Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170
Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170
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DOI：
10.1093/jac/dkx158
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发表时间：
2017-09-01
影响因子：
5.2
通讯作者：
Wu, Tsu-Lan
中科院分区：
文献类型：
--
作者：
Shu, Jwu-Ching;Kuo, An-Jing;Wu, Tsu-Lan
目的：发现携带环7缩短OprD （1a组等位基因）的泛敏感铜绿假单胞菌（PSPA）临床分离株在持续的选择压力下迅速产生碳青霉烯类耐药性。我们进一步研究了OprD多态性是否与潜在的碳青霉烯类耐药性有关。方法：以铜绿假单胞菌PAO1为对照菌株，分析126株PSPA临床分离株的OprD氨基酸序列，测定其STs。在PAO1中进行定点诱变以产生感兴趣的多态性。采用圆盘扩散法从突变株中筛选耐碳青霉烯变异体。采用定量RT-PCR检测oprD的表达水平。采用Etest法测定碳青霉烯类药物的mic。结果：48株（38.1%）携带1a组等位基因。另外两个主要STs C1和C2分别在21个（16.7%）和39个（31.0%）分离株中发现F170L多态性。14株（11.1%）分离出PAO1型。在持续的选择压力下，大多数STs菌株在不同传代次数下都产生了碳青霉烯抗性；只有那些属于PAO1型的人仍然易感。然而，携带oprD group-1A等位基因或oprD - f170l多态性的PAO1突变体能够产生碳青霉烯类耐药性。在PAO1突变体中发现持续亚胺培南刺激引起的oprD表达降低，但在PAO1 WT株中没有发现。结论：OprD多态性，特别是F170L替换和环7的特异性缩短，似乎决定了P. aeruginosa产生碳青霉烯类耐药性的可能性。
Objectives: 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.Methods: 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.Results: 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.Conclusions: 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.