A COMPOSITION AND METHOD FOR THE REMOVAL OF BIOFILM

Abstract

The present invention relates to a synergistic composition and method for removing biofilm using glutaraldehyde and analogs of mannose.

Claims

1. A synergistic composition useful for the removal of biofilm comprising: i) glutaraldehyde; and ii) 2-deoxy-D-glucose.

2. The synergistic composition of claim 1 wherein the weight ratio of glutaraldehyde to 2-deoxy-D-glucose is 2.5:1 to 1:60.

3. A method to remove biofilm comprising: i. providing an aqueous system that comprises a biofilm with at least one sulfate-reducing prokaryote; and ii. contacting the biofilm with the synergistic composition of claim 1.

4. The method of claim 3 wherein the sulfate-reducing prokaryote comprises Desulfovibrio vulgaris.

5. A synergistic composition useful for the removal of biofilm comprising: i) glutaraldehyde; and ii) α-methyl-mannoside.

6. The synergistic composition of claim 5 wherein the weight ratio of glutaraldehyde to α-methyl-mannoside is 2.5:1 to 1:60.

7. A method to remove biofilm comprising: i. providing an aqueous system that comprises at least one sulfate-reducing prokaryote; and ii. contacting the aqueous system with the synergistic composition of claim 5.

8. The method of claim 7 wherein the sulfate-reducing prokaryote comprises Desulfovibrio Vulgaris.

Description

EXAMPLES

Microbicidal Composition

Example: Biofilm Removal Synergy with 2Dg

Stock Culture Preparation

[0014] A lyophilized D. vulgaris pure culture received from ATTCC was resuspended in 500 ul of MB 1249. Aseptically, the content was transferred to a 5¬mL tube of MB1249 medium. The culture was incubated in an anaerobic chamber at 30° C. for 72 hrs. Subsequently, a stock culture with a final concentration of 25% glycerol was prepared by adding equal volumes of culture and 50% glycerol. 1 ml of culture was then transferred to 2-ml cryogenic vials and stored AT-80c. The purity of the stock culture was evaluated through PCR, by amplifying the 16S rDNA region, and thus, it was verified that the original ATCC sample was a pure culture.

[0015] A 10% culture was prepared by taking 1 mL of stock culture and adding it to 9 mL of MB1249, then the culture was placed into a 30° C. side incubator within the chamber and incubated for 72 hours. After 72 hours of incubation, the 10-ml D. vulgaris culture was added to 40 ml of MB1249 medium and incubated for 48 hours. After the incubation time, the optical density of the culture was determined by taking 100 μL of culture and adding it to 900 μL of MB1249 in a cuvette. Another cuvette was filled with 1 mL of MB1249 for a blank. Then, each of culture cuvettes were read and their optical density (O.D.) was recorded by using the Thermofisher Spectronic 200 Spectrophotometer at 620 nm. Using a C1V1=C2V2 calculation and the optical density, the final concentration of culture added to the 96 well plates was ˜0.1 O.D. Then, NUNC Round bottom 96 well plates were filled with culture and anaerobic PBS+10% MB1249. The plates were placed into the side incubator of the anaerobic chamber at 30° C. for 24 hours to form biofilm.

TABLE-US-00001 TABLE 1 Preparation of MB1249 media MB 1249 (pH 7.5) Chemical Amount (g/L) Chemical Source MgSO4 2 Fisher Na-Citrate 5 Fisher CaSO4 × 2H.sub.2O 1 Fisher NH4Cl 1 Fisher K2HPO.sub.4 0.5 Fisher Na-Lactate 3.5 Fisher Yeast Extract 1 Fisher Na-Thioglycolate 0.1 Sigma Aldrich DI H2O 985.9

Removal Biocide Treatment Plate Preparation—Anaerobic

[0016] After 24 hours of incubation at 30° C., all plates were removed from the incubator and the volume from each well was taken out with a multi-channel pipette. Then, the wells were re-filled with PBS+10% MB1249 and treatments.

The sugar analogues used in the treatment were α-Methyl D-Mannoside (αMM) and 2-Deoxy-D-Glucose (2DG). Both αMM and 2DG were purchased from Sigma. Stocks were prepared (W/V) by weighing sugar and adding anaerobic water inside the anaerobic chamber. The Glutaraldehyde, AQUCAR™ GA50 (“Glutaraldehyde”), was prepared outside of the chamber (V/V) as a safety measure. Each of the components were added at their respective concentrations for a final volume of 200 ul per well. In Table 3, the concentrations used for the synergy experiments are listed. Each experiment was done with at least six replicates for different treatments and at least 8 replicates for non-treatment controls.
Plates were then sealed with a titer-top and placed into anaerobic boxes with a gas pack. Then, incubated in an aerobic shaking incubator, 30° C. at 115 RPM. After 24 hours, the plates were processed using a Crystal Violet Assay.

