Investigation of The Presence of Carbapenemase in Carbapenem-resistant Klebsiella pneumoniae Strains by MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization – Time Of Flight Mass Spectrometry) and Comparison with Real-Time PCR Method
Project no. 56 · Status: Kapanmış · 2016
Summary
Aim of the study: The increasing prevalence of bacterial resistance to antibiotics is an important public health problem. Infections due to carbapenem resistant Enterobacteriaceae strains are among the most important health problems in our country as well as in the world. Infections caused by carbapenem resistant bacteria are associated with significant morbidity and mortality worldwide. Carbapenem-resistant Enterobacteriaceae are among the top tier of the WHO list of antibiotic-resistant “priority pathogens” that pose the greatest threat to human health. The aim of this study is to develop a procedure to detect carbapenemase activity by MALD-TOF MS and it is aim to to calculate the sensitivity and specificity of the developed method. The second aim of this study is to determine the distribution of resistance genes of carbapenem-resistant K. penumoniae strains isolated in our hospital using real time PCR. Material and methods: Between January 2017- February 2019 K. pneumoniae strains (n=74) isolated and stocked from blood culture samples in Ege University Hospital Bacteriology Laboratory were included in the study. Klebsiella pneumoniae NCTC 13438 was used as positive control and Escherichia coli ATCC 25922 was used as negative control. First, MIC values of the isolates were determined by imipenem, meropenem and ertapenem gradient test (BioMérieux, France). Then, the strains were investigated by using real time PCR (BD MAX™ CRE Assay, BD Diagnostic Systems, Canada) to detect blaKPC, blaOXA-48 and/or blaNDM carbapenem resistance genes. A method was developed using ertapenem for the determination of carbapenemase activity by MALDI-TOF MS (BioMérieux, France) of all strains. It was evaluated whether two and four hours incubation of the bacteria in ertapenem solution carried out ertapenem hydrolysis with using MALDI-TOF. Sensitivity and specificity of MALDI-TOF MS were determined by PCR. Results: 9 strains were susceptible to the imipenem, meropenem and ertapenem. 65 strains were resistant to imipenem, meropenem and ertapenem. Imipenem, meropenem and ertapenem MIC50 and MIC90 values of all resistant strains were > 32 mg/L. BlaOXA-48, blaNDM and blaKPC were not detected in any of the carbapenem susceptible strains. Of the 65 strains resistant to any of the carbapenems, 57 (87.7%) had blaOXA-48, 15 (23.1%) had blaNDM, and 4 (6.2%) had blaKPC. BlaOXA-48 and blaNDM genes were detected together in 11 (16.9%) strains. The sensitivity and specificity of MALDI-TOF MS at the second hour were 83.1% and 100%,respectively. Sensitivity and specificity at the fourth hour were 100%. MALDI-TOF MS showed that 54 strains resistant to carbapenems hydrolyze ertapenem at second hour; All carbapenem resistant strains were found to hydrolyze ertapenem at the 4th hour. Carbapenem susceptible strains did not hydrolyze ertapenem. Conclusion: In our hospital, blaOXA-48 and blaNDM were detected 87.7% and 23.1% respecively. The sensitivity and specificity of MALDI-TOF MS at the second hour were 83.1% and 100%, respectively. Sensitivity and specificity at the fourth hour were 100%. In the MALDI-TOF MS ertapenem hydrolysis test, false negative results were observed at the second hour. These originated from OXA-48-producing origins. Determination of carbapenemase activity by MALDI-TOF MS is an evolving and promising method. MALDI-TOF MS provides information on the carbapenemase activity of the bacteria in two to four hours. After bacterial identification, it can be used to show carbapenemase activity on the same day. Multiple samples can be tested at the same time. Can be used in the presence of an outbreak. It is an easy test because routine laboratory materials are used in this method. Spectrum analysis is subjective because it is performed manually.