环丙沙星诱导对临床耐药菌株主动外排基因转录的影响

doi:10.11843/j.issn.0366-6964.2017.09.022

摘要/Abstract

摘要：

笔者拟研究环丙沙星诱导对大肠埃希菌临床耐药菌株Y35、J45主动外排基因acrA、acrB、acrD、acrE、acrF、mdtA及外排调控基因marA、robA和soxS mRNA水平的影响。采用微量肉汤稀释法，测定环丙沙星对Y35、J45不同诱导阶段MIC；采用荧光定量PCR检测方法，对Y35、J45及二者诱导至10、20、30代菌株的主动外排及外排调控基因mRNA相对转录量进行测定。结果显示：经过30代诱导，环丙沙星对诱导株MIC均增至原来的2倍，达到256 μg·mL^-1。Y35耐药株诱导至10和30代时，除了mdtA基因外，其他8个基因转录量介于1.20~96.07，与未诱导株相比差异显著（P<0.05或P<0.01），acrD基因转录量增加最为明显；至20代时，acrA、acrB、marA与10代诱导株相比，转录量下降，但高于原代菌株转录量；acrE、robA和soxS基因转录量数值介于1.40~3.81，与未诱导株相比差异显著（P<0.05或P<0.01）。J45耐药株至10代时，acrB、acrF基因转录量增加，转录量分别为原代菌株2.76和1.73倍，其他测定基因转录量下降；至20代时，acrA、acrB、acrE、mdtA基因转录量增加显著，分别为原代菌株的15.35、58.89、31.56、36.50倍，差异极显著（P<0.01）；至30代时，所有检测基因的转录量均大于原代菌株，acrF基因转录量增加最为明显，是原代菌株的102.54倍。本研究表明，长期使用环丙沙星诱导，能够使临床耐药株对其MIC值增加，耐药性增强；环丙沙星诱导能促使临床耐药菌株主动外排基因mRNA转录量增加，更强的耐药性可能是由主动外排泵介导产生。

Abstract:

This study was conducted to explore the effect of ciprofloxacin on the transcription of several active efflux system related genes, acrA, acrB, acrD, acrE, acrF, mdtA, marA, robA and soxS,in clinical E. coli strains Y35 and J45.The minimal inhibitory concentrations (MICs) of ciprofloxacin to E. coli strains Y35 and J45 induced by ciprofloxacin were determined using the standard broth microdilution method. A real-time fluorescent quantitative PCR assay was established to observe the difference of active efflux pumps genes transcription level between parental strains and the 10^th, 20^th, 30^th generation of ciprofloxacin induced strains. Results were as follows:The results showed that, the 30^th generation of ciprofloxacin induced strains exhibited increased MICs of ciprofloxacin (2-fold), which was 256 μg·mL^-1. For Y35, 8 of 9 genes transcription level of the 10^th and 30^th of ciprofloxacin induced strains were increased compared with the parental strain, except gene mdtA, and the values of relative transcription level were between 1.20 and 96.07 (the differences were statistically significant, P<0.05 or P<0.01). The transcription level of acrD gene increased most markedly. The expression level of acrA, acrB and marA in the 20^th generation of ciprofloxacin induced strains were decreased compared with the 10^th generation strain, however,these genes showed increased transcription level compared with the parental strain. The relative transcription level of acrE, robA and soxS were between 1.40 and 3.81 (P<0.05 or P<0.01). For J45, only acrB and acrF gene transcription level were increased in the 10^th generation of ciprofloxacin induced strain, the relative value of transcription level were 2.76 and 1.73, however, the transcription level of other genes were decreased. acrA, acrB, acrE and mdtA gene transcription level were increased in the 20^th generation of ciprofloxacin induced strain, the value of relative transcription level were 15.35, 58.89, 31.56 36.50, respectively (P<0.01). All the genes transcription were increased to a high level in the 30^th generation of ciprofloxacin induced strain,the transcription level of acrF gene increased most markedly, it was 102.54 times of the parental strain. The results of this study suggest that continuous induction of the clinical resistance E. coli strains by subinhibitary concentration of ciprofloxacin might result in stronger resistance to ciprofloxacin, and increased level of transcription of active efflux pump genes. The increased transcription level of efflux pump genes might contribute to the stronger resistance of the induced strains to ciprofloxacin.

中图分类号:

S859.7

刘建华, 付赛赛, 王亚云, 胡功政, 潘玉善, 苑丽. 环丙沙星诱导对临床耐药菌株主动外排基因转录的影响[J]. 畜牧兽医学报, 2017, 48(9): 1753-1760.

LIU Jian-hua, FU Sai-sai, WANG Ya-yun, HU Gong-zheng, PAN Yu-shan, YUAN Li. Transcription Changes of Active Efflux System in Clinical Multidrug-resistant E. coli Strains Induced by Ciprofloxacin Selective Pressure[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1753-1760.

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