Method for biofilm dispersal

Abstract

The present invention relates to a method for dispersing biofilm using mannose and its analogs.

Claims

1. A method to disperse existing biofilm comprising: i. providing a biofilm wherein the biofilm comprises sulfate-reducing prokaryotes; and ii. contacting the biofilm with a compound selected from the group consisting of mannose, 2-deoxy-D-glucose (2DG), methyl α-D-mannopyranoside (αMM), methyl α-D-glucopyranoside (αMG), and mixtures thereof to disperse the biofilm.

2. The method of claim 1 wherein biofilm comprises anaerobic bacteria.

3. The method of claim 1 wherein biofilm comprises sulfate reducing bacteria.

4. The method of claim 2 wherein the biofilm comprises Desulfovibrio vulgaris (ATCC 29579).

5. The method of claim 2 wherein the biofilm comprises Desulfovibrio desulfuricans (DSM 12129).

Description

EXAMPLES

(1) Cultures of Desulfovibrio vulgaris (ATCC 29579) and Desulfovibrio desulfuricans (DSM 12129) were prepared in Modified Baar's medium at 30 C under anaerobic conditions. The bacterial cultures were then used to prepare bacterial suspensions in fresh Modified Baar's medium to a cell density of 0.1 (Table 2 to 6) or 0.05 (Table 1) at 600 nm. The appropriate bacterial suspension was used to fill 96-well plates and the plates were incubated in an anaerobic glove box for 24 h (Table 2 to 6) or 48 h (Table 1) at 30° C. to develop biofilms. Stocks of D-mannose (2.85 M, Alfa Aesar, Cat #A10842), 2DG (1.22 M, Alfa Aesar, Cat #AAAL07338-06), αMM (1.22 M, Acros Organics, Cat #AC229251000), and αMG (1.22 M, Alfa Aesar, Cat #AAA12484-22) were prepared in sterile distilled water and filtered through a 0.22 μm filter. For biofilm dispersal assay, the planktonic cells were removed, and the plates were washed with 150 μL of 1× of phosphate buffered saline (pH 7.4); D-mannose, 2DG, αMM, and αMG were added, the volume was adjusted with 1×PBS, pH 7.4 to 150 μL, and the plates were incubated in the anaerobic glove box for 2 h (Table 1, 2, 4, 5, 6) or 14 h (Table 3) for biofilm dispersal studies. After the treatment, supernatants were discarded; the wells were washed three times with deionized water (DW) by dipping the plates into a 1 L solution of DW, and the plates were dried via a piece of paper towel by patting. 300 μL of 0.1% crystal violet was added to each well, the plates were incubated for 20 minutes at room temperature (25° C.), and the staining solution was discarded. The plates were washed three times with DW by dipping the plates into a 1 L solution of DW, then 300 μL of 95% ethanol was added to each well, and the plates were soaked for 5 min to dissolve the crystal violet. Total biofilm was measured spectrophotometrically at 540 nm using a Sunrise microplate reader (Tecan, Austria Gesellschaft, Salzburg, Austria). Total biofilm remaining after the treatments were summarized in Table 1 to 6.

(2) TABLE-US-00001 TABLE 1 Remaining biofilm of D. vulgaris after 2 h mannose treatment Concentration OD540 Standard deviation 500 mM 0.46 0.04 100 mM 0.48 0.06  30 mM 0.52 0.01 Negative control 0.92 0.02

(3) TABLE-US-00002 TABLE 2 Remaining biofilm of D. vulgaris after 2 h 2DG treatment Concentration OD540 Standard deviation 500 mM 0.18 0.07 100 mM 0.17 0.04  30 mM 0.24 0.06 Negative control 0.35 0.10

(4) TABLE-US-00003 TABLE 3 Remaining biofilm of D. vulgaris after 14 h 2DG treatment Concentration OD540 Standard deviation 10 mM  0.16 0.06 5 mM 0.21 0.01 1 mM 0.24 0.05 Negative control 0.26 0.03

(5) TABLE-US-00004 TABLE 4 Remaining biofilm of D. vulgaris after 2 h αMM treatment Concentration OD540 Standard deviation 500 mM 0.23 0.01 100 mM 0.40 0.11  30 mM 0.42 0.09 Negative control 0.46 0.11

(6) TABLE-US-00005 TABLE 5 Remaining biofilm of D. vulgaris after 2 h αMG treatment Concentration OD540 Standard deviation 500 mM 0.45 0.05 100 mM 0.33 0.05  30 mM 0.39 0.03 Negative control 0.52 0.12

(7) TABLE-US-00006 TABLE 6 Remaining biofilm of D. desulfuricans after 2 h treatment with mannose and its analogs Concentration of treatment OD540 (average) Standard deviation Mannose 500 mM 0.39 0.02 100 mM 0.43 0.02 30 mM 0.49 0.01 2DG 500 mM 0.41 0.02 100 mM 0.49 0.05 30 mM 0.59 0.13 αMM 500 mM 0.50 0.02 100 mM 0.53 0.03 30 mM 0.60 0.12 αMG 500 mM 0.55 0.06 100 mM 0.62 0.06 30 mM 0.75 0.15 Negative control 0.88 0.17