Prompt detection of KPC and CMY by MALDI-TOF MS directly from positive liquid culture media and short-term cultures.

COSTA, AGUSTINA; FIGUEROA ESPINOSA ROQUE A; VAY CARLOS; GABRIEL GUTKIND; DI CONZA JOSÉ

Washington, D.C

Congreso; ASM Microbe 2022.; 2022

ASM

MALDI-TOF MS bacterial identification from short-term cultures obtained from positive blood culture bottles, is widely used among microbiology laboratories around the world. Antimicrobial susceptibility results, however, are available 24 hours later. We developed an easy MALDI-TOF MS-based assay to detect beta-lactamases directly from positive tryptic soy broths and short-term cultures. We evaluated 28 Klebsiella pneumoniae clinical isolates producing KPC (12), KPC+NDM (3), CTX-M-group 1 (2), NDM+CTX-M-group 1 (9), NDM (1) and CMY (1), and 20 Escherichia coli clinical isolates producing KPC (2), CTX-M-group 1 (4), CTX-M-group 2 (1), CMY (12) and NDM-5 (1). Recombinant and transformant strains, and K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as controls. Briefly, 1.5 ml of positive tryptic soy broth (TSB) was pelleted and washed with distilled water after overnight incubation at 37°C. Short-term cultures were performed by culturing 2 drops of positive TSB on tryptic soy agar. Agar plates were incubated at 37°C for 4-5 hours. Protein extraction was performed with organic solvents and the supernatant was used for MALDI-TOF MS analysis. Sinapinic acid was used as matrix for high-molecular weight proteins crystallization. Spectra analysis was performed with Bruker flexAnalysis LT 3.4 (Bruker Daltonics GmbH, Germany) considering 17-50 kDa mass range. Also, bacterial identification was evaluated from all protein extracts, using HCCA as matrix. Bacterial identification was achieved from positive TSB and short-term cultures (scores >2). When comparing the isolates spectra to control strains spectra, KPC and CMY were detected in every K. pneumoniae and E. coli clinical isolate (sensitivity 100%, specificity 100%), from positive TSB and short-term cultures. CTX-M- variants could not be detected unequivocally and NDM was not identified in any isolate. This MALDI-TOF MS procedure could be evaluated in clinic microbiology laboratories for some beta-lactamases detection from positive broth cultures and short-term cultures coming from different clinical samples, complementing susceptibility tests results.