Mutational Variation Analysis of oprD Porin Gene in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa.

The present study deals with the outer membrane OprD porin protein in 29 clinical bacterial isolates of multidrug-resistant Pseudomonas aeruginosa. oprD porin gene expression was investigated using real-time reverse transcription-PCR. Amplicons from oprD and its transcriptional regulator mexT gene were sequenced and analyzed for mutations. Hypothetical models of selected mutant OprD-porin proteins were predicted and refined by homology modeling approach. oprD ampliconic sequences were also screened for restriction fragment length polymorphism (RFLP). The oprD gene was found to be downregulated in 89.7% (n = 26) of the isolates in comparison to the transcript levels in the reference strain P. aeruginosa-PAO (MTCC-3541). Interestingly, all these isolates displayed the presence of a conspicuous 8-bp deletion (GGCCAGCC) at nucleotide position 235 of mexT regulatory gene. Based on the mutational patterns observed in oprD gene, the isolates were classified into categories designated as A, B1-2, C1-4, D1-6, E1-2, and F. Our hypothetical models revealed that mutations were predominantly confined to the extracellular loops emanating from the ß-barrel porin protein. These protein models also enabled clear visualization of loss of substantial portions of the truncated polypeptide. Incidentally, since most of the oprD amplicons of the clinical isolates were found to display distinct RFLP banding patterns, our results also provide a useful diagnostic tool for detection of P. aeruginosa porin mutants.

Mutational analyses of regulatory genes, mexR, nalC, nalD and mexZ of mexAB-oprM and mexXY operons, in efflux pump hyperexpressing multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

The present study deals with membrane-bound efflux pumps, MexAB-OprM and MexXY and their respective regulatory genes mexR, nalC, nalD and mexZ in multidrug resistant (MDR) Pseudomonas aeruginosa. Following antibiotic sensitivity testing and detection of various beta-lactamases, hyperexpression of efflux pump genes, mexB and mexY in the isolates was investigated using semi-quantitative and real-time reverse transcription-PCR. Amplicons from regulatory genes were sequenced and subjected to mutational and phylogenetic analysis. Twenty-nine clinical isolates of P. aeruginosa were obtained from a total of 144 MDR gram-negative bacteria collected from Kerala State, South India. All strains were found to be resistant to ampicillin and nalidixic acid with 13.8, 44.8 and 31% testing positive for extended-spectrum beta-lactamases, metallo-beta-lactamases and AmpC producers respectively. Increased mexB and mexY transcription was detected respectively in 10.3 and 20.7% of the isolates in comparison with P. aeruginosa reference strain, PAO (MTCC). Co-expression of MexY was also observed in MexB overproducers. Various synonymous/and non-synonymous mutations in regulatory gene sequences of efflux pump operons were detected. In the strain designate Pa16, mexR was found to harbour four novel point mutations with one transversion and three transitions which included a substitution of an ochre codon with that for serine. The gene also displayed a novel mutation involving insertion of a cysteine at the 444th base position, followed by an opal codon. The genetic divergence and homogeneity of the concatenated (mexR, nalC and nalD) regulatory gene sequences of mexAB-oprM operon was apparent in the phylogram generated with similar sequences retrieved from public database.