Regulatory Mutations Impacting Antibiotic Susceptibility in an Established Staphylococcus aureus Biofilm

Abstract

We previously determined the extent to which mutations of different Staphylococcus aureus regulatory loci impact biofilm formation as assessed under in vitro conditions. Here we extend these studies to determine the extent to which those regulatory loci that had the greatest effect on biofilm formation also impact antibiotic susceptibility. The experiments were done under in vitro and in vivo conditions using two clinical isolates of S. aureus (LAC and UAMS-1) and two functionally diverse antibiotics (daptomycin and ceftaroline). Mutation of the staphylococcal accessory regulator (sarA) or sigB was found to significantly increase susceptibilities to both antibiotics and in both strains in a manner that could not be explained by changes in the MICs. The impact of a mutation in sarA was comparable to that of a mutation in sigB and greater than the impact observed with any other mutant. These results suggest that therapeutic strategies targeting sarA and/or sigB have the greatest potential to facilitate the ability to overcome the intrinsic antibiotic resistance that defines S. aureus biofilm-associated infections.

FIG 1

Relative daptomycin susceptibility in vitro. Daptomycin susceptibility in LAC, UAMS-1, and the indicated mutants was assessed using a catheter-based model of biofilm formation. The results indicate individual data points. The horizontal bar and error bars indicate the means ± standard errors of the mean (SEM) based on CFU per catheter remaining after antibiotic exposure. An asterisk above an experimental group indicates the statistical significance of each mutant relative to the isogenic parent strain in the absence of antibiotic exposure. An asterisk below a group indicates the significance of each mutant relative to the parent strain after antibiotic exposure. The number above a strain indicates the percentage of catheters cleared of bacteria as defined by the level of detection of our assay. NSR, no significant reduction.

FIG 2

Relative daptomycin susceptibility in vivo. Daptomycin susceptibility was assessed using a murine model of a catheter-based model of biofilm formation. The results indicate individual data points. The horizontal bar and error bars indicate the means ± standard errors of the mean (SEM) based on CFU per catheter remaining after antibiotic exposure. An asterisk below a group indicates the significance after antibiotic exposure. The number above a mutant indicates the percentage of catheters cleared of bacteria below the level of detection. NSR, no significant reduction.

FIG 3

Relative ceftaroline susceptibility in vivo. Ceftaroline susceptibility was assessed using a murine model of a catheter-based model of biofilm formation. The results indicate individual data points. The horizontal bar and error bars indicate the means ± standard errors of the mean (SEM) based on CFU per catheter remaining after antibiotic exposure. An asterisk above an experimental group indicates the statistical significance relative to the isogenic parent strain in the absence of antibiotic exposure. An asterisk below a group indicates the significance after antibiotic exposure. The number above a mutant indicates the percentage of catheters cleared of bacteria below the level of detection. NSR, no significant reduction.

FIG 4

Impact of mutating sarA and sigB on MICs. The MICs of LAC and UAMS-1 sarA and sigB mutants was determined by Etest. DPC, daptomycin; CPT, ceftaroline.