Study On The Role Of Escherichia Coli Movement Regulation In Biomaterial Implantation Infection

[Background and Objective]Biomaterial,is also known as biomedical material,is a kind of special materials that can diagnose,treat,replace,repair,induce regeneration or improve the function of cells,tissues and organs of the body.With the continuous development of science and technology,the implantation of biomaterials has become an indispensable part of modern medical diagnosis and treatment;however,the implantation of biomaterials is a double-edged sword,which not only brings significant benefits to patients,but also increases the risk of infection.Biomaterial implantation infection(BCI)has become a chronic and refractory nosocomial infection,accounting for 50%of nosocomial infections.Biofilm is defined as a highly complex multicellular complex formed by bacteria adhering to the surface of living or inanimate objects and surrounded by extracellular polymers(EPS)secreted by themselves,EPS is the basic structural feature of all biofilms,including proteins,polysaccharides and nucleic acids.The implantation of biomaterials increases the ways for bacteria to invade the body and reduces the minimum number of bacteria to induce infection.Medical biomaterials as foreign bodies can not only affect the immune mechanism of the body,but also provide adhesion sites for free bacteria and promote the formation of bacterial biofilm(BF).Once the biofilm is formed on the surface of biomaterials,the bacterias in the biofilm resist antibiotics and the clearance of the body's immune system,resulting in recurrent and prolonged infection,which is one of the most common and serious complications in the process of clinical diagnosis and treatment.Escherichia coli(E.coli)is a gram-negative,rod-shaped facultative anaerobic bacteria,located in the gastrointestinal tract of warm-blooded animals,is a symbiotic bacteria,generally will not cause infection;in addition,Escherichia coli can also survive in soil and water.It can also cause infection under special circumstances.For example:first,when the skin and mucosa of the body are destroyed,it can enter the body with the implantation of biomaterials and adhere to the surface of the material to form a biofilm;second,in patients with controlled hypotension,hemorrhagic shock,long-term fasting and parenteral nutrition during surgery,intestinal bacterial translocation can cause bacteremia,and bacteria in the blood provide bacteria for the formation of biofilm on the surface of biomaterials.Studies have shown that Escherichia coli accounting for more than half of intestinal bacterial translocation bacteria,and studies have also shown that the detection rate of Escherichia coli in postoperative biomaterial infections such as heart valve replacement is about 3-10%.Cyclicdiguanylatemonophsphate(c-di-GMP)is the second messenger in bacteria.It has been found that cyclic guanosine monophosphate can regulate a variety of functions,including flagellum movement,adhesion,cell cycle initiation and regulation,virulence factor synthesis and biofilm formation.The most important effect of cyclic guanosine monophosphate on many bacteria is to determine the "lifestyle" of bacteria,especially to control the transition between the state of planktonic movement and the state of biofilm.Generally speaking,the increase of the concentration of cyclic guanosine monophosphate in bacterial cells can inhibit the motility of flagella and increase the synthesis of extracellular polymers(EPS),which leads to the formation of biofilm;low levels of cyclic guanosine monophosphate increase the motility of bacteria and disperse the biofilm.It is speculated that the regulation of flagellum movement by cyclic guanosine monophosphate mediates the formation of bacterial biofilm.The level of cyclic guanosine monophosphate in bacteria depends on diguanylate cyclases(DGCs)and phosphodiesterase(PDEs)to modulate and maintain balance.Cyclic guanosine monophosphate inhibits flagellar movement by regulating flagellar immobilization protein YcgR,while YcgR-related dicyclovir cyclase DGCs mainly includes DgcE and DgcQ.It was found that the motility of Escherichia coli strains of pdeH decreased significantly,but the strains with simultaneous deletion of dgcE,dgcQ and pdeH genes could restore the motor ability similar to that of wild-type strains.It can be seen that dgcE and dgcQ are closely related to the movement regulation of flagella.In view of the fact