| PurposeAt present,total knee arthroplasty(TKA)has become a common treatment plan for knee osteoarthritis.However,Periprosthetic Joint Infection(PJI)is the most "catastrophic" complication of artificial knee arthroplasty.It will cause huge pain to the patient,and even amputation in severe cases.Early diagnosis is very important for the treatment and prognosis of PJI.At this time,treatment is relatively easy and the effect is better.Therefore,exploring the early diagnosis method of PJI is one of the key points in the study of PJI diagnosis and treatment.Under the guidance of Quorum Sensing(QS)theory,this study took the signal molecule of Gram-negative bacteria—N-acyl homoserine lactones(AHLs)as the research target,and explored liquid chromatography-mass spectrometry tandem(LC-MS/MS)The detection protocol for AHLs in infected surrounding tissues,and the use of this detection protocol for preliminary exploration of the types of AHLs that promote the biofilm adhesion effect of PA in the initial stage of PA infection caused by PJI,in order to use signal molecules as an early diagnosis method for PJI and through interference AHLs reduce the difficulty of PJI treatment to provide experimental methods and evidence.This study is divided into three parts: 1.Use three drugs(traditional Chinese medicine extracts,antibiotics,AHLs hydrolase)to intervene in the culture of PA bacteria in vitro,and analyze the reasons why the three drugs inhibit the adhesion of biofilms;2.Creation Carry out the pretreatment method and detection conditions of LC-MS/MS detection of AHLs around the infected tissues of the animal PJI model,and conduct the sensitivity test of the pretreatment method and detection conditions;3.Observe the AHLs in the PJI animal model caused by PA The correlation between the amount and the amount of biofilm adhered to the joint prosthesis and the morphology of the biofilm,analyze the QS mechanism of PA promoting the adhesion of biofilm in the early stage of PJI.MethodsThe first part of the experiment:(1)The minimum inhibitory concentration(MIC)of gentamicin,andrographolide and homoserine lactonase on PA was determined by the micro broth dilution method.(2)Intervene PA cultured in 96-well plates with 1/2MIC concentration of gentamicin,andrographolide and homoserine lactonase respectively,select 3 wells every 2 hours for crystal violet experiment,and then use the microplate reader(BIOTCK ELX800)Detect the absorbance at 570 nm once and observe continuously for 24 hours,and draw the curve of the continuous change of absorbance after the PA bacteria liquid crystal violet experiment of three drugs under the action of sub-inhibitory concentration.(3)Put the PA bacteria liquid into the titanium alloy flakes,and incubate them with homoserine lactonase culture medium,observe the morphology of the biofilm attached to the titanium alloy flakes with an environmental scanning electron microscope,and combine them with the LB culture medium PA biofilm for comparison.The second part of the experiment:(1)Using C6-HSL,C8-HSL,C10-HSL,C12-HSL and 3-Oxo-C12-HSL five kinds of AHLs as samples,according to the target precursor ion and product ion,find the appropriate Cluster voltage(DP),collision voltage(CE),entrance voltage(EP)and collision cell exit voltage(CXP)were used to obtain an optimized solution for LC-MS/MS detection of the above five AHLs.(2)Mix animal joint tissues with standard products as samples,collect AHLs in the samples with acetonitrile,purify them with solid phase extraction column,evaporate to dryness at low temperature,and reconstitute them,then perform LC-MS/MS detection to obtain five AHLs from animals The pretreatment plan and the recovery rate after pretreatment in joint tissue.(3)Use the LC-MS/MS detection optimization conditions and pre-processing schemes obtained in this part of the experiment(1)and(2)to process,and LC-MS/MS qualitatively detects the types of AHLs in PA culture in vitro and PJI animal model tissues.The third part of the experiment: Make an animal model of PJI caused by PA.It is divided into two groups.One group is injected with AHLs standard solution in the joint cavity,and the other group is injected with sterile normal saline into the joint cavity.The crystal violet experiment is used to detect joint prostheses.The content of the biofilm on the joint prosthesis was detected by LC-MS/MS and the AHLs content in the joint tissue was detected by the environmental scanning electron microscope.results1.The minimum inhibitory concentration of gentamicin is 1ug/ml,and andrographolide and homoserine lactonase have no obvious inhibitory effect on the growth of PA.2.Gentamicin,andrographolide,homoserine lactonase can inhibit PA biofilm production.Homoserine lactonase and gentamicin have stronger ability to inhibit PA biofilm and have a relatively long duration.Andrographis paniculata The lactone is slightly weaker and has a relatively short duration.3.Electron microscope observation of the biofilm produced by homoserine lactonase intervention in PA shows that only the bacteria adhere loosely,fail to form a complete biofilm,and there are only a few sparse hyphal connections between bacteria.4.The mass spectrometric parameters of C6-HSL,C8-HSL,C10-HSL,C12-HSL and3-Oxo-C12-HSL detected by LC-MS/MS are as follows: The optimal declustering voltages are 60 V,60V,70 V,80V,80V;collision voltages are: 14 V,14V,16 V,15V,20V;quantitative ion pairs are(m/z): 200.1/101.9,228.2/101.7,256.1/101.9,284.0/101.9,298.3/101.9;The recovery rates are: 97.6%,90.2%,95.8%,124.1%,87.6%;5.LC-MS/MS detected PA in vitro culture and PJI animal model tissues and found that in samples with PA,the peak of the signal molecule 3-Oxo-C12-HSL can be detected,while C6-HSL,C8-The peaks of HSL,C10-HSL,and C12-HSL do not correspond to the existence of PA one to one;6.Using LC-MS/MS to test the animal model samples of PA-induced PJI,a clear waveform of AHLs can be detected on the fourth day of infection,at a concentration of2.129115ng/ml.7.In the quantitative observation of the biofilm of the PJI prosthesis caused by the attachment of PA,the amount of biofilm on the joint prosthesis added with AHLs standard was significantly higher than that of the joint injected with saline on the 3rd,4th,and 5th day of the initial infection Prosthesis;8.The biofilm on the PJI prosthesis caused by PA was observed under environmental scanning electron microscopy,and it was found that the biofilm with filamentous,flake and three-dimensional structure appeared more obviously and earlier on the samples added with AHLs standard.ConclusionLC-MS/MS has high sensitivity for detecting AHLs on PJI joint tissues,the signal-tonoise ratio is greater than 10,and the detection recovery rate is greater than 80%,which proves that this experiment explores the pre-treatment plan for PJI joint tissue and LCMS/MS reliable detection scheme of MS optimization scheme;The ability of in vitro culture of PA to produce biofilm adhesion can be inhibited by the hydrolase of QS signaling molecule,and its inhibitory effect is more obvious,and the inhibition time is longer.By inhibiting the signaling molecule,the method to reduce the difficulty of treatment of infection around the joint prosthesis may be A new idea for the future treatment of infections around joint prostheses;In animal experiments,the PJI caused by PA can detect the presence of AHLs molecules from the fourth day,and in the PJI animal group injected with AHLs standard,the amount of biofilm on the joint prosthesis is more than the PJI animal group injected with saline Moreover,the connection of the hyphae and the structure of the biofilm of the former are denser,indicating that the existence of AHLs is conducive to the formation or attachment of more biofilms on the PJI prosthesis by PA,and the development of biofilms is more mature. |
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