Alternative faster MALDi-TOF based procedure to detect KPC-2 carbapenemase directly in clinical isolates.

FIGUEROA ESPINOSA ROQUE A; CEJAS DANIELA; RUMI, VALERIA; BARBERIS CLAUDIA; VAY CARLOS; GUTKIND GABRIEL; DI CONZA JOSÉ

Atlanta

Congreso; ASM MICROBE 2018; 2018

Background: Fast and accurate detection of carbapenemases is critical in clinical microbiology. The KPC is the most prevalent class A carbapenemase in Enterobacterales, worldwide. Rapid methods for KPC detection are needed for guiding antibiotic therapy as early as possible and prevent further dissemination of this resistance marker. MALDI-TOF mass spectrometry (MS) is currently used for species identification. ß-lactamases detection, using the analysis of ß-lactam hydrolysis products, by MALDI-TOF is reliable but time and labour consuming. Detection of KPC has also been inferred by MALDI-TOF after detection of an accompanying protein in KPC-producing microorganisms. The aim of this work was to detect KPC-2 in different enterobacterial isolates applying an easy to perform, fast and direct MALDI-TOF MS procedure.Materials/methods: 117 clinical isolates of K. pneumoniae (65), S. marcescens (24), E. coli (20) and E. cloacae (8) were included in this study. Susceptibility tests were performed according to CLSI. Isolates were analyzed by PCR and grouped according to the presence of ß-lactamases genes. Detection of KPC-2 ß-lactamase was carried out by MALDI-TOF (Bruker) after protein extraction with organic solvents. MALDI-TOF spectra were recorded by MALDI Biotyper system and analyzed using ClinProTool software (Bruker). A KPC-2 producing E. coli XL1 blue transformant was used as a positive control. Those isolates negative for blaKPC (ESBL producers or susceptible strains) and E. coli XL1 blue were included as negative controls.Results: Forty one clinical strains (33 K. pneumoniae, 4 S. marcescens, 3 E. coli and 1 E. cloacae) were categorized as KPC-2 producers. A distinct peak at m/z ~28,477 Da, in agreement with the theoretical MW of the mature protein of KPC-2 was detected in the E. coli XL1 transformant when the instrument operated at a mass range of 17,000 to 50,000 Da. This distinctive peak was present in all KPC-2 producing clinical isolates spectra, and consistently absent in the negative control group (n = 76).Conclusions: Even if our results are preliminary, we here demonstrate that MALDI-TOF MS has the potential to detect the most clinically relevant acquired class A carbapenemase, the KPC-2-enzyme. To the best of our knowledge, this is the first report where mature KPC-2 enzyme was detected by MALDI-TOF in a timely way, substantially less than that needed for ß-lactam hydrolysis. This procedure shows an improvement in the lab abilities due to an easy, fast and direct detection of KPC-2 with extensive clinical implications.