Bacteriostasis Activity Of Recombinant Lysostaphin And Influence On The Structure Of Intestinal Flora In Mice

Lysostaphin can take a specific soluble killing effect on Staphylococcus aureus.Staphylococcus aureus is one of the three pathogenic bacteria causing mastitis, researchershave successfully cultivated transgenic bovine those mammary epithelial cells can secretelysostaphin to against mastitis in dairy cows. However, with the development of transgenicbovine, relevant animal products and food safety issues are getting more and more attention.Presently, research reports about lysostaphin safety on animal organism are limited. As animportant indicator, intestinal microflora are closely related to animals and human health.Therefore, through the detection of the antibacterial properties in vitro of recombinantlysostaphin secreted from bovine mammary epithelial cells, as well as the influence onmouse intestinal microbiota structure, the aim of this research is to provide experimentalbasis of clinical resistance to infection of tran-lysostaphin-gene bovine, and to evaluatefood security of lysostaphin residues on the micro-ecological angle, this study has potentialsignificance on transgenic animal food safety.The size of inhibition zone, the Minimun Inhibitory Concentration (MIC) and theMinimal Bactericidal Concentration (MBC) of the recombinant lysostaphin was assessedby disc agar diffusion test and broth dilution test, through comparing with lysostaphin andtwo kinds of antibiotics, to appraise the bacteriostasis activity of recombinant lysostaphinin vitro; Changes of intestinal microbiota structural before and after lysostaphin taken wasdetected by PCR-DGGE to study its influence on intestinal microbiota in mice, and thesequencing analysis of the main bands was detected. The results as following:1. Disc agar diffusion test showed that recombinant lysostaphin had suppressedstaphylococcus specificity, but was invalid to other strains; The bacteriostasis activity ofrecombinant lysostaphin and lysostaphin was not remarkable(P＞0.05); Regarding thestaphylococcus resisting to penicillin, recombinant lysostaphin still displayed goodbacteriostasis activity. Broth dilution test indicated that the bacteriostasis activity of therecombinant lysostaphin surpassed to hydrochloric acid Vancomycin which was effective toantibiotic resistence staphylococcus, and the MIC was0.1μg/ml~0.2μg/ml.2. Common bands and differential bands existed in the PCR-DGGE profiles of eachgroup; UPGMA cluster analysis showed that the similarity of intestinal microflora of mouse decreased with the increased dose of lysostaphin, the similarities of1μg/mL,5μg/mL and15μg/mL dose of lysostaphin were75%,64%and57%within the group, and5μg/mL of recombinant lysostaphin was71%; Differential analysis showed that, comparedwith the control group, the differences of the similarity between the lanes of each groupbefore and after the test was not significant (P＞0.05); Sequencing analysis of the majorbands in the profiles showed that unknown bacteria accounted for about64%of thesequenced strain; The bacteria identified are most similar to Bacteroides, Aerococcusurinaeequi, Staphylococcus equorum, Lactobacillus taiwanensis, Jeotgalicoccus coquinae,Lactobacillus murinus, Lactobacillus reuteri and Uncultured Firmicutes in more than95%;15μg/mL dose of lysostaphin and5μg/mL dose of recombinant lysostaphin promoted theculture of Lactobacillus taiwanensis.