Use of dicarboxylic and tricarboxylic fatty acids in preventing biofilm formation

Abstract

The present invention relates to the in vitro use of di- or tricarboxylic acids having a chain length of 15 to 23 carbon atoms, such as agaricic acid, for preventing, reducing or delaying the formation of biofilms of bacteria on abiotic surfaces.

Description

Field of the Invention

[0001] The present invention relates to the prevention and treatment of biofilms. Background of the Invention

[0003] Bacteria typically live in dense communities enclosed in a self-produced matrix, often called a biofilm. These biofilms are highly resistant to antibiotics, disinfectants, and mechanical removal, leading to chronic infection or contamination [Costerton et al. (1999) Science 284, 1318-1322; Fux et al. (2005) Trends in microbiology 13, 34-40; Hall-Stoodley and Stoodley (2009) Cellular microbiology 11, 1034-1043]. The highly resistant and persistent nature of biofilms poses a significant problem in a variety of fields, including medicine, the food industry, and agriculture [Galié et al. (2018) Frontiers in microbiology 9, 898-898; Velmourougane et al. (2017) Journal of Basic Microbiology 57, 548-573; Koo et al. (2017) Nature reviews. Microbiology 15, 740-755]. The failure of current strategies to completely prevent or remove biofilms has led to a strong demand for new biofilm inhibitors. Due to the low permeability of biofilms, which has not yet been established, preventive strategies that block initial adhesion appear to be the most promising [Roy et al. (2018) Virulence 9, 522-554].

[0004] One biofilm-forming pathogen that is particularly problematic in the food and feed industry is Salmonella. Globally, there are approximately 94 million cases of Salmonella each year, resulting in 155,000 deaths [Roy et al. (2018) Virulence 9, 522-554]. It is estimated that more than 85% of these cases are foodborne, making Salmonella the most common cause of bacterial foodborne disease outbreaks. In 2017, the European Food Safety Authority reported more than 90,000 cases of Salmonella, resulting in 156 deaths [EFSA (2018) EFSA Journal 2018; 16(12), 5500].

[0005] In the ongoing screening of new anti-biofilm compounds, we identified agaricic acid as an effective Salmonella biofilm inhibitor. Agaricic acid, or 2-hydroxynonadecane-1,2,3-tricarboxylic acid, is a fatty acid naturally produced by certain fungi. It has been reported as an inhibitor of the mitochondrial adenine nucleotide exchange reaction that induces mitochondrial permeability [García et al. (2005) Mitochondrion 5, 272-281]. Historically, this compound has been used as an antiperspirant to symptomatically treat extreme sweating in tuberculosis patients [García et al. (2005) Mitochondrion 5, 272-281]. In addition, at high doses, agaricic acid can suppress the nervous, respiratory, and circulatory systems in lower animals. Therefore, agaricic acid has also been used as a metabolic inhibitor in animal experiments [Freedland and Newton (1981) Methods Enzymol. 72, 497-506].

[0006] Celleno et al. (2019) G Ital Dermatol Venereol. 154, 338-341 describe the use of agaricic acid as an antiperspirant for hyperhidrosis. Summary of the Invention

[0007] The present invention shows that agaricic acid significantly inhibits Salmonella biofilm formation when used in a preventive manner: it significantly reduces the number of bacteria adhering to inanimate surfaces and the amount of biomass by downregulating flagellar rotation genes and inhibiting swimming motility. Importantly, the reduction in biofilm formation allows for more effective treatment with hydrogen peroxide, a disinfectant commonly used in the food industry.

[0008] Agaricic acid or other di- and tricarboxylic acids can be included as anti-biofilm agents in paints and cleaners, or can be added to liquid circuits.

[0009] The present invention also relates to the use of agaricic acid or other dicarboxylic and tricarboxylic acids for inhibiting biofilm formation in mixed species communities.

[0010] The present invention also relates to the use of agaricic acid or other di- and tricarboxylic acids to weaken mixed species biofilm communities and reduce resistance to antimicrobial agents.

[0011] The present invention is further summarized in the following statements:

[0012] 1. Use in vitro of a dicarboxylic or tricarboxylic acid having a chain length of 8 to 41 for treating, preventing, reducing or delaying the formation of biofilms of microorganisms on abiotic surfaces.

[0013] 2. The use according to statement 1, wherein the planktonic growth of the microorganisms is retained.

[0014] 3. The use according to statement 1 or 2, wherein the microorganism is a bacterium.

[0015] 4. Use according to any one of statements 1 to 3, wherein the carboxylic acid is a tricarboxylic acid.

[0016] 5. Use according to any one of statements 1 to 4, wherein the carboxylic acid is substituted by a hydroxyl group.

[0017] 6. Use according to any one of statements 1 to 5, wherein the carboxylic acid is a 1,2,3 tricarboxylic acid.

[0018] 7. Use according to any one of statements 1 to 6, wherein the carboxylic acid is a 2-OH, 1,2,3 tricarboxylic acid.

[0019] 8. Use according to any one of statements 1 to 7, wherein the carboxylic acid has a C chain of 15 to 23 carbon atoms.

[0020] 9. Use according to any one of statements 1 to 8, wherein the carboxylic acid has a C chain of 17 to 21 carbon atoms.

[0021] 10. Use according to any one of statements 1 to 9, wherein the carboxylic acid is selected from the group consisting of: 2-hydroxyheptadecan-1,2,3-tricarboxylic acid-(C 20 H 36 O7); 2-Hydroxynonadecane-1,2,3-tricarboxylic acid (Agaric acid) (C 22 H 40 O7); 2-hydroxyoctadecane-1,2,3-tricarboxylic acid (C 21 H 38 O7); 2-hydroxy-4-oxonadecane-1,2,3-tricarboxylic acid (C 22 H 38 O8) and 2-hydroxy-19-methyleicosane-1,2,3-tricarboxylic acid (C 24 H 44 O7).

