Compounds and compositions for biofilm prevention

Abstract

The present invention relates to compounds and compositions for effective biofilm prevention. Particularly, the invention provides compositions comprising certain cyclic ketones found to be efficient in preventing bacterial biofilm formation.

Claims

1. Composition comprising a compound of General structure 3, ##STR00004## wherein n is 0-2; optionally substituted by 1-3 groups selected from O, OH, C.sub.1-6 alkyl, OC.sub.1-6 alkyl, CHO and C(OH)O; or salts, hydrates, solvates or tautomeres thereof, with the proviso that the compound is not 2-isopropyl-5-methylcyclohexane-1-on; in an amount preventing biofilm formation.

2. Composition as claimed in claim 1 wherein the composition comprises the compound of General structure 3 in an amount of 0.05-500 mg/ml, preferably 0.1-300 mg/ml or at least in a concentration of 1-100 mg/ml.

3. Composition as claimed in claim 1 wherein the composition is a solution, matrix, powder, gel or coating.

4. Composition as claimed in claim 1 wherein the substituted 1-3 groups are independently selected from the group of carbonyl, OH, methyl and ethyl, OC.sub.1-6 alkyl, CHO and C(OH)O.

5. Composition as claimed in claim 1 wherein the compound is substituted by one O; and is optionally substituted by 1-2 groups selected from OH, C.sub.1-6 alkyl, OC.sub.1-6 alkyl, CHO and C(OH)O; or salts, hydrates, solvates or tautomeres thereof.

6. Composition according to claim 1, wherein the compound is selected from the group consisting of: 1,3-cyclohexanedione; 1,4-cyclohexanedione; Cyclopentanone; 5-ethylcyclohexane-1,3 dione and 5-methyl-1,3 cyclohexanedione.

7. Composition as claimed in claim 5 wherein the compound is selected from the group of 1,3-cyclopentadions, a 1,3- or 1,4-cyclohexanedione, and a 1,3- or 1,4-cycloheptadion.

8. Composition as claimed in claim 1, in combination with one or more biocides and/or antibacterial agents.

9. Composition according to claim 8, wherein the biocides and/or antibacterial agent is selected from the group of disinfectants and general biocidal products, preservatives, pest control agents and biocidal products like antifouling agents.

10. Composition according to claim 8 wherein the biocides/or antibacterial agent is selected from the group consisting of 4-hydroxy-3-methoxybenzaldehyde, cetylpyridinium chloride, quorum sensing inhibitors, bioguanides, iodophors, quaternary ammonium compounds, boric acid, cationic tensides, alcohol based, chlorine based, peroxy based and acid based compounds, Tetracyclines, Amphenicols, Beta-lactam antibiotics, Sulphonamides and trimetophrim, macrolides, linkosamides and streptogramins, Aminoglycosides, Quinolones and other antibacterial compounds.

11. Composition according claim 8, wherein the combination with one or more biocides and/or antibacterial agents are for use in a two-step process to prevent biofilm formation.

12. Composition according to claim 1, applicable for either of the following uses: a. A solvent or solvent mixture; b. An industrial paint or varnish; c. Anti-fouling coatings and/or impregnations for marine use; d. Anti-fouling coatings for maritime use; e. A coating bound to the surface of or mixed in polymer material; f. Solutions or coatings attached/linked to inert surfaces; g. A coating bound to the surface of or mixed in fiber glass materials.

13. Composition according to claim 1 for use in medicine.

14. Composition according to claim 13 for use in a solution, ointment or dressing in human or veterinary medicine or for a medical purpose.

15. Use of a composition comprising one or more of the compounds of General structure 3: ##STR00005## wherein n is 0-2; substituted by one O group; and optionally substituted by 1-2 groups selected from OH, C.sub.1-6 alkyl, OC.sub.1-6 alkyl, CHO and C(OH)O; or salts, hydrates, solvates or tautomeres thereof; in preventing biofilm formation.

Description

LIST OF FIGURES

[0080] In the Figures the Y-axis provides the % biofilm formation.

[0081] FIG. 1: 1,3-cyclohexanedione. Gram positive and Gram negative bacteria.

