KR102566917B1 - Composition for inhibiting multi-pathogenic microbial biofilm formation comprising saw palmetto oil and its unsaturated fatty acids as effective ingredients - Google Patents

Abstract

The present invention relates to a composition for inhibiting biofilm formation containing saw palmetto oil and its unsaturated fatty acids as active ingredients. According to the present invention, saw palmetto oil and its unsaturated fatty acids, lauric acid and Myristic acid was found in Staphylococcus aureus, E. coli O157:H7, and Candida albicans alone, dual species, and three species, respectively. biofilm formation is inhibited with minimal cytotoxic effect, and lauric acid or myristic acid among them is treated in combination with antibiotics or antifungal agents to prevent Staphylococcus aureus and pathogenic Escherichia coli (E. coli O157:H7). It has a synergistic antibacterial effect against
Therefore, the composition containing saw palmetto oil and its unsaturated fatty acids as active ingredients can be usefully used as a coating composition for inhibiting biofilm formation of multi-pathogenic microorganisms or as an antibacterial composition.

Description

Composition for inhibiting multi-pathogenic microbial biofilm formation comprising saw palmetto oil and its unsaturated fatty acids as effective ingredients}

The present invention relates to a composition for inhibiting multi-pathogenic biofilm formation containing saw palmetto oil and unsaturated fatty acids thereof as active ingredients.

A biofilm or biofilm is a colony of sessile microorganisms that grow on a surface by self-produced extracellular polymeric material, and these films appear ubiquitously in natural, medical, and engineered environments. Pathogenic biofilms are resistant to conventional antibiotics, host defense systems, and external stresses, which can cause serious problems in human health and persist chronic bacterial infections.

Relatedly, Staphylococcus aureus is the causative agent of various acute and chronic infections. Often resistant to antibiotics, it is responsible for the worldwide outbreak of nosocomial infections. Biofilm formation by Staphylococcus aureus is of particular concern in the medical field because biofilm formation plays an important role in antibiotic resistance. The emergence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus poses a serious threat. Staphylococcus aureus secretes various exotoxins such as α-hemolysin, enterotoxins, coagulase and protein A. In particular, α-hemolysin (also known as α-toxin) is a major virulence factor associated with pathogens of sepsis, pneumonia, and severe skin infections, and is known to contribute to biofilm formation. Therefore, it is important to find a non-toxic compound that does not have drug resistance and can inhibit the biofilm of Staphylococcus aureus.

On the other hand, most antibiotics ideally aim to inhibit microbial growth without harming the host or the environment. However, the overuse of these drugs has led to the emergence of drug-resistant pathogens worldwide. Bacterial and fungal biofilms play an important role in antibacterial resistance and various device-associated infections, and various pathogenic microbes often form multispecies biofilms to further increase resistance to antibiotics.

In other words, when a biofilm is formed, it can be seen that it has become resistant to antibiotics. In this case, the sensitivity of bacteria to antibiotics is lowered, so antibiotics are almost ineffective. In particular, infections caused by bacteria forming biofilms are The problem is more serious because there are many cases caused by multidrug-resistant bacteria that are resistant to antibiotics.

Therefore, alternative non-toxic approaches are needed to control single and multi-microbial biofilm formation. Unlike current antibacterial strategies, it is recognized that it is important to identify biofilm or biofilm inhibitors that do not inhibit planktonic cell growth to reduce the risk of drug resistance.

Currently, the importance of multispecies biofilms is gradually recognized, and there are many research results on single species biofilms, but inhibition of dual species biofilms and three species biofilms has not yet been achieved. Until now, it has hardly been reported.

On the other hand, plants are a major source of antibiotics and other pharmaceuticals, and secondary metabolites of various plants are known to have antibacterial activity against Staphylococcus aureus. As a representative example, saw palmetto (Seronoa repens) fruit extract has been widely used as an alternative treatment for benign prostatic hyperplasia. Pric acid, lauric acid, myristic acid, palmitic acid, oleic acid, linolenic/linoleic acid and stearic acid.

In particular, these fatty acids are widespread in all forms of life, and more than 70 natural fatty acids have been identified. Antibacterial activities of various fatty acids, mostly at doses of 100 µg/ml or higher, have been reported against a variety of microorganisms. However, recent studies have shown that fatty acids exhibit antibacterial and antiviral activity against bacteria and fungi at concentrations well below the minimum inhibitory concentrations (MICs) and promote sporulation of mycorrhizal fungi.

Under this background, the present inventors tried to develop a biofilm inhibitory composition having a dual species or three species biofilm inhibitory effect. Therefore, whether saw palmetto oil and its unsaturated fatty acids can be used as a biofilm inhibitory composition, in detail, for S. aureus, Escherichia coli O157:H7 and Candida albicans, respectively It was confirmed whether there was an inhibitory effect on single, dual species strains, and three species strains of . In addition, the antibacterial effect and cytotoxicity were confirmed through the synergistic effect of fatty acids and antibiotics.

Korean Registered Patent No. 10-1183484 (registered on September 11, 2012)

The present inventors confirmed that saw palmetto oil and lauric acid and myristic acid extracted therefrom inhibit the formation of three species of biofilm by S. aureus, E. coli O157:H7 and C. albicans with minimal cytotoxic effect In particular, it was found that the biofilm formation of S. aureus, E. coli O157:H7, and C. albicans single-, double-, and triple-species pathogenic microorganisms could be inhibited. In addition, it was confirmed that lauric acid or myristic acid had a synergistic antibacterial effect against S. aureus and E.coli O157:H7 by co-treatment with antibiotics.