[0022] 11. Use according to any one of statements 1 to 10, wherein the carboxylic acid is 2-hydroxynonadecan-1,2,3-tricarboxylic acid (agaric acid).

[0023] 12. Use according to any one of statements 1 to 11, wherein the carboxylic acid is used at a concentration of at least 5 μM, at least 10 μM, at least 25 μM, at least 50 μM, at least 100 μM, or at least 500 μM.

[0024] 13. Use according to any one of statements 1 to 12, for preventing Salmonella Typhimurium biofilm formation, Staphylococcus aureus biofilm formation or Escherichia coli biofilm formation.

[0025] 14. The use according to any one of statements 1 to 13, which is followed by the use of an antimicrobial agent, typically an antibacterial agent. Such antimicrobial agents can kill planktonic bacteria, but can also be used against adherent bacteria that are prone to forming biofilms or are in the initial stages of biofilm formation.

[0026] 15. A method according to statement 14, wherein the antimicrobial agent is an oxidizing agent, typically a peroxide such as hydrogen peroxide.

[0027] 16. A di- or tri-carboxylic acid having a chain length of C8 to C41 for use in treating or preventing bacterial biofilm formation.

[0028] 17. A carboxylic acid for use in treatment or prevention according to statement 16, wherein the use is topical.

[0029] 18. A carboxylic acid for use in treatment or prevention according to statement 16 or 17 for preventing or treating biofilm formation on implants.

[0030] 19. A carboxylic acid for use in treatment or prevention according to any one of statements 16 to 18, wherein the carboxylic acid is a tricarboxylic acid.

[0031] 20. A carboxylic acid for use in treatment or prevention according to any one of statements 16 to 19, wherein the carboxylic acid is substituted by a hydroxy group.

[0032] 21. A carboxylic acid for use in treatment or prevention according to any one of statements 16 to 20, wherein the carboxylic acid is a 1,2,3 tricarboxylic acid.

[0033] 22. A carboxylic acid for use in treatment or prevention according to any one of statements 16 to 21, wherein the carboxylic acid is a 2-OH 1,2,3 tricarboxylic acid.

[0034] 23. A carboxylic acid for treatment or prevention according to any one of statements 16 to 22, wherein the carboxylic acid has a C chain of 15 to 23 carbon atoms.

[0035] 24. A carboxylic acid for treatment or prevention according to any one of statements 16 to 23, wherein the carboxylic acid has a C chain of 17 to 21 carbon atoms.

[0036] 25. A carboxylic acid for use in treatment or prevention according to any one of statements 16 to 24, wherein the carboxylic acid is selected from the group consisting of: 2-hydroxyheptadecan-1,2,3-tricarboxylic acid-(C 20 H 36 O7); 2-Hydroxynonadecane-1,2,3-tricarboxylic acid (Agaric acid) (C 22 H 40 O7); 2-hydroxyoctadecane-1,2,3-tricarboxylic acid (C 21 H 38 O7); 2-hydroxy-4-oxonadecane-1,2,3-tricarboxylic acid (C 22 H 38 O8) and 2-hydroxy-19-methyleicosane-1,2,3-tricarboxylic acid (C 24 H 44 O7).

[0037] 26. A carboxylic acid for use in treatment or prevention according to any one of statements 16 to 25, wherein the carboxylic acid is 2-hydroxynonadecan-1,2,3-tricarboxylic acid (agaricic acid). DETAILED DESCRIPTION

[0038] Figure 1 : Agaricic acid has no bactericidal effect but strongly inhibits Salmonella biofilm formation.

[0039] A) Agaricic acid inhibits biofilm formation of Salmonella typhimurium ATCC 14028 in a concentration-dependent manner as measured by crystal violet staining in a Calgary biofilm apparatus. Means and standard deviations of three biological replicates are shown. Significant differences were determined by one-way ANOVA with Bonferroni multiple comparison correction. B) Agaricic acid did not inhibit planktonic growth at concentrations relevant to biofilm inhibition. Planktonic growth was measured as the OD600 of liquid cultures after 48 h of incubation in a Calgary biofilm apparatus. Means and standard deviations of three biological replicates are shown. Significant differences were determined by one-way ANOVA with Bonferroni multiple comparison correction. C) Agaricic acid also reduced the number of cells adhering to the bottom of a glass petri dish. Significant differences were determined by one-way ANOVA with Bonferroni multiple comparison correction. D) Maximum intensity projection top and side views of fluorescently labeled Salmonella biofilms show that the presence of agaricic acid results in a less dense and dispersed biofilm.

[0040] Figure 2: Agaricic acid downregulates the transcription of flagellar genes.

[0041] For Salmonella typhimurium ATCC 14028 (square, light line) or 100 μM agaricic acid (circle, dark line) grown in DMSO, fluorescence as a measure of gene transcription at different time points is shown. The mean value and standard deviation of three biological replicates are depicted. Asterisks represent significant differences (P < 0.05) as determined by two-sided Student's t-test. Unexpectedly, genes important for biofilm formation are upregulated by agaricic acid (Figure A). Agaricic acid indeed reduces the transcription of Class II and Class III flagellar genes (Figure B).

[0042] Figure 3 : Agaricic acid reduces biofilm formation by inhibiting flagellar motility.

