Antibiotic Resistance Analysis And The Transfer Risk Evaluation Of Bifidobacteria

Bifidobacterium is an indigenous microorganism of the human gastrointestinal tract,which is closely related to human health.Owing to its many probiotic functions,it is widely used in infant foods and functional foods.However,due to the irrational use of antibiotics,many bifidobacterial strains exhibit antibiotic resistance and have the potential risk of horizontal transfer of antibiotic resistance genes.At present,the surveillance and safety evaluation system of antibiotic resistance in bifidobacteria at home and abroad is still not perfect.Based on the above problems,this study analyzed the antibiotic resistance towards tetracycline,erythromycin,clindamycin,ampicillin,amoxicillin,trimethoprim,ciprofloxacin,chloramphenicol,rifampicin,and vancomycin of the seven common Bifidobacterium species?including Bifidobacterium animalis,Bifidobacterium bifidum,Bifidobacterium adolescentis,Bifidobacterium breve,Bifidobacterium longum,Bifidobacterium pseudocatenulatum,and Bifidobacterium dentium?in the intestines of the Chinese population.Additionally,the antibiotic resistance genes related to its resistance phenotypes were identified using comparative genomics methods,and finally,the risk of horizontal transfer of its resistance genes was evaluated by the animal models.The main results are as shown below:?1?To establish the antibiotic resistance database of Bifidobacterium at the species level.The data such as the distribution range of its minimum inhibitory concentration?MIC?,the number of antibiotic dilutions covered,model MIC,MIC₅₀,MIC₉₀,and resistance rates were compared to analyze the antibiotic resistance.The results showed that there were obvious interspecies differences in the MIC distribution of different Bifidobacterium species.According to the microbiological cut-off values of the Bifidobacterium genus,there were interspecies differences in the resistance rates of different Bifidobacterium species to ten antibiotics.Compared to the other species,Bifidobacterium dentium shown a higher proportion of antibiotic susceptibility.?2?To formulate the antibiotic species-specific tentative microbiological cut-off values?TMCOFFs?of bifidobacteria.Developing antibiotic resistance determination standards for different Bifidobacterium species to differentiate sensitive strains from acquired resistance strains,the species-specific microbiological cue-off values of bifidobacteria were developed based on the MIC frequency distribution data of 53 bifidobacteria-antibiotic combinations using two statistical methods?Turnidge and Kronvall?and eyeball method and compared with each other.Statistics found that the cut-off values formulated by the three methods had a consistency of 68.42%,overall,the consistency of the calculation results of the three methods was high.The species-specific TMCOFFs were finally defined as the median of cut-off values obtained by the three methods,and it was consistent with 54.69%of the cut-off values of the Bifidobacterium genus.?3?To analyze the antibiotic resistance genes of bifidobacteria.To further analyze the antibiotic resistance mechanism of bifidobacteria,the diversity and abundance of resistance genes,the distribution of acquired resistance genes,the correlation between phenotype and genotype,and the phylogenetic relationship of resistance genes were performed exploration.The study found that there were differences in the abundance of antibiotic resistance genes of seven Bifidobacterium species to tetracyclines,macrolides,?-lactams,glycopeptides,and folic acid metabolism inhibitors,and there was also a gap in the diversity of resistance genes to the ten antibiotics.Tetracycline,erythromycin,and clindamycin resistance phenotypes had higher correlations with genotypes in Bifidobacterium,and the key resistance gene determinants could explain the corresponding resistance phenotype for greater than 80%of the strains,but there was no obvious correlation between the remaining seven antibiotic genotypes and phenotypes.The gene tet?W?was a key antibiotic resistance gene that mediated tetracycline resistance phenotype,and could explain resistance phenotype for 82.2%of the strains for tetracycline.The gene erm?X?was a key gene that mediated simultaneously erythromycin and clindamycin resistance,similarly,could explain resistance phenotype for 84.8%of the strains for erythromycin and clindamycin,what's more,most of the tet?W?and erm?X?genes were acquired resistance genes.Phylogenetic analysis showed that the tet?W?and erm?X?gene sequences of different Bifidobacterium species had high similarity,and the tet?W?and erm?X?gene sequences of different strains belonging to the same Bifidobacterium species had obvious differences.?4?The risk assessment of the horizontal transfer of tetracycline resistance gene tet?W?of bifidobacteria.To further assess the risk of horizontal transfer of tetracycline resistance gene tet?W?in bifidobacteria,the transfer frequency of antibiotic resistance genes in the absence of antibiotics and under the pressure of antibiotic in bifidobacteria was investigated by establishing a mouse model of antibiotic resistance gene transfer.It was found that no transconjugants were detected during the entire experiment,which shows that the gene tet?W?could not be transmitted and transferred between bifidobacterial strains and Enterococcus faecalis.Tetracycline selection pressure did not affect the horizontal transfer frequency of the tet?W?gene,and the concentrations of recipient bacteria in mouse fecal samples and different parts of the intestinal tract were lower than that of donor bacteria.