Detection of AmpC β-lactamase-producing Gram-negative bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Rapid detection of AmpC-producing strains of Gram-negative bacteria is beneficial for patient care. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method that has demonstrated the resistance of a strain to β-lactam antibiotics by determining the molecular structure of an antibiotic and its degradation products.

AIM: To study the detection of AmpC-producing Gram-negative bacteria by MALDI-TOF MS and to determine whether the method can be used in clinical practice.

METHODS: A total of 105 strains were detected by determining their phenotypes and sequence analysis. Sixty-nine well-characterized AmpC-producing and 36 non-AmpC-producing strains were studied. The bacteria were incubated in different reaction buffer solutions (10 mM NH4 HCO3 /0.005% sodium dodecyl sulphate at pH 8.0) containing cefotaxime (0.50 mg/mL), ceftazidime (0.25 mg/mL), ceftriaxone (0.50 mg/mL), cefepime (0.50 mg/mL), and cefoxitin (0.25 and 0.50 mg/mL), respectively. The mixture was centrifuged at 13,000 g for 2 min, and the supernatant analysed by MALDI-TOF MS after incubation for 30, 60, 90, 120, and 240 min. Antibiotic hydrolysed and decarboxylated peaks were identified.

FINDINGS: When incubated for 90 min, hydrolysed cefotaxime formed peaks at 434 and 494 Da, and the sensitivity and specificity for detection of AmpC-producing strains were 85.5% (59/69) and 88.9% (32/36). When incubated for 4 h, hydrolysed ceftazidime formed peaks at 563 and 587 Da, and the sensitivity and specificity were 89.9% (62/69) and 94.5% (34/36), respectively. For hydrolysed ceftriaxone (0.5 mg/mL), cefepime (0.5 mg/mL) and two concentrations of cefoxitin (0.25 and 0.5 mg/mL), no peaks amenable to analysis were identified.

CONCLUSION: This study demonstrated that MALDI-TOF MS can rapidly detect AmpC-producing strains.