[0043] A) Phase contrast microscopy shows no differences between the flagella of Salmonella grown in 100 μM agaricic acid or the corresponding amount of DMSO, as visualized by crystal violet-based flagellar staining. One representative replicate of three biological replicates is shown. B) 100 μM agaricic acid almost completely inhibits motility in soft agar electrophoresis, similar to the motA deletion mutant. One representative replicate of three biological replicates is shown. C) The extent of inhibition of biofilm formation by the deletion of motA is similar to that of preventive treatment with agaricic acid. In addition, agaricic acid does not further reduce biofilm formation of the motA deletion mutant. Three biological replicates are shown. Significant differences were determined by one-way ANOVA with Bonferroni multiple comparison correction.

[0044] Figure 4 Agaricic acid increases sensitivity to H2O2 treatment. Biofilms grown in the presence of 100 μM agaricic acid or the corresponding amount of DMSO were treated with 0.25% H2O2 or 1 μM ciprofloxacin for 1 h. A motA deletion mutant was used as a control. Agaricic acid significantly reduced the number of biofilm cells that survived treatment with 0.25% H2O2. Three biological replicates are shown. Significant differences were determined by two-way ANOVA with Bonferroni multiple comparison correction.

[0045] Figure 5 Agaricic acid also reduces biofilm formation of other pathogens. As measured by crystal violet staining in the Calgary biofilm apparatus, agaricic acid inhibits biofilm formation of Escherichia coli TG1, Pseudomonas aeruginosa PA14, and Staphylococcus aureus SH1000. The OD values ​​of the broth in the wells were measured. 600 Effects on planktonic growth were measured. Means and standard deviations of three biological replicates are shown. Significant differences were determined by one-way ANOVA with Bonferroni multiple comparison correction.

[0046] Figure 6 : Agaricic acid reduces mixed species biofilm formation. A) Agaricic acid significantly reduces biofilm formation in a mixed species community consisting of two Salmonella strains and one Escherichia coli strain. The means and standard deviations of three biological replicates are depicted. Asterisks indicate significant differences (P < 0.05) as determined by two-sided Student's t-test. B) In the mixed species community, only biofilm formation of strain S2 was significantly inhibited by 100 μM agaricic acid. The means and standard deviations of three biological replicates are depicted. Significant differences (P < 0.05) were determined by two-way ANOVA with Sidak's multiple comparison correction. C) In the presence of S2 and E1, agaricic acid attenuated the competition experienced by S1. The means and standard deviations of three biological replicates are depicted. Significant differences (P < 0.05) were determined by paired two-sided Student's t-test.

[0047] Figure 7 Agaricic acid inhibits enhanced tolerance in mixed-species communities. A) Under mixed-species conditions, agaricic acid abolished the increased tolerance of S1 to treatment with ciprofloxacin or H2O2. Means and standard deviations of six biological replicates of DMSO controls and three biological replicates of agaricic acid-treated biofilms are depicted. Significant differences (P<0.05) were determined by two-way ANOVA with Sidak multiple comparison correction. B) Mixed-species communities pretreated with agaricic acid showed the highest sensitivity to antimicrobial treatment. Means and standard deviations of six biological replicates of DMSO controls and three biological replicates of agaricic acid-treated biofilms are depicted. Significant differences (P<0.05) were determined by two-way ANOVA with Sidak multiple comparison correction.

[0048] In the context of the present invention, "biofilm" refers to a syntrophic consortium of microorganisms in which cells adhere to each other and often also to a surface. These adherent cells are embedded in a viscous extracellular matrix composed of extracellular polymeric substances (EPS). The cells within the biofilm produce EPS components, which are typically polymeric aggregates of extracellular polysaccharides, proteins, lipids, and DNA.

[0049] Typical examples of biofilm-producing bacteria are Salmonella, Bacillus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes.

[0050] "Planktonic bacteria" refers to free-living bacteria that exist in the liquid phase and are not attached to surfaces.

[0051] "Agaric acid" or "agaricin" refers to an agaric acid with the chemical formula C22 H 40 2-Hydroxynonadecane-1,2,3-tricarboxylic acid of O7. The structure of agaricic acid is depicted in the following formula I:

[0052]

[0053] Agaricic acid is a specific embodiment of a larger group of di- and tricarboxylic fatty acids.

[0054] With respect to "carbon chain length" or "C-chain", the carbon atoms of COOH groups or other C-containing substituents are not counted in determining the chain length. Thus, a carbon atom having the formula C 22 H 40 Agaricic acid with O7 and three COOH groups has a chain length of 19.

[0055] In the case of a branched C chain, the C-chain refers to the longest branch of the C chain. Examples of dicarboxylic or tricarboxylic fatty acids with a branched C chain are 5-hydroxy-6-octylooctadecane-3,4,5-tricarboxylic acid or 3,5-diethyl-4-hydroxyheneicosane-2,3,4-tricarboxylic acid.

[0056] The compounds used in the process of the present invention are dicarboxylic and tricarboxylic fatty acids, particularly tricarboxylic fatty acids.

[0057] The numbering of the substituents of the dicarboxylic and tricarboxylic fatty acids is consistent with agaricic acid with the COOH groups at positions 1, 2 and 3 and the OH at position 2.

[0058] The COOH groups of the dicarboxylic fatty acids are usually present at the 1- and 2-positions, or the 2- and 3-positions.

[0059] The COOH groups of the tricarboxylic fatty acids are usually present at position 1, 2, 3, or 1, 3, 4, or 2, 3, 4, or 3, 4, 5, most particularly 1, 2, 3.

[0060] The dicarboxylic and tricarboxylic fatty acids have a chain length of 8 to 41 carbon atoms, more specifically 11 to 25 carbon atoms, 13 to 23 carbon atoms, 15 to 23 carbon atoms, 17 to 21 carbon atoms, or a chain length of 19 carbon atoms.

