Effect of DNase and antibiotics on biofilm characteristics

Abstract

The role of extracellular DNA in the maintenance of biofilms formed by gram-positive and gram-negative bacteria was studied. This study evaluated all the bacterial strains that were tested for the presence of extracellular DNA with an average size of 30 kb in the matrix. Our results indicate changes in community biomass, architecture, morphology, and the numbers of CFU in the presence of DNase. This effect seems to be common to biofilms established by various unrelated gram-positive and gram-negative bacteria. The cleavage of extracellular DNA leads to the formation of an altered biofilm that permits the increased penetration of antibiotics. Thus, the addition of DNase enhances the effect of antibiotics, resulting in decreased biofilm biomass and numbers of CFU.

FIG. 1.

Photomicrographs illustrating the effects of different DNase I concentrations on established E. coli ATCC 25922 biofilm formation. Biofilms were formed on the coverslips within 24 h at 37°C. Established biofilms were treated with DNase I at 0.5 μg/ml, 1.0 μg/ml, or 5.0 μg/ml for 24 h at 37°C; stained with 0.1% crystal violet; and photographed at ×100 magnification. The black arrows denote the regions that are free of cells; microcolonies are indicated by white arrows. Bars, 50 μm. (A) Control (untreated); (B to D) treatment with DNase I at 0.5 μg/ml, 1.0 μg/ml, and 5.0 μg/ml, respectively, for 24 h.

FIG. 2.

Quantitative biofilm analysis of the effect of the combined use of DNase I and various antibiotics on 24-h-old biofilms. Preformed 24-h-old biofilms were treated with a particular antibiotic (50 times the MIC), DNase I (5 μg/ml), or the antibiotic (50 times the MIC) plus DNase I (5 μg/ml) in the appropriate medium and were incubated at 37°C for 24 h. The bacteria adhering to the microtiter plates were stained, and the absorbance (the optical density at 570 nm [OD₅₇₀]) was measured as described in Materials and Methods. Each bar represents the average of three or four experiments, with error bars representing the standard errors of the means. A P value of <0.05 was considered statistically significant. (A) Treatment with ampicillin, DNase I, or ampicillin plus DNase I; (B) treatment with cefotaxime, DNase I, or cefotaxime plus DNase I; (C) treatment with levofloxacin, DNase I, or levofloxacin plus DNase I; (D) treatment with rifampin, DNase I, or rifampin plus DNase I; (E) treatment with azithromycin, DNase I, or azithromycin plus DNase I.