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

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.