The carbapenem inoculum effect provides insight into the molecular mechanisms underlying carbapenem resistance in 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 how resistance mechanisms influence the carbapenem inoculum effect (IE), a phenomenon where inoculum size affects antimicrobial susceptibility testing (AST). We demonstrated that seven different carbapenemases impart a meropenem IE in Escherichia coli . Across 110 clinical CRE isolates, the carbapenem IE strictly depended on resistance mechanism: all carbapenemase-producing CRE (CP-CRE) exhibited a strong IE, whereas porin-deficient CRE displayed none. Concerningly, 50% and 24% of CP-CRE isolates changed susceptibility classification to meropenem and ertapenem, respectively, across the allowable inoculum range in clinical 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.