Accordingly, an object of the present invention is to provide a composition for inhibiting biofilm formation of multiple pathogenic microorganisms, a method for inhibiting biofilm formation of multiple pathogenic microorganisms using the same, and a coating composition or antimicrobial composition comprising the composition.

In order to achieve the above object, the present invention is effective at least one selected from the group consisting of saw palmetto oil, its unsaturated fatty acid lauric acid, and myristic acid It provides a composition for inhibiting biofilm formation of multi-pathogenic microorganisms containing as components.

In addition, the present invention is the composition; And antibiotics or antifungal agents; It provides a method for inhibiting multi-pathogenic microbial biofilm formation comprising the step of concurrently treating the surface of an organism or a non-living organism.

In addition, the present invention provides a coating composition for inhibiting multi-pathogenic microorganism biofilm formation comprising the composition for inhibiting biofilm formation.

In addition, the present invention provides (a) lauric acid or myristic acid as an unsaturated fatty acid; And (b) an antibiotic or antifungal agent; it provides an antimicrobial composition containing as an active ingredient.

The composition for inhibiting biofilm formation according to the present invention is Staphylococcus aureus, pathogenic Escherichia coli (E. coli O157:H7), and Candida albicans alone, dual species, and triple species, respectively. (Three species) biofilm formation is inhibited with minimal cytotoxic effect, and lauric acid or myristic acid among them are treated in combination with antibiotics or antifungal agents to inhibit Staphylococcus aureus and pathogenic Escherichia coli (E. coli O157). :H7) has a synergistic antibacterial effect.

Therefore, the composition according to the present invention can inhibit the formation of biofilms of Staphylococcus aureus (S. aureus), pathogenic Escherichia coli (E. coli O157:H7) and Candida (albicans), thereby preventing infectious diseases caused by infection with pathogenic bacteria. It can be used effectively for the prevention and treatment of

1 shows the results of confirming the biofilm inhibitory activity of saw palmetto oil and major fatty acids. Figure 1a is a result of quantifying biofilm formation by each microorganism in the presence of saw palmetto oil after culturing the microorganisms in a 96-well plate for 24 hours, and Figure 1b shows the chemical structure of major fatty acids in saw palmetto oil. 1c to 1f show lauric acid concentrations of S. aureus MSSA 6538 (c), E. coli O157:H7 (d), C. albicans DAY185 (e) and other Staphylococcus strains (f) in 96-well plates. (C12: 0), myristic acid (C14: 0), palmitic acid (C16: 0), and oleic acid (C18: 1) in the presence of 37 ℃ temperature for 24 hours after culturing for 24 hours, the result of quantifying the inhibitory effect on biofilm formation (n = 2 biologically independent samples, error bars indicate standard deviation*, P <0.05 vs. untreated control), Figure 1g shows 100 μg/ml saw palmetto oil, 20 μg/ml lauric acid ( C12: 0) or 20 μg / ml myristic acid (C14: 0) in the presence of biofilm formation on the polystyrene plate was observed by confocal laser microscopy (CLSM). The scale bar represents 100 μm, and for microscopy experiments, two or more independent cultures were performed and more than 10 locations were randomly analyzed.
2 shows the results of confirming the biofilm formation inhibitory effect of saw palmetto oil, lauric acid (C12:0) and myristic acid (C14:0) in polymicrobial models. Figure 2a shows the dual species biofilm inhibitory effect of S. aureus MSSA 6538 and E. coli O157:H7 in LB medium, and Figure 2b shows S. aureus 6538 and C. albicans in a 1:1 mixture of PDB and LB medium. DAY185 confirms the dual species biofilm inhibitory effect, and FIG. 2c shows the dual species biofilm inhibitory effect on E. coli O157:H7 and C. albicans in a 1:1 mixture of PDB and LB medium, and FIG. 2d is the result of confirming the biofilm inhibitory effect on the triple species pathogenic microorganisms S. aureus, E. coli O157: H7 and C. albicans in a 1:2 mixture of PDB and LB media (incubation at 37 ° C. for 24 hours, n = 2 biologically independent samples. *, P < 0.05 vs. untreated control), Fig. 2e shows confocal laser treatment of biofilms of tri-species pathogenic microorganisms of S. aureus, E. coli O157:H7 and C. albicans. 2f is a scanning electron microscope (SEM) observation result of biofilms of three species of pathogenic microorganisms of S. aureus, E. coli O157:H7, and C. albicans. In the inset, the large cell is C. albicans (indicated by a blue triangle), the small round cell is S. aureus (indicated by a red triangle), and the rod type is E. coli O157:H7 (indicated by a yellow triangle). The yellow scale bar represents 100 μm, the white scale bar represents 15 μm and the inset red scale bar represents 3 μm. For microscopy experiments, at least two independent cultures were performed and at least 10 random positions were analyzed.
Figure 3 shows the results of confirming the effect of fatty acids on transcriptional profiles, cell hydrophobicity and hyphal formation. Figures 3a to 3c show the transcriptional profile results, S. aureus MSSA 6538 (a), E. coli O157:H7 (b) or C. albicans DAY185 (c) were treated with 100 μg/ml saw palmetto oil, 20 μg/ml In the presence or absence of ml lauric acid (C12:0) or 20 μg/ml myristic acid (C14:0), shaking at 250 rpm at 37° C. for 6 hours, 4 hours, or 6 hours, respectively, qRT-PCR was performed. This is the result showing the obtained fold changes. Fold results show the transcriptional changes of treated and untreated controls, and experiments were performed in duplicate (four qRT-PCR reactions were performed per gene). *, P < 0.05 vs. Untreated control (None). Figures 3d to 3f are the effects of fatty acids on cell hydrophobicity and hyphal formation, saw palmetto oil and lauric acid in S. aureus 6538 (d), E. coli O157 (e) and C. albicans DAY185 (f). (C12:0) and myristic acid (C14:0), this is the result of confirming the hydrophobicity after culturing at 37°C for 24 hours, regardless of the presence or absence of myristic acid (C14:0). Figure 1g is a result confirming the suppression of hyphal filamentation by fatty acids after culturing C. albicans DAY185 in PDB medium at 37 °C for 24 hours. The scale bar represents 50 μm, and was analyzed by performing at least 2 independent cultures and randomly selecting at least 10 positions for microscopy experiments.
4 shows fatty acids; Or, it is the result of confirming the activity of anti-virulence activities when a fatty acid and antimicrobial agents are co-administered. Figures 4a to 4c are the results showing the survival rate of nematodes, saw palmetto oil, lauric acid (C12: 0) or myristic acid (C14: 0) in the presence of S. aureus MSSA 6538 (a), E This is the result of confirming the survival rate of nematodes after exposure to C. coli O157:H7 (b) and C. albicans DAY185 (c) cells. Figure 4d is a result of chemical toxicity (Chemical toxicities) evaluation obtained by treating uninfected C. elegans for 4 days. A control group that did not do so was indicated as None. *, P < 0.05 vs. Untreated control (None). OP50 represented E. coli OP50. Figure 4e and Figure 4f is a result of confirming the cell viability of the antibiotic or antifungal agent and combined treatment, the antibiotic gentamycin (gentamycin) (10 μg / ml) and the antifungal agent amphotericin B (0.5, 2 and 5 μg / ml) This is the result of measuring the survival rate of S. aureus (e) or E. coli O157:H7 (f) in the presence of * = P < 0.05 vs. untreated control. None; untreated control. SPO: saw palmetto oil (100 μg/ml), fatty acids lauric acid (C12:0) and myristic acid (C14:0) (20 μg/ml), Gen: gentamicin, AMB: amphotericin B n = 2 biologically independent samples.

