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The cefazolin inoculum effect (CzIE) has been associated with poor clinical outcomes in patients with methicillin-susceptible Staphylococcus aureus (MSSA) infections. We aimed to investigate the point prevalence of the CzIE among nasal colonizing MSSA isolates from ICU patients in a multicenter study in Colombia (2019-2023). Patients underwent nasal swabs to assess for S. aureus colonization on admission to the ICU, and some individuals had follow-up swabs. We performed cefazolin MIC by broth microdilution using standard and high inoculum and developed a modified nitrocefin-based rapid test to detect the CzIE. Whole-genome sequencing was carried out to characterize BlaZ types and allotypes, phylogenomics, and Agr-typing. A total of 352 patients were included; 46/352 (13%) patients were colonized with S. aureus and 22% (10/46) and 78% (36/46) with MRSA and MSSA, respectively. Among 36 patients who contributed with 43 MSSA colonizing isolates, 21/36 (58%) had MSSA exhibiting the CzIE. BlaZ type A and BlaZ-2 were the predominant type and allotype in 56% and 52%, respectively. MSSA belonging to CC30 were highly associated with the CzIE, and single-nucleotide polymorphism (SNP) analyses supported possible transmission of MSSA exhibiting the CzIE among some patients of the same unit. The modified nitrocefin rapid test had 100%, 94.4%, and 97.7% sensitivity, specificity, and accuracy, respectively. We found a high point prevalence of the CzIE in MSSA colonizing the nares of critically ill patients in Colombia. A modified rapid test was highly accurate in detecting the CzIE in this patient population., Competing Interests: The authors declare no conflict of interest.