| Enterococcus faecalis is a conditional pathogen,which can cause infection and death in humans and animals.In recent years,E.faecalis infection has become increasingly serious and mixed infections with other pathogenic microorganisms have also gradually increased,but its pathogenic mechanism is still not clear.Especially,the interaction mechanism with other pathogens is still in the initial study.S.aureus,E.coli O157:H7 and Salmonella are also common human and animal pathogenic bacteria.While mixed infections of three bacteria with E.faecalis have also been reported,the exact mechanisms involved are largely unknown.In particular,how it works in a complex environment in the body requires further research.In this study,co-infection with S.aureus,E.coli O157:H7 and Salmonella was performed using a mouse model of peritonitis in vivo.In order to lay the foundation for the study of the mechanism of E.faecalis mixed infection in vivo,we examined the changes in growth gurves of E.faecalis which exist in the peritoneal fluid,spleen,kidney,and liver.Furthermore,changes in virulence genes associated with E.faecalis and analysis of transcriptome changes by transcriptome high-throughput sequencing was observed.Growth Characteristics By comparing the growth curves of intra-abdominal infection and co-infection in E.faecalis N41 mice,we studied the growth characteristics of E.faecalis in the peritoneal fluid,spleen,kidney,and liver,and analyzed the effects of S.aureus,E.coli O157:H7,and Salmonella on the growth characteristics of E.faecalis N41.We compared the growth curve of N41 after co-infection with the growth curve of N41 when infected alone.It was found that the three bacteria after co-infected respectively in the four parts of the mouse's abdominal cavity had a certain role in the growth of N41 strain,in which E.coli O157: H7 and Salmonella could promote the growth of N41 in the peritoneal fluid,spleen,kidney and liver,while S.aureus could only promote the growth of N41 in peritoneal fluid and kidney.Specifically,in peritoneal fluid,all three bacteria could promote the growth of E.faecalis and increase its bacterial concentration by about 2-3 times.Moreover,S.aureus and E.coli O157:H7 also enabled the entire growth curve of N41,and the logarithmic period delayed by 2 hours;In the liver,both E.coli O157:H7 andSalmonella were able to promote the growth of N41 so that the bacterial concentration increased by 57-fold and 44-fold,respectively.But,the concentration of bacteria which N41 co-infected with S.aureus was similar with that of infecton alone.In the spleen,both E.coli O157:H7 and Salmonella promoted the growth of N41,increasing the concentration of the bacteria by about 3 to 5 times,and only E.coli O157:H7delayed the logarithmic phase of the growth curve by 2 hours.S.aureuss lightly inhibited E.faecalis;In the kidney,the bacterial concentration of N41 after co-infection with three bacteria,respectively,was similar to that of infection alone.However,in terms of time,S.aureus could delay the logarithmic phase of N41 by 8hours.E.coli O157:H7 and Salmonella delayed the logarithmic phase of N41 by 4hours.Changes in Virulence Gene mRNA Levels Based on the growth curve of E.faecalis in peritoneal fluid,the total RNA during the mid-log phase(ML)of E.faecalis N41 infection and in pre-log phase(EL),mid-log phase(ML),and late log phase(LL)co-infected with S.aureus and E.coli O157:H7 were extracted.According to the gene sequence of E.faecalis V583(searched in Gen Bank),26 virulence gene primers were designed,and we utilized SYBR Green I real-time quantitative PCR to measure the transcription of 26 virulence genes.We compared the transcriptional levels of the 26 virulence genes in N41 infection alone with that of co-infection.The result showed: Compared with infection alone,S.aureus and E.coli O157:H7increased the transcription levels of eight virulence genes of E.faecalis such as ebp B,rnj B,Cyl L-L,Cyl L-S,gel E,spr E,efa A,and fsr B during the ML period.However,the transcriptional amounts of the eight virulence genes ebp A,ebp C,Cyl I,Cyl R1,Cyl R,AS,Sly A and fsr A did not change significantly.In addition,S.aureus significantly increased the transcription of 8 virulence genes,including fsr C,Cyl M,Cyl B,Cyl A,ace,rpo N,psr and atn.But E.coli O157:H7 only increased the transcriptional quantity of ebp R.Gel E,Spr E and Efa A have a certain role in the formation of enterococci biofilm,and Gel E can enhance the ability of enterococcus faecalis infection.Furthermore,Efa A can also promote the occurrence of endocarditis disease.In this study,both bacteria could increase the transcription of gel E,spr