resistance to chlor amphenicol or tetracycline. All isolates showed similar phenotypes and were resistant to tetracycline, chloramphenicol and norfloxacin. Nineteen rando mly selected isolates were analyzed. Since mutational analysis by direct se quencing of all regions of interest in several strains is time-consuming an d expensive, a screening method, Non-Isotopic RNase Cleavage Assay (NIRCA(T M)), was applied to identify mutant genes so that they could be targeted fo r DNA sequencing. NIRCA is a simple but rapid method for mutational analysi s and can be performed in 3-4 h. Results of NIRCA analysis were compared wi th DNA sequencing. Both NIRCA and DNA sequencing analysis showed mexR gene mutations in 11 of 19 isolates but no alterations in 8 strains. An immunobl ot assay showed overexpression of OprN, a component of another multidrug ef flux pump, MexE-MexF-OprN, in those eight isolates. Nucleotide sequencing o f quinolone resistance-determining regions of DNA gyrase (gyrA) or topoisom erase IV (parC) showed no alterations in any of the 19 mutants. The data in dicate that two efflux pump systems, MexA-MexB-OprM and MexE-MexF-OprN, wer e involved in multidrug resistance including quinolones and that NIRCA is a sensitive method for screening mutations.