Identification And Screening Of Enterococcus Probiotics From Swine Feces

A large number of side effects, such as the emergence of antibiotic resistant in bacteria, the imbalance of normal intestinal flora, antibiotic residues and so on, can be caused by the abuse of antibiotics in livestock production industries. Holding the promises of no toxicity, no residue and no antibiotic resistant, probiotics have aroused wide attention as feed additives, and maybe become an effective alternative of antibiotics.In order to develop Enterococci as probiotics, twenty-two Enterococcus isolates were isolated from anorectal contents of porcine intestinal tracts in this study. The fermentative supernatant fluid of these Enterococci showed obvious inhibitory activity to the indicator bacteria, especially in six Enterococcus isolates TC1, TC3, TC9, HB6, TC12 and TC13. TC3 was identified as Enterococcus faecium, while HB6, TC12 and TC13 were Enterococcus faecalis. Whereas, TC1 and TC9 were uncertain. Based on antimicrobial activity and biochemical characteristics, four Enterococcus isolates TC3, HB6, TC12 and TC13 were screened for the further experiments. Analysis of 16S rRNA gene sequences and specific amplification of fragments intragenic to ddlE.faecalis ddlE.faecium by polymerase chain reaction, they were further confirmed the identification of these four Enterococcus isolates.In order to ensure the safety of bacteria, four Enterococcus isolates were tested for the presence of potential virulence factors and antibiotic resistance. The presence of virulence factors cylA, gelE, esp, agg, ace, efaAfs and efaAfm were evaluated by PCR. As results, Enterococcus faecium TC3 was efaAfm positive, and three virulence genes cylA, esp, efaAfs were detected in Both E. faecalis HB6 and E. faecalis TC13. E. faecalis TC12 carried four virulence genes agg, cylA, esp and efaAfs. Therefore, E. faecium TC3 was chosen to examine antibiotic susceptibility. It showed resistance to penicillin, erythromycin, and intermediately resistant to ciprofloxacin, whereas, be sensitive to ampicillin, vancomycin, tetracycline, norfloxacin, nitrofurantoin, and chloramphenicol. These results suggested that E. faecium TC3 is safe to be used.In order to screen the dominant strains of animal gut bacteria, the growth curve, tolerance characteristics and adhesion to mucin of E. faecium TC3 were studied. Under condition of pH 2.5 and pH 2.0, the survival counts of E. faecium TC3 were 4.10×107 CFU/mL and 2.20×106 CFU/mL respectively after 2h incubation. Futhermore, E.faecium TC3 was able to tolerate bile salt at concentration of 0.5%. These results suggested that E.faecium TC3 was able to successfully go through the gastrointestinal tract. Whereas, E.faecium TC3 unable to survive at the temperature above 70℃. The proliferation speed of E.faecium TC3 was fast without lag phase, and reached logarithmic growth phase in short period(0h～4h). Then, the number of E.faecium TC3 experienced a steadily increase, and reached peak value (3.5×109 CFU/mL) at 18h. Adhesion tests demonstrated that E.faecium TC3 showed significant strong capacity to adhere to mucin(p<0.05).