Table_4_Effect of Benzalkonium Chloride Adaptation on Sensitivity to Antimicrobial Agents and Tolerance to Environmental Stresses in Listeria monocytogenes.DOCX

Listeria monocytogenes is an important food-borne pathogen that can persist in food processing environments and thus contaminate food products. Benzalkonium chloride (BC) is a common disinfectant widely used in food industry. Selective pressure associated with exposure to BC may result in adaptation to this agent in L. monocytogenes. In this study, the effect of BC adaptation on susceptibility to antimicrobial agents and tolerance to environmental stresses, as well as the role of efflux pumps in BC adaptation were investigated in Listeria monocytogenes. Exposure of L. monocytogenes to progressively increasing concentrations of BC led to adaptation not only to BC but also to several other antimicrobial agents with different modes of action, including cefotaxime, cephalothin, ciprofloxacin, and ethidium bromide (EtBr), indicating that the disinfectant BC has the ability to select for antibiotic resistance. Reserpine, an efflux pump inhibitor, reduced minimum inhibitory concentrations (MICs) of cephalosporins, ciprofloxacin, and EtBr in BC adapted strains, indicating that efflux pumps are involved in cross-adaptation to these antimicrobial agents. Our results showed that expression levels of the efflux pump MdrL in the BC adapted strains increased significantly relative to the corresponding wild-type strains (P < 0.05), with the highest increase in one BC adapted strain named HL06BCA. Moreover, the knockout mutant HL06BCAΔmdrL showed impaired growth compared to that of HL06BCA when exposed to 2 μg/ml of BC. It suggests that efflux pump MdrL is associated with BC adaptation in L. monocytogenes. However, we did not find mdrL to be associated with cross-adaptation to cephalosporins, ciprofloxacin, and EtBr in HL06BCA. Additionally, increased sensitivity to acid, alkali, osmotic, ethanol, and oxidative stresses was observed in most strains after repeated exposure to BC. These results suggest rotation of different disinfectant is helpful to maintain high effectiveness of BC toward L. monocytogenes and ethanol and hydrogen peroxide are at least the appropriate candidates.

单核细胞增生李斯特菌（Listeria monocytogenes）是一类重要的食源性致病菌，可在食品加工环境中持久定植并污染食品。苯扎氯铵（Benzalkonium chloride，BC）是食品工业中广泛使用的常见消毒剂。与BC暴露相关的选择压力可促使单核细胞增生李斯特菌对该消毒剂产生适应性。本研究探究了BC适应性对单核细胞增生李斯特菌的抗菌剂敏感性与环境胁迫耐受性的影响，以及外排泵（efflux pumps）在BC适应性中的作用。将单核细胞增生李斯特菌暴露于逐步升高浓度的BC环境中，不仅可使其对BC产生适应性，还可使其对多种作用机制不同的其他抗菌剂产生适应性，包括头孢噻肟、头孢噻吩、环丙沙星与溴化乙锭（Ethidium bromide，EtBr），这表明消毒剂BC具备筛选抗生素耐药菌株的能力。利血平（Reserpine）作为一种外排泵抑制剂，可降低BC适应性菌株对头孢菌素类、环丙沙星与EtBr的最低抑菌浓度（minimum inhibitory concentrations，MICs），提示外排泵参与了针对上述抗菌剂的交叉适应性过程。本研究结果显示，相较于对应的野生型菌株，BC适应性菌株中外排泵基因mdrL的表达水平显著上调（P < 0.05），其中以命名为HL06BCA的BC适应性菌株上调幅度最高。此外，敲除mdrL的突变株HL06BCAΔmdrL在暴露于2 μg/ml的BC环境时，其生长能力较HL06BCA菌株受损，表明外排泵MdrL与单核细胞增生李斯特菌的BC适应性相关。然而，在HL06BCA菌株中，未发现mdrL与针对头孢菌素类、环丙沙星及EtBr的交叉适应性存在关联。此外，经反复BC暴露处理后，多数菌株对酸、碱、渗透压、乙醇与氧化胁迫的敏感性均有所升高。本研究结果提示，轮换使用不同消毒剂有助于维持BC对单核细胞增生李斯特菌的高效抑菌效果，而乙醇与过氧化氢则是较为合适的轮换消毒剂候选。