活性氧与蜡样芽孢杆菌菌膜形成的相关性

夏俊芳; 潘苗; 奇那尔; 玛格撇尔; 古丽娜孜; 武运

doi:10.13386/j.issn1002-0306.2021080341

活性氧与蜡样芽孢杆菌菌膜形成的相关性

The Relationship between Reactive Oxygen Species and Bacillus cereus Biofilms Formation

摘要

摘要: 为阐明活性氧（ROS）与蜡样芽孢杆菌菌膜形成的相关性，以市售原料奶中分离的蜡样芽孢杆菌分离株为目标菌，以细菌中NADPH氧化酶介导产生的ROS为主要靶点，用外源ROS补充剂过氧化氢(H₂O₂)、ROS清除试剂N-乙酰半胱氨酸（NAC）、NADPH氧化酶抑制剂二苯基氯化碘盐（DPI）处理蜡样芽孢杆菌，测定分析ROS变化与菌膜形成之间的关系。结果表明，0.01 μmol/L H₂O₂、1 μmol/L H₂O₂、100 μmol/L H₂O₂处理组平均菌膜形成量均显著高于对照组平均菌膜形成量（P<0.05），0.1 μmol/L H₂O₂、10 μmol/L H₂O₂处理组平均菌膜形成量与对照组平均菌膜形成量相比无显著性差异（P>0.05），随着H₂O₂浓度的升高，ROS逐渐下降。NAC各处理组平均菌膜形成量均显著高于对照组平均菌膜形成量（P<0.05），随着NAC浓度的升高，ROS逐渐下降。DPI浓度≤1 μmol/L时，随着DPI浓度的升高，平均菌膜形成量均显著高于对照组平均菌膜形成量（P<0.05），ROS逐渐下降；DPI浓度>5 μmol/L时，随着DPI浓度的升高，平均菌膜形成量显著低于对照组平均菌膜形成量（P<0.05），ROS含量升高。表明在不影响蜡样芽孢杆菌生长和细胞活性的前提下，一定浓度的H₂O₂、NAC和DPI处理菌株能够诱导ROS减少，增强蜡样芽孢杆菌菌膜的形成。激光共聚焦显微镜结果显示，与对照组相比处理组具有更强的染色效果，呈现红色的网络结构，而未经处理组呈现松散的结构和更少的菌膜生物量，这表明ROS对蜡样芽孢杆菌菌膜的形成存在抑制作用。

Abstract: In order to clarify the correlation between reactive oxygen species (ROS) and Bacillus cereus biofilms, the ROS mediated by NADPH oxidase in the Bacillus cereus that were isolated from commercially available raw milk was used as the main target. The relationship between the formation of Bacillus cereus biofilms and ROS were analyzed by using supplement hydrogen peroxide (H₂O₂) as exogenous reactive oxygen species (ROS), N-acetylcysteine (NAC) as ROS scavenging agent and diphenyl iodide chloride (DPI) as NADPH oxidase inhibitor. The results showed that the treatment groups of 0.01 μmol/L H₂O₂, 1 μmol/L H₂O₂ and 100 μmol/L H₂O₂ average biofilms were higher than the control group average biofilms (P<0.05). There was no significant difference between the treatment groups of 0.1 μmol/L H₂O₂ and 10 μmol/L H₂O₂ and the control group average biofilms (P>0.05). With the increase of H₂O₂ concentration, ROS gradually decreased. The treatment groups of NAC average biofilms were higher than the control group average biofilms (P<0.05). ROS decreased gradually with the increase of NAC concentration. When DPI concentration≤1 μmol/L, with the increase of DPI concentration, the average biofilms were higher than the control group average biofilms (P<0.05), and ROS gradually decreased. When DPI concentration>5 μmol/L, with the increase of DPI concentration, the average biofilms were lower than the control group average biofilms (P<0.05), and the ROS increased. The results showed that without affecting the growth and activity of Bacillus cereus, the reduction of ROS induced by certain concentration of H₂O₂, NAC and DPI enhanced the Bacillus cereus biofilms. Confocal laser scanning microscopy showed stronger staining and red network structures in the treated groups compared to the control groups, while the untreated groups showed loose structures and fewer biofilms, suggesting that ROS inhibits the formation of Bacillus cereus biofilms.