Proteomic Analysis Of Klebsiella Pneumoniae

Objective: Using surface-enhanced laser desorption ionization time of flightmass spectrometry (SELDI-TOF-MS) to detect Klebsiella pneumoniaeprotein,adopting decision tree and cluster analysis analyse the obtained massspectrometry data, to search for simple and fast experimental methods for theKlebsiella pneumoniae strains producing extended-spectrum beta-lactamases(ESBLs) infection and bacterial homology analysis.Methods:1.ESBLs instrument screening test (screening test): analysingKlebsiella pneumoniae isolated from patients drug susceptibility by20kinds ofcommon antibiotic sensitivity testing, for those bacteria which have three ormore than three kinds drug resistance as follow: ceftazidime,cefotaxime,ceftriaxone, aztreonam, cefpodoxime,suggesting these bacteria producingESBLs suspicious strains, further ESBLs confirmatory experiments must bedone.2. ESBLs phenotypic confirmatory test (confirmatory test): antibacterialscephalosporins suspicious on the screening of ESBLs-producing strains tocefotaxime/clavulanate the acid (30μg/10μg) and cefotaxime inhibition zonediameter difference; ceftazidime/clavulanic acid (30μg/10μg) andceftazidime inhibition zone difference to determine when any of the two kindsof antibiotics Jiake clavulanic acid inhibition zone diameter not Jiake clavulanicacid inhibition zone, increasing the value of≥5mm determine positive forESBLs. The above two tests with Klebsiella pneumoniae accompany with bacteria ATCC700603, QC.3.Bacterial protein spectrum analysis:for theKlebsiella pneumoniae strains,through the destruction of bacteria, extractingprotein, and then using the SELDI-TOF-MS and Au protein chip automaticallydetect bacterial protein fingerprint.4.Data analysis:for detection of bacterialprotein fingerprint using Biomarker Wizard3.1software to analyse, screeningpneumoniae Klebsiella ESBLs positive and ESBLs negative strains relateddifferences in protein expression, to establish pneumonia Klebsiellapneumoniae ESBLs stain decision tree diagnosis model and using SPSS clusteranalysis analyse stains protein homology.Results:1.33pneumonia instrument screening of ESBLs in Klebsiellapneumoniae by disk diffusion method phenotype confirmatory test conclusiveevidence of32bacteria ESBLs positive, accounting for96.97%of the totalnumber of these ESBLs positive strains isolated from sputum, followed byurine, patients with newborns and the elderly aged over60.2.32confirmatorythe ESBLs ESBLs and17Klebsiella pneumoniae in the2000-20000range inrelative molecular weight were detected,47protein peaks producing strains anddid not produce the enzyme strains with significant differences a total of11protein peaks (P<0.01).3. The three M/Z of5993.25,8363.60,9215.26proteinsof Klebsiella pneumoniae producing ESBLs strains of the decision treediagnosis model can distinguish between Klebsiella pneumoniae enzymeproduction strains, and enzyme production strains. The test group was100%accuracy, the validation group was94.12%accuracy.4.32Klebsiella pneumoniae producing ESBLs by using cluster analysis results in differencesin the level of the case10is divided into five types (type A, type B, type C,type D, Type E), the B type A family followed, and then followed by type C andE, and D-type at least, each type is divided into2-3subtypes.Conclusion:1.Klebsiella pneumoniae bacteria ESBLs producing strains has its characteristicprotein peak, the decision tree according to the characteristic protein screeningof protein molecular diagnostic model (M/Z:5993.25,8363.60,9215.26) be ableto distinguish between Klebsiella pneumoniae ESBLs positive strains andESBLs negetive strains.2. Klebsiella pneumoniae bacteria cluster analysis and clinical patient clinicalinformation:whether the production of enzymes, and susceptibility information,are basically the same.Through the analysis of bacterial proteins can contributeto homologous Klebsiella pneumoniae clinical infection judgment. Methods ofproteomics and the homology analysis of the bacterial infection has a certainvalue, it is worth further study and use of genetic analysis or amino acidsequence analysis to verify.