The present inventors have found that saw palmetto oil and lauric acid and myristic acid extracted therefrom inhibit biofilm formation of three species by S. aureus, E. coli O157:H7 and C. albicans with minimal cytotoxic effect. In particular, it was found that the biofilm formation of S. aureus, E. coli O157:H7, and C. albicans single-, double-, and triple-species pathogenic microorganisms could be inhibited. In addition, the present invention was completed by confirming that lauric acid or myristic acid combined with an antibiotic or antifungal agent had a synergistic antibacterial effect against S. aureus and E.coli O157:H7.

Accordingly, the present invention contains, as an active ingredient, at least one selected from the group consisting of saw palmetto oil, its unsaturated fatty acid lauric acid, and myristic acid. Provided is a composition for inhibiting biofilm formation of multiple pathogenic microorganisms.

The pathogenic microorganism may be at least one selected from the group consisting of Staphylococcus aureus, pathogenic Escherichia coli (E. coli O157:H7), and Candida albicans.

Preferably, the composition inhibits formation of a dual species biofilm or a three species biofilm.

Specifically, the dual species biofilm is Staphylococcus aureus and pathogenic Escherichia coli (E. coli O157:H7); or Staphylococcus aureus and Candida albicans; or pathogenic Escherichia coli (E. coli O157:H7) and Candida albicans;

The three species biofilm can inhibit biofilms of Staphylococcus aureus, E. coli O157:H7, and Candida albicans.

In the present invention, saw palmetto oil and its abundant lauric acid and myristic acid can inhibit biofilm formation against single, double and triple species pathogenic microorganisms such as S. aureus, E. coli O157:H7 and C. albicans. was experimentally revealed.

In addition, the composition inhibits the expression of hla and hld in Staphylococcus aureus, and csgAB, a fimbriae gene, in pathogenic Escherichia coli (E. coli O157:H7); the motility genes fimH, flhD and motB; and quorum sensing genes, luxR and luxS, and inhibition of HWP1, a cell wall gene in Candida albicans, to inhibit biofilm formation.

The unsaturated fatty acids can inhibit hypha formation and biofilm formation.

The composition may further contain an antibiotic or antifungal agent. The antibiotic may be at least one selected from the group consisting of gentamicin, kanamycin, tetracycline, methicillin, vancomycin, cephalosporin, and rifampicin. Yes, and is not necessarily limited thereto.

In addition, the antifungal agent may be at least one selected from the group consisting of amphotericin B, terbinafine, fluconazole, ketoconazole, and itraconazole, but is not necessarily limited thereto.

In addition, the present invention is a composition for inhibiting the formation of the biofilm; And antibiotics or antifungal agents; It provides a method for inhibiting multi-pathogenic microbial biofilm formation comprising the step of concurrently treating the surface of an organism or a non-living organism.

In addition, the present invention comprises as an active ingredient at least one selected from the group consisting of saw palmetto oil, its unsaturated fatty acid lauric acid, and myristic acid, Provided is a composition for coating for inhibiting multi-pathogenic microbial biofilm formation.

Preferably, the composition may be coated on a medical device, medical material or medical implant, but is not limited thereto.

In addition, the present invention provides (a) lauric acid or myristic acid as an unsaturated fatty acid; And (b) an antibiotic or antifungal agent; it provides an antimicrobial composition containing as an active ingredient.

Preferably, the composition may exhibit antibiofilm and antibacterial activity against Staphylococcus aureus or pathogenic Escherichia coli (E. coli O157:H7).

