Monitoring the Alternative Pathway of Meropenem Degradation by OXA-48-Type Carbapenemases

Abstract

Background:Overuse of carbapenems has been associated with an increasing prevalence of resistance, particularly among the Enterobacterales, Pseudomonasspp., and Acinetobacter spp. Resistance is mediated by the action of carbapenemases, which hydrolytically cleave the carbapenem molecule. In the case of OXA-48-type carbapenemases, an alternative pathway of carbapenem (e.g., meropenem) degradation via lactonization has been observed. According to current studies, this alternative degradation pathway is linked to the occurrence of false-negative results in carbapenemase detection methods based on hydrolytic cleavage of carbapenems. The aim of the study was to investigate the occurrence of β-lactone formation in Gram-negative bacteria and to assess the impact of the SOS response on β-lactone production in OXA-48-type carbapenemases.Materials and Methods: Screening of isolates for carbapenemase production was performed using a meropenem hydrolysis assay by MALDI-TOF MS. The impact of SOS mechanisms on β-lactone formation was investigated in four fully sequenced OXA-48 carbapenemase-producing isolates (Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, and Enterobacter cloacae). For the experiments, Mitomycin C was applied at concentrations of 20, 40, and 60 mg/L, and amikacin was used at subinhibitory levels, as determined by the MIC (25 mg/L and 1 mg/L). Additionally, the effects of EDTA (20 mM), NaCl (150 mM), and pyridine (20 mg/L) on β-lactone formation were evaluated. The effects were monitored by LC-MS using a timsTOF Pro MS instrument (Bruker Daltonics, Germany).Results:A total of 1,543 bacterial isolates were examined and screened for carbapenemase production. A specific β-lactone signal (m/z 362) was detected in 268 out of 272 OXA-48-type carbapenemase producers. The results further demonstrated that in the presence of both OXA-48 and NDM carbapenemases, β-lactone was not detected; 113 bacterial isolates produced these enzymes. Assessment of the impact of SOS-related mechanisms on β-lactone formation following exposure to Mitomycin C, EDTA, NaCl, and pyridine revealed no observable effect on β-lactone production. In contrast, experiments with amikacin consistently resulted in inhibition of β-lactone formation in the C. freundiiisolate, whereas β-lactone production remained preserved in the other bacterial species. Conclusion:Our results confirm that β-lactone formation is a characteristic feature of OXA-48-type carbapenemases as we previously published. The absence of β-lactone in strains producing both OXA-48 and NDM carbapenemases is most likely attributable to the faster kinetics of meropenem degradation by NDM enzymes. The observed inhibition of β-lactone formation in the presence of amikacin in the C. freundiiisolate warrants further investigation.