that the cyclic guanosine acid signal is highly conserved in bacteria,and cyclic guanosine monophosphate promotes the formation of biofilm,the enzymes related to its metabolism are attractive targets for interfering with the formation of bacterial biofilm,especially the dicyclovir cyclase(DGCs),which produces c-di-GMP.To clarify the role of cyclic guanosine acid signal in bacterial biofilm,that is,regulating the level of nucleotides or interfering with the signal pathway may inhibit the formation of biofilm or promote the dispersion of biofilm.In this study,Escherichia coli ATCC25922 strain dgcE and dgcQ negative mutants were constructed by CRISPR/Cas9 technique.Based on the establishment of biomaterial surface biofilm model,the effects of Escherichia coli dgcE and dgcQ on bacterial biofilm formation on biomaterial surface were studied by laser confocal and scanning electron microscopy.Through the establishment of rat bacteremia model,the effects of clearance of different Escherichia coli in rats and the formation of bacterial biofilm on the surface of biomaterials were discussed,and the effective molecular targets for the prevention and treatment of Escherichia coli biofilm formation on the surface of biomaterials were revealed.to provide experimental basis for the prevention and treatment of E.coli.[Methods]This study is divided into four parts:1.Using Crispr/Cas9 technology to construct E.coli dgcE,dgcQ knock-out mutant strains:E.coli ATCC25922 as the research object,using dgcE and dgcQ primer sequences to complete the corresponding gene PCR amplification detection,further completion of dgcE,dgcQ gene sequence Sanger sequencing and comparison with NCBI sequence;reference to gRNA design to complete the design of gRNA,while reference to CRISPR-Vector vector method to construct the vector of the target sequence.The competent state of Enterobacter ATCC25922(E.coli)was prepared,and the competent state of Escherichia coli ATCC25922(E.coli)was prepared by reference to the preparation of Protocol.Crispr-Vector was transformed into Escherichia coli ATCC25922(E.coli),spread to the screening medium,cultured for 3-5 days,and the gene knock-out strains were screened by kan&spe double resistance plate.Strain PCR identification,PCR sequence sanger sequencing to verify the results of DgcQ gene and DgcE gene knockout,respectively,DgcQ gene and DgcE gene knockout E.coli ATCC25922(E.coli)preservation.2.To determine the role of Escherichia coli dgcE and dgcQ in the changes of bacterial biological phenotype and the role of Escherichia coli dgcE and dgcQ in the formation of bacterial biofilm on the surface of biomaterials:to determine the growth curve of Escherichia coli ATCC25922,ATCC25922 ?dgcE and ATCC25922 ?dgcQ,and their movement ability on semi-solid medium.To explore the role of dgcE and dgcQ in the changes of bacterial bio'logical phenotype.The in vitro model of bacterial biofilm on the surface of PVC material was established by using three strains of Escherichia coli(ATCC25922,ATCC25922?dgcE and ATCC25922?dgcQ)as experimental strains.The formation ability of Escherichia coli biofilm was detected by semi-quantitative method,the formation process,the number of bacterial community and the thickness of bacterial biofilm on the surface of PVC material were dynamically observed by laser confocal(CLSM),and the surface structure of Escherichia coli biofilm on PVC material was observed by scanning electron microscope((SEM)).To explore the role of dgcE and dgcQ in the formation of bacterial biofilm on the surface of biomaterials.3.Clearance of Escherichia coli and formation of bacterial biofilm on biomaterial surface in rats:SPF healthy male SD rats were randomly divided into four groups:blank control group,ATCC25922 group,ATCC25922 ?dgcE group and ATCC25922 ?dgcQ group.The PVC material was put into the peritoneal cavity through the median abdominal incision of rats,and the bacteremia model was established by injecting bacterial solution into the tail vein.The rats were killed 24 hours later,and under strict aseptic conditions,the portal vein blood was detected for white blood cell count,PCT detection and the number of bacteria in blood;the colonies on the culture plate were identified by Gram staining and colony PCR at the same time;a piece of biomaterials in vivo was used for bacterial culture,and then colony PCR identification;SEM to observe the surface biofilm structure of in vivo materials;HE staining to observe lung tissue injury.