[0061] Typically, the dicarboxylic and tricarboxylic fatty acids of the present invention are further substituted with one or more hydroxyl groups, typically with one hydroxyl group at the 2-position, as shown in, for example, agaricic acid.

[0062] Further examples of additional substituents are for example CH3, (CH3)2, C2H5, CH3-CO, COOCH3, COOC4H9, COOC5H 11 、CH3(CO2)O、O。

[0063] The C-chains in the dicarboxylic and tricarboxylic fatty acids can be straight or branched. Examples of compounds with branched C chains are 4-butane-2-yl-5-hydroxy-3,7-dimethylnonane-3,4,5-tricarboxylic acid, 5-hydroxy-6-octyloctadecane-3,4,5-tricarboxylic acid or 3,5-diethyl-4-hydroxyheneicosane-2,3,4-tricarboxylic acid.

[0064] Typically, the C-chain is unsaturated.

[0065] Explicitly disclosed herein are dicarboxylic and tricarboxylic fatty acids having any combination of the above-mentioned characteristics.

[0066] The specific compound used in the use of the present invention is: 2-hydroxyheptadecan-1,2,3-tricarboxylic acid-(C 20 H 36 O7); 2-hydroxy-2-(2-methoxy-2-oxoethyl)-3-tetradecylsuccinic acid (C 21 H 38 O7); 2-Hydroxynonadecane-1,2,3-tricarboxylic acid (Agaric acid) (C 22 H 40 O7); 2-hydroxyoctadecane-1,2,3-tricarboxylic acid (C 21 H 38 O7); 2-hydroxy-2-(16-methylheptadecanedioic acid (C 22 H 42 O5); 2-hydroxy-4-oxonadecane-1,2,3-tricarboxylic acid (C 22 H 38 O8); and 2-hydroxy-19-methyleicosane-1,2,3-tricarboxylic acid (C 24 H 44 O7).

[0067] Another specific group of compounds for use in the present invention is 2-hydroxyheptadecan-1,2,3-tricarboxylic acid-(C20H36O7); 2-hydroxynonadecan-1,2,3-tricarboxylic acid (agaric acid) (C22H40O7); 2-hydroxyoctadecane-1,2,3-tricarboxylic acid (C21H38O7); 2-hydroxy-4-oxonadecan-1,2,3-tricarboxylic acid (C22H38O8) and 2-hydroxy-19-methyleicosane-1,2,3-tricarboxylic acid (C24H44O7).

[0068] Table 1: Examples of dicarboxylic acids and tricarboxylic acids

[0069]

[0070]

[0071]

[0072] In the context of the present invention, "prevention" means that the use of the compounds of the present invention results in no biofilm formation at all, or a 75%, 80%, 90% or 95% reduction in the amount of biofilm formed, compared to a reference not using the biofilm preventing compound.

[0073] In the context of the present invention, "delayed" means that the amount of biofilm obtained with the compound of the present invention is obtained 2, 5, 10, or 20 times slower than with a reference not using the biofilm preventing compound.

[0074] Agaricic acid is confirmed as a new inhibitor of Salmonella biofilm that does not reduce planktonic growth. This biofilm-specific effect may be a major advantage, because it has been assumed that if the target virulence traits such as biofilm rather than growth, there is no selection pressure for resistant mutants [Allen et al. (2014) Nature Reviews Microbiology 12,300]. In addition, it has been shown that biofilm-specific inhibitors may increase the risk of contamination diffusion because diffusion is enhanced [Fleming & Rumbaugh (2017) Microorganisms 5,15]. However, in the case of agaricic acid, this potential disadvantage is reduced because flagellar motility is eliminated. In addition, motility itself is also an important virulence factor, further expanding the possible application field of agaricic acid [Josenhan & Suerbaum (2002) Int.J.of Medical Microbiology 291,605-614]. Therefore, agaricic acid illustrates a strong potential for industrial and medical use.

[0075] Example

[0076] Example 1. Agaricic acid reduces Salmonella biofilm formation

[0077] A crystal violet-based screening assay using the Calgary biofilm apparatus revealed agaricic acid as a potent inhibitor of biofilm formation by Salmonella typhimurium. Agaricic acid significantly prevented biofilm formation at concentrations above 100 μM, achieving 99.9% inhibition at 800 μM. Figure 1 A). Crystal violet staining measures the total biomass attached to the surface, thereby binding cells and biofilm matrix. To investigate whether agaricic acid reduces the number of bacteria attached to the surface, the number of CFU in biofilms grown on the bottom of glass petri dishes was determined. This revealed that agaricic acid also significantly inhibited the number of Salmonella cells attached to the surface, but the inhibition was not as strong as the biomass inhibition measured by crystal violet staining ( Figure 1C). Microscopic analysis confirmed that biofilms grown in the presence of agaricic acid were less dense than controls ( Figure 1 D).

[0078] This inhibition was not due to a bactericidal effect, as planktonic growth was not inhibited ( Figure 1 B). Furthermore, planktonic growth was enhanced at the highest concentration, indicating that agaricic acid prevents attachment and results in more bacteria remaining in the planktonic phase. Under the same conditions as the biofilm assay, the minimum inhibitory concentration (MIC) of agaricic acid was determined to be 8 mM, further confirming that no bactericidal effect occurred at concentrations relevant for biofilm inhibition.

