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
发布时间：2018-08-18 18:12
【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189bp的序列。但临床分离的6株耐药菌株(MIC=4~8μg·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(P0.01)或显著(P0.05)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(P0.05),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16μg·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 reported that PmrA-PmrB binary regulatory system plays an important role in the process of Gram-negative bacteria resistance to polymyxin B. based on this understanding, the author intends to explore the possibility of Escherichia coli resistance to myxin B mediated by PmrA-PmrB from two aspects of gene mutation and mRNA expression. The sensitivity of 52 clinical isolates of avian pathogenic Escherichia coli to myxin was detected, and the drug-resistant strains were screened out, and then the artificially induced strains were obtained by step-wise method. Finally, the pmrA-pmrB of all drug-resistant strains was amplified by PCR, and the mutation sites were analyzed by Mega software, and the changes of mRNA transcription level of pmrA-pmrB in all resistant strains were detected by real-time fluorescence quantitative PCR (qRT-PCR) technique. To elucidate the effect of PmrA-PmrB binary control system on myxin resistance of avian pathogenic Escherichia coli. The results showed that although most strains (88.546 / 52) were still sensitive to myxin, a few resistant strains (11.5%) were isolated. The results of mutation locus analysis showed that there were no mutations in pmrA of 5 strains of drug-resistant strains (9Rn36RN53RN91R and 107R) induced by artificial induction, but pmrB had different degrees of mutation. The resistant strains 9Rn36R and 53R had point mutations in G55A (G19R) T500C (L167P) and T263A (V88E), respectively. While 91R and 107R insert 30 and 189bp sequences at 229 and 478 positions, respectively. However, no mutation was found in the pmrA-pmrB of the 6 clinically isolated drug-resistant strains (MIC=4~8 渭 g mL ~ (-1). The results of qRT-PCR showed that the pmrA-pmrB transcripts of the three artificially induced drug-resistant strains with point mutation were significantly increased (P0.01) or significantly (P0.05). The pmrA-pmrB transcription of the two drug-resistant strains with insertion mutation showed an upward trend, but the change was not significant (P0.05), while the pmrA-pmrB transcription of clinical drug-resistant strain (NN6) did not change. The mutation sites of pmrA-pmrB in the resistant strains of different MIC were further detected. It was found that when the MIC reached 16 渭 g mL ~ (-1), the mutation of pmrA-pmrB. PmrA and PmrB mediated the high resistance of Escherichia coli to myxin. The point mutation of PmrB accompanied with the high expression of PmrA-PmrB or the insertion of (HAMP) domain of histidine kinase adenylate cyclase-methyl-binding protein-phosphorylase is one of the mechanisms leading to the high resistance of avian pathogenic Escherichia coli to myxin.
【作者单位】： 南京农业大学动物医学院;
【基金】：江苏省自然基金(BK2012771) 江苏省高校"青蓝工程"中青年学术带头人项目
【分类号】：S852.61
本文编号：2190281
[Abstract]:It has been reported that PmrA-PmrB binary regulatory system plays an important role in the process of Gram-negative bacteria resistance to polymyxin B. based on this understanding, the author intends to explore the possibility of Escherichia coli resistance to myxin B mediated by PmrA-PmrB from two aspects of gene mutation and mRNA expression. The sensitivity of 52 clinical isolates of avian pathogenic Escherichia coli to myxin was detected, and the drug-resistant strains were screened out, and then the artificially induced strains were obtained by step-wise method. Finally, the pmrA-pmrB of all drug-resistant strains was amplified by PCR, and the mutation sites were analyzed by Mega software, and the changes of mRNA transcription level of pmrA-pmrB in all resistant strains were detected by real-time fluorescence quantitative PCR (qRT-PCR) technique. To elucidate the effect of PmrA-PmrB binary control system on myxin resistance of avian pathogenic Escherichia coli. The results showed that although most strains (88.546 / 52) were still sensitive to myxin, a few resistant strains (11.5%) were isolated. The results of mutation locus analysis showed that there were no mutations in pmrA of 5 strains of drug-resistant strains (9Rn36RN53RN91R and 107R) induced by artificial induction, but pmrB had different degrees of mutation. The resistant strains 9Rn36R and 53R had point mutations in G55A (G19R) T500C (L167P) and T263A (V88E), respectively. While 91R and 107R insert 30 and 189bp sequences at 229 and 478 positions, respectively. However, no mutation was found in the pmrA-pmrB of the 6 clinically isolated drug-resistant strains (MIC=4~8 渭 g mL ~ (-1). The results of qRT-PCR showed that the pmrA-pmrB transcripts of the three artificially induced drug-resistant strains with point mutation were significantly increased (P0.01) or significantly (P0.05). The pmrA-pmrB transcription of the two drug-resistant strains with insertion mutation showed an upward trend, but the change was not significant (P0.05), while the pmrA-pmrB transcription of clinical drug-resistant strain (NN6) did not change. The mutation sites of pmrA-pmrB in the resistant strains of different MIC were further detected. It was found that when the MIC reached 16 渭 g mL ~ (-1), the mutation of pmrA-pmrB. PmrA and PmrB mediated the high resistance of Escherichia coli to myxin. The point mutation of PmrB accompanied with the high expression of PmrA-PmrB or the insertion of (HAMP) domain of histidine kinase adenylate cyclase-methyl-binding protein-phosphorylase is one of the mechanisms leading to the high resistance of avian pathogenic Escherichia coli to myxin.
【作者单位】： 南京农业大学动物医学院;
【基金】：江苏省自然基金(BK2012771) 江苏省高校"青蓝工程"中青年学术带头人项目
【分类号】：S852.61
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