摘要
Background: The aim of this study was to investigate the intergeneric transfer of vancomycin resistance gene vanA between probiotic enterococci in the fermentation progress of soybean meal and in the digestive tract of growing pigs.One vanA genotype vancomycin resistant E.faecium strain,Efm4,and one chloramphenicol-resistant E.faecalis strain,Efs2,were isolated from twenty-nine probiotic basis feed material/additive samples.For in vitro conjugation,Efm4 and Efs2 were used as starter to ferment soybean meal.For in vivo conjugation,thirty growing pigs were randomly assigned to five groups(n = 6),treated with a basic diet,or supplemented with 10% fermented soybean meal,1% Efm4,5% Efs2 or a combination of 1% Efm4 + 5% Efs2 for 7 d,respectively.Fecal samples of pigs in each group were collected daily for the isolation and dynamic analysis of Efm4,Efs2 and transconjugants.The sequence types(STs) of Efm4,Efs2 and transconjugants were analyzed by multilocus sequence typing(MLST).The vanA harboring plasmid in Efm4 and transconjugants was analyzed by S1-pulsed field gel electrophoresis(PFGE)and further verified by multiple alignments.Results: The results showed that,in FSBM,transconjugants were detected 1 h after the fermentation,with a conjugation frequency of ~ 10^-3 transconjugants/recipient.Transconjugants proliferated with Efm4 and Efs2 in the first 8 h and maintained steadily for 10 d till the end of the experiment.Additionally,in vivo experiment showed that transcojugants were recovered in one of six pigs in both FSBM and Efm4 + Efs2 groups,with conjugation frequency of ~ 10^-5 and ~ 10^-4,respectively.MLST revealed the ST of Efm4,Efs2 and transconjugants was ST1014,ST69 and ST69,respectively.S1-PFGE confirmed the existence of the vanA-harboring,142,988-bp plasmid,which was also a multi-drug resistant plasmid containing Tn1546-like transposon.Conclusions: The findings revealed the potential safety hazard existing in the commercial probiotic enterococci in China,because the horizontal transfer from farm to fork could potentially pose a safety risk to the public.
Background: The aim of this study was to investigate the intergeneric transfer of vancomycin resistance gene vanA between probiotic enterococci in the fermentation progress of soybean meal and in the digestive tract of growing pigs. One vanA genotype vancomycin resistant E. faecium strain, Efm4, and one chloramphenicol-resistant E. faecalis strain, Efs2, were isolated from twenty-nine probiotic basis feed material/additive samples. For in vitro conjugation,Efm4 and Efs2 were used as starter to ferment soybean meal. For in vivo conjugation, thirty growing pigs were randomly assigned to five groups(n = 6), treated with a basic diet, or supplemented with 10% fermented soybean meal, 1% Efm4, 5% Efs2 or a combination of 1% Efm4 + 5% Efs2 for 7 d, respectively. Fecal samples of pigs in each group were collected daily for the isolation and dynamic analysis of Efm4, Efs2 and transconjugants. The sequence types(STs) of Efm4, Efs2 and transconjugants were analyzed by multilocus sequence typing(MLST). The vanA harboring plasmid in Efm4 and transconjugants was analyzed by S1-pulsed field gel electrophoresis(PFGE)and further verified by multiple alignments.Results: The results showed that, in FSBM, transconjugants were detected 1 h after the fermentation, with a conjugation frequency of ～ 10-3 transconjugants/recipient. Transconjugants proliferated with Efm4 and Efs2 in the first 8 h and maintained steadily for 10 d till the end of the experiment. Additionally, in vivo experiment showed that transcojugants were recovered in one of six pigs in both FSBM and Efm4 + Efs2 groups, with conjugation frequency of ～ 10-5 and ～ 10-4, respectively. MLST revealed the ST of Efm4, Efs2 and transconjugants was ST1014, ST69 and ST69, respectively. S1-PFGE confirmed the existence of the vanA-harboring, 142,988-bp plasmid,which was also a multi-drug resistant plasmid containing Tn1546-like transposon.Conclusions: The findings revealed the potential safety hazard existing in the commercial probiotic enterococci in China, because the horizontal transfer from farm to fork could potentially pose a safety risk to the public.
基金
funded by National Key Research and Development Program of China(Grant number 2016YFD0501308)
Agro-scientific Research in the Public Interest(Grant number 201403047)