[0079] Example 2. Agaricic acid inhibits flagellar motility

[0080] In order to reveal the mechanism by which agaricic acid inhibits biofilm formation, an internally developed reporter gene GFP-promoter fusion library was screened. The library contains reporter genes for 130 Salmonella genes associated with biofilm formation, including genes regulating matrix production, pili and flagella synthesis, quorum sensing, and c-di-GMP regulation (Table 2) [Robijns et al. (2014). Biofouling 30, 605-625.]. The first 24 h of biofilm formation in microtiter plates was delayed to confirm genes that were differentially transcribed in the presence of 100 μM agaricic acid. Since these reporter gene fusions express stable GFPmut3 variants as fluorophores, the fluorescence values ​​measured are the accumulation of fluorescence over time [Robijns et al. (2014). Biofouling 30, 605-625] ( Figure 2 ).

[0081] Notably, transcription of central biofilm regulatory genes such as CsgD and RpoS was not downregulated in the presence of agaricic acid. In addition, transcription of downstream genes such as csgB and adrA, responsible for the production of curli pili and cellulose, respectively, was not affected by agaricic acid [Steenackers et al. (2012) Food Research International 45, 502-531; Simm et al. (2014) Future microbiology 9, 1261-1282]. However, from 12 h onwards, transcription of the flagellar sigma factor fliA was significantly inhibited by agaricic acid. This downregulation was not caused by decreased transcription of flhDC, a master regulator of motility in Salmonella [Das et al. (2018) Front Cell Infect Microbiol 8, 36-36], as transcription of flhDC increased between 9 and 15 h compared to the control. Inhibition of fliA significantly reduces transcription of the downstream class III flagellar genes flgM, flgK, and motA, whereas transcription of the adjacent genes tdcA, flgB, fljB, and fliC is not reduced. FlgM encodes an anti-sigma factor that directly binds to and represses transcription of class III genes. The FlgM protein is secreted by the flagellar-specific export apparatus, effectively coupling flagellar assembly with transcriptional regulation. FlgK is a hook-associated protein that, together with FlgL, stabilizes the hook-filament junction, while the motor protein MotA is essential for driving flagellar rotation.

[0082] Therefore, the reporter gene fusion data show that agaricic acid inhibits flagellar motility. Motility and biofilm formation in Salmonella are reversed by the second signal molecule c-di-GMP. However, although the expression of genes necessary for flagellar motility is downregulated during biofilm maturation [Steenackers et al. (2012) Food Research International 45,502-531], initial adhesion on plastic or glass surfaces requires both flagella and active motility [Prouty and Gunn (2003) Infection and immunity 71,7154-7158; Mireles et al. (2001) Journal of bacteriology 183,5848-5854]. Therefore, inhibiting flagellar motility may explain the biofilm inhibition of agaricic acid.

[0083] To confirm that these changes in gene transcription also lead to phenotypic changes in motility, the flagella of Salmonella grown in the presence and absence of agaricic acid were visualized by staining. However, no difference in flagellar appearance was observed between these two conditions ( Figure 3A). Soft agar electrophoresis assays were then performed to verify whether downregulation of flagellar genes in the presence of agaricic acid resulted in reduced motility. In the absence of agaricic acid, Salmonella formed halos with an average diameter of 195 mm, indicating migration from the starting position by flagellar motility ( Figure 3 B). Agaricic acid completely abolished this migration, as no halo formed, indicating a clear effect on the motility phenotype. Concurrently, it was also found that the motA deletion mutant did not show any migration, confirming that motA expression is necessary for flagellar motility under the conditions tested. These results indicate that agaricic acid can completely inhibit motility, most likely not by inhibiting flagellar synthesis but due to downregulation of the flagellar motor protein MotA. Consistent with our hypothesis, the deletion of motA inhibited biofilm formation to a similar extent as agaricic acid. Importantly, the addition of agaricic acid did not further reduce biofilm formation in this deletion mutant, providing strong support for the mechanism by which agaricic acid reduces biofilm formation by inhibiting flagellar motility ( Figure 3 C).

[0084] Example 3. Salmonella biofilms are more sensitive to antimicrobial treatment in the presence of agaricic acid

[0085] It is well known that biofilms are extremely resistant to antimicrobial treatment. To test whether the prophylactic addition of agaricic acid makes Salmonella biofilms more sensitive to treatment with conventional disinfectants or antibiotics, biofilms grown for 48 hours in the presence and absence of agaricic acid were treated with 0.25% H2O2 or 1 μM ciprofloxacin for 1 hour. Hydrogen peroxide is a commonly used disinfectant in the food industry [Meireles et al. (2016) Food Research International 82, 71-85; Meireles et al. (2016) Food Research International 82, 71-85], while ciprofloxacin is a fluoroquinolone antibiotic frequently used to treat Salmonella infections [Tabak et al. (2009) FEMS microbiology letters 301, 69-76]. Biofilm formation has been shown to strongly protect Salmonella from the effects of either compound [González et al. (2018) Sci Rep 8, 222-222].

[0086] Biofilms grown in the presence of agaricic acid were significantly more sensitive to hydrogen peroxide. The increased sensitivity to treatment may be due to the presence of fewer bacteria, i.e., the result of the inoculum effect. In addition, crystal violet staining has shown that agaricic acid has a stronger inhibitory effect on the biofilm matrix than on the cell number, indicating that the remaining attached cells are less protected by the matrix. Agaricic acid also further increases the effect of ciprofloxacin treatment, but not significantly.

[0087] Example 4. Agaricic acid has a broad spectrum of activity

[0088] The Calgary biofilm apparatus was used to test whether agaricic acid could also inhibit biofilm formation of other opportunistic pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. It was found that agaricic acid inhibited biofilm formation of all three species. E. coli TG1 was even more sensitive to agaricic acid than Salmonella, as significant inhibition occurred already at 12.5 μM. Similar to the case of Salmonella, planktonic growth was unaffected, except for an increase at the highest concentration. In contrast, agaricic acid inhibited planktonic growth and biofilm formation of P. aeruginosa PA14 and S. aureus SH1000. However, S. aureus biofilm inhibition occurred already at concentrations lower than the bactericidal effect, indicating that some biofilm-specific effects occurred. In contrast, the main effect on P. aeruginosa was bactericidal, as planktonic growth decreased at concentrations below biofilm inhibition. However, at high concentrations of agaricic acid, biofilm formation was inhibited to a greater extent than planktonic growth.

