The Roles Of C-di-GMP In The Formation Of Pseudomonas Aeruginosa Biofilm In New Zealand Rabbit Septic Arthritis

Septic arthritis（SA）is a type of joint space infection that destroys articular cartilage,causes synovitis,and affects joint function.Because the bacteria are not easily killed,SA can become chronic and linger.The recurrence rate of chronic SA is extremely high and treatment is difficult.Pseudomonas aeruginosa（PA）is one of the main bacteria responsible for SA nosocomial infection,accounting for 6%.PA can cause SA through hematogenous infection and infection of joint cavity by surgical operation.PA has two ways of life:plankton and Biofilm（BF）.PA-BF is a microbial aggregate formed by the adhesion of PA to the surface of the medium through the extracellular polymeric matrix secreted by PA and wrapping itself in it.More than 80%of PA infections are known to be associated with PA-BF.Once formed,PA-BF will protect bacteria and reduce the damage of immune cells and antibiotics,leading to clinically refractory infections.Some cases have reported that SA in wrist joint is related to PA-BF.In order to further study the relationship between SA and PA-BF,animal models need to be established for scientific research.Although Jin T et al.established a mouse model of SA by injecting PA,PA-BF was not involved in this study.At present,no studies on PA-BF have been found in animal models of PA induced SA,and the mechanism of PA-BF infection in SA is not clear.Cyclic diguanylate（C-di-GMP）is the second messenger of intracellular signal transducing in Pseudomonas aeruginosa.Current studies indicate that C-di-GMP has a regulatory effect on THE formation of PA-BF,but whether C-di-GMP has a regulatory effect onthe formation of PA-BF in animal models of SA has not been reported.In thisstudy,we first established the SA model of New Zealand rabbits by injectingPA into the knee cavity of healthy New Zealand rabbits.Then,staining with crystal violet,Peptide nucleic acid-Fluorescence in situ hybridization（PNA-FISH）and Scanning electron microscope（SEM）were used to verify the formation of PA-BF in the articular cavity and synovium of SA model in New Zealand rabbits.Finally,New Zealand rabbit SA models were established by intra-joint injection of PAO1/P_lac-yhj H strain with low intracellular C-di-GMP,PAO1 strain with normal intracellular C-di-GMP,and PAO1Δwsp F strain with high intracellular C-di-GMP.The PA-BF formation ability of the above three strains in SA model was compared by PNA-FISH test and C-di-GMP test,and the effect of C-di-GMP on PA-BF formation in SA model of New Zealand rabbits was investigated.Part Ⅰ:Establishment of New Zealand rabbit SA modelObjective The New Zealand rabbit SA model was constructed.The existence of PA-BF was verified by traditional culture counting method,PNA-FISH,SEM observation and other methods.Methods 1.Experimental animals:Male New Zealand rabbits,weighing2.5±0.3kg,were divided into experimental group,control group and blank group.He was fed in the animal center of Guangxi Medical University under clean condition for 1 week.2.Preparation of suspended bacterial solution in vitro:the experimental strain PAO1 was resuspended,then the bacteria were added,the concentration of the solution was adjusted by OD600 value,and the solution was diluted 10-fold to the required concentration for reserve.3.Operation:The New Zealand rabbits in each group were anesthetized,skin preparation,positioning,disinfection,towel laying,puncture with a syringe in the space of the right knee joint,and the bacterial solution was injected,and the control group was injected with LB and talc solution.The SA model was raised for 8 days in a clean grade environment.4.Sample collection:The SA model of New Zealand rabbits was established by injecting PA bacteria through the joint cavity.On the eighth day after the operation,the model knee was dissected,and the synovial tissues of the joint were collected and fixed in 10%formaldehyde solution and 3%glutaraldehyde solution,respectively,for HE staining and SEM examination of pathological sections.The Whole knee joint was cut and fixed in 10%formaldehyde solution for HE staining of pathological sections,and the joint cavity floc was collected and fixed in 3%glutaraldehyde solution for SEM examination.5.Verification of the formation of SA model:The basic situation of rabbits was observed.If the skin temperature of the contralateral knee joint was increased,the joint circumference was significantly increased（the joint circumference was greater than 1cm）,the experimental animals were limited in movement（limping）,and their diet was reduced.Gram-negative bacilli were found in the joint fluid by Gram-staining,which proved the formation of SA model.6.Proof of the formation of PA-BF in joint cavity:Wash off the floc and homogenate,and observe PA-BF by PNA-FISH;The synovial tissues and flocs of the joints fixed in glutaraldehyde solution were prepared by SEM,and THE BF was observed by SEM.The floc was washed away and immersed in sterile normal saline.After eddy oscillation and ultrasonic oscillation,the leaching solution was taken for colony counting.Results 1.On the 8th day after the operation,the New Zealand rabbits were sacrificed and the knee joint was dissected.It could be seen that the New Zealand rabbits in the experimental group had uneven floccule and turbidity articular cavity effusion formed in the knee cavity,the synovial membrane was significantly thickened,and there were floccule attached on the synovial surface.HE staining of synovial pathological sections showed synovial thickening and inflammatory cell infiltration.Gram-negative bacilli can be seen in the articular effusion/pus floccus.2.PA-BF can be observed in the joint cavity floccule by PNA-FISH.3.The formation of PA-BF on synovial surface and floccule was observed by SEM.4.The viable bacteria count and crystal violet staining of floccule in the joint cavity proved that PA-BF was formed in the knee cavity of SA model.Conclusion The SA model of New Zealand rabbits can be successfully established by injecting PA suspension into the joint cavity.PA-BF was found in synovium and floccule of SA model of New Zealand rabbits infected with PA.Part Ⅱ Effect of C-di-GMP on PA-BF formation in the joint cavity of SA model of PA infected New Zealand rabbitsObjective The effect of C-di-GMP on PA-BF formation in vitro was analyzed.To investigate the regulatory effect of C-di-GMP on PA-BF formation in SA model of New Zealand rabbits.Methods1.Modeling:The SA models of New Zealand rabbits were established using PAO1/P_lac-yhj H、PAO1、PAO1Δwsp F strains with low to high intracellular concentration of C-di-GMP.2.Sample collection:Under aseptic operation,the synovium、total knee and floc in the knee cavity of SA model samples in each group were collected and fixed in 10%formaldehyde solution and 3%glutaraldehyde solution,respectively,for HE staining and SEM examination of pathological sections.Flocs were collected for PNA-FINA examination to compare the differences in PA-BF.3.Comparison of PA-BF formation:floc were counted by colony count,determined by C-DI-GMP and detected by PNA-FISH.4.Statistical analysis:SPSS22.0 software package was used to analyze the experimental data,and univariate ANOVA was used to analyze the mean between groups.Results 1.The bacterial populations of the three strains with different levels of intracellular C-di-GMP were similar at the 24-hour stage.2.PAO1/P_lac-yhj H<PAO1<PAO1Δwsp F of intracellular C-di-GMP.3.In the animal experiment,the amount of PA-BF formation in flocs of three different strains from low to high was PAO1/P_lac-yhj H<PAO1<PAO1Δwsp F.Conclusion The formation of PA-BF in New Zealand rabbit SA model was regulated by intracellular C-di-GMP level of bacteria.