Decoding the cefazolin inoculum effect: phenotypic and genotypic insights beyond the MIC

The cefazolin inoculum effect (CzIE) is a clinically relevant in vitro phenomenon that may compromise cefazolin efficacy in high-inoculum methicillin-susceptible Staphylococcus aureus (MSSA) infections such as bacteremia and infective endocarditis. We aimed to define mechanisms underlying CzIE beyond BlaZ-dependent enzymatic pathways. We evaluated CzIE in 198 MSSA isolates (single center) by comparing minimum inhibitory concentrations at standard and high inoculum. The inoculum effect index was used as a quantitative measure of the CzIE to validate the operational definition and assess its concordance with the classical definition of CzIE. Whole-genome sequencing was used to define β-lactamase gene (blaZ) allotypes, accessory gene regulator (agr) genotypes (I-IV), and clonal lineages. CzIE was observed in 16.16% of isolates, with Type A (71.9%) and Type C (28.1%) blaZ allotypes predominating among CzIE-positive strains. These isolates were enriched within clonal complex CC8 and CC30. Twenty-four-hour time-kill experiments combining cefazolin with BlaZ inhibitor (sulbactam) demonstrated initial killing followed by regrowth in CzIE-positive isolates, while complete eradication was observed in CzIE-negative isolates. Population analysis profiles (PAP) revealed the expansion of cefazolin-non-susceptible subpopulations at high inoculum, consistent with phenotypic heterogeneity. agr typing identified agrIII as the predominant genotype among CzIE-positive isolates, and agr dysfunction was significantly associated with CzIE-positive phenotype. Together, these findings suggest that CzIE is driven not only by BlaZ-mediated activity but also by agr-dependent regulatory pathways. These observations highlight the potential clinical importance of CzIE and support further investigation into its implications for therapeutic optimization and improved patient outcomes.IMPORTANCECefazolin inoculum effect (CzIE) represents an underrecognized potential determinant of therapeutic failure in methicillin-susceptible Staphylococcus aureus (MSSA) infections, yet it remains absent from routine susceptibility testing frameworks. We demonstrate that CzIE is not solely driven by blaZ-mediated activity but is strongly associated with agr-dependent regulation and population heterogeneity. Our findings suggest that standard MIC testing can underestimate cefazolin failure under high-inoculum conditions commonly encountered in invasive diseases. Importantly, identification of CzIE-positive isolates could inform therapeutic decision-making, including selection of alternative β-lactams or adjunctive strategies to suppress tolerant subpopulations. Integration of CzIE assessment with genomic markers such as blaZ allotype and agr genotype may enable more precise prediction of treatment response. These data provide a framework for incorporating CzIE into diagnostic workflows and guide future studies evaluating its clinical utility. Ultimately, recognizing CzIE as a multifactorial phenotype may improve antibiotic selection, reduce treatment failure, and enhance outcomes in serious MSSA infections across patient populations.