The first of these mechanisms suggests that the biofilm glycocalyx prevents the perfusion of biocides to cellular targets, while the second recognizes the nearly dormant growth pattern of bacterial populations within the biofilm that renders organisms indifferent to antibiotic activity. The third proposes that the microenvironment of the biofilm adversely affects the activity of the antimicrobials (
4,
9,
40,
85).
In support of the first of these proposals, Farber et al. (
35) found that cell extracts of the slime polysaccharide of
S. epidermidis interfered with the antimicrobial activity of glycopeptide antibiotics. The addition of 0.5% slime extract to broth microdilution susceptibility plates increased the MIC of both vancomycin and teichoplanin approximately fivefold versus both slime-positive and -negative strains. Slime also negated the synergistic effects of both vancomycin and gentamicin while having no effect on the activity of clindamycin, rifampin, and cefazolin. The authors suggested that the slime either physically complexes with and inactivates glycopeptides or coats the cell wall to create a permeability barrier. In an in vitro model, however, Dunne et al. (
31) showed that a slime-positive
S. epidermidis biofilm did not prevent the perfusion of vancomycin or rifampin, nor could it be sterilized in the presence of either or both antibiotics at concentrations exceeding bactericidal levels (Fig.
2). Nickel and colleagues (
72) created an artificial
Pseudomonas aeruginosa biofilm on urinary catheter material using a modified Robbins device and showed that exposure to 1 g of tobramycin/ml for 12 h did not sterilize the biofilm. Interestingly, the MIC of tobramycin for the surviving organisms was not affected. In a related study, Nichols et al. (
71) investigated the binding of [
3H]tobramycin to the alginic acid exopolysaccharide produced by mucoid strains of
Pseudomonas aeruginosa and to commercially prepared alginate. The authors were able to show concentration-dependent binding of tobramycin to both. As further evidence of this activity, the addition of 1% alginate to tobramycin in a well diffusion assay demonstrated reduced zones of inhibition versus
Escherichia coli and
Staphylococcus aureus, indicating that this exopolysaccharide interferes with either the antimicrobial action of the drug or the perfusion of tobramycin through the medium.
These observations were supported by the report of Coquet et al. (
15), who exposed alginate-embedded biofilms of
P. aeruginosa to 15 times the MIC of tobramycin, 20 times the MIC of imipenem, or both and compared the results to those for planktonic cultures of the same organism. While the planktonic cultures showed an approximately 100,000-fold reduction in the viable cell count after 6 h of exposure to either tobramycin or imipenem, neither drug produced more than a 1,000-fold reduction in the viable cell count of embedded organisms after 10 h of incubation. Furthermore, while the combination of tobramycin and imipenem demonstrated a synergistic effect against planktonic organisms, no such effect was observed with alginate-embedded bacteria. Hoyle et al. (
49) showed that biofilms of
P. aeruginosa established on dialysis membranes retarded the diffusion of piperacillin. In the presence of Ca
2+, the diffusion of piperacillin was completely prevented, presumably by creating an alginic acid-Ca
2+ barrier matrix.