In addition, the composition for inhibiting biofilm formation according to the present invention prevents infectious diseases caused by biofilms of Staphylococcus aureus, E. coli O157:H7, and Candida albicans. Alternatively, it may be provided as a health food composition for improvement.

In addition, the composition for inhibiting biofilm formation according to the present invention may provide an antibacterial cosmetic composition. When the composition of the present invention is a cosmetic composition, the cosmetic composition may include, in addition to the components, a stabilizer, a solubilizer, common adjuvants such as vitamins, pigments and fragrances, and a carrier. In addition, the cosmetic composition may further include a skin absorption accelerator to enhance its effect.

Therefore, the composition for inhibiting biofilm formation according to the present invention includes a composition for preventing or treating infectious diseases caused by biofilms of multiple pathogenic microorganisms, an antibacterial composition, and a coating composition for inhibiting biofilm formation of pathogenic microorganisms. can be put to good use.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Preparation of strains, chemicals, and culture materials

Table 1 shows the 11 microorganisms used in this study, media, minimum inhibitory concentration (MIC), and inoculum sizes for minimum inhibitory concentration (MIC) determination. All media [Luria-Bertani (LB), tryptic soy broth (TSB), potato dextrose broth (PDB), nutrient broth and agar were prepared by Becton Dickinson (Franklin Lakes) , NJ, USA). Minimum inhibitory concentrations (MICs) were determined according to the Clinical Laboratory Standards Institute (CLSI) for bacteria and yeast (Table 1). Table 1 is a table showing the minimum inhibitory concentrations (MICs) of the strains and culture media used in the present invention and saw palmetto oil, lauric acid and myristic acid.

The minimal inhibitory concentration (MIC) was defined as the lowest concentration that inhibited cell growth by 80%, as assessed by spectrophotometry and colony counting. Experiments were performed with at least three independent cultures. Four fatty acids, dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid), 9-octadecenoic acid (oleic acid), and antibiotics, namely gentamicin and amphotericin B was purchased from Sigma-Aldrich (St. Louis, USA) or TCI Co. (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was used to dissolve all fatty acids, and 0.1% (v/v) DMSO was used as a negative control. For reference, this concentration did not affect bacterial growth or biofilm formation.

Example 2: Biofilm formation assay in 96-well plate

Biofilm formation of various microorganisms was produced in 96-well polystyrene plates as previously described. Briefly, overnight culture at an initial turbidity of OD 0.05 (~ 108 or 107 CFU/ml) for bacteria and 600 nm OD 0.1 (~ 105 CFU/ml) for Candida at an initial turbidity. It was inoculated into an appropriate culture medium in a final volume of 300 μL with or without fatty acids, and incubated at 37° C. for 24 hours without shaking (Table 1). Biofilm cells attached to a 96-well plate (SPL Life Sciences, Pocheon, Korea) were stained with 0.1% crystal violet Sigma-Aldrich (St. Louis, USA) for 20 minutes, washed repeatedly with sterile distilled water and 95% ethanol. was resuspended in The plate was read at 570 nm and the results were performed in at least 6 replicates in two independent cultures. The percentage of inhibition ratio was expressed as a relative biofilm value (compound-treated biofilm/compound-untreated control x 100).

Example 3: Gas Chromatography/Mass Spectrometry (GC-MS)

The components of saw palmetto oil were separated and gaseous using an Agilent 6890N GC and SP-2560 (Supelco, Sigma-Aldrich, St. Louis, USA) with a silica capillary column (100 mx 0.25 mm id., film thickness 0.25 mm). It was analyzed by chromatography/mass spectrometry (GC-MS). Column temperature conditions and derivatization (methylation) of fatty acids were previously known Front. Microbiol. 9, 1241 (2018). Quantification was performed using triundecanoin (C11: 0) as an internal standard and integrating regions and correcting for fatty acid methylation. Supelco 37 component FAME Mix (Supelco) was used as a reference standard.

Example 4: Observation of biofilm formation using a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM)

Single-, double-, or triple-species biofilms were formed in 96-well polystyrene plates in the presence or absence of fatty acids at 37° C. for 24 hours without agitation. Planktonic cells were removed by washing with distilled water three times, and single- or double-species biofilms were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular Probes, Inc, Eugene, USA). The plate base was visualized using a 488 nm argon (Ar) laser (emission 500-550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo, Japan). Two independent cultures were performed under each experimental condition and at least 10 sites were randomly analyzed. The presence or absence of biofilm formation of various species was observed on the nylon membrane using scanning electron microscopy (SEM). Briefly, nylon membranes (Merck Millipore, Burlington, USA) were cut into 0.5 x 0.5 cm pieces and placed in 96-well plates containing single or mixed species grown with or without fatty acids and incubated at 37°C for 24 hours. For the C. albicans biofilm, PDB medium was used, and for the dual biofilm of C. albicans and S. aureus, a 1:1 mixture of PDB and LB medium was used. For double biofilms of C. albicans and E. coli O157:H7, use a 1:1 mixture of PDB and LB medium as medium, or for triple biofilms of S. aureus, C. albicans, and E. coli O157:H7 A 1:2 mixture of PDB and LB medium was used. Cells attached to the nylon membrane were fixed with glutaraldehyde (2.5%) and formaldehyde (2%) for 24 h, then post-fixed using osmium and ethanol series (50%, 70%, 80%, 90%, 95%). %, 100%) and dehydrated using isoamyl acetate. After critical point drying, cells were examined and imaged using an S-4100 scanning electron microscope (SEM, Hitachi, Tokyo, Japan) at a voltage of 15 kV.