[Results]1.Construction of Escherichia coli dgcE and dgcQ knock-out mutants:the results of colony PCR and Sanger sequencing showed that the internal sequences of dgcE and dgcQ genes had been deleted,and there were no corresponding kanamycin and spectinomycin resistance genes,and the genes were knocked out successfully.2.The role of dgcE and dgcQ in the phenotypic transformation of Colorectal bacilli and the role of Escherichia coli dgcE and dgcQ in the biofilm formation of bacteria indicated by biomaterials:there was no significant difference in the growth of ATCC25922,ATCC25922 dgcE and ATCC25922 dgcQ Escherichia coli in TSB medium;compared with ATCC25922,the movement ability of ATCC25922 dgcE and ATCC25922 dgcQ were stronger,and dgcQ was stronger than that of dgcE mutant(P<005).The biofilm forming ability of gene knoce-out strain was higher than that of standard strain at 4-8 h,and it was statistically significant at 8 h(P<0.05).The peak biofilm formation time of Escherichia coli ATCC25922 was 12 h,but the time for gene knoce-out strain to form biofilm peak was 8 h,which was earlier than that of standard strain.the growth kinetics of Escherichia coli ATCC25922 standard strain was significantly higher than that of gene knoce-out strain at 8 h,12h and 24h,and there was significant difference compared with dgcQ at 8 h,12 h and 24 h(P<0.5).The growth kinetics of E.coli ATCC25922 standard strain was significantly higher than that of mutant strain at 8 h,12 h and 24 h(P<0.5).The number of bacteria per unit area of Escherichia coli ATCC25922 was significantly higher than that of dgcE and dgcQ gene knoce-out strain,and there was no significant difference among mutants.Escherichia coli ATCC25922 standard strain biofilm thickness was.thicker than that of mutants,and at 8 h,ATCC25922-dgcE was thicker than ATCC25922-dgcQ.3.The clearance of Escherichia coli in rats and the formation of bacterial biofilm on the biomaterial surface:the white blood cell count in the bacteremia model group was significantly lower than that in the blank control group(P<0.05),but there was no significant difference among the three bacteremia model groups(P>0.05),For the plasma PCT level,the bacteria in the bacteremia model group was significantly higher than that in the control group,and the pairwise comparison among the three bacteremia model groups was statistically significant(P<0.05).At 24 hours of infection,the number of living bacteria in the ATCC25922 group was the highest,reaching 3×103CFU/ml.There was significant difference between the bacteremia animal model group and the control group,and there was significant difference between the ATCC25922-dgcE group,the ATCC25922-dgcQ group and the ATCC25922-WT group,but there was no significant difference between the two groups.The number of bacteria on the surface of biomaterials in ATCC25922-WT,ATCC25922-dgcE and ATCC25922-dgcQ groups was significantly higher than that in control group,at the same time,the number of bacteria on the surface of biomaterials in ATCC25922 group was more than that in ATCC25922-dgcE and ATCC25922-dgcQ groups,and there was significant difference compared with ATCC25922-dgcQ group.The injury of lung tissue in ATCC25922-WT group was more severe than that in ATCC25922-dgcE group and ATCC25922-dgcQ group.[Conclusions]1.Using CRISPR/Cas9 technology,dgcE and dgcQ gene knockout strains of Escherichia coli were successfully constructed,which laid a foundation for subsequent gene function studies.dgcE and dgcQ gene knockout can enhance the motor ability of Escherichia coli,but does not affect the growth of bacteria.2.dgcE and dgcQ have dual functions in biofilm formation,namely,low levels of c-di-GMP are important for effective early adhesion,while high levels of c-di-GMP are required in mature biofilms to promote matrix production,which is consistent with adhesion and aggregation in biofilm formation.dgcE and dgcQ gene knockout affects the production of extracellular polymeric substances,leading to the instability of biofilm structure.Therefore,it may be a potential therapeutic target for biomaterial implantation infection.3.The deletion of dgcE and dgcQ genes in Escherichia coli resulted in the decrease of c-di-GMP level,which resulted in the increase of bacterial clearance efficiency of the immune-system in rats.In addition,during bacteremia in rats,the formation of biofilm on the surface of biomaterials in vivo was reduced,and the damage to lung tissue of rats was alleviated.