| Lactic acid bacteria are commonly utilized in food due to their long history of safety;nevertheless,there is a danger of gene transfer in lactic acid bacteria harboring antibiotic resistance genes,which could pose a food safety problem.Tetracycline resistance genes are generally found on mobile genetic elements,thus they have a high chance of horizontal transfer and should be given special attention.Although there is an international standard system for evaluating the antibiotic resistance of lactic acid bacteria,the antibiotic resistance of different lactic acid bacteria species to the same antibiotic is different.At present,there are still many kinds of lactic acid bacteria species without species-specific cut-off values,and the mechanism of antibiotic resistance in many lactic acid bacteria strains with antibiotic resistant phenotypes is unknown.Based on the above problems,this study analyzed the tetracycline resistance of lactic acid bacteria from different sources in China,identified the drug resistance genes in the strains based on comparative genomics and analyzed their correlation with their drug resistance phenotypes.Finally,their possible tetracycline resistance genes were mined and verified using transcriptome technology and gene overexpression technology for strains with drug resistance phenotype but not related with known drug resistance genes.The main results are as follows:（1）To establish the tetracycline resistance database of lactic acid bacteria at the species level.The minimum inhibitory concentration（MIC）of 477 strains of 8 kinds of lactic acid bacteria to tetracycline was measured.The antibiotic resistance of different kinds of lactic acid bacteria species to tetracycline was analyzed by comparing their MIC distribution range,number of dilution,model MIC,MIC50 and MIC90.The results showed that there were significant interspecific differences in MIC distribution of different lactic acid bacteria species.The tetracycline resistance phenotype of lactic acid bacteria was determined according to the cut-off values formulated by EFSA,and the tetracycline resistance rates of eight lactic acid bacteria species were counted.The drug resistance rates of Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus to tetracycline were the lowest,and the drug resistance rates of Lactobacillus johnsonii and Lactobacillus crispatus to tetracycline were the highest.Compared with other lactic acid bacteria species,the lactic acid bacteria species without cut-off values showed a higher proportion of tetracycline resistance.（2）Develop species-specific Tentative Microbiological Cut-off Values（TMCOFFs）for Lactobacillus gasseri/paragasseri,Lactobacillus johnsonii,and Lactobacillus crispatus to tetracycline.In order to better distinguish the sensitive strains and acquired resistant strains,based on the MIC frequency distribution data of Lactobacillus gasseri/paragasseri,Lactobacillus johnsonii,and Lactobacillus crispatus to tetracycline,two statistical methods and eyeball method were used to formulate the species-specific TMCOFFs of these lactic acid bacteria species,species-specific TMCOFFs is finally defined as the median of the cut-off values formulated by the three methods,and the subsequent genotype-phenotype association results also verify the accuracy of the species-specific TMCOFFs.（3）Genotype-phenotype association analysis to explore the mechanism of tetracycline resistance of lactic acid bacteria.In order to further explore the tetracycline resistance mechanism of lactic acid bacteria,the tetracycline resistance genes of 477 strains of lactic acid bacteria were identified by comparative genomics method.The presence or absence of drug resistance genes was correlated with drug resistance phenotype to explore the key genes mediating resistance,and the distribution of tetracycline resistance genes in lactic acid bacteria and the phylogenetic relationship of tetracycline resistance genes were explored.It was found that there was a high correlation between tetracycline resistance phenotype and genotype in lactic acid bacteria.Genes tet（M）、tet（W/N/W）、tet（L）、tet（S）and tet（45）were the key tetracycline resistance genes mediating tetracycline resistance in lactic acid bacteria.The genes of tet（M）and tet（W/N/W）were mediated tetracycline resistance in most lactic acid bacteria strains.Phylogenetic analysis showed that the gene sequences of tet（M）and tet（W/N/W）in lactic acid bacteria had high intraspecific homology and interspecific variability.（4）The genes yjc A and ABC-SBP which encode efflux pump proteins can mediate the resistance of lactic acid bacteria to tetracycline.In order to explore the tetracycline resistance mechanism of strains with tetracycline resistance phenotype and without known tetracycline resistance genes,the transcriptome technology was used to explore their resistance basis.The strain can be resistant to tetracycline by reducing its own metabolism,increasing ribosomal protein synthesis,improving translation level and using ABC efflux pump.Overexpression of suspected tetracycline resistance genes verified that the gene yjc A encoding the efflux pump protein may mediate the tetracycline resistance phenotype of Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus,and the gene ABC-SBP encoding the efflux pump protein could mediate the tetracycline resistant phenotype of Lactiplantibacillus plantarum and Limosilactobacillus reuteri. |
