The Applied Research Of MALDI-TOF Mass Spectrometry In The Matter Of Carbapenemase Rapid Screening And Epidemiology Of Enterobacteriaceae

Objective 1.Detecting the ability to hydrolyze ertapenem of Enterobacteriaceae from blood culture directly using MALDI-TOF MS.According to this method,carbapenemase producers can be screened rapidly.2.Establishing the method to cluster carbapenem-resistant Klebsiella pneumoniae using MALDI-TOF MS,in order to proceed epidemiological analysis rapidly.3.Screening the specific peak of KPC-2 producing Klebsiella pneumoniae ST11 using MALDI-TOF MS.And establishing the rapid method to detect this type Klebsiella pneumoniae with this specific peak in order to provide evidence for clinical treatment and nosocomial infection control rapidly and accurately.Methods 1.We selected 32 carbapenemae-produced and 32 non-carbapenemase produced enterobacteriaceae strains to simulate positive blood cultures and detected the ability of hydrolyze ertapenem of these enterobacteriaceae strains in positive blood culture simulated.LogRQ values were calculated with the the peaks intensity of ertapenem and its hydrolysate and the cutoff value was acquired to diagnose the results of hydrolysis assay.Then,we collected 385 positive clinical blood cultures which contained Enterobacteriaceae strains to be detected with the MALDI-TOF MS based ertapenem hydrolysis assay.And the carbapenemase genes of the strains that the results of hydrolysis assay were positive and the carbapenem-resistant strains that the results of hydrolysis assay were negative were detected.2.Picking 52 CRKP strains contained 25 ST types for confirm the standard of specific peak selection,and screening a series of specific peaks.Then,using these peaks to cluster 24 CRKP strains which contained 5 common ST types,and comparing this method with the MLST.3.First,a total of 118 Klebsiella pneumoniae(67 KPC-2 producing Klebsiella pneumoniae ST11,and 51 other types Klebsiella pneumoniae)were selected as screening group.Clinpro Tools 3.0 and Flex analysis 3.0 software were used to screen the specific peak from the mass spectrums with the score above 2.30 which were acquired from the strains growned on Columbia blood plates.Second,other 89 Klebsiella pneumoniae(37 KPC-2 producing Klebsiella pneumoniae ST11,and 52 other types Klebsiella pneumoniae)were selected as verification group.Mass spectrums were acquired under experimental conditions strictly and the specificity and repeatability of the specific peak were detected.Finally,95 Klebsiella pneumoniae clinical isolates which growned on MacConkey agar plate and their mass spectrums with the score above 2.00 were collected to evaluate the efficiency of the specific peak in the clinical routine.Results 1.The mean logRQ value of 32 noncarbapenemase producers was-0.85 ± 0.14 while the logRQ value of 32 carbapenemase producers was 0.87± 0.55 in 64 simulated blood cultures.The cutoff value of logRQ was set at-0.45 with sensitivity of 100% and specificity of 100%.Then,in 385 clinical positive blood culture,the logRQ values of all carbapenem-susceptible Enterobacteriaceae(81.3%,313/385)were <-0.45 with the 100% coincidence rate.In carbapenem-resistant Enterobacteriaceae strains(18.7%,72/385),the logRQ values of 62 strains were >-0.45,and the logRQ values of 10 strains were <-0.45.And the results of carbapenemase genes detection of 67 CRE were positive,the other 5 CRE were negative.Comparing with the detection of carbapenemase genes,carbapenem-resistant Enterobacteriaceae strains were well distinguished by MALDI-TOF MS based ertapenem hydrolysis assay with a sensitivity of 92.5%(62/67)and specificity of 100%(5/5).2.According to the standard(1)S/N ? 4(2)S/N ratio ? 1.5(3)CV ? 40%,45 peaks were selected to cluster 29 CRKP strains(only the ST types which contain 3 or more strains)with the highest coincidence rate of 82.8% with MLST.So this standard was considered as the best.Then,using these specific peaks to cluster other 24 CRKP strains for verification and the coincidence rate was 83.3% with MLST.3.In the experimental conditions,the specific peak of KPC-2 producing Klebsiella pneumoniae ST11 was m/z 4521.The specificity and sensitivity of identifying this subtype by the specific peak was 98.1% and 97.3%,and the repeatability of the specific peak was 97.4%.In the clinical condition,the specificity and sensitivity was 95.2% and 96.9%.Conclusion 1.Our datas showed that MALDI-TOF MS is a rapid and accurate method to detect the carbapenemase activity of Enterobacteriaceae isolated from blood culture,and can provide evidence for clinicians to select useful antibiotics.2.We screened a series specific peaks using MALDI-TOF MS to cluster CRKP strains.It can provide evidence for controling the spread of hospital infection.3.In the clinical routine work,staff of microbiological laboratory can directly identify KPC-2 producing Klebsiella pneumoniae ST11 according to the specific peak with high specificity and sensitivity,in order to provide evidence for clinical treatment and nosocomial infection control rapidly and accurately.