24株鸡源丁酸梭菌的分离鉴定及耐药基因与毒力基因携带情况

Detection of resistance and virulence genes from 24 Clostridium butyricum strains isolated from chickens

摘要:

[目的] 旨在对鸡源丁酸梭菌进行分离鉴定与安全性评估。[方法] 利用厌氧培养方法对源自汶上芦花鸡与SPF鸡粪便样品进行丁酸梭菌的分离与纯化，挑选可疑菌落进行微生物质谱鉴定，进一步通过16S rRNA基因测序进行鉴定，16S rRNA测序结果与NCBI核苷酸数据库中丁酸梭菌的16S rRNA序列进行同源性分析；同时，进行所有分离株对氧氟沙星、头孢吡肟等9种药物的药敏试验，利用PCR方法进行mefA等23种耐药基因扩增，基于益生菌安全要求对样品进行alpha等4种梭菌毒素基因以及typeA等4种肉毒毒素基因的测定。[结果] 共分离鉴定了24株丁酸梭菌。24株均对氧氟沙星等7种抗生素表现为敏感，L-1、L-6、L-12仅对新霉素表现为中介，L-19仅对头孢吡肟表现为中介。全部菌株的mefA等16种耐药基因结果全部为阴性，sul2、flor、bla_TEM 3种耐药基因全部呈阳性，tetC携带率为79.2%，cmlA携带率为45.8%，bla_OXA携带率为37.5%，aadB携带率为12.5%，qnrA携带率为4.2%。PCR结果显示所有分离菌株的alpha、beta、epsilon、iota等4种梭菌毒素基因携带率0%。全部分离菌株均未携带typeA、typeB、typeE、typeF 4种肉毒毒素基因。[结论] 结果表明，从未饲喂抗生素和丁酸梭菌的汶上芦花鸡与SPF鸡群中获得的24株丁酸梭菌分离株达到预期的安全性要求，可作为益生添加菌的筛选参考株。

关键词:丁酸梭菌;药敏试验;耐药基因;梭菌毒素基因;肉毒毒素基因

Abstract:

[Objective] To isolate, identify and evaluate the safety of Clostridium butyricum from two breeding hen farms in Shandong province. [Methods] Anaerobic culture was used to isolate anaerobic bacterial strains from both Luhua chicken and SPF bird droppings originated in Shandong Province. Suspicious colonies were selected for mass spectrometry and then identified by 16S rRNA gene sequencing. The 16S rRNA sequencing results were analyzed for homology with 16S rRNA sequence of Clostridium butyricum in NCBI nucleotide data. Meanwhile, all isolates were tested for susceptibility to 9 drugs, such as ofloxacin and cefepime. PCR was used for the determination of 23 antimicrobial resistance genes such as mefA. Four clostridium toxin genes including alpha and four botulinum toxin genes including typeA were determined based on probiotic safety requirements. [Results] A total of 24 strains of Clostridium butyricum were identified, and they were sensitive to 7 antibiotics such as ofloxacin. L-1, L-6 and L-12 were moderated only for neomycin and L-19 was moderated only for cefepime. All strains of 16 drug-resistant genes were negative, and 3 drug-resistant genes of sul2, flor and bla_TEM were positive, tetC carrying rate is 79.2%, cmlA carrying rate is 45.8%, bla_OXA carrying rate is 37.5%, aadB carrying rate is 12.5%, qnrA carrying rate is 4.2%. PCR results showed that the alpha, beta, epsilon, iota clostridium toxin gene carrying rates of all isolated strains were 0%. The carrying rate of botulinum toxin genes typeA, typeB, typeE and typeF of all isolates was 0%. [Conclusion] The 24 isolates of Clostridium butyricum from tested chickens never fed with antibiotics and Clostridium butyricum met the expected safety requirements and could be used as a screening reference strain for probiotic added bacteria.

Key words:Clostridium butyricum;drug sensitivity test;resistance gene;clostridium toxin gene;botulinum toxin gene