Therapeutic Effects Of Bacteroides Fragilis For Vibrio Parahaemolyticus Infection In Vitro And In Vivo Experiments

Bacteroides fragilis is the one of the commensal bacteria in human's gut.Recently,many studies have found Bacteroides fragilis to play an important role in promoting the maturation of the immune system,suppressing abnormal inflammation and improve intestinal microflora.For this,it may have great prospects when Bacteroides fragilis has been developed as a probiotic strain.Since the inhibition of intestinal pathogen is an important characteristic for probiotic,We tested whether Bacteroides fragilis has the ability to inhibit pathogenic bacteria.Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide.When infected with Vibrio parahaemolyticus,symptoms can be as mild as self-limiting diarrhea to as severe as cholera-like-to-moderate,there will be abdominal pain,diarrhea,abdominal cramps,nausea,vomiting,fever and so on.In most cases patients' feces will be watery,or the blood sample when severe.The resistance and immunity of patients after infection are so weak that people can be repeated infection by Vibrio parahaemolyticus.The aim of this study was to evaluate the probiotic effects of Bacteroides fragilis against Vibrio parahaemolyticus in intestinal epithelial cells and mouse model.This paper includes the following two parts:Part 1 The inhibition effects of Bacteroides fragilis for Vibrio parahaemolyticus infection in vitro experimentIn the oxford plate assay we found that the cultures of Bacteroides fragilis could inhibit the growth of Vibrio parahaemolyticus in vitro.Bacteroides fragilis was added to the tumor-derived human colonic epithelial cells,the LoVo cells,trying to simulate the relationship between normal intestinal epithelial cells and intestinal bacterium.Then we infected the cell with Vibrio parahaemolyticus to explore whether could protect LoVo cells from being damaged caused by Vibrio parahaemolyticus.We combined the real-time cell analysis techniques(Real-Time Cell Analysis,RTCA)and the method of observing cell morphology under a microscope to detect toxic effects on the cells infected with bacteria.LoVo cells or raw 264.7 cells was incubated with Bacteroides fragilis for 8 hours,the normal cell index(NCI)curves of the two groups were similar with the control group while the NCI curve of the Vibrio parahaemolyticus group declined quickly;The morphology of the two cells showed a little change compared with the control group while the Vibrio parahaemolyticus groups cell morphology became wrinkled and broken.However,when the two cells were infected with Vibrio parahaemolyticus after incubated with Bacteroides fragilis for 3 hours,the NCI crave decline more slowly than the Vibrio parahaemolyticus group,and the morphology of the two groups were much better than the Vibrio parahaemolyticus group.All the results above indicated that Vibrio parahaemolyticus had obvious toxicity for LoVo cells and raw 264.7 cells while Bacteroides fragilis could protect both the raw264.7 cells and the LoVo cells from being damaged caused by Vibrio parahaemolyticus.Part 2 The treatment effects of mice infected with Vibrio parahaemolyticus by Bacteroides fragilisThe light-emitting plasmid pXEN-luxCDABE was successfully transduced into Vibrio parahaemolyticus,and the grow rate of the lux strain was similar with the wide-type strain.The released lactate dehydrogenase and the NCI crave of LoVo cells caused by the lux strain were also similar with the wide-type strain.The transduction of the light-emitting plasmid showed little effect on the growth and cytotoxicity of Vibrio parahaemolyticus.Thanks to the technology of in-vivo imaging,we can trace the real-time dynamic proliferation and displacement process of the lux strains in vivo without damaging the experimental mice.Mice were treated with Bacteroides fragilis twice on 3 and 14 hours post-infection by the lux-Vibrio parahaemolyticus.To 14s hour,the luminous intensity of the treatment mice was weaker than the control group.To 26s hour,the luminous intensity of the treatment group was nearly impossible to detect while the luminous intensity of the control still had significant bioluminescence,although there was a marked decline.When we counted the number of Vibrio parahaemolyticus in the stool of two groups using TCBS agar which was a selective medium for Vibrio parahaemolyticus,we found that the number of Vibrio parahaemolyticus in the stool from the treatment group was significantly less than the control group.These results suggest that the treatment with Bacteroides fragilis might assist mice in shortening the time of Vibrio parahaemolyticus colonization.In conclusion,Vibrio parahaemolyticus can be inhibited by the culture of Bacteroides fragilis,and it has obvious toxicity for LoVo cells and Raw264.7 cells.Bacteroides fragilis could protect both the Raw264.7 cells and the LoVo cells from being damaged caused by Vibrio parahaemolyticus.The transduction of the light-emitting plasmid pXEN-luxCDABE shows little effect on the growth and cytotoxicity of Vibrio parahaemolyticus.When infected with the light-emitting strain,compared with the mice which were not gavaged with Bacteroides fragilis after being infected,the luminous intensity of the mice which were gavaged with Bacteroides fragilis after being infected was much lower,indicating Bacteroides fragilis could help mouse to remove Vibrio parahaemolyticus.Our results indicated Bacteroides fragilis might provide assistance in clinical for the treatment of gastroenteritis caused by Vibrio parahaemolyticus.