Example 5: Quantitative real-time PCR analysis (qRT-PCR)

Three sets of transcriptome analyzes were performed using S. aureus, E. coli O157:H7 and C. albicans cells. For S. aureus , S. aureus MSSA 6538 cells were cultured at 37 °C in the presence or absence of saw palmetto oil (100 μg/ml), lauric acid (20 μg/ml) and myristic acid (20 μg/ml). °C, inoculated in 25 ml of LB medium in a 250 ml flask, and cultured at 250 rpm while shaking for 6 hours starting at an OD ₆₀₀ of 0.05. In the case of E. coli O157:H7, E. coli O157:H7 cells were inoculated into 25 ml of LB medium in a 250 ml flask at 37°C, then saw palmetto oil (100 μg/ml) and lauric acid (20 μg/ml) and in the presence or absence of myristic acid (20 μg/ml) for 4 hours with shaking at 250 rpm. Then, RNase inhibitor (RNAlater, Ambion, TX, USA) was added and the cells were immediately chilled for 30 seconds in a dry ice bath with 95% ethanol to prevent RNA degradation.

For C. albicans , 25 ml of C. albicans DAY185 was inoculated into 250 ml of PDB medium at an initial turbidity of 0.1 (~ 10 ⁵ CFU/ml) at OD ₆₀₀ , saw palmetto oil (100 μg/ml), and It was incubated for 6 hours at 37° C. with agitation (250 rpm) in the presence or absence of uric acid (20 μg/ml) and myristic acid (20 μg/ml).

To prevent RNA degradation, an RNase inhibitor (RNAlater, Ambion, TX, USA) was added to the cells and cells were immediately incubated using the hot acidic phenol method. Separated. RNA was purified using the Qiagen RNeasy mini Kit (Valencia, USA).

The expression of various biofilm-related genes was confirmed using qRT-PCR: agrA, aur, hla, hld, icaA, sarA and sigB in S. aureus ; csgA, csgB, fimH, flhD, luxR, luxS and motB for E. coli O157:H7; For C. albicans , ALS1, ALS3, CDR1, CDR2, CHT4, CYR1, ECE1, ERG9, ERG10, HWP1, MTS1, RAS1 , RBT5 and UME6

Table 2 shows the specific primers used in the qRT-PCR. Three housekeeping genes, 16s rRNA, rrsG or RDN18 were used respectively and the expression of housekeeping genes was not affected by fatty acids.

For the qRT-PCR method, SYBR Green master mix (Applied Biosystems, Foster City, USA) and ABI StepOne Real-Time PCR System (Applied Biosystems) were used, and at least two independent cultures were performed.

Example 6: Cell-Surface Hydrophilicity Assay

Cell surface hydrophobicity was analyzed. Specifically, the cell culture was cultured in LB medium for 20 hours, centrifuged at 7,000 xg for 5 minutes, and harvested by shaking at 250 rpm at 37°C. The harvested cells were mixed with hexadecane (TCI chemicals, Tokyo, Japan) at a ratio of 6:1 by vortexing for 90 seconds. The mixture was left at room temperature for 30 min to allow phase separation. Aqueous phase absorbance was measured at OD ₆₀₀ . Four independent cultures were used for each material.

Example 7: Candida ( C. albicans ) Observation of hyphae formation

To observe hyphal formation, a microscopic imaging system was used. Briefly, C. albicans DAY185 cells were re-inoculated in 2 ml PDB medium with or without fatty acids (0, 10, 20 or 100 μg/ml) at a 1:50 dilution and incubated for 24 hours at 37°C without shaking.

After culturing, the cells were mixed and visualized using the iRiS ^™ Digital Cell Imaging System (Logos Bio Systems, Anyang, Korea). A minimum of 4 independent cultures were used.

Example 8: Analysis of antiviral and cytotoxicity of fatty acids in nematode models

To investigate the toxicity of S. aureus, E. coli O157:H7 or C. albicans to fatty acids, the present inventors used C. elegans strain fer-15 (b26) ; fem-1 (hc17) was used. Briefly, each well of a 96-well plate containing M9 for S. aures and E. coli O157:H7 infection, and PDB:M9 (20:80) mixture for C. albicans infection. About 30 nematodes were put in. In addition, untreated control and saw palmetto oil (20 or 100 μg/ml), lauric acid (10 or 20 μg/ml) or myristic acid (10 or 20 μg/ml) treated S. aures , E. coli O157:H7 (~ 10 ⁸ , 10 ⁷ CFUml ^{−1 ,} respectively) or C. albicans (~ 10 ⁵ CFU/ml) were added to the wells containing the nematodes. The plates were then incubated at 25° C. for 10 days without shaking. For the cytotoxicity assay, 96- Thirty uninfected nematodes were pipetted into a single well of a well plate.

The plates were then incubated at 25° C. for 4 days without shaking. Three independent experiments were performed in triplicate. Results are expressed as percentage of viability of live nematodes determined in response to platinum wire contact. Observations were made using an iRiS ™ digital cell imaging system (Logos Bio Systems, Anyang, Korea).

Example 9: Combined treatment of fatty acids and antimicrobial agents

The effects of fatty acids and antibiotics were analyzed against S. aureus, E. coli O157:H7 and C. albicans . Briefly, overnight cultures at an initial turbidity of OD 0.05 for bacteria and OD 0.1 for Candida at 600 nm were cultured in appropriate culture medium (final volume 1000 μl) with antibiotics, gentamicin (10 μg/ml) or amphotericin. Inoculate with B (0.5, 2, 5 μg/ml) and/or fatty acids (saw palmetto oil 100 μg/ml, lauric acid (C12:0) or myristic acid (C14:0) (20 μg/ml) did

Cells were incubated for 1 hour at 37°C with shaking. Cell mixtures were serially diluted in PBS and plated in appropriate culture media. CFU was determined by colony counting after 24 hours of incubation at 37°C. Two independent cultures were repeated at least three times.

