ABSTRACT Staphylococcus aureus is one of the most important pathogens responsible for a wide range ofillnesses including life-threatening infections. An important number of these infections are caused bymethicillin-susceptible S. aureus (MSSA) and in many parts of the world (including the US), MSSA infectionsare more common than those caused by methicillin-resistant S. aureus (MRSA). Indeed, MSSA represent amajor burden of S. aureus infections and are important contributors to mortality. The treatment of MSSAinfections relies on the use of ?-lactams. Indeed, the first line of therapy for severe MSSA infections are theisoxazolyl penicillins (e.g., nafcillin, oxacillin). However, recent data suggest that clinical outcomes in MSSAbacteremia are similar in patients treated with nafcillin/oxacillin vs cefazolin, a first generation cephalosporinwith activity against MSSA that appears to be less toxic. Moreover, treatment with nafcillin seem to beassociated with increased costs, more drug reactions (including hepatotoxicity, interstitial nephritis andneutropenia) and, possibly, higher mortality. Due to these concerns, an important shift in the treatment ofMSSA is occurring whereby clinicians are now using cefazolin as first line of therapy for severe MSSAinfections. An important concern of using cefazolin and other cephalosporins as primary therapy for theseserious infections is the occurrence of the cefazolin inoculum effect (CzIE), defined as a cefazolin minimalinhibitory concentration of > 16 Âµg/ml when a high inoculum (107 CFU/ml) is used. The CzIE has beenassociated with failures in the treatment of deep-seated MSSA infections and with the production of certainisotypes of the staphylococcal ?-lactamase. However, the molecular basis and the actual clinical impact ofthis phenomenon are unknown. Our published and preliminary clinical data indicate that the CzIE is animportant contributor to clinical outcomes of severe MSSA infections. Our Prelim. Data also provide evidencethat the CzIE is mediated, at least in part, by changes in the processing and extracellular release of the ?-lactamase (BlaZ) enzyme and it is influenced by the catalytic activity of the ?-lactamase isotype. Ourhypotheses are that, i) the CzIE is primarily mediated by the activity of extracellular ?-lactamase released intothe extracellular milieu, ii) the ability to hydrolyze cefazolin effectively depends on the catalytic activity of theenzyme isotype, and iii) a rapid phenotypic test can be developed to identify isolates with the CzIE and helpclinicians in the treatment of MSSA infections. In two specific aims, we will define the molecular andbiochemical bases of the CzIE exploring the genetic factors influencing this phenomenon and characterizingnovel structural and biochemical isoforms of BlaZ identified via our genomic analyses. We will also aim todevelop a rapid assay to identify MSSA strains with the CzIE. Our findings are likely to influence the manner bywhich MSSA infections are treated and will provide the basis to develop novel diagnostic tools to advance thetreatment of these life-threatening infections.