[0082] The graph shows a decrease in biofilm formation of 74 and 70% produced by Gram positive and Gram negative bacteria respectively, when using 1,3-cyclohexanedione in a concentration of 1 mg/ml. Further, a decrease of 98% was seen at a concentration of 10 mg/ml in Gram positive bacteria and 87% in Gram negative bacteria. The asterisks show that the reduction is statistically significant (p<0.05).

[0083] FIG. 2: 1,4-cyclohexanedione. Gram positive and Gram negative bacteria. The graph shows a 53% decrease in biofilm produced by Gram positive bacteria when using 10 mg/ml 1,4-cyclohexanedione. Similarly, a decrease of 91% was seen in biofilm formation by Gram negative bacteria. The asterisks show that the reduction is statistically significant (p<0.05).

[0084] FIG. 3: Cyclopentanone. Gram negative bacteria. The graph shows a decrease of 77% in biofilm formation by Gram negative bacteria using cyclopentanone at a concentration of 10 mg/ml. The asterisk shows that the reduction is statistically significant (p<0.05).

[0085] FIG. 4: 5-ethylcyclohexane-1,3-dione. Gram positive bacteria. The graph shows a decrease of 55% in biofilm production using 5-ethylcyclohexane-1,3-dione at 1 mg/ml, and 74% decrease at 2 mg/ml and 98% decrease at 10 mg/ml. The asterisks show that the reduction is statistically significant (p<0.05).

[0086] FIG. 5: 5-methyl-1,3-cyclohexanedione. Gram positive bacteria. The graph shows a decrease of 32% in biofilm formation using 5-methyl-1,3-cyclohexanedione at 1 mg/ml, 85% at 2 mg/ml and 98% at 10 mg/ml. The asterisks show that the reduction is statistically significant (p<0.05).

[0087] FIG. 6: 1,3-cyclohexanedione. Listeria monocytogenes. The graph shows a decrease of 27%, 40%, 53% and 70% using 1,3-cyclohexanedione at 0,625 mg/ml, 2.5 mg/ml, 5 mg/ml and 10 mg/ml respectively. The asterisks show that the reduction is statistically significant (p<0.05).

[0088] FIG. 7: 1,4-cyclohexanedione. Sulphate reducing bacteria (SRB). Arrow A in the picture points to the slide with SRB bacterial biofilm included as a control. Arrow B points to a slide that has been immersed in medium containing 1,4-cyclohexanedione at a concentration of 10 mg/ml.

[0089] FIG. 8: Cyclopentanone. Sulphate reducing bacteria (SRB). Arrow A in the picture points to the slide with SRB bacterial biofilm included as a control. Arrow B points to a slide that has been immersed in medium containing cyclopentanone at a concentration of 10 mg/ml.

[0090] FIG. 9: 1,3-cyclohexanedione. Resistance to increasing temperature. The graph shows biofilm formation after heating the solutions containing 1,3-cyclohexanedione compound to 80 C., 90 C. and 100 C. The graph shows a decrease in biofilm formation of 38%, 40% and 28% respectively, compared to the control (0 mg/ml 1,3-cyclohexanedione). The unheated control solution containing 1,3-cyclohexanedione (1 mg/ml) shows a decrease of 46% in biofilm formation, compared to the sample without the substance added.

[0091] FIG. 10: 1,3-cyclohexanedione. Stainless steel. The figure shows the average of log values of the parallel experiments using 1,3-cyclohexanedione in two concentrations. A decrease in biofilm formation was seen with 1,3-cyclohexanedione at a concentration of 1 mg/ml. At 10 mg/ml, we could not detect any colony forming units (CFU's).

[0092] FIG. 11: 1,3 Cyclohexanedione and menthone. E. coli. The graph shows a decrease of 46 and 70% in biofilm formation using 1,3-cyclohexanedione at 1 mg/ml, and 97% and 100% at 10 mg/ml. For menthone, the decrease is seen to be 27% and 48% at 1 mg/ml, and 6% and 26% at 10 mg/ml.