Example 10: Statistical Analysis

Replication numbers for the assay are provided above and results are presented as means ± SDs. Statistical analysis was performed using one-way ANOVA followed by Dunnett's test using SPSS version 23 (SPSS Inc., Chicago, USA). A P value of less than 0.05 was considered significant, and an asterisk indicates a significant difference between treated and untreated samples.

Experimental Example 1: Confirmation of inhibitory effect on biofilm formation of microorganisms

As shown in FIG. 1, the biofilm inhibitory effect of saw palmetto oil on 11 microorganisms was first investigated. Saw palmetto oil at a concentration of 50 μg/ml significantly inhibited biofilm formation by 4 S. aureus strains including 2 methicillin-resistant strains, Staphylococcus epidermidis strains, 2 pathogenic E. coli strains, and C. albicans strains. suppressed. On the other hand, biofilm formation by A. baumannii strains and P. aeruginosa strains was not inhibited (FIG. 1a).

In particular, saw palmetto oil inhibited biofilm formation by more than 85% of C. albicans strains even at 20 μg/ml. In addition, as a result of GC-MS analysis, 25 fatty acids were identified in saw palmetto oil (Table 3). The main components of saw palmetto oil were lauric acid (34.3%), myristic acid (14.3%), palmitic acid (9.6%), and oleic acid (29.1%) (FIG. 1b). The components of saw palmetto oil identified by GC-MS analysis are consistent with the results of previous studies ( Nutrients 5, 3617-3633 (2013) and J. Pharm. Pharmacol. 66, 811-822 (2014)).

The antimicrobial membrane efficacy of four major fatty acids, lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and oleic acid (C18:1), was compared to three strains ( S. aureus) . , E. coli , and C. albicans .) were further investigated. Lauric acid and myristic acid appear to be the most active against the three microorganisms. For example, 20 μg/ml of lauric acid and myristic acid inhibited biofilm formation by more than 50% of five strains of Staphylococcus, E. coli O157:H7 and C. albicans (FIGS. 1c to 1f). In the case of the S. aureus strain, oleic acid at 20 μg/ml or more significantly reduced biofilm formation, but palmitic acid did not (Fig. 1c). However, palmitic acid and oleic acid did not change biofilm formation by E. coli O157:H7 and C. albicans (Fig. 1d and 1e). Similar to the results observed with saw palmetto oil, lauric acid, and myristic acid, most C. albicans biofilm formation was inhibited at 5 μg/ml (Fig. 1e). It was confirmed that lauric acid and myristic acid showed biofilm inhibitory activity against E. coli O157:H7. Therefore, it was confirmed that the biofilm inhibitory activity of saw palmetto oil against S. aureus, E. coli O157:H7 and C. albicans was mainly due to lauric acid and myristic acid.

On the other hand, the minimum inhibitory concentration (MIC) of saw palmetto oil and two active fatty acids against 11 microorganisms was found to be > 500 μg/ml (Table 1), which indicates that saw palmetto oil, lauric acid and Myristic acid inhibited microbial biofilm formation without affecting planktonic cell growth, suggesting that, unlike conventional antibiotics, these fatty acids may be less susceptible to developing drug resistance.

Biofilm inhibition at the bottom of the 96-well plate was analyzed by confocal laser microscopy (CSLM). While the three microorganisms in the untreated control group formed dense biofilms (thickness > 40 μm, and nearly 100% surface coverage), the 100 μg/ml saw palmetto oil-treated group The thickness was significantly reduced (Fig. 1g). In addition, both lauric acid and myristic acid significantly inhibited the biofilm formation of S. aureus, E. coli O157:H7 and C. albicans at a concentration of 20 μg/ml.

Experimental Example 2: Multiple microorganisms of saw palmetto oil and major fatty acids ( S. aureus, E. coli O157:H7 and C. albicans ) Confirmation of biofilm inhibitory activity

The biofilm inhibitory efficacy of saw palmetto oil and fatty acids was confirmed in relation to biofilm formation of S. aureus, E. coli O157:H7 and C. albicans dual species and three species strains. . In detail, the present inventors first searched for an appropriate medium and inoculum for biofilm formation for a total of three sets for dual species strains and one set for three strains. For example, LB medium for S. aureus and E. coli 0157:H7, S. aureus and C. albicans ; A 1:1 mixture of PDB and LB for E. coli O157:H7 and C. albicans ; In the case of three species, a 1:2 mixture of PDB and LB was used as a medium.

Dual species strains ( S. aureus and E. coli O157:H7, S. aureus and C. albicans , E. coli O157:H7 and C. albicans ) or three strains ( S. aureus , E. coli O157 :H7 and C. albicans ), it was confirmed that appropriate biofilms were formed (1 to 2 in OD ₅₇₀ ) in all four cases (FIGS. 2a to 2d). Lauric acid and myristic acid inhibited biofilm formation in a concentration-dependent manner for dual species or three species (FIGS. 2a to 2d). In particular, lauric acid and myristic acid inhibited biofilm formation by about 90% for the three strains after culturing at a concentration of 10 μg/ml for 24 hours.

On the other hand, similar to the biofilm for a single strain, it was confirmed that saw palmetto oil and two fatty acids, lauric acid and myristic acid, most significantly inhibited multiple biofilms including C. albicans (FIGS. 2b to 2d). .

