PmrA-PmrB二元調(diào)控系統(tǒng)介導(dǎo)大腸桿菌對黏桿菌素耐藥的機(jī)制研究
發(fā)布時間：2018-08-18 18:12
【摘要】：已報道PmrA-PmrB二元調(diào)控系統(tǒng)在革蘭陰性菌對多黏菌素B耐藥過程中起重要作用,基于此認(rèn)識,作者擬從基因突變和mRNA表達(dá)兩個方面探討PmrA-PmrB介導(dǎo)大腸桿菌對黏桿菌素耐藥的可能性。首先檢測了臨床分離的52株禽致病性大腸桿菌對黏桿菌素的敏感性,篩選出耐藥菌株;然后采用step-wise方法對黏桿菌素敏感菌株進(jìn)行誘導(dǎo),獲得人工誘導(dǎo)的耐藥菌株;最后通過PCR擴(kuò)增所有耐藥菌株的pmrA-pmrB后采用Mega軟件進(jìn)行突變位點分析,并采用實時熒光定量PCR(qRT-PCR)技術(shù)檢測所有耐藥菌株中pmrA-pmrB的mRNA轉(zhuǎn)錄水平的變化,擬闡明PmrA-PmrB二元調(diào)控系統(tǒng)對禽致病性大腸桿菌黏桿菌素耐藥性產(chǎn)生的作用。MIC結(jié)果顯示,52株大腸桿菌中,雖然大多數(shù)菌株(88.5%,46/52)仍對黏桿菌素敏感,但也分離到少數(shù)耐藥菌株(為11.5%)。突變位點分析表明人工誘導(dǎo)成功的5株耐藥菌(9R、36R、53R、91R和107R)pmrA未發(fā)生突變,而pmrB均有不同程度的突變,其中耐藥菌株9R、36R和53R各在G55A(G19R)、T500C(L167P)和T263A(V88E)發(fā)生點突變,而91R和107R在229位和478位各插入長為30和189bp的序列。但臨床分離的6株耐藥菌株(MIC=4~8μg·mL~(-1))的pmrA-pmrB均未發(fā)生突變。qRT-PCR結(jié)果顯示發(fā)生點突變的三株人工誘導(dǎo)耐藥菌pmrA-pmrB轉(zhuǎn)錄量均極顯著(P0.01)或顯著(P0.05)上升,發(fā)生插入突變的兩株耐藥菌pmrA-pmrB轉(zhuǎn)錄量雖有上升趨勢,但變化不顯著(P0.05),而臨床耐藥菌株(n=6)的pmrA-pmrB轉(zhuǎn)錄量均無變化。進(jìn)一步檢測人工誘導(dǎo)不同MIC的耐藥菌株中pmrA-pmrB的突變位點,發(fā)現(xiàn)菌株MIC達(dá)16μg·mL~(-1)時pmrA-pmrB才會發(fā)生突變。PmrA和PmrB介導(dǎo)了大腸桿菌對黏桿菌素的高度耐藥,其中PmrB的點突變伴隨PmrA-PmrB的高表達(dá)或者PmrB的組氨酸激酶-腺苷酰環(huán)化酶-甲基結(jié)合蛋白-磷酸化酶(HAMP)結(jié)構(gòu)域的插入突變是導(dǎo)致禽致病性大腸桿菌對黏桿菌素高度耐藥的機(jī)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.
【作者單位】： 南京農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院;
【基金】：江蘇省自然基金(BK2012771) 江蘇省高校"青藍(lán)工程"中青年學(xué)術(shù)帶頭人項目
【分類號】：S852.61
本文編號：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.
【作者單位】： 南京農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院;
【基金】：江蘇省自然基金(BK2012771) 江蘇省高校"青藍(lán)工程"中青年學(xué)術(shù)帶頭人項目
【分類號】：S852.61
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