DEFINITIONS

[0093] In the present context, the disclosed compounds or molecules are of any of the formulas above, and are typically compounds such as 1,3-cyclohexanedione (C.sub.6H.sub.8,O.sub.2), 1,4-cyclohexanedione (C.sub.6H.sub.12O.sub.2), cyclopentanone (C.sub.5H.sub.8O), 5-ethylcyclohexane-1,3-dione (C.sub.8H.sub.12O.sub.2) and 5-methyl-1,3 cyclohexanedione (CH.sub.3C.sub.6H.sub.7(O).sub.2) for use as anti-biofilm agent in solutions, or incorporated into or onto materials. Examples, but not limited thereto are paint, antifouling coating, polymer, glass or metal surfaces.

[0094] When referring to general structure any compound falling within either of the formulas shown above are encompassed, such as compounds of general structure 1 and general structure 2, and of General Structure 3.

[0095] In the present context, a biofilm is an extracellular matrix community of sessile, stable attached microorganisms, such as bacteria, embedded in a self-produced matrix consisting of various components, including extracellular polymeric substances. Biofilm formation consists of three steps; attachment, growth and detachment in order to recolonize another surface. Extracellular matrix is continuously formed during the first two steps.

[0096] In the present context, a biofilm is considered to have been established from the moment when one or more microorganisms is/are irreversibly attached to a surface. Examples, but not limited thereto, of a surface, is a wound and wound area.

[0097] The term surface is intended to relate to any surface which may be partially or fully covered by a biofilm. Examples, but not limited to, of surfaces are metal, polymer, fibre glass, human skin, epithelial cells, muscle tissue and surgical suture material or any coated or impregnated area.

[0098] The term effective amount as used herein refers to an amount effect, at dosages and for periods of time necessary to achieve a desired result.

[0099] In the present context, for medical use, the term effective amount refers to an amount of a compound or compounds that is sufficient to effect treatment when administered to a subject in need of such treatment.

EXPERIMENTAL

[0100] The following examples are illustrations within the scope of the claims.

Example 1

Biofilm Formation Experiments

[0101] All experiments were performed by using sterile 96-wells polystyrene microtiter plates (Nunc, Nuncleon, Roskilde, Denmark) under conditions promoting biofilm formation by the different bacterial genera. The Inhibio compound preparations of the invention; 1,3-cyclohexanedione, 1,4-cyclohexanedione, cyclopentanone, 5-ethylcyclohexane-1,3-dione and 5-methyl-1,3 cyclohexanedione 98% were solved directly in Tryptic Soy Broth (TSB) 1+1 or Liquid Microbiology Broth (LB) broth without NaCl (LB.sup.wo/NaCl) to obtain the test concentrations, i.e. from 1 mg/ml to 10 mg/ml.

Gram Positive Bacteria

[0102] Three Staphylococcus aureus strains of animal origin were used in this study. All strains were stored at 80 C. in Brain Heart Infusion Broth (BHI) (Difco, BD, Franklin Lakes, N.J., USA) supplemented with 15% glycerine (Merck KGaA, Darmstadt, Germany) and were recovered on blood agar at 37.01.0 C. The bacterial cultures were then transferred into TSB and were incubated statically overnight at 37.01.0 C. to obtain an overnight working culture. A total of 2 l of this suspension was transferred to each 96 wells of polystyrene microtiter plates (Nunc, Nuncleon, Roskilde, Denmark) containing 198 l TSB 1+1 with dissolved Inhibio compounds in the following concentrations: 0 mg/ml, 1 mg/ml and 10 mg/ml for 1,3-cyclohexanedione; 0 mg/ml and 10 mg/ml for 1,4-cyclohexanedione; and 0 mg/ml, 1 mg/ml, 2 mg/ml and 10 mg/ml for 5-ethylcyclohexane-1,3-dione and 5-Methyl-1,3-cyclohexanedione in the total amount of broth. Three parallels of each strain were used and the microtiter plates were incubated statically for one day, at 371.0 C. After incubation, OD.sub.595 was measured in a microplate photometer (Multiscan EX; Thermo Fisher Scientific Inc, Waltham, Mass., USA) before the plates were gently washed three times with 290 l tap water. The plates were dried in room temperature before addition of 220 l 1% crystal violet (Sigma-Aldrich, St. Louis, Mo., USA). After 30 minutes incubation in room temperature, the plates were washed three times with tap water before the addition of 220 l ethanol:acetone (70:30 w:w) to dissolve the bound dye. The plates were incubated for 10 minutes in room temperature before OD.sub.595 was measured after the bound dye was dissolved using ethanol:acetone. For each strain, the result was calculated by subtracting the median OD.sub.595 of the three parallels of the control (test broth only) from the median OD.sub.595 of the three parallels of sample. Further, the average result of all three Gram positive strains included in the study were calculated. Three independent experiments were performed and the average was evaluated.

