28 - regulation of the Staphylococcal β-lactamase Plays a Major Role in the cefazolin Inoculum Effect

Background: Cefazolin (Cz) is commonly used to treat methicillin-sensitive Staphylococcus aureus (MSSA) bacteremia. Yet, some MSSA isolates producing the staphylococcal β-lactamase (BlaZ) exhibit the Cz inoculum effect (CzIE), defined as an increase in the minimum inhibitory concentration (MIC) to ≥ 16 µg/mL at high inoculum (10⁷ CFU/mL, HI-MIC). Retrospective clinical data linked the CzIE to increased 30-day mortality and Cz treatment failure in patients with MSSA bacteremia, yet the mechanistic bases of this phenomenon are unknown. We aimed to explore the contribution of blaZ regulation, via BlaR (antibiotic sensor) and BlaI (transcriptional repressor) (Fig 1) to the CzIE by i) in trans expression assays and ii) analysis of their sequences in a set of isolatesFigure 1. Structure of the Staphylococcal bla Operon


Methods: The blaZ genes (with putative promoters) of strains exhibiting and lacking the CzIE (TX0117 and ATCC29213, respectively) were expressed in trans in RN4220 (blaZ neg) using the promotor-less vector pWM401 (Figure 2). We subsequently cloned the blaR and blaI genes of each TX0117 and ATCC29213 upstream of each blaZ allele (Figure 3). The presence of the CzIE was assessed in transformants using broth microdilution at standard (10⁵ CFU/mL, SI-MIC) and high inoculum. We also performed whole-genome sequencing (WGS) in 104 MSSA isolates exhibiting and lacking the CzIE to compare the sequences of BlaZ, BlaR, and BlaI and classified them by allotypes (unique amino acid sequences) using ATCC29213 as reference.Figure 2. In trans expression of blaZ genes from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220
Figure 3. In trans expression of the bla Operons from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220


Results: Expression of blaZ_TX0117 and blaZ_ATCC29213 with their native promoters in RN4220 resulted in the CzIE with Cz HI-MICs ≥ 64 µg/mL regardless of the origin of the allele (Table 1). Inclusion of the regulatory elements blaR and blaI from TX0117 (CzIE+) did not change the phenotype. In contrast, addition of blaR and blaI from ATCC29213 (CzIE-) led to a marked decrease in the Cz HI-MIC (Table 1). Sequence analyses of 104 MSSA isolates revealed 10, 17 and 6 BlaZ, BlaR and BlaI allotypes, respectively (Table 2). BlaZ-2 and BlaR-4 were linked to the CzIE in 90% of isolates.Table 1. MIC values of transformans after In trans expression of blaZ genes and bla Operons from a S. aureus CzIE+ strain(TX0117) and a CzIE- strain (ATCC29213) in RN4220
Table 2. BlaZ allotypes of 104 Staphylococcus aureus isolates and their association with the CzIE


Conclusion: Our results suggest that overexpression of blaZ can lead to the CzIE in any MSSA strain. Thus, the regulation of blaZ expression via BlaR and BlaI seem to play a major role in the CzIE. Identification of specific BlaR and BlaI allotypes could predict the presence of the CzIE.

Presenting Author(s)

• 

  Sara I. Gomez-Villegas, MD

  Post Doctoral Research Fellow
  University of Texas Health Sciences Center at houston
  Houston, Texas

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.

  Slides Slides and Audio

Co-Author(s)

• AD

  An Q. Dinh, BS

  Research Engineer
  Center for Antimicrobial Resistance and Microbial Genomics, UTHealth, Houston, TX,
  Houston, Texas

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• JR

  Jinnethe Reyes, MSc, PhD

  Postdoctoral fellow
  Molecular Genetics and Antimicrobial Resistance Unit and International Center for Microbial Genomics, Universidad El Bosque, Bogota, Colombia
  Bogota, Distrito Capital de Bogota, Colombia

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• DP

  Diana Panesso-Botero, PhD

  Assistant Professor
  UTHSC at Houston Mc Govern Medical School
  Houston, Texas

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• MR

  Maria I. Reyes, n/a

  Research Assistant
  UTHSC at Houston Mc Govern Medical School
  Houston, Texas

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• RR

  Rafael Rios, MSc

  Assistant Professor
  Universidad El Bosque
  Bogota, Distrito Capital de Bogota, Colombia

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• OO

  Oscar Ortega-Recalde, MD, MSc, PhD

  Assistant Professor
  Universidad El Bosque
  Bogota, Distrito Capital de Bogota, Colombia

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• LD

  Lorena Diaz, Ph.D.

  Associate Professor
  Universidad El Bosque
  Bogota, Cundinamarca, Colombia

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• SR

  Sandra Rincon, PhD

  Professor
  Universidad el Bosque
  Bogota, Distrito Capital de Bogota, Colombia

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• BM

  Barbara E. Murray, M.D.

  Professor and Division Director
  Center for Antimicrobial Resistance and Microbial Genomics, UTHealth, Houston, TX,
  Houston, Texas

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• KS

  Kavindra V. Singh, Ph.D.

  Professor
  Center for Antimicrobial Resistance and Microbial Genomics, UTHealth, Houston, TX,
  Houston, Texas

  Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.
• CA

  Cesar A. Arias, M.D., MSc, Ph.D., FIDSA

  Professor
  CARMiG, UTHealth and Center for Infectious Diseases, UTHealth School of Public Health, HOU, TX ; Molecular Genetics and Antimicrobial Resistance Unit and International Center for Microbial Genomics, Universidad El Bosque, BOG, COL
  Houston, Texas

  Disclosure: Entasis Therapeutics : Scientific Research Study Investigator; MeMed : Scientific Research Study Investigator; Merck : Grant/Research Support