Mesaros Narcisa, Glupczynski Youri, Avrain Laëtitia, Caceres Nancy E, Tulkens Paul M, Van Bambeke Françoise
Unité de Pharmacologie cellulaire et moléculaire, Brussels, Université catholique de Louvain, UCL 7370 avenue E. Mounier 73, B-1200 Bruxelles, Belgium.
J Antimicrob Chemother. 2007 Mar;59(3):378-86. doi: 10.1093/jac/dkl504. Epub 2007 Feb 8.
Mex efflux pumps contribute to multidrug resistance in Pseudomonas aeruginosa. Evidencing their expression in clinical isolates would help in rationalizing antibiotic selection.
We have developed a combined phenotypic and genotypic approach for the differential diagnosis of resistance mediated by four major transporters (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM). The methodology was validated using reference strains harbouring only one specific transporter and its applicability evaluated towards seven selected clinical isolates, the resistance mechanisms of which could not be assigned by conventional techniques. Phenotypic detection used MIC measurements with reporter antibiotics [carbenicillin (MexAB-OprM); erythromycin (MexCD-OprJ); norfloxacin and imipenem (MexEF-OprN); gentamicin (MexXY-OprM)] with and without Phe-Arg-beta-naphthylamide. Genotypic detection was made by semi-quantitative reverse transcription PCR (RT-PCR) for mexC and mexE, and by quantitative competitive RT-PCR and real-time PCR for mexA and mexX (correlation between both methods: > 88 % ; overexpression levels ranging between 4.8 and 8.1).
Convergence between phenotypic and genotypic methods was observed in control strains for all pumps. For clinical isolates, convergence was obtained in 6 of 7 strains for MexXY-OprM and MexEF-OprM, and in 5 of 7 for MexAB-OprM and MexCD-OprJ, mostly due to hard to interpret phenotypic data.
The data plead for combining phenotypic and genotypic approaches in the diagnosis of efflux-mediated resistance in P. aeruginosa.
Mex外排泵与铜绿假单胞菌的多药耐药性有关。证明其在临床分离株中的表达有助于合理选择抗生素。
我们开发了一种结合表型和基因型的方法，用于鉴别由四种主要转运蛋白（MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM）介导的耐药性。使用仅携带一种特定转运蛋白的参考菌株对该方法进行验证，并评估其对七株选定临床分离株的适用性，这些分离株的耐药机制无法通过传统技术确定。表型检测使用含和不含苯丙氨酸-精氨酸-β-萘酰胺的报告抗生素[羧苄青霉素（MexAB-OprM）；红霉素（MexCD-OprJ）；诺氟沙星和亚胺培南（MexEF-OprN）；庆大霉素（MexXY-OprM）]进行MIC测量。基因型检测通过对mexC和mexE进行半定量逆转录PCR（RT-PCR），以及对mexA和mexX进行定量竞争性RT-PCR和实时PCR来进行（两种方法之间的相关性：>88%；过表达水平在4.8至8.1之间）。
在所有泵的对照菌株中观察到表型和基因型方法之间的一致性。对于临床分离株，MexXY-OprM和MexEF-OprM在7株中有6株获得了一致性，MexAB-OprM和MexCD-OprJ在7株中有5株获得了一致性，这主要是由于表型数据难以解释。
这些数据支持在铜绿假单胞菌外排介导的耐药性诊断中结合表型和基因型方法。
