Horizontal transfer of vanA between probiotic Enterococcus faecium and Enterococcus faecalis in fermented soybean meal and in digestive tract of growing pigs

Abstract

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, E fm 4, and one chloramphenicol-resistant E. faecalis strain, E fs 2, were isolated from twenty-nine probiotic basis feed material / additive samples. For in vitro conjugation, E fm 4 and E fs 2 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% E fm 4, 5% E fs 2 or a combination of 1% E fm 4 + 5% E fs 2 for 7 d, respectively. Fecal samples of pigs in each group were collected daily for the isolation and dynamic analysis of E fm 4, E fs 2 and transconjugants. The sequence types (STs) of E fm 4, E fs 2 and transconjugants were analyzed by multilocus sequence typing (MLST). The vanA harboring plasmid in E fm 4 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 − 3 transconjugants / recipient. Transconjugants proliferated with E fm 4 and E fs 2 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 E fm 4 + E fs 2 groups, with conjugation frequency of ~ 10 − 5 and ~ 10 − 4 , respectively. MLST revealed the ST of E fm 4, E fs 2 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. 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.