Impact of Carbapenemase Mechanism on the Clinical Detection of Carbapenem-Resistant Bacteria

Abstract

Production of β-lactamases by multidrug-resistant bacteria is one of the most common mechanisms of antibiotic resistance. A subclass of β-lactamases called carbapenemases threaten the clinical viability of carbapenems, a group of antibiotics used as last-resort therapeutics. Rapid detection of carbapenemase-producing bacteria is critical when determining the appropriate therapeutic to treat an infection. Serine β-lactamases with carbapenemase activity employ a two- step mechanism that involves the formation of an acyl-enzyme intermediate, which then gets hydrolyzed by a nucleophilic water molecule, ultimately rendering the antibiotic inactive. As carbapenem hydrolysis results in a decrease in pH, many current detection approaches use colorimetric pH indicators to monitor carbapenem degradation. However, although carbapenem- hydrolyzing class D β-lactamases (CHDLs) are becoming of increasing clinical significance, they are substantially more difficult to detect through pH assays when compared to other carbapenemases. We recently found that CHDLs can degrade carbapenems through an alternative mechanism involving the formation of carbapenem-derived β-lactones We hypothesize that this mechanism impacts the clinical detection of bacteria that produce these enzymes. To investigate whether lactone formation impacts the detection of CHDL-producing bacteria, we adapted a clinically used pH assay. In combination with NMR spectroscopic studies, our assay results indicate that carbapenem degradation through β-lactone formation results in a smaller change in pH than hydrolysis. In addition, we have shown that conservative substitutions in the active sites of CHDLs have a dramatic impact on the relative levels of β-lactone and hydrolysis products formed. The V120L variant of the CHDL OXA-48 catalyzes carbapenem degradation almost exclusively through lactone formation, with little impact on pH; notably, this enzyme has been identified in a clinical context. As our results demonstrated that clinically used pH assays have significant limitations in detecting CHDLs, we have also been exploring alternatives like surface enhanced Raman spectroscopy (SERS). Our results reveal that CHDL-producing bacteria are likely missed by current pH-based detection strategies due to the mechanism by which the CHDLs degrade carbapenems.