In addition, through confocal laser microscopy (CLSM) observation, saw palmetto oil or lauric acid and myristic acid at a concentration of 20 μg/ml significantly reduced the biofilm formation of three species at the bottom of a 96-well plate. It was confirmed (Fig. 2e). In addition, through scanning electron microscopy (SEM) observation, it was confirmed that saw palmetto oil, lauric acid, or myristic acid had inhibitory effects on the biofilm formation of the three species (FIG. 2f).

We observed multi-microbial associations of S. aureus , E. coli O157:H7 and C. albicans in the untreated control group. C. albicans appears to have formed hyphae and few yeast cells that are much larger than round S. aureus cells and rod-shaped E. coli O157:H7 cells. In particular, it was found that saw palmetto oil, lauric acid, or myristic acid clearly reduced hyphal formation of C. albicans , S. aureus , and E. coli O157:H7 cells.

Experimental Example 3: Differential gene expressions induced by saw palmetto oil, lauric acid and myristic acid

In order to investigate the molecules responsible for the inhibition of biofilm formation by saw palmetto oil, lauric acid and myristic acid, qRT-PCR was performed on the above three microorganisms having various biofilm-related genes.

Specifically, in S. aureus strains, saw palmetto oil and lauric acid, but not myristic acid, reduced the expression of the alpha-hemolysin hla gene 20- and 32-fold, respectively, and delta-hemolysin (hld, also known as RNAIII) agrA , other genes such as aur, icaA and sigB were unaffected (Fig. 3a). On the other hand, hld (RNAIII) is known to stimulate hla translation. hla is a major cytotoxic substance and is associated with biofilm formation in S. aureus, which is supported by the inhibition of hla and hld by fatty acids (Fig. 3a). Thus, hla inhibition was partly due to the downregulation of hld by saw palmetto oil and lauric acid. Interestingly, saw palmetto oil and lauric acid upregulated sarA, a positive regulator of S. aureus biofilms. Adjusted. In addition, two omega fatty acids are known to inhibit the expression of hla and hld20, and lauric acid and other omega fatty acids are considered to inhibit biofilm formation because they down-regulate the expression of hla and hld genes.

In addition, it was confirmed that the effect of lauric acid and myristic acid on biofilm-related gene expression was more significant than that of saw palmetto oil in E. coli O157:H7 strain. In particular, lauric acid and myristic acid inhibited the expression of two fimbriae genes (csgAB), three motility genes (fimH, flhD, and motB), and two quorum sensing genes (luxR and luxS) (Fig. 3b). .

In C. albicans strains, both saw palmetto oil and lauric acid suppressed the expression of the HWP1 gene, which is essential for hyphal development, and their expression correlated with cell elongation and biofilm formation (Fig. 3c).

In addition, saw palmetto oil and two fatty acids in particular induced the expression of CHT4 encoding chitinase. CHT4 transcription is known to be reduced upon conversion from yeast to mycelia, suggesting that saw palmetto oil and two fatty acids can inhibit mycelial formation. In addition, it has recently been known that medium-chain fatty acids mimic farnesol, a quorum sensing molecule, to suppress the expression of the HWP1 gene. partially supports the hypothesis.

Experimental Example 4: Confirmation of mycelial inhibition effect of C. albicans by hydrophobicity change and fatty acid of S. aureus

Cell surface hydrophobicity and biofilm formation are closely linked in many bacteria because hydrophobic cells adhere more to hydrophobic surfaces. Therefore, the hydrophobicity of the cell surface was measured in the presence of fatty acids of the three strains (FIG. 3d-f). Interestingly, saw palmetto oil, lauric acid, and myristic acid increased hydrophilicity, indicating that cells were less hydrophobic in S. aureus and did not alter hydrophilicity in E. coli O157:H7 or C. albicans. (FIGS. 3e and 3f). These results may partially explain the inhibition of biofilm formation by S. aureus by fatty acids on the hydrophobic polystyrene surface. Since yeast-to-hyphal transition is known to be a prerequisite for biofilm development by C. albicans, the effect of fatty acids on hyphal formation was observed. Saw palmetto oil, lauric acid, and myristic acid substantially inhibited mycelial transition, whereas untreated control cells were predominantly hyphal cells (Fig. 3g).

These results support that lauric acid and myristic acid strongly inhibit hypha formation, reduce biofilm formation by C. albicans, and even reduce multispecies biofilm formation including C. albicans.

Experimental Example 5: Confirmation of Anti-virulence activities activity in nematode model

Biofilm or biofilm formation and cell adhesion are major virulence factors in animal hosts. The Caenorhabditis elegans model is used as an alternative to the use of animal hosts in various studies in biology, particularly in the field of bacterial infection. Pathogenic microorganisms such as S. aureus, E. coli O157:H7 and C. albicans can kill nematodes. C. elegans survived well with E. coli OP50, a common food source for the worms. On the other hand, the lifespan of C. elegans is greatly reduced in the presence of each pathogen. Through the results of FIG. 4, saw palmetto oil, lauric acid, and myristic acid inhibited C. aureus (FIG. 4a), E. coli O157:H7 (FIG. 4b) or C. albicans (FIG. 4c) in the presence of . elegans survival significantly. For example, nematode survival was almost 100% without pathogens for 10 days, but more than 55% of nematodes survived in the presence of 100 μg/ml saw palmetto oil or 20 μg/ml myristic acid. These results are consistent with the observed down-regulation of biofilm formation (FIG. 1) and suppression of the hemolysin gene (FIG. 3a).

In addition, to investigate the cytotoxicity of saw palmetto oil, lauric acid, and myristic acid, only E. coli OP50 was fed and the survival rate of C. elegans was investigated without pathogens.

