In this study, we compared and evaluated the antimicrobial effect of dapsone along with the other clinically tested antibiotics (doxycycline, rifampin, azithromycin, cefuroxime) on the growth and viability of attached B. burgdorferi biofilms using standard crystal violet biofilm mass and dimethylmethylene blue glycosaminoglycan assays combined with BacLight Live/Dead microscopic analysis. As in the previous studies from our group and others [30, 35], we tested two different antibiotic concentrations (10µM and 50µM) against attached B. burgdorferi biofilm structures. The 10µM in vitro concentration corresponds well to the achievable serum level after administration of the antibiotics tested in this study [37, 38, 39, 40, 41]. Recent studies showed however that higher 50µM concentrations for these antibiotics could be very effective against persister cells [27, 35], therefore this higher concentration was also tested.
First, the effectiveness of the single and combination antibiotic treatments on attached Borrelia biofilm was quantified by crystal violet biofilm mass assay after 72h treatments with various antibiotics. The most significant results were achieved with individual and combination treatments with dapsone, as listed in Table 1. The best reduction in biofilm mass by a single antibiotic was achieved with dapsone at 10µM and 50µM concentrations resulting in 69% and 58% residual viability respectively, when compared to the PBS treated control (p value <0.01). Rifampin at both 10µM and 50µM concentrations also resulted in a significant decrease in biofilm mass (76% and 60% respectively, p values <0.01) compared to the PBS treated negative control samples, although not quite as effective as dapsone alone at comparable concentrations. Treatments with other single antibiotics including doxycycline, cefuroxime, and azithromycin were less effective and, in some cases, even increased biofilm size when compared to the PBS treated control (Table 1).
The most effective dual combination was dapsone+doxycycline at both 10µM and 50µM concentrations (68% and 65% residual viability respectively) significantly decreasing biofilm size compared to the PBS treated control (p value <0.01; Table 1). However, when compared to dapsone alone treated samples, this dual combination was not more effective than dapsone alone (p value >0.05)
The triple combination treatment of dapsone+doxycycline+rifampin (52% residual viability) and quadruple combination of dapsone+doxycycline+rifampin+azithromycin (58% residual viability) treatments both at 50µM concentration were the most effective when compared to the PBS treated control (p values < 0.01), however, the effect of three and four drug combinations at 50µM was not significantly better than the 50µM concentration treatments of dapsone alone or dapsone+doxycycline (p values > 0.05).
Crystal violet biofilm assay only measures the cellular mass (both live or dead) and does not provide information about the viability and the individual sizes of the antibiotic treated biofilm aggregates. Therefore, Live/Dead fluorescent microscopy techniques were used to visualize the effect of the most effective single and combination treatments after 72h with different antibiotics and represented images are presented in Figure 1. Biofilm cultures treated only with PBS show live (green) and compact morphology (Figure 1; Panels A and B). For single and dual antibiotic treatments, the microscopy data were in good agreement with the crystal violet data. For example, it confirmed that single treatment dapsone and dual treatment dapsone+doxycycline were indeed effective in reducing Borrelia biofilm size, resulting in very small aggregates (less than 20 µm aggregates [Figure 1, Panel C]).
However, for triple and quadruple antibiotic treatments, such as dapsone+doxycycline+rifampin and dapsone+doxycycline+rifampin+azithromycin, the microscopy images suggest a more significant effect of these triple and quadruple combinations than dapsone or dapsone+doxycycline as demonstrated by very small (less than 10 µm) and disorganized biofilms structures (Figure 1, Panels K and L).
In order to further evaluate the effectiveness of the antibiotics on the attached biofilm form of B. burgdorferi, the amount of the protective layers of biofilm polysaccharide matrix of these aggregates were measured using the DMMB glycosaminoglycan (GAG) assay before and after 72h of antibiotic treatments. Biofilms treated with negative control, PBS, showed no reduction in the amounts of GAG, when compared with the untreated biofilm control. The most significant results with the different single and combination of antibiotics are summarized in Table 2. Single antibiotic treatments at 10µM of rifampin and doxycycline were the most effective resulting in 75% and 76% residual GAG amounts compared to the PBS treated control (p value <0.01). However, at 50µM, dapsone showed the most significant effects with 68% residual GAG amounts of Borrelia biofilm compared to the untreated control (p value <0.01). For combination therapies, dapsone+doxycycline+rifampin at 10µM and dapsone+doxycycline+rifampin+azithromycin at 50µM were the most effective agents when compared with the untreated control with 69% and 62% residual GAG amounts respectively. The 50µM quadruple combination data were found to be significant not just in comparison to the negative control but to any single treatment result (p value <0.05).