Gram Negative Bacteria

[0103] One Salmonella ser. Typhimurium (S. Typhimurium), two Salmonella ser. Agona (S. Agona) and two Escherichia coli (E. coli) isolates were used in these studies. The studies performed using 1,3-cyclohexanedione and 1,4-cyclohexanedione were done on Salmonella isolates only. The studies comparing Menthone to 1,3-cyclohexanedione were using two E. coli strains, E. coli 1242 and E. coli 1153, see FIG. 11. The Salmonella isolates were isolated from Norwegian feed factories except one S. Typhimurium strain which was a culture collection strain (ATCC 14028). The E. coli isolates were of animal origin. All strains were stored at 80 C. in BHI supplemented with 15% glycerine and were recovered on blood agar at 37.01.0 C. The bacterial cultures were then transferred into LB broth and incubated statically overnight at 37.01.0 C. to obtain an overnight working culture. A total of 30 l of this suspension was transferred to each well in 96 wells polystyrene microtiter plates (Nunc, Nuncleon, Roskilde, Denmark) in triplets. The wells were containing 100 l LB.sup.wo/NaCl (bacto-tryptone 10 g/l, yeast extract 5 g/l) with 1,3-cyclohexanedione dissolved in concentration 0 mg/ml, 1 mg/ml and 10 mg/ml in the total amount of broth and 1,4-cyclohexanedione and cyclopentanone at 0 mg/ml and 10 mg/ml. The microtiter plates were incubated statically for two days, at 201.0 C. After incubation, OD.sub.595 was measured before the plates were gently washed one time with 150 l tap water. The plates were dried in room temperature before addition of 140 l 1% crystal violet (Sigma-Aldrich). After 30 minutes incubation in room temperature, the plates were washed three times with tap water before addition of 140 l ethanol:acetone (70:30 w:w) and incubated for another 10 minutes in room temperature. OD.sub.595 was measured in a microplate photometer (Multiscan EX) after the bound dye was dissolved using ethanol:acetone. For each strain, the result was calculated by subtracting the median OD.sub.595 of the three parallels of the control (test broth only) from the median OD.sub.595 of the three parallels of sample. Further, the averages of the Gram negative strains were calculated. Three independent experiments were performed and the average was evaluated.

Results:

[0104] The results are expressed as a decrease in biofilm formation calculated in percentage of the control (without compound of the invention). A decrease of 74 and 70% in biofilm production was found using a concentration of 1 mg/ml 1, 3-cyclohexanedione on biofilm produced by Gram positive and Gram negative bacteria, respectively. Further, a decrease of 98% was seen at a concentration of 10 mg/ml 1,3-cyclohexanedione in Gram positive bacteria and 87% in Gram negative bacteria. For 1,4-cyclohexanedione, the results show a 53% decrease in biofilm produced by Gram positive bacteria at 10 mg/ml. Similarly, a decrease of 91% was seen in biofilm formation by Gram negative bacteria. Using cyclopentanone, a decrease of 77% in biofilm formation by Gram negative bacteria was detected at a concentration of 10 mg/ml. Further, a decrease of 55% of biofilm formation was found using 5-ethylcyclohexane-1,3 dione in 1 mg/ml, 74% in 2 mg/ml and 98% in 10 mg/ml in Gram positive bacteria. Similarly, a decrease of 32% was found using 5-methyl-1,3-cyclohexanedione 98% at 1 mg/ml, 85% decrease at 2 mg/ml and 98% at 10 mg/ml. The studies comparing 1,3-cyclohexanedione and menthone showed a decrease of 46 and 70% using 1,3-cyclohexanedione at a concentration of 1 mg/ml and a decrease of 97 and 100% at 10 mg/ml. On the contrary, menthone showed a decrease of 27 and 48% at 1 mg/ml and at 10 mg/ml the decrease was 6 and 26% (FIG. 11). This shows that menthone fails to show a dose dependent effect, which is a known response by plant based compounds. All results were statistically significant (shown with asterisk) at confidence interval 95% except 5-methyl-1,3-cyclohexanedione 98% at 1 mg/ml. A student's T-test was not performed on the comparative studies with menthone. See FIGS. 1, 2, 3, 4, 5 and 11.

