Pheno-Molecular Testing Enabled by Multiplex Droplet Digital PCR for Rapid and High-Content Detection of Carbapenem-Resistant <i>Enterobacterales</i>

Pheno-molecular testing evaluates bacterial responses to antibiotics via quantitative nucleic acid detection, thus enabling rapid antibiotic susceptibility testing by eliminating the time-consuming isolation culture step. Carbapenems exert their antibacterial effects by disrupting cell wall synthesis, which requires prolonged antibiotic exposure to identify carbapenem-resistant Enterobacterales (CRE) by pheno-molecular testing. To overcome this limitation, we developed a bacterial integrity evaluation-based pheno-molecular testing (Baci-PmT) assay using multiplex droplet digital PCR (mddPCR). This assay features a low-osmolarity culture system supplemented with Meropenem and DNase. Its underlying principle lies in the carbapenem-induced cell wall weakening combined with osmotic pressure-induced outer membrane rupture, resulting in bacterial DNA leakage and subsequent enzymatic degradation. Experimental results show that after 30 min antibiotic exposure, the DNA levels of carbapenem-susceptible Enterobacterales (CSE) were significantly lower than those of CRE. Moreover, the types of carbapenemases were identified by evaluating the synergistic effects of carbapenemase inhibitors and Meropenem on bacterial integrity disruption. Using a color and spatial resolution-based mddPCR system, the Baci-PmT assay enabled the simultaneous identification of CRE-associated bacterial species, CRE phenotypes, and types of carbapenemases. The entire testing process can be completed within 3 to 4.5 h. When analyzing a cohort of 51 clinical samples, the Baci-PmT assay achieved 100% sensitivity and 93.1% specificity in identifying CRE. The concordance rates with conventional assays for determining CRE MIC and the types of carbapenemases were 100% and 87.5%, respectively. The developed Baci-PmT assay can achieve rapid and high-content detection of CRE, offering timely and accurate guidance for clinical interventions in CRE infections.