长双歧杆菌(Bifidobacterium longum)是食品中常用的益生菌,对其耐药性的判定关系着食品安全。为制定长双歧杆菌抗生素耐药性判定标准,探究其抗生素耐药性的关键基因,参照ISO 10932标准方法,测定了52株B. longum菌株对四环素、红霉素、克林霉素、氨苄青霉素、氯霉素和万古霉素的最小抑菌浓度(minimal inhibitory concentration, MIC),采用Turnidge(T)和Kronvall(K)两种统计学方法制定B. longum六种抗生素的种特异性初始微生物折点值(tentative microbiological cut-off values, TMCOFFs),并从功能基因层面进行耐药机制研究。结果发现:B. longum对四环素、红霉素、克林霉素、氨苄青霉素、氯霉素和万古霉素的种特异性TMCOFFs分别为8、8、0.25、8/2(T/K)、8和2 μg/mL;相应折点值下的耐药率分别为28.85%、25%、28.85%、3.85%/7.69%(T/K)、0和19.23%。通过功能基因和比较基因组分析发现,tet(W)是B. longum四环素耐药性的主要抗性基因,erm(X)则介导菌株对红霉素和克林霉素的耐药性,并且耐药基因位于可移动遗传元件上;系统进化分析发现不同耐药型菌株的tet(W)和erm(X)基因序列存在显著差异。该研究对B. longum在食品中的安全应用以及益生菌耐药性评价标准的制定具有指导意义和参考价值。
Bifidobacterium longum is a commonly used probiotic in food industry, and its drug resistance is related to food safety issues. The objective of this study was to formulate antibiotic resistance determination criteria for B. longum and to explore the key genes of antibiotic resistance through functional gene analysis. The minimum inhibitory concentrations of 52 B. longum strains towards tetracycline, erythromycin, clindamycin, ampicillin, chloramphenicol, and vancomycin were determined with reference to ISO standard methods (ISO 10932). The species-specific microbiological cut-off values of these six antibiotics of B. longum were evaluated by Turnidge (T) and Kronvall (K) statistical methods and analyzed the antibiotic resistance mechanism from the molecular genetic basis. The results showed that the species-specific microbiological cut-off values for tetracycline, erythromycin, clindamycin, ampicillin, chloramphenicol, and vancomycin of B. longum were 8, 8, 0.25, 8/2(T/K), 8 and 2 μg/mL respectively, and the drug resistance rates were 28.85%, 25%, 28.85%, 3.85%/7.69%(T/K), 0 and 19.23%. Functional gene and comparative genomic analysis revealed that tet(W) and erm(X) both located on mobile genetic elements were the main resistance genes for tetracycline resistance and erythromycin and clindamycin resistancein B.longum.Phylogeneticanalysis indicated that the tet(W) and erm(X) gene sequences of different phenotypic resistance strains possessed significant variation. This study has certain guiding significance and reference value for the safety application of B. longum in food and the development of evaluation criteria for probiotics resistance.
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