Terzi Huseyin Agah, Kulah Canan, Ciftci Ihsan Hakkı
Department of Medical Microbiology, Sakarya University Training and Research Hospital, Sakarya, Turkey,
World J Microbiol Biotechnol. 2014 Oct;30(10):2681-7. doi: 10.1007/s11274-014-1692-2. Epub 2014 Jun 26.
In this study, we investigated the roles of active efflux pumps in antibiotic resistance. The transcription efflux pump genes were analyzed by real-time polymerase chain reaction (qPCR) to determine their role in drug resistance. Antibiotic sensitivity testing was carried out using the Vitek 2 automated system (bioMérieux, France). Isolates were divided into four groups according to their resistance status: multiple-drug resistant (MDR), isolated carbapenem resistant (ICR), isolated quinolone resistant (IQR), and carbapenem and quinolone resistant (CQR). Transcript levels of mexB, mexD, mexF, and mexY were analyzed by qPCR using a LightCycler instrument (Roche, Germany). The genetic similarity between isolates was determined using arbitrarily primed PCR (AP-PCR). Among the 50 isolates investigated, the frequency of genes classified as overexpressed were 88 % for mexD, 76 % for mexB, 46 % for mexF, and 40 % for mexY. Within the MDR group, mexB was overexpressed in 15 of 22 isolates, mexD in 20 of 22, mexF in 15 of 22, and mexY in 19 of 22. In the ICR group, isolates mexB and mexD were each overexpressed in five isolates. mexD overexpression was observed in all seven CQR isolates. Within the IQR group, mexB and mexD were overexpressed in all 12 isolates. mexF overexpression was detected in 7 of 12 isolates in this group. 18 distinct banding patterns were determined by AP-PCR. Increased transcription of mexB was directly correlated with meropenem resistance in the majority of isolates tested, while MexCD-OprJ and MexEF-OprN were related to quinolone resistance; the MexCD-OprJ efflux pump was also related to multidrug resistance. Increased transcription of mexY may contribute to the gentamicin resistance.
在本研究中，我们调查了主动外排泵在抗生素耐药性中的作用。通过实时聚合酶链反应（qPCR）分析转录外排泵基因，以确定它们在耐药性中的作用。使用Vitek 2自动化系统（法国生物梅里埃公司）进行抗生素敏感性测试。根据分离株的耐药状态将其分为四组：多重耐药（MDR）、单独碳青霉烯耐药（ICR）、单独喹诺酮耐药（IQR）以及碳青霉烯和喹诺酮耐药（CQR）。使用LightCycler仪器（德国罗氏公司）通过qPCR分析mexB、mexD、mexF和mexY的转录水平。使用任意引物PCR（AP-PCR）确定分离株之间的遗传相似性。在所研究的50株分离株中，分类为过表达的基因频率分别为：mexD为88%，mexB为76%，mexF为46%，mexY为40%。在MDR组中，22株分离株中有15株mexB过表达，20株mexD过表达，15株mexF过表达，19株mexY过表达。在ICR组中，mexB和mexD在5株分离株中各自过表达。在所有7株CQR分离株中均观察到mexD过表达。在IQR组中，所有12株分离株中mexB和mexD均过表达。该组12株分离株中有7株检测到mexF过表达。通过AP-PCR确定了18种不同的条带模式。在所测试的大多数分离株中，mexB转录增加与美罗培南耐药直接相关，而MexCD-OprJ和MexEF-OprN与喹诺酮耐药有关；MexCD-OprJ外排泵也与多重耐药有关。mexY转录增加可能导致庆大霉素耐药。
