Modified Carba PBP test for rapid detection and differentiation between different classes of carbapenemases in Enterobacterales

An advanced biochemical assay named modified Carba PBP test was innovated to identify and differentiate distinct categories of clinically significant carbapenemases (Ambler classes A, B, and D) within the Enterobacterales. The mechanism of mCarba PBP hinges on two core attributes: (i) the hydrolysis of the meropenem substrate by various carbapenemases, (ii) the immobilized penicillin and free meropenem in their affinity to interact with a limited quantity of penicillin-binding protein (PBP). Specific inhibitors for class A (phenylboronic acid, PBA) and class B (ethylenediaminetetraacetic acid, EDTA) were employed to inhibit the hydrolysis activity of carbapenemase and facilitate the classification of carbapenemase classes within 25 min. A comprehensive evaluation was undertaken using 94 clinical Enterobacterales isolates, comprising 75 carbapenemase-producing strains and 19 non-carbapenemase-producing strains. Its overall specificity and sensitivity were 100% and 97.3%, respectively, including detection of all types of OXA-48-like carbapenemases. For precise carbapenemase type identification, the assay exhibited remarkable sensitivities for class A, class B, and class D detection at 94.7%, 100%, and 100%, respectively. This user-friendly test presents a promising tool for carbapenemase identification, refining the selection of β-lactam/β-endoenzyme inhibitor combinations for effectively treating infections due to carbapenemase-producing organisms.