| Enterococcus faecalis,a opportunistic pathogen in the gastrointestinal tract of human and animal,however,it has become a common phenomenon that E.faecalis are becoming increasingly for people and animals infection in the hospital in recent years.The existence of the more common mixed infection of bacteria and bacteria as well as bacteria and viruses,however,the process of interaction in mixed infection between various microorganisms is unclear,especially the process of interaction in this less virulent enterococci bacteria.E.coli O157: H7,salmonella,staphylococcus aureus are similar to E.faecalis who are human and animal intestinal or skin common pathogens.It is not clear that how E.faecalis influence on this three kinds of bacteria in the process of enterococcus pathogenic especially in molecular level.In this study we through coculture to E.faecalis N41 in vitro,observe the bacterial growth curve,and the changes of N41 virulence gene transcription level,and then observed biofilm formation ability,adhesion,resistance to phagocytosis of N41 after coculture in vitro.Which laid a good foundation for the mechanism of interaction between bacteria and the prevention and treatment of E.faecalis mixed infection.The influence on growth characteristics Compared the growth characteristics of N41 in individual culture with E.coli O157: H7,salmonella,staphylococcus aureus and cocultivation with N41 separately,all kinds of bacteria which its growth characteristics are affected by different degree.After cocultivation,the growth of the three kinds of bacteria have been suppressed by N41 in different degree.The peak concentration of E.coli O157: H7 dropped about 30 times,the peak concentration of salmonella dropped about 110 times,the peak concentration of staphylococcus aureus dropped about 8 times,In addition,after cocultivation,E.coli O157: H7 and salmonella have weaker inhibition to N41,and when staphylococcus aureus co-culture with N41,the peak concentration of N41 strains rose about 967 times.This indicated that staphylococcus aureus have a stronger role in promoting for N41 strains.The influence on transcription level of virulence gene According to coculture,the growth curves were drawn and bacterial total RNA were extracted at given time points.Acording to the genes sequences of E.faecalis V583 in Gen Bank,the primer pairs of 26 kinds of virulence gene were designed by using Primer Premier 5.0.Using SYBR Green I real-time fluorescence quantitative polymerase chain reaction to analyze the m RNA transcription levels of 26 virulence genes of N41.The results showed that when E.coli O157: H7 coculture with N41,E.coli O157: H7 promoted the m RNA transcription levels of 13 kinds of virulence genes such as ebp ABC,fsr AB,gel E,ace,esp and so on in four different growth period,however,Cyl R1,Cyl R2,psr,atn,efa A and AS had no significant changes.When salmonella coculture with N41,salmonella promoted the m RNA transcription levels of 12 kinds of virulence genes such as ebp A,ebp C,gel E,ace,esp,fsr A,psr and so on in four different growth period,however,Cyl R1,Cyl R2,efa A and AS had no significant changes.When staphylococcus aureus coculture with N41,staphylococcus aureus promoted the m RNA transcription levels of 22 kinds of virulence genes such as ebp ABC,fsr ABC,gel E,ace,esp,cyl L-L and so on in four different growth period,however,efa A and AS had no significant changes.In addition,the m RNA transcription levels of virulence genes Sly A and spr E had a downward trend after coculture with three kinds of bacteria.This shows that E.coli O157: H7,salmonella and staphylococcus aureus could promote the transcription levels of virulence genes of N41 as a whole.The influence on biofilm formation,adhesion and resistance to phagocytosis In vitro cocultivation,we observing the biofilm formation,adhesion and resistance to phagocytosis.The results showed that the biofilm production had increased dramatically when coculture with staphylococcus aureus.Bacteria adhesion test results show the CFU counts of N41 strains had increased dramatically when coculture with salmonella or staphylococcus aureus respectively.The survival of N41 in macrophages assays shown that the survival of N41 strains had significant difference when coculture with staphylococcus aureus.This suggests that the adhesion ability of N41 have increase under the coexisting of salmonella,as well as the adhesion ability and the resistance to phagocytosis of N41 have increase under the coexisting of staphylococcus aureus. |
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