Bactroban

Atlanta, Ga. -- Biofilm formation may lead to increased antimicrobial resistance and create a significant impediment to wound healing, according to Stephen C. Davis, research associate professor in the department of dermatology and cutaneous surgery at the University of Miami School of Medicine. Biofilms have been implicated in several chronic diseases associated with bacterial infection, such as recurrent urinary tract infection and otitis media.

Antimicrobial efficacy is typically determined using in vitro assays that assess the effect of the antimicrobial agent on growth of planktonic, or free-floating, bacteria. However, bacteria in wounds can form colonies that have different properties from planktonic bacteria. "Bacterial biofilms are tissue-like and resemble multicellular structures, whereas planktonic bacteria are free-floating, independent cells" Professor Davis says. "Recent studies have demonstrated that biofilm-associated bacteria have certain physiologic and molecular differences from their planktonic counterparts."

Professor Davis and colleagues used a porcine model to examine whether biofilms formed in acute wounds infected with Staphylococcus aureus and, if so, whether conventional antimicrobial therapy was effective in treating biofilm-associated bacteria, compared to planktonic bacteria.

In one set of experiments, partial thickness wounds were created and infected with S. aureus. Wounds in the experimental group were covered with a polyurethene film to encourage biofilm growth. Freshly infected wounds served as controls representing planktonic bacterial growth. Scanning electron microscopy (SEM), light microscopy (LM), and epiluminescent microscopy (EpiLM) were used to visualize bacterial growth in biopsy specimens from both types of wounds.

Wounds were made on the backs of three young pigs using a modified electrokeratome at a setting of 0.3 mm (depth) x 5 mm x 7 mm. For wound inoculation, a fresh culture of S. aureus was grown from a clinical isolate obtained from the American Type Culture Collection. Bacteria from overnight growth were scraped from culture plates and suspended in saline solution at a density of approximately 107 CFU/ml. The partial thickness wounds were infected by lightly scrubbing a 5 ml aliquot of the bacterial suspension into the wounds.

For visualization of planktonic bacteria, three 3 mm punch biopsies (for SEM) and four incisional biopsies (for LM and EpiLM) were taken immediately after inoculation of the control wounds. The remaining wounds were covered with a polyurethane film held in place with self-adherent bandages. After 48 hours, the film was removed and biopsy specimens were taken for microscopic examination.

Aggregates of microcolonies of cocci bacteria, embedded within and attached to the wound bed, were clearly visible in the biopsy specimens after 48 hours. "The colonies of bacteria found within our wound colonization model represent the biofilm-like structures," Professor Davis says.

In a second set of experiments, 93 partial thickness wounds were made on the paravertebral area of each of three young pigs. The wounds were infected with S. aureus as before. Cultures were obtained immediately from three wounds on each animal, to provide baseline cultures before antimicrobial treatment. The remaining wounds were either treated immediately (planktonic group, n=45) or covered with polyurethane film dressing for 48 hours before treatment (biofilm group, n=45). Wounds in each group were randomly assigned to one of three treatment groups: mupirocin cream (Bactroban, GlaxoSmithKline), triple antibiotic ointment containing bacitracin zinc, polymyxin B sulfate and neomycin (Neosporin, Pfizer), or no treatment. Treatment was applied twice daily for 5 days.

Neither treatment was able to completely eliminate the biofilm-associated S. aureus. At 72 hours after the initial treatment, bacterial load in the biofilm group treated with mupirocin cream was 5.3 [+ or -] 0.4 log CFU/ml, compared to 4.9 [+ or -] 0.7 for the biofilm group treated with triple antibiotic cream and 6.7 [+ or -] 0.4 for the untreated biofilm group. By contrast, no bacteria were detected in either of the two active treatment planktonic groups after 72 hours. These data show that both topical antimicrobial agents were much less effective in reducing S. aureus counts in the biofilm group.

"The role biofilms play in the pathogenesis of chronic wound colonization/infection, acute wound infection and surgical site infections has yet to be determined. What we anticipate to see in the future is more rational use of antimicrobial therapy based on assays using the biofilm phenotype of bacteria," Professor Davis says.

BARBARA J. RUTLEDGE, PH.D. STAFF CORRESPONDENT

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