PmrA-PmrB二元调控系统介导大肠杆菌对黏杆菌素耐药的机制研究

doi:10.11843/j.issn.0366-6964.2016.04.022

摘要/Abstract

摘要：

已报道PmrA-PmrB二元调控系统在革兰阴性菌对多黏菌素B耐药过程中起重要作用，基于此认识，作者拟从基因突变和mRNA表达两个方面探讨PmrA-PmrB介导大肠杆菌对黏杆菌素耐药的可能性。首先检测了临床分离的52株禽致病性大肠杆菌对黏杆菌素的敏感性，筛选出耐药菌株；然后采用step-wise方法对黏杆菌素敏感菌株进行诱导，获得人工诱导的耐药菌株；最后通过PCR扩增所有耐药菌株的pmrA-pmrB后采用Mega软件进行突变位点分析，并采用实时荧光定量PCR（qRT-PCR）技术检测所有耐药菌株中pmrA-pmrB的mRNA转录水平的变化，拟阐明PmrA-PmrB二元调控系统对禽致病性大肠杆菌黏杆菌素耐药性产生的作用。MIC结果显示，52株大肠杆菌中，虽然大多数菌株（88.5%，46/52）仍对黏杆菌素敏感，但也分离到少数耐药菌株（为11.5%）。突变位点分析表明人工诱导成功的5株耐药菌(9R、36R、53R、91R和107R) pmrA未发生突变，而pmrB均有不同程度的突变，其中耐药菌株9R、36R和53R各在G55A (G19R)、T500C（L167P）和T263A（V88E）发生点突变，而91R和107R在229位和478位各插入长为30和189 bp的序列。但临床分离的6株耐药菌株(MIC=4~8 μg•mL^-1)的 pmrA-pmrB 均未发生突变。qRT-PCR结果显示发生点突变的三株人工诱导耐药菌pmrA-pmrB转录量均极显著（P<0.01）或显著(P<0.05)上升，发生插入突变的两株耐药菌pmrA-pmrB转录量虽有上升趋势，但变化不显著（P>0.05），而临床耐药菌株（n=6）的pmrA-pmrB转录量均无变化。进一步检测人工诱导不同MIC的耐药菌株中pmrA-pmrB的突变位点，发现菌株MIC达16 μg•mL^-1时pmrA-pmrB才会发生突变。PmrA和PmrB介导了大肠杆菌对黏杆菌素的高度耐药，其中PmrB的点突变伴随PmrA-PmrB的高表达或者PmrB的组氨酸激酶-腺苷酰环化酶-甲基结合蛋白-磷酸化酶（HAMP）结构域的插入突变是导致禽致病性大肠杆菌对黏杆菌素高度耐药的机制之一。

Abstract:

It has been recently reported that PmrA-PmrB are associated with polymyxin B resistance.To determine whether PmrA-PmrB is contributing to colistin resistance in E.coli，we investigated amino acid alterations and transcription level of pmrA-pmrB in colistin resistance strains.All clinical resistance isolated strains were screened for colistin resistance by using MIC determinations and step-wise method was used to induce colistin resistant strains. pmrA-pmrB genes were amplified by PCR from resistant strain and their sequences were characterized by Mega software.Simultaneously，expression levels of PmrA-PmrB were detected by quantitive Real-time PCR.MIC (minimum inhibitory concentration) results showed that 88.5% of 52 clinic resistance strains tested in this study were susceptibility to colistin and 11.5% of the strains were resistant.Five mutations were identified in pmrB in induced colistin-resistant mutants 9R (G55A)，36R (T500C)，53R (T263A)，91R (insertion of 30 bp at position of 229) and 107R (insertion of 189 bp at position of 478).However，six clinical resistance isolates (MIC=4-8 μg•mL^-1) did not show any mutations in pmrA-pmrB.Further study showed that pmrA-B expression levels were significantly activated in three lab-derived colistin resistant strains with point mutations of pmrB.The expression level of pmrA-pmrB genes tended to increase but not significant changed in resistant strains with insertion mutations compared with its correspond parent strains.The transcription level of pmrA-pmrB in 6 clinical resistance isolates remained unchanged compared with susceptible strains.In addition，when MICs were over than 16 μg•mL^-1，mutations in pmrA-pmrB would occur.Mutations in pmrB or high expression levels in pmrA-pmrB contribute to high-level colistin resistance in E.coli strains.

中图分类号:

S859.7

葛琳，郭大伟，何方，黄金虎，王丽平. PmrA-PmrB二元调控系统介导大肠杆菌对黏杆菌素耐药的机制研究[J]. 畜牧兽医学报, 2016, 47(4): 812-819.

GE Lin，GUO Da-wei，HE Fang，HUANG Jin-hu，WANG Li-ping. Resistance Mechanism of Escherichia coli to Colistin Mediated by PmrA-PmrB[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 812-819.

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