No toxic effects were observed when uninfected nematodes were exposed to saw palmetto oil at concentrations up to 200 μg/ml, and lauric and myristic acids at 100 μg/ml or 200 μg/ml had mild titers against nematodes ( titer) was shown (Fig. 4d). However, lauric acid and myristic acid showed antibiofilm activity at concentrations of 10 μg/ml or 20 μg/ml, well below the cytotoxic level.

Therefore, it was confirmed that the biofilm formation inhibitory composition according to the present invention has an anti-virulence activity activity effect with minimal cytotoxicity.

Experimental Example 6: Effect of combined treatment of fatty acids and antimicrobial agents

Combinatory efficacies of fatty acids and antimicrobial agents on microbial growth have been investigated for individual microorganisms, since combination treatments have been proposed to improve antibacterial efficacy in the past. First, 10 μg/ml of gentamicin, an antibiotic agent, was used alone to test against S. aureus or E. coli O157:H7, and the antifungal agent, amphotericin B 0.5, 2 or 5 μg/ml alone was tested against C. albicans .

As expected, it was confirmed that aminoglycoside gentamicin partially killed more than 90% of the two bacteria within 1 hour (FIGS. 4e and 4f). In addition, amphotericin B at 2 μg/ml or more almost inhibited the growth of C. albicans (FIG. 4g).

Next, the effects of the combined use of fatty acids and antimicrobial agents on three individual microorganisms were confirmed. When 20 μg/ml lauric acid (C12:0) or 20 μg/ml myristic acid (C14:0) was added, the viability of S. aureus (Fig. 4e) and E. coli O157:H7 (Fig. 4f) was increased. got much lower Treatment with 20 μg/ml lauric acid or 20 μg/ml myristic acid alone did not affect cell growth at all (MICs > 500 μg/ml). The antibacterial efficacy of gentamicin was significantly enhanced in the presence of lauric acid or myristic acid. On the other hand, saw palmetto oil showed a marginal effect on antibiotic efficacy at a concentration of 100 μg/ml. On the other hand, in the case of C. albicans , the addition of saw palmetto oil, lauric acid, or myristic acid greatly increased the survival rate of C. albicans , resulting in an unexpected result (FIG. 4g). Through these results, it was confirmed that the combined treatment of lauric acid or myristic acid with the antibiotic gentamicin exhibited synergistic antibacterial effects against S. aureus and E.coli O157:H7.

Taken together, In the present invention, saw palmetto oil and its abundant lauric acid and myristic acid can inhibit biofilm formation against single, double and trispecies pathogenic microorganisms such as S. aureus, E. coli O157:H7 and C. albicans , which did not affect planktonic cell growth. In addition, it was confirmed that lauric acid or myristic acid had a synergistic antibacterial effect against S. aureus and E.coli O157:H7 by concurrent treatment with gentamicin, an antibiotic. Therefore, the composition for inhibiting biofilm formation according to the present invention is a composition for preventing or treating infectious diseases caused by biofilms of multiple pathogenic microorganisms, an antibacterial composition, and a coating composition for inhibiting biofilm formation of pathogenic microorganisms. can be put to good use.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (14)

unsaturated fatty acids such as lauric acid or myristic acid; And a composition for inhibiting biofilm formation of multiple pathogenic microorganisms containing gentamicin as an active ingredient, wherein the pathogenic microorganism is Staphylococcus aureus or pathogenic Escherichia coli (E. coli O157:H7). A composition for inhibiting biofilm formation of multi-pathogenic microorganisms. According to claim 1,
The unsaturated fatty acid is a composition for inhibiting biofilm formation of multi-pathogenic microorganisms, characterized in that derived from saw palmetto oil. According to claim 1,
The composition is a composition for inhibiting biofilm formation of multi-pathogenic microorganisms, characterized in that for inhibiting the formation of a dual species biofilm (dual species biofilm). According to claim 3,
The dual species biofilm is a biofilm of Staphylococcus aureus and pathogenic Escherichia coli (E. coli O157: H7), characterized in that the composition for inhibiting biofilm formation of multiple pathogenic microorganisms. According to claim 1,
The composition is a composition for inhibiting biofilm formation of multi-pathogenic microorganisms, characterized in that it further comprises saw palmetto oil (saw palmetto oil). According to claim 1,
The composition inhibits the expression of hla and hld in Staphylococcus aureus, and csgAB, a fimbriae gene, in pathogenic Escherichia coli (E. coli O157:H7); the motility genes fimH, flhD and motB; and suppressing the expression of luxR and luxS, which are quorum sensing genes, to suppress biofilm formation. According to claim 1,
The unsaturated fatty acid is a composition for inhibiting biofilm formation of multi-pathogenic microorganisms, characterized in that for inhibiting hypha formation and biofilm formation. delete delete delete A method for inhibiting biofilm formation of multi-pathogenic microorganisms, comprising the step of treating the surface of a living organism or a non-living body with the composition of any one of claims 1 to 7. unsaturated fatty acids such as lauric acid or myristic acid; And a coating composition for inhibiting multi-pathogenic microbial biofilm formation comprising gentamicin as an active ingredient, wherein the pathogenic microorganism is Staphylococcus aureus or pathogenic Escherichia coli (E. coli O157:H7). A composition for coating, characterized in that. (a) lauric acid or myristic acid as an unsaturated fatty acid; And (b) an antibacterial composition containing gentamicin as an active ingredient, wherein the composition has an antibacterial effect against Staphylococcus aureus or pathogenic Escherichia coli (E. coli O157:H7) antibacterial composition. delete