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 β-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.

中文翻译：

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铜绿假单胞菌多药耐药临床分离株中oprD孔蛋白基因的突变变异分析。

本研究涉及多药耐药性铜绿假单胞菌的29种临床细菌分离物中的外膜OprD孔蛋白。使用实时逆转录PCR研究oprD孔蛋白基因表达。对来自oprD的扩增子及其转录调节子mexT基因进行了测序，并分析了其突变。通过同源建模方法预测和完善了所选突变OprD-孔蛋白的假设模型。还筛选了oprDampliconic序列的限制性片段长度多态性（RFLP）。该OPRD发现基因在89.7％被下调（ñ 与参考菌株铜绿假单胞菌-PAO（MTCC-3541）中的转录水平相比，分离株的分离度为26 ）。有趣的是，所有这些分离株在mexT调节基因的核苷酸位置235处都显示出明显的8 bp缺失（GGCCAGCC）。根据oprD基因中观察到的突变模式，将分离株分为A，B1-2，C1-4，D1-6，E1-2和F。我们的假设模型表明，突变主要局限于β-桶孔蛋白的细胞外环。这些蛋白质模型还使得能够清楚地看到截短的多肽的实质部分的损失。顺便说一句，由于大多数操作发现临床分离株的扩增子显示出不同的RFLP谱带模式，我们的结果也为检测铜绿假单胞菌孔蛋白突变体提供了有用的诊断工具。