Carbapenem-resistantEnterobacterales(CRE) are important pathogens that can develop resistance via multiple molecular mechanisms, including enzymatic hydrolysis or reduced antibiotic influx. The identification of these mechanisms is crucial for effective pathogen surveillance, infection control, and patient care. However, many clinical laboratories do not test for the molecular basis of resistance. In this study, we investigated whether we could gain insight into resistance mechanisms by using the inoculum effect (IE), a phenomenon where the inoculum size used in antimicrobial susceptibility testing (AST) affects the minimum inhibitory concentration (MIC) measured. We demonstrated that seven different carbapenemases impart a meropenem IE when expressed inEscherichia coli. Next, we measured the meropenem MIC as a function of inoculum size for 110 clinical CRE isolates. We found that the carbapenem IE was strictly dependent on the resistance mechanism: carbapenemase-producing CRE (CP-CRE) exhibited a strong IE, whereas porin-deficient CRE (PD-CRE) displayed none. Strains with both carbapenemases and porin deficiency had higher MICs at low inoculum and also an IE; we termed these "hyper-CRE". Concerningly, 50% and 24% of CP-CRE isolates changed susceptibility classification to meropenem and ertapenem, respectively, across the allowable inoculum range in clinical guidelines, with 42% testing meropenem susceptible at some point in this range. The meropenem IE and the ratio of ertapenem to meropenem MIC at standard inoculum reliably distinguished CP-CRE and hyper-CRE from PD-CRE. Understanding how molecular mechanisms of resistance affect AST could improve diagnosis and guide therapies for CRE infections.Significance StatementInfections caused by carbapenem-resistantEnterobacterales(CRE) pose a significant threat to public health worldwide. Carbapenem resistance can occur through several molecular mechanisms, including enzymatic hydrolysis by carbapenemases and reduced influx via porin mutations. Knowing the mechanisms of resistance informs therapies and infection control measures to prevent further spread of these deadly pathogens. In a large collection of CRE isolates, we found that only carbapenemase-producing CRE exhibit an inoculum effect, in which their measured resistance varies markedly with cell density, which risks misdiagnosis. Measuring this inoculum effect, or integrating other data from routine antimicrobial susceptibility testing, enhances the detection of carbapenem resistance and thus paves the way for more effective strategies to combat this growing public health crisis.