Increased Temperature Enhances the Antimicrobial Effects of Daptomycin,Vancomycin, Tigecycline,Fosfomycin, and Cefamandole on Staphylococcal Biofilms

Implant-related infections are serious complications of trauma and orthopedic surgery and are most difficult to treat. The bacterial biofilms of 34 clinical Staphylococcus sp. isolates (Staphylococcus aureus, n = 14; coagulase-negative staphylococci, n = 19) were incubated with daptomycin (DAP; 5, 25, or 100 mg/liter), vancomycin (VAN; 5, 25, or 100 mg/liter), tigecycline (TGC; 1, 5, or 25 mg/liter), fosfomycin (FOM; 100, 250, or 1,000 mg/liter), and cefamandole (FAM; 50, 100, or 500 mg/liter) for 24 h at three different ambient temperatures: 35°C, 40°C, and 45°C. To quantify the reduction of the biomass, the optical density ratio (ODr) of stained biofilms and the number of growing bacteria were determined. Increasing the temperature to 45°C or to 40°C during incubation with FAM, FOM, TGC, VAN, or DAP led to a significant but differential reduction of the thickness of the staphylococcal biofilms compared to that at 35°C (P < 0.05). Growth reduction was enhanced for DAP at 100 mg/liter at 35°C, 40°C, and 45°C (log count reductions, 4, 3.6, and 3.3, respectively; P < 0.05). A growth reduction by 2 log counts was detected for FAM at a concentration of 500 mg/liter at 40°C and 45°C (P = 0.01). FOM at 1,000 mg/liter reduced the bacterial growth by 1.2 log counts (not significant). The antibacterial activity of antimicrobial agents is significantly but differentially enhanced by increasing the ambient temperature and using high concentrations. Adjuvant hyperthermia may be of value in the treatment of biofilm-associated implant-related infections.Implant-related infections are severe complications of trauma and orthopedic surgery that frequently require long-term antimicrobial treatment, supportive management, and multiple additional surgical procedures (53). Staphylococcus aureus and coagulase-negative staphylococci (CoNS), primarily Staphylococcus epidermidis, are the most common organisms associated with implant-related infections after trauma and orthopedic surgery (13).Bacterial biofilms develop on the surfaces of the implants (12, 13, 30, 41). The biofilm consists of a structured community of bacterial cells enclosed in a self-produced polymeric matrix that adheres to an inert or living surface. The bacteria embedded in the biofilm are quasiprotected by this self-made polymeric matrix; thus, resistance to antimicrobials is increased such that the concentrations needed to kill the biofilm bacteria are 500 to 5,000 times higher than the levels needed to kill planktonic bacteria (8, 12, 13). Although removal of the colonized foreign material may be the most effective means to treat biofilm-associated infections (14), the implant must sometimes be retained because of technical or physiological complications. In this case, any hope for cure or at least the stability of the patient lies in antimicrobial treatment alone.Antimicrobial agents for the treatment of staphylococcal infections include beta-lactam antibiotics and, in the case of methicillin resistance, vancomycin (VAN), fosfomycin (FOM), tigecycline (TGC), or daptomycin (DAP) (32, 38). FOM is a small-molecule antibiotic with a wide antibacterial spectrum and excellent tissue penetration, representing an excellent alternative antimicrobial agent for the treatment of deep-seated infections (18, 27). Both TGC and DAP are highly active against Gram-positive cocci resistant to commonly used antibiotics, including methicillin-resistant staphylococci (18, 43). Antimicrobial agents may reduce biofilm and bacterial growth (35, 39). In a previous study, we demonstrated that the antibiotic concentrations achieved under normal physiological conditions did not decrease the growth of established staphylococcal biofilms (21). Bacterial biofilm growth was significantly reduced by the use of antimicrobials at excessive concentrations or when antimicrobials were used in combination with azithromycin (21, 37).Because the effects of antibiotics on established biofilms are unsatisfactory, other measures to reduce biofilm thickness and to kill biofilm bacteria may be helpful (37). Adjunctive therapy to enhance the activity of an antimicrobial to save an infected but unremovable implant or to improve the engraftment of a new implant after reimplantation may be a benefit for the patient (13, 16). Experimental measures taken to reduce bacterial biofilms included antibiotic combination therapy, particularly with rifampin; therapy with a combination of antibiotics and chemicals like EDTA or N-acytylcysteine; or therapy with a combination of antibiotics and physical measures, such as ultrasound or an electric current (7, 15, 39, 51). Low-frequency ultrasound was used to prevent uncontrolled heating and associated tissue damage (5, 6). For physical therapy, however, ultrasound of 1 MHz (unpulsed) had been described to have a beneficial effect on osteoarthritis (45). Further, application of heat has traditionally been used in physical therapy with various rates of success and minimal harm (9).Increasing the temperature induced the formation of thicker biofilms (40). Although the amount of heating was never measured in the ultrasound or electric current experiments, heating may have an influence on the effects of antibiotics on biofilms. Thus, we investigated the effects of stepwise increases in the ambient temperature together with antibiotic treatment on biofilm thickness and bacterial growth. Static biofilms of clinical isolates from orthopedic implant infections or preoperative skin isolates, including S. aureus and CoNS (S. epidermidis, S. lugdunensis, S. hominis, and S. capitis), were incubated at an ambient temperature of 35, 40, or 45°C for 24 h with DAP, VAN, FOM, TGC, or FAM. Three concentrations of each of the antimicrobial agents were investigated.(Some of the data presented here were presented as poster K2069 at the 48th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy-Infectious Diseases Society of America 46th Annual Meeting, San Francisco, CA, 2009.)