4种藏药提取物对产气荚膜梭菌生物被膜及孢子形成的影响  

Effects of four Tibetan medicine extracts on the biofilm and spore formation of Clostridium perfringens

作　　者：罗润波 蔡重振 黄家旗 吴丹 郭晓庆 白吉 格桑卓玛 边巴央拉 索朗斯珠 LUO Runbo;CAI Chongzhen;HUANG Jiaqi;WU Dan;GUO Xiaoqing;Baiji;Gesangzhuoma;Bianbayangla;Suolangsizhu(College of Animal Science,Xizang Agricultural and Animal Husbandry University,Nyingchi 860000,China;Xizang Key Laboratory of Hydatid Disease Control,Ministry of Agriculture and Rural Affairs,Nyingchi 860000,China;Xizang Center for Animal Disease Control and Prevention,Lhasa 850000,China)

机构地区：[1]西藏农牧学院动物科学学院,西藏林芝860000 [2]农业农村部西藏包虫病防治重点实验室,西藏林芝860000 [3]西藏自治区动物疫病预防控制中心,拉萨850000

出　　处：《黑龙江畜牧兽医》2024年第12期101-107,129,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：财政部和农业农村部:国家现代农业产业技术体系项目(CARS-37)。

摘　　要：为了探究4种藏药提取物对产气荚膜梭菌生物被膜及孢子形成的影响,试验利用银染法鉴定产气荚膜梭菌Nyingchi-C20株的生物被膜,采用结晶紫法检测生物被膜形成能力,采用微量肉汤稀释法检测4种藏药提取物的最小抑菌浓度(MIC)。选取抑菌效果较好的藏药提取物稀释后分为6组,分别为液体硫乙醇酸盐培养基(TSB)组、1/8 MIC组、1/4 MIC组、1/2 MIC组、MIC组和抗生素万古霉素MIC(VAN)组,每组接种菌悬液300μL后加入各组药液至30 mL,培养后取各组生物被膜悬液,采用实时荧光定量PCR检测其对生物被膜形成及孢子形成相关基因表达的影响。结果表明:马兜铃甲醇提取物及诃子、波棱瓜子、白豆蔻75%乙醇提取物MIC分别为0.2 mg/mL、0.8 mg/mL、0.4 mg/mL、1.6 mg/mL。与TSB组相比,马兜铃甲醇提取物各MIC组agrB基因与htrA基因(除1/8 MIC组和1/4 MIC组外)的表达水平显著或极显著降低(P<0.05或P<0.01),1/2 MIC组和MIC组sigK基因的表达水平显著或极显著升高(P<0.05或P<0.01);诃子75%乙醇提取物1/8MIC组(除ribD、codY、spo0A基因外)、1/4MIC组(除ribD基因外)、1/2MIC组、MIC组agrB、htrA、abrB、ribD、codY、spo0A基因表达水平显著或极显著降低(P<0.05或P<0.01);波棱瓜子75%乙醇提取物1/4 MIC组、1/2 MIC组及MIC组agrB基因相对表达水平极显著降低(P<0.01),1/8MIC组、1/4 MIC组、1/2 MIC组及MIC组minD基因相对表达水平极显著升高(P<0.01);白豆蔻75%乙醇提取物1/2 MIC组argG、ribD基因及MIC组argG、ribD、ribE基因相对表达水平显著或极显著升高(P<0.05或P<0.01)。说明4种藏药提取物对Nyingchi-C20株具一定的抑制作用,对其生物被膜及孢子形成相关基因表达有不同程度的影响。In order to investigate the effects of four Tibetan medicine extracts on the biofilm and spore formation of Clostridium perfringens,in the test,the silver staining method was used to identify the biofilm of Clostridium perfringens strain Nyingchi-C20,the crystal violet method was used to detect the biofilm forming ability,and the minimum inhibitory concentration(MIC)of four Tibetan medicine extracts was detected by micro broth dilution method.The extracts of Tibetan medicines with good antibacterial effect were diluted and divided into 6 groups,which were liquid thioglycolate medium(TSB)group,1/8 MIC group,1/4 MIC group,1/2 MIC group,MIC group and antibiotic vancomycin MIC(VAN)group,respectively.After each group was inoculated with 300μL of bacterial suspension,the drug solution of each group was added to 30 mL.After incubation,the biofilm suspensions of each group were taken to detect their effect on the expression of genes related to biofilm formation and spore formation by real-time fluorescence quantitative PCR.The results showed that the minimum inhibitory concentrations of Aristolochia methanolic extract and 75%ethanol extracts of Terminalia,Herpetospermum seed,and Amomum were 0.2 mg/mL,0.8 mg/mL,0.4 mg/mL,and 1.6 mg/mL,respectively.Compared with the TSB group,the expression levels of agrB gene and htrA gene(except for the 1/8 MIC group and 1/4 MIC group)in each MIC group of Aristolochia methanol extract were significantly or extremely significantly lower(P<0.05 or P<0.01),and the expression levels of sigK gene in the 1/2 MIC group and MIC group were significantly or extremely significantly higher(P<0.05 or P<0.01).The expression levels of agrB,htrA,abrB,ribD,codY,and spo0A genes were significantly or extremely significantly reduced in the 1/8 MIC group(except ribD,codY,and spo0A genes),the 1/4 MIC group(except ribD gene),the 1/2 MIC group,and the MIC group of Terminalia 75%ethanol extract(P<0.05 or P<0.01).The relative expression levels of agrB gene in the 1/4 MIC group,1/2 MIC group and MIC group of 75%e

关 键 词：产气荚膜梭菌 藏药 生物被膜 孢子 基因 

分 类 号：S859.3[农业科学—临床兽医学]