The carbapenem inoculum effect provides insights into the molecular mechanisms underlying carbapenem resistance in the Enterobacterales

Carbapenem-resistant Enterobacterales (CRE) are important pathogens that can develop resistance via multiple molecular mechanisms, including hydrolysis or reduced antibiotic influx. Identifying these mechanisms can improve pathogen surveillance, infection control, and patient care. We investigated, both phenomenologically and mechanistically, how resistance mechanisms influence the carbapenem inoculum effect (IE), a phenomenon where inoculum size affects antimicrobial susceptibility testing (AST). We demonstrated that any of seven different carbapenemases were sufficient to impart a meropenem IE when transformed into a laboratory strain of <i>Escherichia coli</i>. Across 106 clinical CRE isolates spanning 6 genera and 12 species, the carbapenem IE strictly depended on resistance mechanism: all 36 carbapenemase-producing CRE (CP-CRE) exhibited a clear IE, whereas 43 porin-deficient CRE displayed none. Twenty-seven isolates with both carbapenemase production and porin deficiency exhibited high-level resistance at all inocula and still displayed an IE, albeit smaller in magnitude than CP-CRE with intact porins. Mechanistically, we found that CP-CRE released carbapenemase activity into the culture supernatant to protect other cells, and this released activity markedly increased upon exposure to lethal antibiotic doses. Concerningly, 50% and 24% of CP-CRE isolates changed susceptibility classification to meropenem and ertapenem, respectively, across the allowable inoculum range in clinical testing guidelines. The meropenem IE and the ratio of ertapenem to meropenem minimal inhibitory concentration (MIC) at standard inoculum reliably identified CP-CRE. Understanding how resistance mechanisms affect AST could improve diagnosis and guide therapies for CRE infections.IMPORTANCEInfections caused by carbapenem-resistant Enterobacterales (CRE) pose significant threats to patients and public health worldwide. Carbapenem resistance can occur through several molecular mechanisms, including enzymatic hydrolysis by carbapenemases and reduced influx via porin mutations. Identifying carbapenemase-producing isolates could enable tailored antibiotic selection to improve patient outcomes and infection control measures to prevent further carbapenemase transmission. In a large collection of CRE isolates, we found that only carbapenemase-producing CRE exhibited an inoculum effect, in which their measured resistance varies markedly with cell density, which risks misdiagnosis. Further, this inoculum effect occurred under conditions where bacteria released carbapenemases to the community upon exposure to antibiotics that resulted in cell death. Measuring this inoculum effect, or integrating other data from routine antimicrobial susceptibility testing, enhanced carbapenem resistance detection, paving the way for more effective strategies to combat this growing public health crisis.