Department of Laboratory Medicine, Chang Gung Memorial Hospital Linkou, Taoyuan, Taiwan.
Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
J Antimicrob Chemother. 2017 Sep 1;72(9):2489-2495. doi: 10.1093/jac/dkx158.
Pan-susceptible Pseudomonas aeruginosa (PSPA) clinical isolates carrying an OprD with loop 7 shortening (the group-1A allele) were found to rapidly develop carbapenem resistance under continuous selection pressure. We further studied whether OprD polymorphisms are associated with the potential to develop carbapenem resistance.
OprD amino acid sequences of 126 PSPA clinical isolates were analysed to determine their STs using P. aeruginosa strain PAO1 as the control strain. Site-directed mutagenesis was performed in PAO1 to generate polymorphisms of interest. A disc diffusion method was used to select carbapenem-resistant variants from the mutant strains. Expression levels of oprD were determined by quantitative RT-PCR. MICs of carbapenems were determined by Etest.
Forty-eight (38.1%) of the tested isolates carried the group-1A allele. Another two major STs, C1 and C2, both of which harboured an F170L polymorphism, were found in 21 (16.7%) and 39 (31.0%) isolates, respectively. The PAO1 type was also found in 14 (11.1%) isolates. Under continuous selective pressure, isolates of most STs developed carbapenem resistance at different numbers of passaging events; only those belonging to the PAO1 type remained susceptible. However, PAO1 mutants carrying either the oprD group-1A allele or the OprD-F170L polymorphism were able to develop carbapenem resistance. Reduced oprD expression triggered by continuous imipenem challenge was found in PAO1 mutants, but not in the PAO1 WT strain.
OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.
携带 OprD 环 7 缩短（组 1A 等位基因）的泛敏感铜绿假单胞菌（PSPA）临床分离株在持续选择压力下迅速发展为碳青霉烯类耐药。我们进一步研究了 OprD 多态性是否与产生碳青霉烯类耐药的潜力相关。
分析了 126 株 PSPA 临床分离株的 OprD 氨基酸序列，以铜绿假单胞菌 PAO1 株作为对照菌株确定其 STs。在 PAO1 中进行定点突变以产生感兴趣的多态性。采用纸片扩散法从突变株中筛选出碳青霉烯类耐药变体。通过定量 RT-PCR 测定 oprD 的表达水平。采用 Etest 法测定碳青霉烯类的 MIC。
在检测的 126 株分离株中，有 48 株（38.1%）携带组 1A 等位基因。另外两种主要的 ST，C1 和 C2，都携带 F170L 多态性，分别在 21 株（16.7%）和 39 株（31.0%）分离株中发现。PAO1 型也在 14 株（11.1%）分离株中发现。在持续的选择压力下，大多数 ST 型的分离株在不同的传代次数下产生了碳青霉烯类耐药性；只有属于 PAO1 型的分离株仍然敏感。然而，携带 oprD 组 1A 等位基因或 OprD-F170L 多态性的 PAO1 突变株能够产生碳青霉烯类耐药性。在 PAO1 突变株中发现持续亚胺培南挑战引起的 oprD 表达减少，但在 PAO1 WT 株中没有发现。
OprD 多态性，特别是 F170L 取代和环 7 的特定缩短，似乎决定了铜绿假单胞菌产生碳青霉烯类耐药的潜力。
