The carbapenem inoculum effect provides insight into the molecular mechanisms underlying carbapenem resistance in...

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 Escherichia coli. 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. 27 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 clinical CP-CRE release carbapenemase activity into culture supernatant to protect other cells. Further, this released activity markedly increased upon exposure to lethal antibiotic doses, functionally consistent with altruism. 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.