Abstract Background The cefazolin inoculum effect (CzIE), defined as Cz minimum inhibitory concentration ≥ 16 µg/ml at high inoculum (HI-MIC), has been associated with poor clinical outcomes in patients with MSSA bacteremia or osteomyelitis. The CzIE is correlated with the presence of the blaZ gene, one of the components of the bla operon encoding the BlaZ β-lactamase (type A, B, C or D). Other portions of the bla operon include blaR and blaI (encoding the antibiotic sensor and transcriptional repressor, respectively) and the intergenic region with operator and promoter sequences (Figure 1). In BlaR, residue 293 mediates signal transduction, and the Z and R dyads in the intergenic region are the DNA-binding sites for BlaI (Figure 2). Previous experiments have shown that the regulatory portions of the bla operon play a key role in the CzIE. Here, we investigated the association between the CzIE and specific variations in the regulatory sequences of the bla operon. Figure 1. Functioning of the bla operon and the production of the staphylococcal β-lactamase BlaZ. Figure 2. Structure and key regions of the intergenic region of the bla operon, incluiding the promoter and the BlaI DNA-binding regions (Z and R dyads). Methods A total of 437 MSSA containing blaZ were evaluated for the CzIE using broth microdilution at high inoculum. Using whole genome sequencing, the sequences of the bla operons were classified into cassettes based on unique changes in predicted amino acid sequences of BlaZ, BlaR and BlaI paired with specific nucleotide alterations in the intergenic region. The bla operon sequence of S. aureus ATCC29213 was used as reference (cassette 0). Results Among 437 MSSA isolates, 46% exhibited the CzIE. We identified 55 unique bla cassettes. The bla cassettes were phylogenetically grouped in 7 clusters (Figure 3) which grouped cassettes with different BlaZ types and variations in the Z dyad, the -35 box, residue 293 of BlaR, and the blaI ribosomal binding site. Each cluster had an association to the CzIE and distinct Cz HI-MICs. The combination of: a BlaZ type A, C or D, an adenine in the position -66 of blaZ (-35 box of blaZ), a cytosine in the position -22 of blaZ(Z dyad), and either an arginine or a serine in position 293 of BlaR was a very strong predictor of the CzIE (Figure 4). Figure 3. Phylogenetical organization of bla operon cassettes into clusters, their association with polymorphisms in key regulatory regions and the CzIE. GM Cz-MIC: Geometric mean of the Cefazolin MIC at high Inoculum. Figure 4. Variations of the bla operon, their association with the CzIE, their GM (Geometric Mean) of the Cefazolin MIC and the MIC distribution of the strains with each specific combination of polymorphisms. Conclusion Specific variations in regulatory portions of the bla operon, which are likely to influence BlaZ expression, are highly associated with the CzIE, supporting the notion that regulation of blaZ is the key factor responsible for the CzIE in MSSA. Disclosures Lorena Diaz, PhD , Nothing to disclose William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)
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