单核细胞增生李斯特菌喹诺酮耐药机制研究

Study on the Quinolone-resistant Mechanisms of Listeria monocytogenes

以食源性单核细胞增生李斯特菌(LM)环丙沙星耐药株为研究对象,旨在调查LM对喹诺酮产生耐药的分子机制。采用琼脂二倍稀释法测定菌株对环丙沙星的最小抑菌浓度(MIC);通过PCR检测喹诺酮耐药决定区(QRDR)基因突变以及质粒介导喹诺酮耐药(PMQR)基因的分布;实时荧光定量PCR检测lde基因在LM菌株中的相对表达量;利用SOE-PCR技术构建lde基因缺失突变株,调查外排泵Lde的作用。结果表明,15株LM耐药菌株GyrA、GyrB、ParC和ParE亚基的氨基酸序列与敏感株100%相似;所有菌株中均未检出PMQR基因。加入外排泵抑制剂利血平后,菌株L28和L47对环丙沙星的MIC值分别下降为原来的1/8和1/4。lde基因在耐药株和敏感株中均有表达,且表达量相近;在环丙沙星的作用下,lde基因在耐药株中的相对表达量增加显著,而在敏感株中的表达量基本没有变化;加入利血平后,lde在耐药株中的表达明显受到抑制。缺失lde基因后,菌株对环丙沙星由耐药转为敏感;利血平存在下突变株对环丙沙星的MIC值不受影响。本研究证明外排泵Lde介导LM对喹诺酮类药物耐药。

Using ciprofloxacin-resistant strains of Listeria monocytogenes（LM）isolated from food as research object,this study aimsto understand the quinolone-resistant mechanisms of LM.The minimum inhibitory concentration（MIC）of ciprofloxacin by the strain wasdetermined by the agar dilution method.The mutations in the quinolone resistance-determining region（QRDR）and the presence of plasmidmediated quinolone resistance（PMQR）genes were investigated using PCR.Expression levels of lde gene under different conditions weredetected by q RT-PCR.To address the role of efflux pump Lde in ciprofloxacin resistance,deletion mutant in lde was constructed using SOEPCR.Results showed that the amino acid sequences of GyrA,GyrB,ParC,and ParE in 15 resistant strains showed 100% similarity with thesequences of sensitive strains,and no PMQR genes were detected in any of the resistant strains.In the presence of reserpine,an efflux pumpinhibitor,the MICs of ciprofloxacin by strain L28 and L47 reduced to the 1/8 and 1/4 of original one.The expression of lde was observed in allsensitive and resistant strains by similar values.After treatment with ciprofloxacin,the resistant strains showed significant increases in lde,however,no obvious changes in expression of lde were observed in the sensitive strains.In the presence of reserpine,the expression of ldesignificantly inhibited in resistant strains.The mutant lacking the lde gene was susceptible to ciprofloxacin and its MIC of ciprofloxacin did notchange in the presence of reserpine.Our data demonstrated that efflux pump Lde mediated quinolone resistance in L.monocytogenes.