The Study Of Adherence Properties Of Lactobacillus To The Endometrium Epithelial Cell

Lactic acid bacteria (LAB) is the normal flora in genital tract. It plays the vital role in adjustmenting the balance of microecology of the organism genital tract through producing materials such as H₂O₂, lactic acid, acetic acid and so on, which can inhibit the growth of pathogenic bacteria. It has been proved that LAB preparations can effective prevent and treat cows endometritis. Since the adhesion of LAB is one of the important indexes as it works as a probiotics, adherence experriment In vivo or in vitro becomes the main way to screening the candidate probiotic strains, in vitro study on cell level is the main method in present as the limitation of in vivo study. Meanwhile, due to the limitation of appropriate cell model, the relevant research about the adhesion of LAB to goat endometrial epithelial cells (EEC) is rare. Consequently, the study of adhesion of LAB to EEC will be with potential value both on the further research and the development of microecologics which can prevent and treat organism genital tract bacterial infection.In this study, Immortalized EEC obtained by transfection hTERT enzyme as an in vitro model. The adherence properties of LAB to EEC and its antagonistic activity against pathogens were studied through Gram's staining and plate counting. Then the adhesion components of Lactobacillus acidophilus was indentified preliminary.The results as following:1. All of the three LAB strains: Lactobacillus acidophilus,Enterococcus faecalis and Enterococcus cecorum screened out from juice of healthy cow showed strong adhesive activity with the counting results were 87.00±15.00 CFU/cell,45.33±7.506 CFU/cell and 35.33±6.110 CFU/cell respectively. The adherence ability of Enterococcus faecalis and Enterococcus cecorum was not significantly different (P>0.05), but they were significantly lower than Lactobacillus acidophilus(P<0.05).2. Common pathogenic bacterias that lead to cows endometritis showed strong adhesive activity to EEC. The adherence rate of pathogenic Escherichia coli C 83903 was 38.33%, pathogenic Escherichia coli ATCC 25922 was 65.42% and Staphylococcus aureus CVCC 2258 was 86.18%. The adherence rate of enteropathogens was significantly lower than control when the LAB strains: Lactobacillus acidophilus or Enterococcus faecalis or Enterococcus cecorum was added as to test their antagonistic activity against enteropathogens. The antagonistic activity of different LAB strains against enterpathogens adhesion was not significantly different. While Lactobacillus acidophilus showed the strongest antagonistic activity against Escherichia coli making the adherence rate of C 83903 decreased from 38.33% to 9.95% and ATCC 25922 from 65.42% to 8.27% and the adherence rate of Staphylococcus aureus decreased from 86.18% to 8.63% and from 86.18% to 8.71% when cocultured with Enterococcus cecorum and Enterococcus faecalis respectively.3. The adhesion of bacteria was affected by the growth phase of EEC. Cells cultured for 48 h have the most bacterias to adhered to with the counting result as to 97 CFU/cell. The adhesion of Lactobacillus acidophilus to EEC dependent on bacteria concentration and incubation time obviously. A significant rise in the number of bacteria adhesion on cells was observed during the 30-120 min incubation time and the number was not changed after 120 min. As bacteria concentration increasing, the number of adhered bacteria rise accordingly and the number was 82.33±2.333 CFU/cell when the concentration was 10⁹-10¹⁰ CFU/mL. While the number of adhered bacteria decreased significantly with the rise of the pH value. When the pH value was 4-5, the number of bacteria adhesion on cells was as most as about 91.67±1.764 CFU/cell. While the batercia in the stationary phase takes up the most of the adhesion bacteria with their number as to 93.33±5.686 CFU/cell.4. The adherence ability of Lactobacillus acidophilus is significant decreased when treated with trypsin,LiCl,phenol or sodium periodate. Further analysis showed that a number of ingredients of Lactobacillus acidophilus participate in the adherence progress and the surface proteins play a significant role.5. The surface proteins of cell wall of Lactobacillus acidophilus was extracted through the LiCl assay and the SDS-PAGE analysis showed a protein of 48 ku in size as the main surface protein. The surface proteins-mediate adherence inhibition assays showed that the surface proteins play a vital role in inhibition of the Lactobacillus acidophilus adhesion to EEC and the effect of adherence inhibition was concentration-dependent.