单增李斯特菌inlK基因缺失株的构建及其生物被膜与消毒剂抗力分析

【目的】 研究inlK基因对Lm90SB2菌株生物被膜形成能力的影响及其生物被膜与消毒剂抗力的关系，以期为有效防控单增李斯特菌污染提供参考。【方法】 以单增李斯特菌Lm90SB2为试验菌，根据GenBank中公布的单增李斯特菌F2365 inlK基因序列(登录号:AE017262)，应用Primer Premier 5.0软件设计用于扩增inlK基因上、下游同源臂片段及验证缺失株的特异性引物，以同源重组技术构建inlK基因缺失株，并通过旁外侧引物运用PCR方法进行缺失株检测。将标准菌株Lm90SB2和构建的缺失株分别培养8、12、24、48 h后进行结晶紫染色，在倒置显微镜下观察形态变化，并用酶标仪测定生物被膜形成能力;用含3 g/L卵磷脂+3 g/L吐温80的PBS溶液和含10 g/L卵磷脂+20 g/L吐温80的PBS溶液作为新洁尔灭消毒剂的中和剂，含5 g/L硫代硫酸钠+5 g/L卵磷脂+20 g/L吐温80的PBS溶液和含10 g/L硫代硫酸钠+30 g/L卵磷脂+20 g/L吐温80的PBS溶液84消毒剂的中和剂，设消毒剂+菌悬液、消毒剂+菌悬液+中和剂、中和剂+菌悬液、消毒剂+中和剂+菌悬液、稀释液+菌悬液(阳性对照)、稀释液+中和剂+培养基(阴性对照)6个试验组，进行中和剂的筛选，并检测不同浓度新洁尔灭(1∶15、1∶30)和84消毒剂(1∶50、1∶100)分别作用1、5、10、20 min时对2株菌的灭菌率。【结果】 PCR结果表明，成功构建了缺失株Lm90SB2ΔinlK，且inlK基因的缺失导致Lm90SB2菌株生物被膜形成能力显著或极显著下降(P<0.05;P<0.01);含3 g/L卵磷脂+3 g/L吐温80的PBS溶液构成的中和剂可有效中和新洁尔灭消毒剂，5 g/L硫代硫酸钠+5 g/L卵磷脂+20 g/L吐温80的PBS溶液可有效中和84消毒剂。不同比例的新洁尔灭(1∶15、1∶30)和84消毒剂(1∶50、1∶100)消毒剂在1、5、10 min对Lm90SB2ΔinlK株的灭菌率均显著或极显著高于Lm90SB2株(P<0.05;P<0.01)，且在20 min时灭菌率均为100%。【结论】 inlK基因的缺失导致Lm90SB2菌株生物被膜形成能力下降，且对消毒剂抗力减弱。

Construction of Listeria monocytogenes inlK Gene Deletion Strain and Analysis of Its Biofilm and Disinfectant Resistance

【Objective】 The aim of this expriment was to study the effect of inlK gene on biofilm formation ability of Lm90SB2 strain and the relationship between biofilm and disinfectant resistance,in order to provide reference for effective prevention and control of Listeria monocytogenes contamination.【Method】 Listeria monocytogenes Lm90SB2 was used as the test strain.According to the inlK gene sequence (accession number:AE017262) of Listeria monocytogenes F2365 published in GenBank,specific primers for amplifying the upstream and downstream homology arm fragments of inlK gene and verifying the deletion strains were designed using Primer Premier 5.0 software.The inlK gene deletion strain was constructed by homologous recombination technology,and the deletion strain was detected by PCR with paralateral primers.The standard strain Lm90SB2 and the constructed deletion strain were cultured for 8,12,24 and 48 h and stained with crystal violet.Morphological changes were observed under an inverted microscope,and biofilm formation ability was measured with a microplate reader.PBS solution containing 3 g/L lecithin+3 g/L Tween 80 and PBS solution containing 10 g/L lecithin+20 g/L Tween 80 were used as neutralizers of bromogeramine,PBS solution containing 5 g/L sodium thiosulfate+5 g/L lecithin+20 g/L Tween 80 and PBS solution containing 10 g/L sodium thiosulfate+30 g/L lecithin+20 g/L Tween 80 were used as neutralizers of 84 disinfectant,disinfectants+bacterial suspension,disinfectant+bacterial suspension+neutralizer,neutralizer+bacterial suspension,disinfectant+neutralizer+bacterial suspension,diluent+bacterial suspension (positive control),diluents+neutralizer+culture medium (negative control) groups designed to screen neutralizers.And the sterilization rates of biofilm of two strains treated with different concentrations of bromogeramine (1∶15,1∶30) and 84 disinfectant (1∶50,1∶100) for 1,5,10 and 20 min were detected,respectively.【Result】 PCR results showed that the deletion strain Lm90SB2ΔinlK was successfully constructed,and the deletion of inlK gene resulted in significant or extremely significant decrease in the biofilm formation ability of Lm90SB2 strain (P<0.05;P<0.01).The neutralizer composed of PBS solution containing 3 g/L lecithin+3 g/L Tween 80 could effectively neutralize the bromogeramine,and PBS solution containing 5 g/L sodium thiosulfate+5 g/L lecithin+20 g/L Tween 80 could effectively neutralize 84 disinfectant.The sterilization rate of different proportions of bromogeramine (1∶15,1∶30) and 84 disinfectant (1∶50,1∶100) disinfectant on the biofilm of Lm90SB2ΔinlK strain was higher than that of Lm90SB2 strain at 1,5 and 10 min (P<0.05;P<0.01),and the sterilization rate was 100% at 20 min.【Conclusion】 The biofilm formation ability of Lm90SB2 strain and its resistance to disinfectant were decreased by deleting inlK gene.