Pathogenicity Of Multi-bacterial During Co-infection And Its Effect On Transcriptional Level Of Enterococcus Faecalis

At present,the more multi-microbial co-infection is reported in the clinic,in which multi-bacterial co-infection accounts for 25%of bacterial infection.Multi-bacterial co-infection aggravates the incidence and severity of the disease,especially after the formation of biofilm,generating a significant impact on the occurrence,development,and clinical treatment of some diseases,but also increases the difficulty of clinical treatment.Enterococcus faecalis(E.faecalis)is an important conditional pathogen,belonging to foodborne pathogens together with Salmonella enteritidis and Escherichia coli O157:H7.They may co-exist in intestinal and food habitats,and adhere to and invade intestinal epithelial cells,causing enteritis,toxicity,and other diseases.Therefore,understanding the interactions between bacteria and the results of these interactions is vital for understanding the occurrence,development,and clinical treatment of diseases.To understand the interaction and results of E.faecalis,an increasingly opportunistic pathogen infected in hospitals and animals,with S.enteritidis and E.coli O157:H7 as two pathogens,these three bacteria were used as test strains.Their pathogenicity and antibiotic resistance were analyzed by the interaction of three kinds of bacteria in the state of plankton(liquid culture)and biofilm.The reasons for the increase of E.faecalis infection were analyzed through the effects of S.enteritidis and E.coli O157:H7 on the expression of virulence genes and glucose metabolism genes of E.faecalis in the state of biofilm,attempting to lay a foundation for the mechanism and clinical treatment of multi-bacterial infection.The effect of co-infection on pathogenicity in the state of plankton.Three kinds of bacteria showed the same performance in the process of adhesion and invasion of porcine intestinal epithelial cells IPEC-J2.Compared with the corresponding single bacterial infection,E.faecalis and S.enteritidis decreased the amount of adhesion and invasion.E.faecalis decreased by 0.40 log and 0.56 log,Salmonella decreased by 1.09 log and 0.12 log,while E.coli O157:H7increased by 0.47 log and 1.53 log,respectively.However,in co-infection inoculated with the same number of three kinds of bacteria,although the amount of adhesion and invasion of E.faecalis was lower than that of single inoculation,the amount of E.faecalis was still higher than that of S.enteritidis and E.coli O157:H7,which was consistent with the adhesion results observed by scanning electron microscope.In the abdominal cavity assay of mice co-infected with three kinds of bacteria,by evaluating the survival curve and health score of mice,it was found that the death time of co-infection group was shorter and the health score was higher than that of each single inoculation group.Moreover,in the determination of MIC in co-culture and single culture of three kinds of bacteria,except for the increased resistance to tetracycline,the other 11 kinds of antimicrobials were basically unchanged.As a whole,co-infection in the state of planktonic enhanced the pathogenicity.The effects of co-culture of three kinds of bacteria on the formation of multi-species biofilm.The biofilm biomass formed by three kinds of bacteria co-cultured at 1:1:1 was significantly higher than that of single culture,and the biofilm biomass at 24 h was higher than that at 48 h.Among them,the biofilm biomass of 24 h co-culture was 1.7 times higher than that of E.faecalis,8.4 times higher than that of E.coli,and 48.2 times higher than that of Salmonella.To determine the number of bacteria in the biofilm of 24 h and 48 h co-culture,the results of qPCR detection and CFU count showed that the number of E.faecalis was the most in the biofilm at 24 h,followed by E.coli and Salmonella.Compared with the biofilm cultured alone,E.faecalis had no significant change,while Salmonella and E.coli increased significantly,which was also reflected in the intuitive results of the scanning electron microscope.At 48 h,the results of qPCR detection and CFU count showed that the microflora in the biofilm was the same as that at 24 h.However,the copy number of E.faecalis significantly decreased,but the copy number of E.coli and Salmonella increased by qPCR.In contrast,the number of E.faecalis was significantly increased,while the number of E.coli and Salmonella was significantly decreased by CFU count.To detect the possibility that multi-species biofilm can be removed,the two main components of the biofilm,eDNA and protein,were first treated with DNase I and protease K,respectively.Compared with E.faecalis biofilm,the concentration of DNase I treatment,which resulted in a significant decline in multi-species biofilms,doubled.In addition,the biomass of multi-species biofilm treated with protease K was still relatively high.The results of treatment with vancomycin showed that vancomycin increased biofilm biomass when E.faecalis were cultured alone,instead of decreasing,and the difference was significant,while multi-species biofilm was not affected by vancomycin treatment.However,when the effect of vancomycin was evaluated by CFU counting,the number of bacteria in the biofilm of E.faecalis significantly decreased with the increase of vancomycin concentration from 1024μg/mL to 4096 μg/ml.Although the number of E.faecalis in multi-species biofilm decreased,there was no significant difference.In the treatment of biofilm with vancomycin and DNase I,the results showed that the biomass of E.faecalis biofilm and multi-species biofilm significantly decreased,but considering the higher biomass of multi-species biofilm,this treatment had little effect on multi-species biofilm.To determine the metabolic activity of bacteria in multi-species biofilm,XTT and biofilm reaction method was used.The results showed that the metabolic activity of bacteria in multi-species biofilm was significantly different from that of bacteria in single-species biofilm,even 1.24 times more than E.faecalis biofilms with relatively strong metabolic activity.The effects of E.coli and Salmonella on E.faecalis transcriptome in the state of biofilm.The transcriptome analysis of E.faecalis which formed 24 h multi-species biofilm(BC)and E.faecalis biofilm(BN)was carried out by RNA-seq technique.The results showed that there were 1487 differentially expressed genes(DEG)between BC and BN,including 780 up-regulated genes and 707 down-regulated genes,of which 29.6%up-regulated genes and 28.3%down-regulated genes changed more than 4 times.Among the 29 virulence genes identified in these DEGs,the up-regulated 15 virulence genes varied 2.2-7.9 times,while the down-regulated 14 virulence genes also changed 2.0-10.5 times.By GO functional enrichment analysis,it was found that these DEGs were mainly distributed in organic cyclic compound binding(317),heterocyclic compound binding(317),cell metabolism process(283),nitrogen compound metabolism process(275),and macromolecular metabolism process(227).KEGG pathway enrichment analysis of DEGs showed that there were significant differences in biosynthetic of secondary metabolites(115),microbial metabolism in diverse environments(64),ribosome(43),biosynthesis of amino acids(38),glycolysis/gluconeogenesis pathway(26),pentose phosphate pathway(16)and some other pathways.In conclusion,the co-infection of E.faecalis,S.enteritis,and E.coli O157:H7 enhanced pathogenicity,environmental resistance,and clinical treatment difficulty.In the co-infection of three kinds of bacteria,especially in chronic infection,E.faecalis played a leading role in multi-species biofilm,which not only protected E.coli and Salmonella from external adverse factors,but also increased their number,which may make the infection chronic and easy to relapse.At the same time,E.coli and Salmonella may also increase the virulence of E.faecalis,which needs further research.