Listeria

[0105] Six strains of Listeria monocytogenes were used in this study isolated from food research. The strains were recovered in 200 l TSB broth in a microtiter plate and incubated at 37 C. for 24 hours. A total of 5 l of the bacterial suspension was transferred to a 96 wells polystyrene microtiter plate (Thermo scientific Nucleon Delta surface) together with 200 l medium (LB.sup.wo/NaCl) with 1,3-cyclohexanedione (dissolved to 0,625 mg/ml, 2.5 mg/ml, 5 mg/ml and 10 mg/ml) to each well. Each strain was added in duplex and incubated at 35 C. for 24 hours. After incubation, OD.sub.595 was measured before the plates were gently washed one time with 200 l tap water. This was repeated once. The plates were dried in room temperature before addition of 200 l 0, 1% crystal violet (Sigma-Aldrich). After 30 minutes incubation in room temperature, the plates were washed twice with 200 l tap water and once with 240 l. This was followed by the addition of 200 l ethanol:acetone (70:30 w:w) and incubated for another 10 minutes in room temperature. OD.sub.595 was measured in a microplate photometer (Multiscan EX) after the bound dye was dissolved using ethanol:acetone. For each strain, the result was calculated by subtracting the average OD.sub.595 of the two parallels of the control (test broth only) from the average OD.sub.595 of the two parallels of sample. Three independent experiments were performed. The average of the 6 strains as well as the average between the experiments was calculated.

Results:

[0106] The results from the studies with 1,3-cyclohexanedione are shown as a decrease in biofilm formation in percentage of the control (no claimed compound present). A decrease of 27%, 40%, 53%, 70% was found using 1, 3-cyclohexanedione at 0.625 mg/ml, 2.5 mg/ml, 5 mg/ml and 10 mg/ml respectively. All results were statistically significant at confidence interval 95% (marked with asterisk). See FIG. 6.

Example 2

The Effect of 1,4-Cyclohexanedione and Cyclopentanone on Biofilm Produced by SRB (Sulphate Reducing Bacteria) in Fluids.

[0107] One SRB isolate (the culture collection strain: ATCC 29579 Desulfovibrio vulgaris subspecies vulgaris) was tested. 1,4-cyclohexanedione and cyclopentanone were diluted in the culture medium ATCC 1249 to a concentration of 10 mg/ml (1%) and added to each their 50 ml Falcon centrifugal tube. A third tube, with 10 ml of medium only (ATCC 1249) was included as a control. 50 l SRB starting culture was added to each of the three tubes together with a carbon steel coupon. Biofilm was formed on the coupon by incubating at 20 C. for 12 days. After incubation, the coupons were washed in 40 ml sterile saline to remove loosely adhered cells. A sample from this fluid was injected into SRB medium and blackening of the medium was visualized, showing that there were still free-floating bacteria present.

Results:

[0108] The characteristic black biofilm was seen on the coupon that is not treated. In contrast, on the coupon treated with 1,4-cyclohexanedione or cyclopentanone at a concentration of 10 mg/ml only scarce amount of biofilm could be visualized. See FIGS. 7 and 8.

Example 3

The Effect of Increased Temperature on 1,3-Cyclohexanedione

[0109] The experiment was performed using 1,3-cyclohexanedione at a concentration of 1 mg/ml. One strain of Salmonella was used in this study. All strains were stored at 80 C. in BHI (Difco, BD, Franklin Lakes, N.J., USA) supplemented 220 l etanol:aceton 70:30 with 15% glycerine (Merck KGaA, Darmstadt, Germany) and were recovered on blood agar at 37.01.0 C. The bacterial culture was transferred into LB broth and was incubated statically overnight at 37.01.0 C. to obtain an overnight working culture. 1,3-cyclohexanedione was diluted in LB wo/NaCl at a concentration of 1 mg/ml and divided into 4 tubes. The tubes were heated, for two minutes, to 80 C., 90 C., 100 C. and the last tube was not heated and included as a control. 100 l from each tube was added to a sterile 96-wells polystyrene microtiter plates (Nunc, Nuncleon, Roskilde, Denmark) together with 30 l bacterial culture or 30 l LB.sup.wo/NaCl in the case of the blank controls. The plates were incubated for 72 hours at 20 C. The plate was emptied and washed twice using 200 l tap water in each well each time. This was followed by the addition of 140 l 1% Crystal violet to each well and, after 30 minutes, the plate was again emptied and washed 3 times using 200 l tap water. 140 l etanol:acetone 70:30 was added and OD.sub.595 was measured after 10 minutes. For each strain, the result was calculated by subtracting the median OD.sub.595 of the three parallels of the control (test broth only) from the median OD.sub.595 of the three parallels of sample. At least two experiments were performed and the average was estimated.