E and efa A,which may also promote the formation of N41 biofilm and enhance the infection ability of N41.At the same time,S.aureus can increase the transcription level of ace,and ace-encoded collagen adhesin can promote colonization and adhesion of E.faecalis.This demonstated that both S.aureus and E.coli O175:H7 in animals couldpromote the transcription of many virulence genes in E.faecalis N41,and co-infection with these two bacteria might increase the pathogenicity of E.faecalis.The transcript levels of virulence genes in the three stages of co-infection EL,ML,and LL were analyzed.The 19 virulence genes of E.faecalis such as ebp A,ebp B,ebp C,and rnj B when mixed with S.aureus were highest in the ML stage,and the transcriptions of ebp A and esp were decremented in the three periods EL,ML,and LL.However,the transcription amounts of ebp R,Cyl I,fsr A,sly A and AS were not significantly different in the three periods.Genes such as gel E,spr E,rnj B,ace,and efa A can promote the formation of E.faecalis biofilms and increase their ability to survive and infect.These demonstrated that the transcript of virulence genes when E.faecalis N41 co-infected S.aureus in animals was greatest in ML,but the pathogenicity was probably the strongest at this time.The 16 virulence genes such as ebp A,ebp B,ebp C,rnj B,esp and atn when E.faecalis co-infected with E.coli O175:H7 had the lowest transcription level in ML,but they showed a rise again the LL period.However,the transcription amounts of ebp R,Cyl L-L,and Cyl L-S increased sequentially during the three periods,and the amount of ace transcription gradually decreased in the three periods.In addition,the virulence gene changes were not significant.The decrease in ace may be due to the fact that the ace-encoded collagen adhesin plays a major role in colonization and adhesion when N41 is co-infected with E.coli O175:H7,and therefore has a stronger effect in the EL period.These demonstrated that the two bacteria had different effects on N41 when they were co-infected in animals,especially in the time of action.And the transcriptional level of most of the N41 virulence genes was suppressed to a certain degree in the mid-log phase when N41 was co-infected with E.coli O175:H7.Changes of Transcriptome Level In order to explore pathogenic genes other than virulence genes of E.faecalis,we performed three treatments for E.faecalis strain N41,including in vitro culture alone,infection in mice in vivo and co-infection with S.aureus in mice.Then,we selected E.faecalis under three conditions in mid-logarithmic phase and compared transcriptome levels of E.faecalis by transcriptome high-throughput sequencing.The result showed: when E.faecalis N41 infected alone in vivo compared to the E.faecalis N41 cultured in vitro,3083 genes were detected at the transcriptome level,of which 1054 were differential genes.And compared to N41 cultured in vitro when infected in vivo alone,75% of differentially expressed genes were up-regulated,including genes for metabolic processes(302/402:a total of 402 differential genes,of which 302 were up-regulated),membranes(87/104),cell adhesion(4/5),bacterial defense response(4/4),pathogenesis(2/4),etc.These upregulated genes such as the glycosyl transferase family,alkaline phosphatase3,and methylated chemotactic proteins may promote bacterial infection by promoting bacterial colonization or disrupting normal function in the infected host.This showed that E.faecalis live and infect in the body and made corresponding adjustments at the transcriptome level,indicating that the complex environment in the body would have a huge impact on E.faecalis.At the same time,we found that 3,083 genes were detected at the transcriptome level in E.faecalis N41 co-infected with S.aureus in vivo and E.faecalis N41 infected in vivo,among which there were 582 differentially expressed genes.Co-infection compared to N41 when infected alone,only 25% of differentially expressed genes were shown to be upregulated,including metabolic processes(47/230),membranes(17/63),cell killing(1/1),and detoxification(1/1)other genes.These up-regulated genes such as the rod determinant protein Mre C,bacterial sterol glucosyltransferase-like protein,etc,may promote the survival and infection of N41 by acting on the bacteria or the infected host.However,many down-regulated genes such as glycosyl hydrolase and silencing information regulator Sir2 may be inhibited by S.aureus,or they may be due to competitive survival of the two bacteria in a difficult in vivo environment,and the transcription of more genes was inhibited. |
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