Application Of Matrix-assisted Laser Desorption Ionization Time-offlight Mass Spectrometry In The Rapid Detection Of Carbapenem-resistant Klebsiella Pneumoniae

BackgroundAt present,bacterial-caused drug-resistance has become a global problem.The wide spread of carbapenem resistant Enterobacteriaceae（CRE）,especially carbapenem-resistant Klebsiella pneumoniae（CRKP）,has posed a great threat to global public health.Carbapenem antibiotics（including meropenem,imipenem,ertapenem,etc.）are the most effective drugs for Extendedspectrum β-lactamases producing Escherichia coli and Klebsiella pneumoniae infection treatment,and are still considered as the final-line antibiotics.With the increasing use of these antibiotics in clinical practice,number of CRKP isolates is increasing rapidly year by year.European Antibiotic Resistance Monitoring Network recorded a sharp increase in CRKP.The proportion of CRKP isolates increased from 2% in 2009 to 15% in 2010,and 35% in 2013,respectively.According to the data of CHINET bacterial resistance monitoring network,the resistance rate of Klebsiella pneumoniae to carbapenems increased rapidly from 3.0% in 2005 to more than 25% in 2018.The mortality of CRE infection in China is as high as 33.5%.Compared with the infection of carbapenem sensitive Enterobacteriaceae（CSE）,the effective antibiotics for CRKP treatment are more limited and the prognosis of CRKP infection is worse.The production of carbapenemase is the most common mechanism of carbapenem resistant Enterobacteriaceae in clinical isolates.Therefore,it is very important and urgent to detect CRKP quickly and accurately.Matrix assisted laser desorption ionization time of flight mass spectrometry（MALDI-TOF MS）is a technology widely used in the identification of microorganisms.In recent years,the application of MALDI-TOF MS in the detection of bacterial drug resistance has aroused researchers’ great interests.Therefore,in this study,MALDI-TOF MS was used to investigate its ability to detect CRKP in clinic,so as to apply it to the early screening and diagnosis of CRKP.ObjectiveMALDI-TOF MS was used to quickly detect of meropenem hydrolysis capacity of Carbapenem-resistant Klebsiella pneumoniae.By quantitatively calculating hydrolysis index（HI）of meropenem,we aimed to establish a rapid and accurate detection procedure for screening CRKP in our laboratory,thus providing support for guiding the rational selection of antiinfection treatment and the correct implementation of sensing and control measures in the early stage of clinical practice.Methods1.Klebsiella pneumoniae was isolated and frozen from various clinical specimens in the clinical microbiology laboratory of our hospital from June 2020 to December 2020.Then all strains were resuscitated and identified again;2.A total of 206 clinical isolates and 2 quality control strains were screened by modified carbapenemase inactivation method（m CIM）and combined disc test（CDT）;3.The detected these 206 isolates’ carbapenemase by MALDI-TOF MS meropenem hydrolysis assay,to evaluate its reliability in carbapenemase detection.Results1.Both MALDI-TOF MS and BD Phoenix M50 automatic microbial identification drug sensitivity analyzer were used,and those with consistent results were identified as Klebsiella pneumoniae.A total of 206 strains of non-repetitive Klebsiella pneumoniae were screened,with156 CRKP strains and 50 non-CRKP strains;2.In MALDI-TOF MS meropenem hydrolysis assay,137 CRKP strains were found producing carbapenemase within 2 hours;the other 19 CRKP strains and 50 non-CRKP were not observed to produce carbapenemase.The average hydrolysis index of carbapenem producing Klebsiella pneumoniae strains and non-carbapenem-producing Klebsiella pneumoniae strains were 0.11 and 1.05,respectively.3.m CIM results showed 137 of 156 CRKP strains were positive and the other 19 were negative.And CDT results found 135 CRKP strains produced carbapenem and the other 21 CRKP strains were negative.50 non-CRKP strains were observed negative results in both m CIM and CDT,suggesting none of them producing carbapenemase.;4.Using modified Carbapenem inactivation method as reference,sensitivity,specificity and kappa value of matrix-assisted laser desorption ionization-time of flight mass spectrometry were 99.3%,98.6% and 0.978,respectively;while using Combined disc test as reference the sensitivity,specificity and kappa value of matrix assisted laser desorption ionization-time of flight mass spectrometry were 99.3%,95.8% and 0.957,respectively.Conclusion1.MALDI-TOF MS meropenem hydrolysis test can quickly and accurately distinguish between Klebsiella pneumoniae producing carbapenem and others,so as to quickly and accurately provide an important basis for reasonable anti infection treatment in the early stage of clinical practice.2.MALDI-TOF MS meropenem hydrolysis test showed high sensitivity,specificity in the detection of carbapenem.It can be used as a routine method for rapid and accurate screening of carbapenem producing Klebsiella pneumoniae in our laboratory.