Novel Insights Into The Classification of Staphylococcal β-Lactamases In Relation To The Cefazolin Inoculum Effect

Cefazolin has become a prominent therapy for methicillin-susceptible Staphylococcus aureus (MSSA) infections. However, an important concern is the cefazolin-inoculum effect (CzIE), a phenomenon mediated by staphylococcal beta-lactamases. Four variants of staphylococcal beta-lactamases have been described based on serological methodologies and limited sequence information. Here, we sought to reassess the classification of staphylococcal beta-lactamases and their correlation with the CzIE. We included a large collection of 690 contemporary bloodstream MSSA isolates recovered from Latin America, a region with high prevalence of the CzIE. We determined cefazolin MICs at standard and high-inoculum by broth-microdilution. Whole-genome-sequencing was performed to classify the beta-lactamase based on the predicted full sequence of BlaZ. We used the classical schemes for beta-lactamase classification and compared it to BlaZ allotypes found in unique sequences using the genomic information. Phylogenetic analyses were performed based on the BlaZ and core-genome sequences. The overall prevalence of the CzIE was 40%. Among 641 genomes, type C was the most predominant beta-lactamase (37%), followed by type A (33%). We found twenty-nine allotypes and 43 different substitutions in BlaZ. A single allotype, designated BlaZ-2, showed a robust and statistically significant association with the CzIE. Two other allotypes (BlaZ-3 and BlaZ-5) were associated with the lack of the CzIE. Three amino acid substitutions (A9V, E112A and G145E) showed statistically significant association with CzIE (P =<0.01). CC30 was the predominant clone among isolates displaying CzIE. Thus, we provide a novel approach to the classification of the staphylococcal beta-lactamases with the potential to more accurately identify MSSA strains exhibiting the CzIE.