Li Ning, Yu Haitao, Liu Hongbin, Wang Yuming, Zhou Junyan, Ma Xi, Wang Zheng, Sun Chengtao, Qiao Shiyan
1State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 10093 China.
2Beijing Key Laboratory of Bio-feed Additives, China Agricultural University, Beijing, 10093 China.
J Anim Sci Biotechnol. 2019 Apr 12;10:36. doi: 10.1186/s40104-019-0341-x. eCollection 2019.
The aim of this study was to investigate the intergeneric transfer of vancomycin resistance gene between probiotic enterococci in the fermentation progress of soybean meal and in the digestive tract of growing pigs. One genotype vancomycin resistant strain, E4, and one chloramphenicol-resistant strain, E2, were isolated from twenty-nine probiotic basis feed material / additive samples. For conjugation, E4 and E2 were used as starter to ferment soybean meal. For conjugation, thirty growing pigs were randomly assigned to five groups ( = 6), treated with a basic diet, or supplemented with 10% fermented soybean meal, 1% E4, 5% E2 or a combination of 1% E4 + 5% E2 for 7 d, respectively. Fecal samples of pigs in each group were collected daily for the isolation and dynamic analysis of E4, E2 and transconjugants. The sequence types (STs) of E4, E2 and transconjugants were analyzed by multilocus sequence typing (MLST). The harboring plasmid in E4 and transconjugants was analyzed by S1-pulsed field gel electrophoresis (PFGE) and further verified by multiple alignments.
The results showed that, in FSBM, transconjugants were detected 1 h after the fermentation, with a conjugation frequency of ~ 10 transconjugants / recipient. Transconjugants proliferated with E4 and E2 in the first 8 h and maintained steadily for 10 d till the end of the experiment. Additionally, experiment showed that transcojugants were recovered in one of six pigs in both FSBM and E4 + E2 groups, with conjugation frequency of ~ 10 and ~ 10, respectively. MLST revealed the ST of E4, E2 and transconjugants was ST1014, ST69 and ST69, respectively. S1-PFGE confirmed the existence of the -harboring, 142,988-bp plasmid, which was also a multi-drug resistant plasmid containing -like transposon.
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.
本研究旨在调查在豆粕发酵过程以及生长猪消化道中，益生菌肠球菌之间万古霉素抗性基因的属间转移情况。从29份益生菌基础饲料原料/添加剂样本中分离出一株基因型为万古霉素抗性的菌株E4和一株氯霉素抗性菌株E2。为进行接合试验，以E4和E2作为发酵豆粕的起始菌。为进行接合试验，将30头生长猪随机分为五组（每组6头），分别用基础日粮处理，或补充10%发酵豆粕、1% E4、5% E2或1% E4 + 5% E2的组合，持续7天。每天收集每组猪的粪便样本，用于分离和动态分析E4、E2及接合子。通过多位点序列分型（MLST）分析E4、E2及接合子的序列类型（STs）。通过S1-脉冲场凝胶电泳（PFGE）分析E4及接合子中携带的质粒，并通过多重比对进一步验证。
结果显示，在发酵豆粕中，发酵1小时后检测到接合子，接合频率约为每受体10个接合子。接合子在最初8小时内与E4和E2一起增殖，并在实验结束前的第10天保持稳定。此外，实验表明，在发酵豆粕组和E4 + E2组的6头猪中，各有1头猪检测到接合子，接合频率分别约为每受体10个和每受体10个。MLST显示E4、E2及接合子的ST分别为ST1014、ST69和ST69。S1-PFGE证实存在携带142,988 bp质粒，该质粒也是含有类似转座子的多药耐药质粒。
研究结果揭示了中国市售益生菌肠球菌存在的潜在安全隐患，因为从农场到餐桌的水平转移可能会对公众构成安全风险。
