Impact of sarA on Antibiotic Susceptibility of Staphylococcus aureus in a Catheter-Associated In Vitro Model of Biofilm Formation

Abstract

Mutation of the staphylococcal accessory regulator (sarA) in Staphylococcus aureus limits but does not abolish the capacity of the organism to form a biofilm. As a first step toward determining whether this limitation is therapeutically relevant, we carried out in vitro studies comparing the relative susceptibility of an S. aureus clinical isolate (UAMS-1) and its isogenic sarA mutant (UAMS-929) in the specific context of a catheter-associated biofilm. The antibiotics tested were daptomycin, linezolid, and vancomycin, all of which were evaluated by using concentrations based on the MIC defined as the breakpoint for a susceptible strain of S. aureus (<1.0, <2.0, and <4.0 g/ml for daptomycin, vancomycin, and linezolid, respectively). Mutation of sarA had no significant impact on the MIC of UAMS-1 for any of the targeted antibiotics, as defined by Etest antimicrobial susceptibility testing. However, mutation of sarA did result in a significant increase in antimicrobial susceptibility to all targeted antibiotics when they were tested in the specific context of a biofilm. Additionally, whether susceptibility was assessed by using UAMS-1 or its sarA mutant, daptomycin was found to be more effective against established S. aureus biofilms than either linezolid or vancomycin.Staphylococcus aureus is a devastating human pathogen, with the death toll from invasive S. aureus infections recently having passed that from AIDS in the United States (9, 18). These infections range from acute toxemias and septic shock to more chronic infections, including osteomyelitis and infections associated with indwelling medical devices. The latter are characterized by the formation of a biofilm, which has a significant impact not only on the disease process itself but also on the ability of clinicians to effectively treat the infection. This is true because biofilm-associated infections are recalcitrant to antimicrobial therapy, irrespective of the resistance status of the offending strain or the ability to achieve what would otherwise be therapeutic serum levels of antibiotic (10,21,30). For this reason, the resolution of biofilm-associated staphylococcal infections often requires surgical debridement to remove infected tissues and/or indwelling devices (5,20).An alternative approach to the therapeutic problem of biofilm-associated infection would be the development of methods that specifically prevent or at least limit biofilm formation. This could be done by targeting either the substrate (e.g., by developing novel biomaterials that are less conducive to biofilm formation) or the bacterium (e.g., by developing novel agents capable of limiting biofilm formation). The latter approach requires the identification of those bacterial targets that are most relevant in the specific context of a biofilm. Studies focusing on the staphylococci have led to the identification of many potential targets (24). However, in almost every case, there are conflicting reports about the roles of different genes and gene products in biofilm formation (12,24,35)...

Cis-2-decenoic Acid Inhibits S. aureus Growth and Biofilm In Vitro: A Pilot Study

“…1). Each of these C2DA concentrations was also combined with twofold dilutions of three different antibiotics to which UAMS-1 was susceptible, daptomycin, vancomycin, and linezolid [46], and the dependent variables turbidity and biofilm mass were measured (n = 3). We then added C2DA, dissolved in the same ethanol concentration as bacteriological studies, to a culture of normal human dermal fibroblasts to evaluate cell number after 24 and 48 hours of exposure to C2DA (n = 4).…”

Novel Antibiotic-loaded Point-of-care Implant Coating Inhibits Biofilm

“…The limitations of the animal model include the small size, soft tissue instead of orthopaedic implantation, and limited diffusion of physiologic substances within the sequestrum of the catheter. The animal model is useful as a screening tool for antibiofilm activity in vivo [54,74,75], where the catheter serves as a sequestrum for localizing the bacterial inoculum and as a nidus for biofilm formation. Because of difficulty in applying coatings directly to the catheter, we modified the catheter-associated biofilm model by including an easily coated stainless steel wire.…”

“…One-way Kruskal-Wallis analysis of variance with Student-Newman-Keuls test post hoc were performed using Sigma Plot (Systat Software, Inc, San Jose, CA, USA) to determine whether colony counts in coated coupons or wires differed from those in control groups. Using results of previous studies determining log-fold reduction of CFUs in response to antimicrobials [74], an a priori power analysis was performed using SigmaPlot. Assuming a SD of 1 unit in log-transformed CFU counts, five animals per group were required for 80% power to detect a difference of 3 units between the control group and each of the four experimental treatment groups at a significance level of 5%.…”

“…An established catheter biofilm model, using a subcutaneous dorsal implantation of a catheter segment [74], was modified to include a stainless steel wire (0.015-inch diameter, type 316L) that could be coated with antibiotic-loaded coating. To place the wire within the catheters without sharp edges exposed, the stainless steel wires were bent into loop shapes ( Fig.…”

Could Targeted, Antibiotic-Loaded Gold Nanoconstructs be a New Magic Bullet to Fight Infection?

“…The first and arguably most important of these is that, since the advent of the 'antibiotic era', the emergence of antibiotic resistance has quickly out-paced the development of novel traditional antimicrobial drugs, a fact that is exacerbated by the decreased effort by the pharmaceutical industry to develop new antibiotics [9]. This problem is further exacerbated by the fact that many pathogenic bacteria are capable of forming a biofilm, an 'alternative lifestyle' that confers a therapeutically relevant level of intrinsic resistance to all currently available antibiotics [10,11].…”