Epidemiology and impact of methicillin-sensitive Staphylococcus aureus with β-lactam antibiotic inoculum effects in adults with cystic fibrosis

Abstract

Staphylococcus aureus is the most prevalent cystic fibrosis (CF) pathogen. Several phenotypes are associated with worsened CF clinical outcomes including methicillin-resistance and small-colony-variants. The inoculum effect (IE) is characterized by reduced β-lactam susceptibility when assessed at high inoculum. The IE associates with worse outcomes in bacteremia and other high-density infections, and may therefore be relevant to CF. The prevalence of IE amongst a CF cohort (age ≥18 years), followed from 2013 to 2016, was investigated. Yearly methicillin-sensitive S. aureus (MSSA) isolates were screened at standard (5 × 10⁵ CFU/mL) and high (5 × 10⁷ CFU/mL) inoculum against narrow-spectrum anti-Staphylococcal β-lactams and those with anti-pseudomonal activity common to CF. A ≥ 4-fold increase in minimum inhibitory concentration between standard and high inoculum defined IE. Isolates underwent blaZ sequencing and genotyping and were compared against published genomes. Fifty-six percent (99/177) of individuals had MSSA infection. MSSA was observed at ≥10⁵ CFU/mL in 44.8% of entry sputum samples. The prevalence of the IE was 25.0%-cefazolin; 13.5%-cloxacillin; 0%-meropenem; 1.0%-cefepime; 5.2%-ceftazidime; and 34.4%-piperacillin-tazobactam amongst baseline MSSA isolates assessed. blaZ A associated with cefazolin IE (P = 0.0011), whereas blaZ C associated with piperacillin-tazobactam IE (P < 0.0001). Baseline demographics did not reveal specific risk factors for IE-associated infections, nor were long-term outcomes different. Herein, we observed the IE in CF-derived MSSA disproportionally for cefazolin and piperacillin-tazobactam and this phenotype strongly associated with underlying blaZ genotype. The confirmation of CF being a high density infection, and the identification of high prevalence of MSSA with IE in CF supports the need for prospective pulmonary exacerbation treatment studies to understand the impact of this phenotype.

Keywords: MSSA; antimicrobial resistance; bacteremia; blaZ; bronchiectasis; cefazolin; exacerbation; piperacillin-tazobactam.

Fig 1

Distribution of minimum inhibitory concentrations (MICs) of 223 CF-derived MSSA isolates tested with (A) cefazolin, (B) piperacillin-tazobactam, and (C) meropenem, at standard (SI; grey) and high (HI; black) inoculum. CLSI breakpoints for non-susceptibility are noted for each antibiotic on respective panels by a diamond. Panel B shows piperacillin MIC as a varying concentration, but tazobactam was kept at a constant 4 µg/mL. Statistical significance (t-test) is shown between the MIC₅₀ and MIC₉₀ of isolates tested at standard versus high inoculum with cefazolin and piperacillin-tazobactam (*P < 0.0001).

Fig 2

(A) Distribution of blaZ genotypes across 96 MSSA isolates as a function of the occurrence of inoculum effects (IE) against relevant β-lactam antibiotics. (B) The cefazolin (CZ) IE is associated with type A blaZ (*P = 0.0011), and (C) the piperacillin-tazobactam (TZP) IE is associated with type C blaZ (**P < 0.0001). Associations with blaZ were not noticed for IEs in cloxacillin (CLO), ceftazidime (CAZ), or cefepime (FEP).

Fig 3

Multilocus sequence types (MLSTs), cefazolin (CZ), and piperacillin-tazobactam (TZP) inoculum effect phenotypes amongst 223 MSSA strains, derived from 96 individuals with CF over the course of four years (2013–2016). The TZP inoculum effect was found to be relatively consistent over time, where the re-occurrence of the TZP IE was common in isolates of the same strain type previously having the TZP IE. All three isolates of patient 94 were identified as ST-97, though clustered with ST-1, and were therefore noted as such.