Abstract: 【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.

Key words: Listeria monocytogenes; inlK gene; biofilm; disinfectant