食品源单增李斯特菌喹诺酮类抗生素耐药机制研究

以945株食品源单核细胞增生李斯特菌(Listeria monoocytogens,LM)作为研究对象,分析其对喹诺酮类抗生素耐药的分子机制。采用KB法筛选出喹诺酮类耐药的LM,利用PCR检测喹诺酮耐药决定区(quinolone resistance-determining region,QRDR)的基因突变以及质粒介导喹诺酮耐药(plasmid-mediated quinolone resistance,PMQR)基因的分布情况,结合最小抑菌浓度(MIC)值和外排泵抑制剂利血平分析其外排泵的作用,多位点序列分型(multilocus sequence typing, MLST)结合血清组对耐药菌株进行遗传多样性分析。结果表明:32株耐药菌株中gyrA、gyrB、parC与parE的QRDR均未发生氨基酸突变,且未检测到PMQR相关基因；而7株fepR基因发现新的氨基酸突变位点,其作用机制有待阐明；外排泵基因lde在耐药LM中皆有检出,78.1%(25/32)LM加入利血平后MIC值降低至原MIC的1/2以下,lde可能是导致LM对喹诺酮耐药的重要因素。血清组分析发现32株耐药菌株分属血清组I.1、I.2、II.1、II.2和III,MLST共分为10种ST型,其中ST87、ST9和ST8为优势株,具有一定的致病能力。结果表明,lde是LM产生喹诺酮耐药的重要因素,但LM潜在喹诺酮耐药分子机制仍有待阐明,同时其潜在的致病性应引起重视。

Exploring Potential Mechanism of Quinolone Resistance of Foodborne Listeria monocytogenes Isolates

The quinolone-resistant isolates were screened from 945 foodborne Listeria monocytogenes, aimed to explore the potential molecular mechanisms of quinolone resistance in Listeria monocytogenes. A total of 32 isolates exhibited quinolone resistance by the KB method. Gene mutation in quinolone resistance-determining region(QRDR) and the distribution of plasmid-mediated quinolone resistance (PMQR)genes were detected by PCR. The MIC values of ciprofloxacin with or without efflux pump inhibitor reserpine were measured. Compared to QRDR of these genes in quinolone-sensitive Listeria monocytogenes EGDe, no amino acid substitution was observed in 32 quinolone-resistant isolates, and no PMQR gene was also found in all these strains. However, seven kinds of amino acids substitutions of fepR were found, whether these mutations were critical to the quinolone resistance still to be elucidated. The presence of efflux pump lde gene was 100% in all 32 strains of Listeria monocytogenes by PCR. However, after the efflux pump inhibitor reserpine addition, the MIC value of ciprofloxacin in 25 (78.1%) isolates were reduced to more than 1/2 of MIC value before the reserpine addition. These results demonstrated that efflux pump lde may be an important factor in mediating quinolone resistance, but the resistance level may be determined by the expression of lde gene in Listeria monocytogenes. The 32 isolates belonged to 10 sequence types (STs) by multilocus sequence typing, ST8(serogroup I.1), ST9(serogroup I.2) and ST87 (serogroup II.2) were predominant in quinolone-resistant isolates, which posed a potential public health risk to consumers.