Authors: Smeltzer, MS, Weiss, EC, Beenken, KE, UAMS, Little Rock, AR
Title: Impact of sarA on antibiotic susceptibility in staphylococcal biofilm-associated infection
Purpose: The purpose of these experiments was to assess the impact of the sarA locus on antibiotic susceptibility in the specific context of a Staphylococcus aureus biofilm.
Methods: We used a murine model of catheter-associated biofilm formation to determine whether mutation of the staphylococcal accessory regulator (sarA) has an impact on the susceptibility of established Staphylococcus aureus biofilms to treatment with daptomycin in vivo. The experiments were done with two clinical isolates, one of which (UAMS-1) was obtained from the bone of a patient suffering from osteomyelitis while the other (UAMS-1625) is an isolate of the USA300 clonal lineage of community-acquired methicillin-resistant S. aureus (CA-MRSA). Specifically, a 1 cm FEP catheter segment was placed into a subcutaneous pocket generated in each flank of NIH Swiss mice. After implantation of all catheters (approximately 1 hr), 105 colony-forming units (cfu) of the test strain in a total volume of 100µl of phosphate-buffered saline (PBS) was introduced directly into the lumen of each catheter. After 24 hrs, mice were randomly divided into experimental groups (n = 15). In the untreated groups, 100µl of sterile PBS was injected in the lumen of each catheter at daily intervals. In the treated groups, 100µl of sterile PBS containing the indicated amounts of daptomycin was injected into the lumen, also at daily intervals. In all cases, treatment was continued daily for 7 days, at which point catheter segments were harvested, rinsed in sterile PBS to remove non-adherent bacteria, and sonicated in a total volume of 5.0 ml of PBS to remove adherent bacteria. The total number of viable bacteria colonizing colonizing each catheter was then determined by plating appropriately diluted samples on TSA as described above. Bacterial count data was analyzed using both parametric and nonparametric methods. Specifically, Wilcoxon rank-sum tests were used to make comparisons between untreated samples, while Fisher's exact tests were used to compare clearance percentages among the daptomycin-treatment groups.
Results: Mutation of sarA limited biofilm formation in both strains by comparison to the corresponding parent strain (p = 0.001). It did not affect the minimum inhibitory concentration (MIC) of either strain to daptomycin, but it did result in increased susceptibility in vivo in the context of an established biofilm. Specifically, daptomycin treatment resulted in clearance of detectable bacteria from < 10% of the catheters colonized with the parent strains, while treatment with an equivalent daptomycin concentration resulted in clearance of 46.4% of the catheters colonized with the UAMS-1 sarA mutant and 69.1% of the catheters colonized with the UAMS-1625 sarA mutant. Mice with catheters colonized with the UAMS-1625 parent strain also developed skin lesions in the region adjacent to the implanted catheter. No such lesions were observed in any other experimental group including untreated mice containing catheters colonized with the UAMS-1625 sarA mutant.
Discuassion and Conclusion: the results we present provide support for the hypothesis that inhibitors of sarA would have the capacity to enhance the efficacy of conventional antimicrobial agents, perhaps to the point that at least some biofilm-associated infections could be effectively resolved without surgical intervention and/or removal of the offending device. They also indicate that such inhibitors would not only increase antimicrobial susceptibility in the context of an established biofilm but may also limit the pathology of at least some S. aureus biofilm-associated infections including those caused by CA-MRSA isolates of the USA300 clonal lineage.