Results:

[0110] The results show a decrease of 38%, 40% and 28% in biofilm formation after heating the solutions containing 1,3-cyclohexanedione compound to 80 C., 90 C. and 100 C., respectively. The unheated control showed a decrease of 46%. Considering normal variations, this shows that the 1,3-cyclohexanedione compound was still effective in reducing biofilm formation at higher temperatures. See FIG. 9.

Example 4

The Effect of 1,3-Cyclohexanedione on Biofilm Formed on Stainless Steel Coupons by Gram Negative Bacteria

[0111] One strain of S. Agona (FIG. 10) and one strain of E. coli (FIG. 10) was used in this study. The study was performed using 1,3-cyclohexanedione in a concentration of 1 and 10 mg/ml. The strain was stored at 80 C. in BHI (Difco, BD, Franklin Lakes, N.J., USA) supplemented with 15% glycerine (Merck KGaA, Darmstadt, Germany) and was recovered on blood agar at 37.01.0 C. The bacterial culture was then transferred into LB broth and was incubated statically overnight at 37.01.0 C. to obtain an overnight working culture. The Inhibio compound was dissolved in LB.sup.wo/NaCl to a concentration of 10 mg/ml and then further diluted to a concentration of 1 mg/ml. 10 ml of each solution was added to a 50 ml Falcon tube and, in a third tube, only LB broth.sup.wo/NaCl was added as a control. To each tube, 200 l bacterial culture and an autoclaved stainless steel coupon was added and the tubes were incubated at 20 C. for 72 hours.

[0112] Following incubation, each coupon was dipped three times in three different tubes containing physiological saline and further transferred to a tube containing 5 ml cold physiological saline as well as 20 sterile silica glass beads. Each coupon was further scraped with a sterile cell scraper before the coupon was removed and the solution was vortexed at 2000 rpm for one minute. A 10-fold dilution was made in a Nunc microtiterplate (kept on ice) with 180 l physiological saline and 20 l of the previous dilution for each well. 100 l were spread on a blood agar plate and incubated on 37 C. for 24 hours. After incubation, the bacterial colonies were counted. If more than 200 colonies on a plate it was considered overgrown. At least two experiments were performed.

Results:

[0113] The Colony forming units (CFU) on the plate was calculated into CFU in biofilm by multiplying with a factor for each dilution. Further, the log value was calculated of the average of each dilution. A significant decrease was seen with 1,3-cyclohexanedione in a concentration of 1 mg/ml. At 10 mg/ml, there were no bacteria found in the biofilm. See FIG. 10.

Example 5

InSilico Studies to Assess Biodegradability

[0114] For the compounds 1,3-Cyclohexanone and 1,4-Cyclohexanone InSilico studies were performed using EPIWEB 4.1 and BIOWIN v4.10. The Biowin 3 (the ultimate biodegradability Timeframe) and Biowin 4 (The primary Biodegradation Timeframe) were evaluated together with the Biowin5 (MITI Linear model prediction). These results were again used to obtain a YES or NO Ready Biodegradability Prediction.

Results:

[0115] The Biowin 3 of 1,3-Cyclohexanone and 1,4-Cyclohexanone were both estimated to 2,90 (weeks). The Biowin 4 of 1,3-Cyclohexanone and 1,4-Cyclohexanone were both estimated to be 3,64 (days-weeks) and the Biowin 5 was estimated to be 0,69. Their Ready Biodegradability Prediction was therefore YES (readily biodegradable).