The widespread application of triclosan contributes to its residual deposition in urine, which provides an environment of long-term exposure to triclosan for the intestinal Escherichia coli. We determined the triclosan and antibiotic resistance characteristics of E. coli strains isolated from urine samples and further investigated the resistance mechanism and molecular epidemic characteristics of triclosan-resistant E. coli isolates. A total of 200 non-repetitive E. coli strains were isolated from urine samples and then identified. The minimum inhibitory concentrations (MICs) of triclosan and antibiotics, fabI mutation, efflux pump activity, the expression of 14 efflux pump encoding genes, and epidemiological characteristics were determined by the agar dilution method, polymerase chain reaction (PCR), carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibition test, quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE) for all triclosan-resistant isolates. Furthermore, we also investigated the effect of triclosan exposure in vitro on antibiotic susceptibility and the efflux pump encoding gene expressions of triclosan-susceptible strains via serial passage experiments. Of the 200 E. coli isolates, 2.5% (n = 5) were found to be resistant to triclosan, and multidrug resistance (MDR) and cross-resistance phenotypes were noted for these triclosan-resistant strains. The triclosan-sensitive strains also exhibited MDR phenotypes, probably because of the high resistance rate to AMP, CIP, LVX, and GEN. Gly79Ala and Ala69Thr amino acid changes were observed in the triclosan-resistant strains, but these changes may not mediate resistance of E. coli to triclosan, because mutations of these two amino acids has also been detected in triclosan-susceptible strains. Moreover, except for DC8603, all other strains enhanced the efflux pumps activity. As compared with ATCC 25922, except for fabI, increased expressions were noted for all efflux pump encoding genes such as ydcV, ydcU, ydcS, ydcT, cysP, yihV, acrB, acrD, and mdfA among the studied strains with varying PFGE patterns and STs types. Unexpectedly, 5 susceptible E. coli isolates showed rapidly increasing triclosan resistance after exposure to triclosan in vitro for only 12 days, while MDR or cross-resistance phenotypes and the overexpression of efflux pump genes were recorded among these triclosan-induced resistant isolates. This is the first study to report that short-term triclosan exposure in vitro increases triclosan resistance in susceptible E. coli isolates. After acquiring resistance, these strains may present MDR or cross-resistance phenotypes. Moreover, triclosan resistance mainly involves the overexpression of fabI and efflux pumps in E. coli isolates.

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温州地区尿样大肠埃希菌对三氯生耐药的流行情况及机制

三氯生的广泛应用导致其在尿液中残留沉积，为肠道大肠杆菌提供了长期接触三氯生的环境。我们确定了从尿液样本中分离的大肠杆菌菌株的三氯生和抗生素耐药性特征，并进一步研究了三氯生耐药大肠杆菌菌株的耐药机制和分子流行特征。从尿样中分离出总共 200 株非重复性大肠杆菌菌株，然后进行鉴定。通过琼脂稀释法、聚合酶链反应 (PCR)、羰基氰化物 3-氯苯腙 (CCCP) 抑制试验、实时定量聚合酶链反应 (RT-qPCR)、多位点序列分型 (MLST) 和脉冲场凝胶电泳 (PFGE) 对所有三氯生耐药菌株。此外，我们还通过连续传代实验研究了体外暴露于三氯生对三氯生敏感菌株的抗生素敏感性和外排泵编码基因表达的影响。在 200 株大肠杆菌中，发现 2.5% (n = 5) 对三氯生具有耐药性，并且注意到这些三氯生耐药菌株具有多重耐药性 (MDR) 和交叉耐药性表型。三氯生敏感菌株也表现出 MDR 表型，可能是因为对 AMP、CIP、LVX 和 GEN 的高耐药率。在三氯生耐药菌株中观察到 Gly79Ala 和 Ala69Thr 氨基酸变化，但这些变化可能不会介导大肠杆菌对三氯生的耐药性，因为在三氯生敏感菌株中也检测到这两个氨基酸的突变。此外，除 DC8603 外，所有其他菌株都增强了外排泵活性。与 ATCC 25922 相比，除了 fabI，在具有不同 PFGE 模式和 STs 的研究菌株中，所有外排泵编码基因（例如 ydcV、ydcU、ydcS、ydcT、cysP、yihV、acrB、acrD 和 mdfA）的表达均有所增加类型。出乎意料的是，在体外暴露于三氯生仅 12 天后，5 种易感大肠杆菌分离株显示出快速增加的三氯生耐药性，而在这些三氯生诱导的耐药菌株中记录了 MDR 或交叉耐药表型和外排泵基因的过度表达。这是第一项报告短期体外暴露于三氯生会增加易感大肠杆菌分离株对三氯生的耐药性的研究。在获得耐药性后，这些菌株可能会出现 MDR 或交叉耐药表型。此外，三氯生抗性主要涉及大肠杆菌分离株中 fabI 和外排泵的过度表达。