[0089] The flagellar systems of E. coli and Salmonella show a high degree of similarity at the genetic and functional levels [Albanna et al. (2018) Sci Rep 8, 16705]. In addition, E. coli also requires normal flagellar function in order to successfully adhere to abiotic surfaces [Pratt & Kolter (1998) Molecular microbiology 30, 285-293]. Therefore, the specific biofilm inhibition of agaricic acid on Salmonella and E. coli further supports our hypothesis that agaricic acid prevents biofilm formation by inhibiting flagellar rotation. In contrast, Staphylococcus aureus does not show flagellar motility, but moves by extension or sliding [Pollitt & Diggle (2017) Cell Mol Life Sci 74, 2943-2958]. Therefore, agaricic acid cannot inhibit Staphylococcus aureus biofilm formation by interfering with the expression of genes responsible for flagellar rotation. Furthermore, although Pseudomonas has flagella that are involved in adhesion and biofilm formation [O'Toole & Kolter (1998) Molecular microbiology 30, 295-304], the primary bactericidal effect of agaricic acid suggests that agaricic acid has different targets in Pseudomonas. Therefore, the mode of action of agaricic acid is species-dependent.

[0090] Example 5. Agaricic acid inhibits biofilm formation in mixed species communities

[0091] In situ biofilms are typically complex communities composed of various bacterial strains and species [Elias & Banin (2012) FEMS Microbiol Rev 36, 990-1004; et al. (2014) Trends Microbiol 22, 84-91; Giaouris et al. (2015) Front Microbiol 6, Article 841; Mitri & Richard Foster (2013) Communities. Annual Review of Genetics 47, 247-273]. Since these bacterial consortia are often reported to show enhanced biofilm formation compared to their single-species counterparts, it was determined whether agaricic acid also inhibits biofilm formation of mixed-species communities [Oliveira et al. (2015) PLoS Biol 13, e1002191-e1002191; Lories et al. (2020) Current Biology 30, 1231–1244; et al. (2006) Appl Environ Microbiol 72, 3916-3923; Ren et al. (2014). Microbial ecology 68, 146-154]. To this end, a previously characterized model community consisting of two Salmonella and one E. coli strain (S. typhimurium SL1344 (S1), S. typhimurium ATCC14028 (S2) and E. coli MG1655 (E1)) was utilized. In this mixed species consortium, agaricic acid also significantly reduced the number of biofilm cells attached to abiotic surfaces ( Figure 6 A). The reduction in biofilm productivity in mixed-species communities was mainly due to the inhibition of strain S2 ( Figure 6 B) The lack of S1 inhibition under mixed species conditions may be due to agaricic acid weakening the competitive interaction experienced by S1 ( Figure 6 C). Previous studies have shown that the presence of S2 and E1 significantly inhibits the growth of S1. Therefore, S1 detects damage caused by competition through its stress response system and induces a competitive response. This competitive response includes upregulation of tolerance- and virulence-related phenotypes and leads to increased tolerance of S1 to antimicrobial treatment. Therefore, we subsequently investigated whether agaricic acid could interfere with the increased tolerance under mixed-species conditions by reducing competition and preventing the induction of this competitive response.

[0092] Example 6. Agaricic acid eliminates the enhanced tolerance of S1 under mixed species conditions

[0093] It was confirmed that the presence of S2 and E1 increased the resistance of S1 to antimicrobial treatment in DMSO control ( Figure 7 A). These findings are consistent with previous reports showing that bacterial interactions within mixed-species biofilms can strongly enhance tolerance [Harriott & Noverr (2009). Antimicrob Agents Chemother 53, 3914-3922; Jahid et al. (2015). Food microbiology 46, 383-394; Parijs & Steenackers (2018) ISME J 12, 2061-2075; Schwering et al. (2013) Biofouling 29, 917-928; Lee et al. (2014). ISME J 8, 894-907]. As previously shown using a slightly modified setup, preventive treatment with agaricic acid reduced the tolerance of the remaining biofilm cells to ciprofloxacin and H2O2. In addition, S1 co-cultured with the other two strains no longer survived the antimicrobial treatment to a greater extent than the monocultured S1 biofilm. Therefore, agaricic acid completely eliminates the tolerance that S1 enhances under mixed species conditions. These results show that the reduction of competition prevents competitive reaction and suppresses the increase of tolerance relevant to this reaction. However, weakening competition can increase the viability of unwanted bacteria at first. Therefore, it is crucial that the sensitivity to the increase of antimicrobial agents is enough to compensate for this initial increase in cell number and cause the absolute reduction of remaining cells after treatment. Importantly, in the presence of agaricic acid, the mixed species community shows the minimum number of S1 cells that survive in antimicrobial treatment ( Figure 7 B) Thus, agaricic acid also shows a strong potential to attenuate mixed-species biofilm communities and reduce resistance to antimicrobial agents.

[0094] Example 7. Materials and Methods

[0095] Bacterial strains and culture conditions

[0096] Overnight cultures (ONC) of Salmonella enterica serovar Typhimurium ATCC14028, Escherichia coli TG1, Pseudomonas aeruginosa PA14, and Staphylococcus aureus SH1000 were grown in the presence of 100 μg ml -1 The cells were grown in Luria–Bertani (LB) broth containing 1% ampicillin (if appropriate) at 37°C with shaking and aeration.

