Rapid Detection Of Drug Resistance Of Klebsiella Pneumoniae Based On MALDI-TOF MS

Part Ⅰ Rapid detection of ESBLs-producing Klebsiella pneumoniae by MALDI-TOF MSObjective: To study the detection of ESBLs-producing Klebsiella pneumoniae through antibiotic hydrolysis assay using MALDI-TOF MS and to evaluate the sensitivity and specificity of this method.Methods: Fifty-four ESBLs-producing strains and 24 cephalosporins-sensitive strains of Klebsiella pneumoniae were randomly collected.Third-generation cephalosporin(cefotaxime and ceftriaxone)and its enzyme inhibitor clavulanic acid were used as indicators for antibiotic hydrolysis assay.The experimental bacteria were incubated with0.5mg/ml cephalosporin and 0.5mg/ml cephalosporin /0.5mg/ml clavulanic acid at 35℃,respectively.After incubation for 1 h,2 h and 3 h,MALDI-TOF MS was used to detect and analyze the alteration of the mass peaks of antibiotics in the solution.The PCR was perfomed to screen the resistance mechanism and the results were confirmed by DNA sequencing.And the susceptibility tests were performed by automatic micro-dilution method(Vitek-2 Compact system)and disc diffusion method.Results: ESBLs-producing Klebsiella pneumoniae showed high resistance to a variety of antibiotics,and ESBLs genes of bla TEM,bla SHV,bla CTX-M were detected.When the incubation time was 1 h for cefotaxime and was 2 h for ceftriaxone,54 strains of ESBLs-producing bacteria were able to hydrolyze antibiotics with two hydrolysis peaks of 414 Da and 370 Da appeared.When clavulanic acid was added,the hydrolysis peaks of 52 strains disappeared.The hydrolysis assay of cefotaxime and ceftriaxone could successfully identify ESBLs-producing Klebsiella pneumoniae after incubation of 1h and 2h,respectively.Comparing with genotype and phenotype confirmations,the specificity and sensitivity of this method were 100% and 96.3%(52/54),respectively.Conclusion: MALDI-TOF MS has been introduced to the clinical laboratory workflow for rapid identification of microbes,and its great potential in the identification of antimicrobial resistance needs more explorations.MALDI-TOF MS-based cephalosporins hydrolysis assay can quickly detect ESBLs-producing Klebsiella pneumoniae.Part Ⅱ Rapid Antimicrobial Susceptibility Test for Klebsiella pneumoniae by using Broth Micro-dilution Method combined with MALDI-TOF MSObjective: To explore the ability of MALDI-TOF MS-based broth micro-dilution method in detecting the MIC values of Klebsiella pneumoniae to ceftriaxone and imipenem.Methods: Sixty strains of K.pneumoniae presenting different resistance to carbapenems and cephalosporins were randomly collected.PCR and DNA sequencing were used to screen the genotype of resistance mechanism.The bacterial suspension was incubated with different concentrations of antibiotics in 96-well plates.After incubation for 4 hours,the bacteria suspension were centrifuged and the bacterial cells were lysed with formic acid.Then the lysed cells were used for acquiring mass spectra to detect the invisible growth of bacteria at different concentrations of antibiotics,so as to detect the susceptibility of bacteria to antibiotics.In parallel,broth micro-dilution method was referred as the standard for the determination of minimum inhibitory concentration(MIC).The results were interpreted by recognizing the visible bacterial growth after incubation of 18-20 hours,and the differences between the MIC values determined by the two methods were compared.Results: The resistance genes of bla KPC,bla FOX,bla DHA,bla CTX-M and bla TEM were detected in the thirty carbapenems-resistance strains.The agreement on MIC values derived from MALDI-TOF MS and reference broth micro-dilution method were61.7% and 71.7% of ceftriaxone and imipenem,respectively.According to the CLSI breakpoint of resistance to ceftriaxone and imipenem,The sixty isolates were accurately classified as resistance and susceptibility with 100% sensitivity and 100% specificity.Conclusion: The transmission and infection of multi-drug resistant bacteria could be better managed and treated with rapid identification as well as earlier antimicrobial susceptibility results.The MALDI-TOF MS-based susceptibility test could identify resistance of bacteria to some critical antibiotics for the pathogenic infections in a short time.This approach is possible to be used as a supplemented antimicrobial susceptibility test in routine workflow of laboratories in the near future.