Effects of Azithromycin in Combination with Vancomycin,Daptomycin, Fosfomycin,Tigecycline, and Ceftriaxone on Staphylococcus epidermidis Biofilms

Staphylococcal biofilms on surgical implants are the underlying cause of a lack of response to antimicrobial treatment. We investigated the effects of vancomycin (VAN), daptomycin (DAP), fosfomycin (FOS), tigecycline (TGC), and ceftriaxone (CRX), alone and in combination with azithromycin (AZI), on established biofilms of Staphylococcus epidermidis. Biofilms were studied using the static microtiter plate model with established S. epidermidis biofilms, with an initial inoculum of 10⁶/ml in 96-well polystyrene flat-bottom microtiter plates. Biofilms were inoculated with VAN, DAP, FOS, TGC, or CRX at two concentrations, alone or in combination with AZI (2, 512, or 1,024 mg/liter). To assess the reduction in biomass, the optical density ratio (ODr), calculated as (optical density [OD] of the treated biofilm)/(OD of the untreated biofilm, taken as 1), was used. For antibacterial efficacy, the viable bacterial count was used. Reductions in the biofilm ODr were observed for VAN (15 and 40 mg/liter) and FOS (200 mg/liter) only (ODr [mean ± standard deviation] for VAN at 15 and 40 mg/liter, 0.77 ± 0.32 and 0.8 ± 0.35, respectively; ODr for FOS at 200 mg/liter, 0.78 ± 0.26; P < 0.05), but not for DAP (2 and 5 mg/liter), TGC (0.2 and 2 mg/liter), or CRX (600 and 2,400 mg/liter). The addition of AZI had no further effect on the ODr, but a significant reduction of bacterial growth was achieved with high doses of AZI plus TGC or AZI plus CRX (a 3-log count reduction for AZI at 1,024 mg/liter plus CRX at 600 mg/liter and for AZI at 512 or 1,024 mg/liter plus CRX at 2,400 mg/liter; a 2-log count reduction for AZI at 512 or 1,024 mg/liter plus TGC at 2 mg/liter [P < 0.05]). No significant reduction in bacterial growth was observed for FOS (50 and 200 mg/liter), DAP (2 and 5 mg/liter), or TGC (0.2 mg/liter) in combination with AZI. None of the antibiotics at either concentration reduced the bacterial count of the biofilms when used alone. Thus, the use of a combination of AZI plus TGC, FOS, or CRX at high concentrations has little effect on biofilm density but significantly reduces bacterial growth.Staphylococcus epidermidis is the leading pathogen causing infections of surgical implants. Given the high incidence of fracture fixation devices, 2 million per annum, the number of implant infections amounts to 100,000 per year in the United States (10). Many of these infections are associated with biofilms formed by staphylococci on implant surfaces (9, 26, 34). The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. The two consequences of biofilm formation on implant surfaces are increased resistance to antimicrobial agents and frequent failure of conventional antimicrobial therapy. This resistance of bacteria within biofilms is attributed to a possible barrier function of the biofilm, binding of the antimicrobial agents within the matrix, and the metabolic change in the bacterial cells. Thus, infection of medical implants is associated with considerable morbidity and costs due to loss of mobility, nonproductive time, and health care (10, 18, 35).The antimicrobial agents most widely used for staphylococcal infections are beta-lactam antibiotics, primarily cephalosporins, and in the case of beta-lactam resistance, vancomycin (23, 28). Alternative agents are intravenous fosfomycin, a small-molecule antibiotic with a wide antibacterial spectrum and excellent tissue penetration, and two newer antibiotics, the glycylcycline tigecycline and the cyclic lipopeptide daptomycin (14). Daptomycin, which is highly active against gram-positive cocci resistant to commonly used antibiotics, including methicillin (meticillin)-resistant staphylococci, causes membrane changes in the bacteria, whereas tigecycline inhibits protein synthesis (20). Although macrolides are not commonly used in the treatment of staphylococcal infections, clinical and experimental data suggest that azithromycin decreases biofilm formation and enhances the efficacy of other antibiotics for patients with cystic fibrosis and Pseudomonas infections (17, 19).The MICs of antimicrobial agents tested on bacterial biofilms are dramatically increased, up to concentrations >1,000 times the MICs for staphylococci under planktonic conditions (5). However, there is some evidence that higher antibiotic concentrations reduce biofilms and bacterial growth, whereas standard concentrations are not effective (24, 30). In a previous study, we demonstrated that antibiotic concentrations equivalent to 100 times the MIC under planktonic conditions did not decrease established staphylococcal biofilms (16). To investigate if even higher concentrations will overcome the bacterial resistance within biofilms and reduce biofilm thickness and bacterial growth, static biofilms of clinical S. epidermidis isolates causing implant infections and catheter-associated bacteremia were incubated with vancomycin, daptomycin, fosfomycin, tigecycline, or ceftriaxone at two concentrations. To explore the additional effect of the macrolide azithromycin (22) on S. epidermidis biofilms, we treated the biofilms with vancomycin, daptomycin, fosfomycin, tigecycline, or ceftriaxone in combination with azithromycin at three concentrations (2, 512, and 1,024 mg/liter).(Part of this research was presented as a poster at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 [29a].)