[0097] Anti-biofilm assay

[0098] As previously described, a static PEG assay was used to prevent bacterial biofilm formation. A lid with 96 polystyrene PEGs was assembled into a microtiter plate, with a PEG suspended in each well. Two-fold serial dilutions of the compound in 100 μl of liquid broth per well were prepared in the microtiter plate. Subsequently, the overnight culture was diluted 1:100 into the corresponding liquid broth, and 100 μl (~10 6 Cells) were added to each well of the microtiter plate to give a total of 200 μl of culture medium per well. After the lid was placed on the microtiter plate, samples containing Salmonella, Pseudomonas, or E. coli were statically cultured in TSB 1 / 20 at 25°C for 48 h, while Staphylococcus aureus was incubated in undiluted TSB at 37°C for 48 h. After incubation, the lid was removed from the microtiter plate and the liquid culture was transferred to a new microtiter plate, which was then analyzed by OD using a Synergy MX multimode reader (Biotek, Winooski, VT). 600 The planktonic growth in each well was measured. The pegs were washed once in 200 μl PBS and the remaining attached bacteria were stained with 200 μl 0.1% crystal violet in isopropanol-methanol-PBS solution (1:1:18) for 30 min. Excess stain was washed off by placing the pegs in a 96-well plate filled with 200 μl distilled water per well. The pegs were then air-dried for 30 min and the dye bound to the adherent cells was dissolved in 200 μl 30% glacial acetic acid. The OD of each well was measured using a Synergy MX multimode plate reader. 570 Data were analyzed using GraphPad Prism 6 software.

[0099] Minimum inhibitory concentration (MIC) determination

[0100] MIC values ​​were determined in 96-well plates. Two-fold serial dilutions of agaricic acid or DMSO were prepared in 100 μl of TSB 1 / 20, and 100 μl of the inoculum diluted 1 / 100 in TSB 1 / 20 was added. The plates were covered with a breathable seal and lid and incubated at 25°C with shaking at 200 rpm for 24 hours. The MIC was defined as the lowest concentration of compound at which Salmonella growth was below the upper limit of the 95% confidence interval of the negative control.

[0101] Petri dish biofilm assay

[0102] The ONC of Salmonella typhimurium ATCC 14028 was normalized to an OD of 3.2. 595 , and in a small tube containing 10 ml of 1 / 20 TSB Agaricic acid was diluted 1 / 100 in a glass Petri dish and a final concentration of 100 μM or a corresponding amount of DMSO was added. 7 cells ml -1 , and incubated at 25°C under static conditions for 48 h. The liquid above the biofilm was then poured off and the biofilm was scraped from the bottom of the plate in 1 ml of PBS, passed through a syringe (25G) and vortexed to disrupt the biofilm structure and ensure uniform suspension during dilution [Hermans et al. (2011). Journal of microbiological methods 84, 467-478]. The number of colony forming units (CFU) of the biofilm was determined by plating.

[0103] Microscopic analysis

[0104] The ONC of Salmonella typhimurium ATCC 14028 containing the pFPV25.1 plasmid encoding constitutive GFPmut3 production was normalized to an OD of 3.2. 595 , and 20 μl was added to a solution containing 2 ml 1 / 20 TSB and 100 μg ml -1 glass-bottom microwell culture dishes ( Petri dish, Microwell). Inoculate about 12*10 7 cells ml -1 , and incubated at 25° C. under static conditions for 48 h. After incubation, the planktonic phase was gently decanted and the biofilm was washed with 1 ml of PBS. The biofilm was visualized.

[0105] Microtiter plate-based GFP promoter fusion assay

[0106] 1.5 μl of reporter gene-fused ONC was transferred in triplicate to a black polystyrene, clear-bottom microtiter plate (Greiner bio-one 655096) containing 200 μl of 1 / 20 TSB and agaricic acid at a final concentration of 100 μM or the corresponding amount of DMSO. The microtiter plate was then incubated statically at 25°C for 24 h. Every 3 h, fluorescence (excitation 488 nm, emission 511 nm) and absorbance at 600 nm (OD) were measured using a Synergy MX multimode plate reader. 600 For data analysis, fluorescence (using a promoter-less pFPV25 vector as a control) and OD 600 Subtract the blank measurement from both. Use different OD values ​​for strains / conditions 600Any effect on fluorescence caused by differences in bacterial growth was normalized by the ratio between the values ​​obtained. Significant differences in fluorescence levels between treatments and controls were determined using a two-sided Student's t-test (P < 0.05).

[0107] Flagella staining

[0108] The ONC of Salmonella typhimurium ATCC 14028 was diluted 1 / 100 in 5ml TSB1 / 20 with a final concentration of 100μM agaricic acid or the corresponding amount of DMSO as a control. The planktonic culture was incubated at 25°C with 200rpm shaking for 24h. According to Kearns and Losick (2003) Molecular microbiology 49, 581-590, flagella were stained. In brief, the stain consisted of ten parts of mordant (2g tannic acid, 10ml 5% phenol, 10ml saturated AlK (SO4) 2 aqueous solution) mixed with one part of stain (12% crystal violet in ethanol). 3μl of sample was applied to a microscope slide and covered with a 22mm × 40mm cover glass. After the slide was placed vertically, 10μl was applied to the top edge of the cover glass so that the sample was stained due to capillary forces. Samples were visualized using phase contrast using a Zeiss Axio Imager Z1 microscope with an EC Plan Neofluar (x100 magnification / 1.3 numerical aperture) objective.

