UCD Centre for Food Safety & Centre for Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland.
Appl Environ Microbiol. 2011 Oct;77(20):7113-20. doi: 10.1128/AEM.00600-11. Epub 2011 Aug 19.
Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.
从都柏林爱尔兰大学兽医医院获得的一份采集样本中选择了 11 株多药耐药性大肠杆菌分离株（包括 6 株猪源性和 5 株牛源性田间分离株），这些分离株对氟喹诺酮类（FQ）具有耐药性。通过 Etest 法测定萘啶酸和环丙沙星的 MIC。所有分离株的萘啶酸 MIC 均>256μg/ml，环丙沙星 MIC 范围为 4 至>32μg/ml。通过 DNA 测序鉴定了目标基因喹诺酮耐药决定区的突变，并通过定量实时 PCR（qRT-PCR）评估了主要孔蛋白 OmpF 的表达以及 AcrAB-TolC 外排泵及其相关调节基因座的组成基因。在某些但不是所有分离株中，在存在外排泵抑制剂苯丙氨酸-精氨酸-β-萘基酰胺（PAβN）的情况下，观察到对萘啶酸和/或环丙沙星的 MIC 值降低。在编码喹诺酮靶酶的基因中发现了几种突变（每种菌株 3 至 5 个突变）。所有分离株均在 GyrA 基因的 83 和 87 位氨基酸取代。观察到新的 GyrA（Asp87→Ala）、ParC（Ser80→Trp）和 ParE（Glu460→Val）取代。使用半自动化溴化乙锭（EB）摄取测定法评估了这些分离株的外排活性。与野生型大肠杆菌 K-12 AG100 相比，分离株积累的 EB 较少，并且在存在 PAβN 的情况下，EB 的积累增加。在 11 个分离株中的 5 个中观察到 acrB 基因（编码 AcrAB-TolC 外排泵的泵组件）的上调，而 10 个分离株显示 OmpF 表达降低。这项研究确定了多种机制，这些机制可能有助于研究中田间分离株对基于喹诺酮类药物的耐药性。
