MdrL外排泵在单核细胞增生李斯特菌对苯扎氯铵耐受中的作用

Role of efflux pump MdrL in tolerance to benzalkonium chloride in Listeria monocytogenes

摘要:

【背景】季铵盐类消毒剂特别是苯扎氯铵(Benzalkonium chloride，BC)在食品工业中的广泛应用导致单核细胞增生李斯特菌(Listeria monocytogenes，Lm)对BC的敏感性下降。外排泵是介导Lm对BC耐受的主要机制。【目的】调查MdrL外排泵在Lm对BC耐受中的作用。【方法】利用同源重组技术构建mdrL基因缺失株。比较野生株EGD-e和突变株ΔmdrL在对BC的耐受性、亚致死浓度BC (2 μg/mL)胁迫下的生长情况以及致死浓度BC (16 μg/mL)下的存活率等方面的差异。【结果】构建mdrL基因缺失株ΔmdrL及回复突变株CΔmdrL。mdrL的缺失不影响菌株对BC的最小抑菌浓度。生长曲线测定结果显示，与野生株相比，突变株在亚致死浓度BC作用下的生长迟滞期延长、平均最大生长率和平均最大光密度值均降低。当BC浓度为4 μg/mL时，梯度稀释后的野生株和突变株在平板上的生长显现出明显的差异。与野生株相比，突变株在致死浓度BC作用下的存活率降低2个log值。透射电镜的观察结果显示，加入BC后，突变株的细胞明显变细长。此外，野生株和突变株对溴化乙锭(Ethidium bromide，EB)的积累和外排无明显差异。【结论】MdrL外排泵介导Lm对BC的耐受，但是与Lm对EB的外排无关。

Abstract:

[Background] Quaternary ammonium compounds, especially benzalkonium chloride (BC), are widely used in food industry, leading to decreased sensitivity to BC in Listeria monocytogene. Efflux pumps are recognized as an important mechanism for BC tolerance. [Objective] To investigate the role of efflux pump MdrL in tolerance to BC in Listeria monocytogenes. [Methods] The mutant strain ΔmdrL was constructed from the wild-type strain EGD-e by homologous recombination. The differences in BC tolerance, growth in sub-lethal concentration of BC (2 μg/mL), and survival in lethal concentration of BC (16 μg/mL) between the mutant strain ΔmdrL and the wild-type strain EGD-e were investigated. [Results] The mutant strain ΔmdrL and its complemented strain were constructed in this study. The mutant and wild-type strains showed the same minimum inhibitory concentrations to BC. The results show that compared with the wild-type strain EGD-e, the lag-phase duration of the mutant strain ΔmdrL was prolonged and the mean maximum growth rate and the mean maximum optical density were decrease in the presence of sub-lethal concentration of BC. Significantly difference was observed in the serial diluted culture between the mutant strain ΔmdrL and the wild-type strain EGD-e on the plates in the presence of BC (4 μg/mL). Compared with the wild-type strain EGD-e, two log decrease were observed in the mutant strain ΔmdrL in the presence of the lethal concentration of BC. The results of electron microscopy showed that the mutant strain ΔmdrL became longer when exposed to BC. Besides, no differences were observed in the accumulation and efflux to ethidium bromide (EB) between the mutant strain ΔmdrL and the wild-type strain EGD-e. [Conclusion] Efflux pump MdrL plays an important role in tolerance to BC in Listeria monocytogenes, however, this efflux pump is not associated with efflux of EB.