Crystal Violet Assay

[0017] After 24 hours of treatment, all the treatment solution was gently removed from plates using a multi-channel pipette. Then, 250 μL of DI water was re-filled into the wells. This step was then repeated so the plates were each rinsed twice. If residual DI water was in plates, they were inverted and gently patted onto a thick paper towel. Then, the plates were air dried in a ventilated hood.
To fix the biofilm in the wells, 250 μL per well of methanol was used, waiting time 5 minutes. This methanol was then removed and allowed to air dry. Lastly, 250 μL per well of 0.1% Crystal Violet Solution was used to stain the biofilm in the plates for 5 minutes. Then, using a multi-channel pipette, the stain was removed, and the rinse step was repeated. The plates were allowed to air dry one final time. Then, 250 μL of methanol was used to extract stained biofilm from well walls, mixing gently to break up clumps. Then, the plates were placed into the Biotek Cytation 5, and the optical density (OD) was read at 600 nm. OD measurements were used to determine % biomass against control by calculating the ratio of sugar and glutaraldehyde combinations against respective no-treatment control within the same plate to minimize plate to plate variations. These % biomass values of sugar and glutaraldehyde combos were compared against glutaraldehyde-alone treatment, and success criterion was determined based on p-value calculations. P-values were calculated by comparing at-least 6 replicates for various combinations against glutaraldehyde-alone replicates, and glutaraldehyde combinations with p-values less than 0.05 demonstrated a statistically significant removal of biofilm.
Table 2 summarizes the efficacy of αMM and glutaraldehyde and their combinations, as well as the Synergy Index of each combination, while Table 3 summarizes the efficacy of 2DG and glutaraldehyde and their combinations. One measure of synergism is the industrially accepted method described by Kull, F. C.; Eisman, P. C.; Sylwestrowicz, H. D. and Mayer, R. L., in Applied Microbiology 9:538-541 (1961), using the ratio determined by the formula:

Q.sub.a/Q.sub.A+Q.sub.b/Q.sub.B=Synergy Index (“SI”)

[0018] Wherein: [0019] Qa=Concentration of biocide A required to achieve significant biomass reduction when used in combination with B [0020] QA=Concentration of biocide A required to achieve significant biomass reduction when used alone [0021] Qb=Concentration of biocide B required to achieve significant biomass reduction when used in combination with A [0022] QB=Concentration of biocide B required to achieve significant biomass reduction when used alone
When the sum of Qa/QA and Qb/QB is greater than 1.0, antagonism is indicated. When the sum is 1.0, additivity is indicated, and when less than 1.0, synergism is demonstrated.

TABLE-US-00002 TABLE 2 Concentrations of αMM and Glutaraldehyde in the synergy experiments. (Desulfovibrio vulgaris) In Standalone experiments Concentration % OD compared to Chemical (ppm) no-treatment control Glutaraldehyde 2500 97.4 αMM 2000 100.3
In experiment combinations (listed in ppm)

TABLE-US-00003 % OD compared to Glutaral- no-treatment p- Synergy dehyde αMM Ratio control value index 25 2000 1/80 90.7 0.00264 1.01 25 1500 1/60 87.4 0.00010 0.76 25 1250 1/50 93.4 0.02036 0.64 25 1000 1/40 83.1 <.00001 0.51 25 750 1/30 80.7 <.00001 0.39 25 500 1/20 65.6 <.00001 0.26 25 400 1/16 65.5 <.00001 0.21 25 300 1/12 62.9 <.00001 0.16 25 250 1/10 78.3 <.00001 0.14 25 200 1/8  59.2 <.00001 0.11 25 100 1/4  63.0 <.00001 0.06 25 75 1/3  80.9 <.00001 0.05 25 50 1/2  66.8 <.00001 0.04 25 25 1/1  82.2 <.00001 0.02 25 10  1/0.4 82.0 <.00001 0.02 25 0 99.3 0.742879 —

TABLE-US-00004 TABLE 3 Concentrations of 2DG and Glutaraldehyde in the synergy experiments. (Desulfovibrio vulgaris) In Standalone experiments Concentration % OD compared to Chemical (ppm) no-treatment control Glutaraldehyde 2500 97.4 2DG 2000 101.7
In experiment combinations (listed in ppm)

TABLE-US-00005 Glutaral- % OD compared to Glutaral- dehyde/ no-treatment p- Synergy dehyde 2DG 2DG ratio control value Index 25 2000 1/80 79.1 0.0001 1.01 25 1500 1/60 76.4 0.0001 0.76 25 1250 1/50 84.2 0.0001 0.635 25 1000 1/40 75.2 <.00001 0.51 25 800 1/32 72.8 <.00001 0.41 25 750 1/30 79.8 0.0001 0.385 25 600 1/24 68.5 <.00001 0.31 25 500 1/20 75.5 0.0001 0.26 25 400 1/16 69.3 <.00001 0.21 25 250 1/10 69.8 <.00001 0.135 25 200 1/8  72.0 <.00001 0.11 25 100 1/4  61.5 <.00001 0.06 25 75 1/3  77.7 0.0001 0.0475 25 50 1/2  71.2 <.00001 0.035 25 25 1/1  78.7 0.0001 0.0225 25 10 2.5/1   75.3 0.0001 0.015 25 0 92.7 0.023 —