单增李斯特菌(Listeria monocytogenes)是一种常见的食源性致病菌,其生物被膜是导致食品污染和疾病传播的重要原因。该研究选用枯草芽孢杆菌(Bacillus subtilis)无细胞上清液(cell free supernatant,CFS)作为抑制剂,探究B.subtilis CFS在抑制L.monocytogenes野生菌株118(以下简称No.118)生物被膜方面的作用和潜力。首先测定了B.subtilis CFS的最小抑菌浓度(minimum inhibitory concentration,MIC),接着采用结晶紫染色法测定了不同亚最小抑菌浓度下B. subtilis CFS对No.118的生物被膜抑制率,发现不同亚抑菌浓度下的CFS对No.118生物被膜均具有显著的抑制作用,且在1/64 MIC时生物被膜抑制率仍可达到47%;同时B. subtilis CFS对No.118生物被膜代谢活性、自聚集率、表面疏水性、群集泳动能力和膜外聚合物分泌也均有显著抑制作用;最后,通过激光共聚焦显微镜观察了在CFS作用下,No.118生物被膜结构和细胞分布的变化规律,经CFS处理后,No.118生物被膜结构松散,细菌数量明显减少。综上,B. subtilis CFS可通过影响No.118生物被膜细胞的代谢活性、自聚集能力和群集泳动能力以及膜外聚合物的分泌来抑制其生物被膜形成,对探究益生菌代谢产物作为生物被膜抑制剂方面的应用具有重要的参考价值。
Listeria monocytogenes is a common foodborne pathogen, and its biofilm is an important cause of food contamination and disease transmission.In this study, the cell free supernatant (CFS) of Bacillus subtilis was selected as an inhibitor to explore the role and potential of B. subtilis CFS in inhibiting L.monocytogenes 118 (No.118 for short) biofilm.Firstly, the minimum inhibitory concentration (MIC) of B. subtilis CFS was determined, and then the biofilm inhibition rate of B. subtilis CFS to No.118 was determined by using crystal violet staining method.Results showed that CFS at different sub-MICs had a significant inhibitory effect on No.118 biofilm, and the biofilm inhibition rate could still reach 47% at 1/64 MIC.Meanwhile, B. subtilis CFS also significantly inhibited the metabolic activity, self-aggregation rate, surface hydrophobicity, swaming and extramembrane polymer secretion of No.118 biofilm cells.Finally, the changes of No.118 biofilm structure and cell distribution under the action of CFS were observed by confocal laser scanning microscope.After CFS treatment, the structure of No.118 biofilm was loose, and the number of bacteria was significantly reduced.Consequently, this study found that B. subtilis CFS can inhibit the biofilm formation of No.118 biofilm cells by affecting their metabolic activity, self-aggregation ability and swaming, as well as the secretion of extramembrane polymers.This has important reference value for exploring the application of probiotic metabolites as biofilm inhibitors.
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