鹅粪便中特基拉芽孢杆菌和类肠膜魏斯氏菌的分离鉴定

doi:10.16431/j.cnki.1671-7236.2022.07.036

摘要/Abstract

摘要： 【目的】从鹅粪中分离芽孢杆菌属和乳杆菌属细菌，为鹅源功能菌研究和合理用药提供参考。【方法】收集30只50周龄健康北方白鹅的直肠粪便，利用特异性培养基和生化反应试验分离筛选菌株，筛选的菌株通过DNA提取、PCR扩增16S rDNA测序鉴定后，与GenBank中参考菌株进行比对构建进化树，进行相似性比对确定菌株种类。培养48 h后检测细菌的生长特性、最适酸碱度和温度，以pH 7.0、无胆盐环境下细菌生长状态为对照，检测其耐酸耐胆盐能力，并进行药敏试验检测其对抗菌药的耐受性。【结果】经分离筛选共获得9株细菌，通过生化试验最终选出2株菌，根据进化树的进化距离和相似性比对确定其分别是特基拉芽孢杆菌CC2FG2和类肠膜魏斯氏菌JY0R2。生长特性结果显示，特基拉芽孢杆菌CC2FG2在培养基pH为6.5、温度为30 ℃条件下培养16 h的增殖效果最好，可以耐受1.2%胆盐，活菌数是无胆盐的69.2%，耐酸特性比较强，培养基pH为3.5的强酸环境下活菌数是pH为7.0的81.5%；抗菌药耐受性结果显示，其对氧氟沙星、诺氟沙星、卡那霉素、链霉素、克拉霉素、红霉素、复方新诺明和万古霉素敏感，对头孢呋辛，头孢唑林、头孢噻肟、氨苄西林、呋喃妥因、环丙沙星、庆大霉素和四环素不敏感。类肠膜魏斯氏菌JY0R2在培养基pH为6.0、温度为36.5 ℃的环境中培养18 h增殖效果最好，在1.2%胆盐下活菌数是无胆盐的50.6%，在pH为3.5的强酸环境下活菌数是pH为7.0的87.6%，对环丙沙星、氧氟沙星、诺氟沙星、克拉霉素和红霉素敏感，对卡那霉素、呋喃妥因和四环素中度敏感，对头孢呋辛、头孢唑林、头孢噻肟、氨苄西林、复方新诺明、万古霉素、庆大霉素和链霉素不敏感。【结论】鹅源特基拉芽孢杆菌CC2FG2和类肠膜魏斯氏菌JY0R2具有很好的耐酸、耐胆盐特性，且特基拉芽孢杆菌可以与部分β-内酰胺类、喹诺酮类、氨基糖苷类、硝基呋喃类和四环素类抗菌药联合使用，类肠膜魏斯氏菌可以与部分β-内酰胺类和氨基糖苷类抗菌药联合使用。该结果可为鹅源功能菌的研发提供参考。

关键词: 特基拉芽孢杆菌; 类肠膜魏斯氏菌; 生长特性; 酸碱耐受性

Abstract: 【Objective】 The aim of this study was to isolate Bacillus and Lactobacillus from goose feces,so as to provide references for the study of goose-derived functional bacteria and rational drug use.【Method】 The rectal feces of 30 50-week-old healthy Northern White geese were collected,and the strains were isolated and screened by specific culture medium and biochemical reaction test.The selected strains were identified by DNA extraction and PCR amplification 16S rDNA sequencing.The phylogenetic tree was established by comparing with the reference strains in GenBank and the strains were identified by homology comparison.After 48 h of culture,the growth characteristics,optimum pH and temperature of bacteria were detected.The growth status of bacteria under pH 7.0 and no bile salt environment was used as control.The ability of acid and bile salt tolerance was detected,and the tolerance of antibiotics was detected by drug sensitivity test.【Result】 After isolation and screening,9 strains of bacteria were obtained,and 2 strains were finally selected by biochemical test.According to the evolutionary distance and homology comparison of the phylogenetic tree,they were Bacillus tequilensis CC2FG2 and Weissella paramesenteroides JY0R2.The results of growth characteristics showed that Bacillus tequilensis CC2FG2 had the best proliferation effect under the condition of medium pH 6.5 and temperature 30 ℃ for 16 h,which could tolerate 1.2% bile salt,the number of viable bacteria was 69.2% of that without bile salt,and the acid resistance was strong.The number of viable bacteria under the strong acid environment of medium pH 3.5 was 81.5% of that of pH 7.0.The results of antibiotic tolerance showed that it was sensitive to ofloxacin,norfloxacin,kanamycin,streptomycin,clarithromycin,erythromycin,compound sulfamethoxazole and vancomycin,but not sensitive to cefuroxime,cefazolin,cefotaxime,ampicillin,nitrofurantoin,ciprofloxacin,gentamicin and tetracycline.In the medium with pH of 6.0 and temperature of 36.5 ℃,Weissella paramesenteroides JY0R2 had the best prolifervation effect when cultured for 18 h.Under 1.2% bile salt,the number of viable bacteria was 50.6% of that without bile salt.Under strong acid environment with pH 3.5,the number of viable bacteria was 87.6% of that with pH 7.0.It was sensitive to ciprofloxacin,ofloxacin,norfloxacin,clarithromycin and erythromycin,moderately sensitive to kanamycin,nitrofurantoin and tetracycline,and insensitive to cefuroxime,cefazolin,cefotaxime,ampicillin,compound sulfamethoxazole,vancomycin,gentamicin and streptomycin.【Conclusion】 The results of the above studies showed that Bacillus tequilensis CC2FG2 and Weissella paramesenteroides JY0R2 isolated from geese had good acid resistance and bile salt resistance,and Bacillus tequilensis could be used in combination with some β-lactams,quinolones,aminoglycosides,nitrofurans and tetracyclines,and Weisseria paramesenteroides could be used in combination with some β-lactams and aminoglycosides,providing a reference for the development of goose-derived functional bacteria.

Key words: Bacillus tequilensis; Weissella paramesenteroides; growth characteristics; acid-alkali tolerance

中图分类号:

S835

魏笑, 王秋菊, 孙愉, 张昊, 李畅洋, 陶燕子. 鹅粪便中特基拉芽孢杆菌和类肠膜魏斯氏菌的分离鉴定[J]. 中国畜牧兽医, 2022, 49(7): 2778-2787.

WEI Xiao, WANG Qiuju, SUN Yu, ZHANG Hao, LI Changyang, TAO Yanzi. Isolation and Identification of Bacillus tequilensis and Weissella paramesenteroides from Goose Feces[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2778-2787.

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