Preferred β-lactone synthesis can explain high rate of false-negative results in the detection of OXA-48-like carbapenemases

The resistance to carbapenems is usually mediated by enzymes hydrolyzing beta-lactam ring. Recently, an alternative way of the modification of the antibiotic, a beta-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC-MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived beta-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), beta-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z=340.5 and 362.5 representing decarboxylated beta-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z=358.5) and its sodium adduct (m/z=380.5) have been detected. The peak of m/z=362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC-MS, we were able to identify meropenem-derived beta-lactone directly according to the different retention time. All strains with a predominant beta-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived beta-lactone. We also identified beta-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC-MS to detect meropenem-derived beta-lactone.

Automated summary

This study focused on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. A new alternative way of antibiotic modification by OXA-48-like enzymes was identified. Various methods were used to determine the carbapenemase activity, and it was found that meropenem-derived beta-lactone was preferentially produced by strains that showed false-negative results using Carba NP test.