Rapid detection of bacteria that produce extended-spectrum β-lactamase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Abstract Objectives Proper use of antibacterial agents is required to prevent the spread of drug-resistant bacteria. To support clinicians, laboratories need to rapidly determine bacterial drug susceptibility/resistance. We have established a method to distinguish extended-spectrum β-lactamase (ESBL)-producing clinical isolates by capturing structural changes in β-lactam antibiotics using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, classified into ESBL-producing strains and sensitive strains based on the presence or absence of a CTX-M-type gene, were used. Test bacteria were cultured aerobically in solid-phase wells of “Eiken” DPD-1 dry plates at 35°C for 15 or 30 min with antibiotics [cefotaxime (CTX), cefpodoxime (CPDX), or piperacillin (PIPC)]. Then, the culture supernatants were used for analysis with a MALDI Biotyper. Results Signals derived from non-hydrolyzed products of antibiotics were observed in all strains. In the case of ESBL-producing strains, signals derived from the hydrolysis products of antibiotics were observed. Since the ratio of signal intensity derived from hydrolysis products divided by the total signal intensity detected was more than 11% for CTX and more than 6% for CPDX and PIPC, all strains were determined to be ESBL-producing bacteria. Conclusion The short incubation time of 15 minutes suggests that this method can identify ESBL-producing strains much more rapidly than conventional methods.