Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China.
Vet Microbiol. 2012 Dec 28;161(1-2):159-68. doi: 10.1016/j.vetmic.2012.07.019. Epub 2012 Jul 20.
Multidrug resistance (MDR) is associated with fluoroquinolone (FQ) resistance in companion animal Escherichia coli (E. coli). In this study, gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced among uropathogenic E. coli isolates with different resistant phenotypes. Also determined were porin, efflux pump and regulatory gene expression based on quantitative real-time reverse transcriptase PCR (qRT-PCR), the impact of efflux pump inhibition (Phe-Arg-β-naphthylamide) and the presence of plasmid-mediated quinolone resistance (PMQR). Using enrofloxacin as the prototypic FQ, we found that (i) the number of mutations in target genes correlate well with minimum inhibitory concentrations (MICs). A single mutation (Ser83Leu) in gyrA increases FQ MIC in susceptible isolates; subsequent mutations result in resistance that increases from low (enrofloxacin MICs 4-16 μg/ml) to high level (enrofloxacin MICs≥128 μg/ml) with each progressive mutation. (ii) as MIC increase, acrB activity and the number of drug classes contributing to the MDR phenotype increases; (iii) a consistent relationship between regulatory gene expression and MIC could not be identified; and (iv) qnrS and aac(6')-Ib-cr gene were detected in 14 and 5 ENR(R)-MDR isolates containing the target mutation, respectively. Of 13 isolates expressing PDR isolates, 10 (77%) were positive for qnrS gene, and 4 (40%) carried both qnrS and aac(6')-Ib-cr gene. These findings demonstrated that MDR-associated FQ resistance in canine and feline uropathogenic E. coli reflects a combination of point mutations, enhanced efflux pump activities, and PMQR mechanisms. Point mutations in DNA gyrase, however, are necessary to achieve a clinical level of FQ resistance.
多药耐药性（MDR）与伴侣动物大肠杆菌（E. coli）中的氟喹诺酮（FQ）耐药性有关。在这项研究中，对具有不同耐药表型的尿路致病性大肠杆菌分离株的喹诺酮耐药决定区（QRDR）中的gyrA、gyrB、parC 和 parE 进行了测序。还根据定量实时逆转录 PCR（qRT-PCR）确定了孔蛋白、外排泵和调节基因的表达，以及外排泵抑制（Phe-Arg-β-萘基酰胺）的影响和质粒介导的喹诺酮耐药（PMQR）的存在。使用恩诺沙星作为原型 FQ，我们发现：（i）靶基因中的突变数量与最小抑菌浓度（MIC）密切相关。gyrA 中的单个突变（Ser83Leu）可增加敏感分离株的 FQ MIC；随后的突变导致耐药性增加，从低水平（恩诺沙星 MIC 4-16 μg/ml）到高水平（恩诺沙星 MIC≥128 μg/ml），每个渐进性突变都会导致耐药性增加。（ii）随着 MIC 的增加，acrB 活性和导致 MDR 表型的药物种类增加；（iii）无法确定调节基因表达与 MIC 之间的一致关系；（iv）在含有靶突变的 14 个 ENR（R）-MDR 分离株中检测到 qnrS 和 aac（6'）-Ib-cr 基因，分别为 14 和 5。在 13 个表达 PDR 分离株的分离株中，有 10 个（77%）qnrS 基因阳性，有 4 个（40%）同时携带 qnrS 和 aac（6'）-Ib-cr 基因。这些发现表明，犬和猫尿路致病性大肠杆菌中与 MDR 相关的 FQ 耐药性反映了点突变、增强的外排泵活性和 PMQR 机制的组合。然而，DNA 回旋酶中的点突变是实现临床水平 FQ 耐药性的必要条件。