[0109] Soft agar electrophoresis assay

[0110] Based on Kim & Surette (2003) Biol Proced Online 5, 189-196, swimming plates were prepared by mixing 30 ml of TSB 1 / 20 with 0.25% agar. These plates contained agaricic acid at a final concentration of 100 μM or the corresponding amount of DMSO. After drying at room temperature for 2 h, 3 μl of overnight culture was inoculated by piercing the agar surface with a pipette tip. The plates were incubated upright at 25°C for 24 h, after which the size of the halo was measured and recorded visually.

[0111] Tolerance determination

[0112] To determine the tolerance of mature biofilms, biofilms were grown on microscope glasses (75 mm x 25 mm) placed vertically in a 50 ml falcon container filled with 30 ml TSB 1 / 20. This setup allowed for easy transfer of mature biofilms because the top of the vertical slide protruded from the culture medium, which allowed the slide to be gripped with tweezers without damaging the biofilm. The ONC of Salmonella was normalized to an OD of 3.2. 600, and diluted 1 / 100 into a broth containing a final concentration of 100 μM agaricic acid or the corresponding amount of DMSO. After static incubation at 25°C for 48 h, the slides were transferred to new 50 ml falcon containers containing 0.25% H2O2, 1 μM ciprofloxacin, or PBS and incubated for 1 h. The biofilms were then scraped from the slides in 10 ml of PBS, vortexed with a 25G syringe to disrupt the biofilm structure and ensure uniform suspension during the dilution period. The number of colony-forming units (CFU) of the biofilms was determined by plating.

[0113] Mixed species biofilm assay

[0114] Normalize ONCs of S1, S2, and E1 to an OD of 3.2 595 And add to a small amount of 1 / 20 TSB containing 10 ml The three strains were inoculated in a 1:1:1 ratio in glass Petri dishes and incubated at 25°C under static conditions for 48 h. The same total number of cells (~12*10 7 cells ml -1 ). The liquid above the biofilm was then poured off and the biofilm was scraped from the bottom of the plate in 1 ml of PBS, passed through a syringe (25G) and vortexed to disrupt the biofilm structure and ensure uniform suspension during dilution. The number of colony forming units (CFU) of the biofilm was determined by plating. To distinguish between strains, S1 was labeled with the constitutive gfpmut3 on the plasmid, while S2 and E1 were labeled with the constitutive dsRed.T4 encoded by the plasmid. The differences in colony shape and size allowed S2 to be distinguished from E1 during CFU counting.

[0115] Mixed species tolerance assay

[0116] As described above, single-species and mixed-species biofilms were grown on Petri dishes at 25°C for 48 h. The medium was then replaced with 5 ml of PBS containing 0.25% H2O2 or 1 μM ciprofloxacin, and the biofilms were incubated for an additional 1 h at 25°C. Subsequently, the biofilms were scraped off and plated on solid LB agar plates for CFU determination. To differentiate between strains, S1 was labeled with the constitutive gfpmut3 on the plasmid, while S2 and E1 were labeled with the constitutive dsRed.T4 encoded by the plasmid. Differences in colony shape and size allowed S2 to be distinguished from E1 during CFU counts.

[0117] Table 2: Overview of all genes tested during the GFP promoter fusion assay

[0118]

[0119]

Claims

1. In vitro use of a dicarboxylic or tricarboxylic acid having a chain length of 17 to 21 carbon atoms for preventing, reducing or delaying the formation of biofilms of bacteria on abiotic surfaces.

2. The method according to claim 1, wherein the carboxylic acid is substituted with a hydroxyl group.

3. The method according to claim 1 or 2, wherein the carboxylic acid is a 1,2,3 tricarboxylic acid.

4. Use according to any one of claims 1 to 3, wherein the carboxylic acid is a 2-OH, 1,2,3 tricarboxylic acid.

5. The use according to any one of claims 1 to 4, wherein the carboxylic acid is selected from the group consisting of: 2-hydroxyheptadecan-1,2,3-tricarboxylic acid-(C 20 H 36 O7); 2-Hydroxynonadecane-1,2,3-tricarboxylic acid (Agaric acid) (C 22 H 40 O7); 2-hydroxyoctadecane-1,2,3-tricarboxylic acid (C 21 H 38 O7); 2-hydroxy-4-oxonadecane-1,2,3-tricarboxylic acid (C 22 H 38 O8) and 2-hydroxy-19-methyleicosane-1,2,3-tricarboxylic acid (C 24 H 44 O7).

6. The use according to any one of claims 1 to 5, wherein the carboxylic acid is 2-hydroxynonadecane-1,2,3-tricarboxylic acid (C 22 H 40 O7)(Agaricic acid).

7. Use according to any one of claims 1 to 6, wherein the carboxylic acid is used in a concentration of at least 5 μM.

8. Use according to any one of claims 1 to 6, wherein the carboxylic acid is used in a concentration of at least 10 μM.

9. Use according to any one of claims 1 to 6, wherein the carboxylic acid is used in a concentration of at least 25 μM.

10. Use according to any one of claims 1 to 6, wherein the carboxylic acid is used in a concentration of at least 50 μM.

11. Use according to any one of claims 1 to 6, wherein the carboxylic acid is used in a concentration of at least 100 μM.

12. Use according to any one of claims 1 to 6, wherein the carboxylic acid is used in a concentration of at least 500 μM.

13. The use according to any one of claims 1 to 12, for preventing Salmonella typhimurium biofilm formation, Staphylococcus aureus biofilm formation or Escherichia coli biofilm formation.

14. Use according to any one of claims 1 to 13, followed by the use of an antimicrobial agent.

15. The use according to claim 14, wherein the antimicrobial agent is a peroxide.

16. The use according to claim 14, wherein the antimicrobial agent is hydrogen peroxide.

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