Composition for preventing biofilm formation and antibacterial adjuvent composition, which comprise tyrosol compound

Abstract

The present invention relates to a composition for preventing biofilm formation and an antibacterial adjuvent composition for enhancing biofilm antibiotic sensitivity, which comprise a tyrosol compound, and, more specifically, to a composition for preventing biofilm formation and an antibacterial adjuvent composition for enhancing biofilm antibiotic sensitivity, which comprise the compound of chemical formula (1) as an active ingredient. In chemical formula (1), R3 is a hydroxyl group, each of R1, R2, R4 <sp / > and R5 is independently hydrogen, a hydroxyl group or hydroxyalkyl (C1-C10) group.

Description

Composition for inhibiting biofilm formation and composition for antibacterial assistance containing tyrosol compound

[0001] The present invention relates to a composition for inhibiting biofilm formation and an antimicrobial auxiliary composition comprising a tyrosol compound, and more particularly, to a composition for inhibiting biofilm formation and an antimicrobial auxiliary composition that inhibit biofilm formation and reduce antibiotic resistance based on the mechanism of action of a tyrosol compound that inhibits biofilm formation by inducing excessive production of nitrogen monoxide.

[0002] When environmental conditions deteriorate, pathogenic bacteria secrete extracellular polymeric substances to protect themselves, forming an extracellular matrix within which they can survive in colonies, forming biofilms. Bacteria living in biofilms are 100 to 1,000 times more resistant to antibiotics than free-living bacteria and are also resistant to host defense mechanisms, leading to chronic infections that are difficult to treat.

[0003] Antibiotic resistance in biofilms has been reported to be caused by several factors, including reduced metabolic rate, reduced growth due to nutrient deficiency, the production of persister bacteria, and reduced antibiotic penetration into the biofilm. Antibiotic resistance in biofilms is also influenced by the developmental stage of the biofilm, which is formed through five developmental stages: initial reversible attachment, irreversible attachment, maturation-I, maturation-II, and dispersal. During this process, antibiotic resistance begins to develop through the expression of antibiotic resistance genes, and during the maturation stage, the anaerobic state within the biofilm has been reported to be a cause of antibiotic resistance.

[0004] Meanwhile, the emergence and increase of antibiotic-resistant bacteria, which have acquired drug resistance and can survive exposure to antibiotics, is posing a significant global problem. The spread of antibiotic resistance can lead to significant economic losses as well as human health risks. In particular, the damage caused by antibiotic-resistant bacteria is expected to increase exponentially due to the emergence of multidrug-resistant bacteria (super-bacteria) that are resistant to all existing antibiotics simultaneously.

[0005] Addressing the problem of antibiotic-resistant bacteria requires the development of new antibiotics. However, identifying targets for new drug development is difficult, and the significant cost and time required to develop new drugs poses significant challenges. A more serious problem is that even if new antibiotics are developed by identifying new targets that can bypass previously known resistance mechanisms, resistant bacteria will rapidly emerge. Therefore, developing substances that can increase the effectiveness of existing antibiotics in terms of cost and time is an effective strategy.

[0006] Accordingly, there is a growing need to develop technologies that can suppress biofilm formation based on a new mechanism and inhibit antibiotic resistance, thereby killing bacteria by increasing susceptibility to antibiotics when treated together with antibiotics.

[0007] One aspect of the present invention is to provide a composition for inhibiting biofilm formation, which can inhibit biofilm formation by inducing excessive production of nitrogen monoxide.

[0008] Another aspect of the present invention is to provide an antimicrobial adjuvant composition capable of increasing susceptibility to antibiotics.

[0009] According to one aspect of the present invention, a composition for inhibiting biofilm formation is provided, which comprises a compound of the following chemical formula (1) as an active ingredient.

[0010] According to another aspect of the present invention, an antibacterial auxiliary composition is provided, which comprises a compound of the following chemical formula (1) as an active ingredient and is used in combination with an antibiotic to enhance antibacterial activity against a biofilm.

[0011]

[0012] In the above chemical formula (1), R3 is a hydroxy group, and R1, R2, R4 and R5 are each independently hydrogen, hydroxyl group, or hydroxyalkyl (C1-C 10 ) is unique.

[0013] The tyrosol compound provided by the present invention is a safe component contained in olive oil and white wine, and can eliminate multidrug-resistant biofilms, a cause of chronic, intractable infections caused by major pathogenic microorganisms, through a novel mechanism. Therefore, it is expected to be widely applied as an antibacterial agent and antimicrobial adjuvant. Furthermore, by enhancing antibiotic susceptibility, it can significantly reduce side effects such as renal toxicity caused by antibiotic overdose, and can prevent sepsis and septic shock caused by antibiotic overdose.

[0014] Figure 1 shows the results of biofilm inhibition activity of tyrosol compounds on the production of nitric oxide in E. coli, P. aeruginosa, and A. baumannii. The results show that tyrosol and 3-hydroxytyrosol inhibit biofilm formation in E. coli BW25113 (Figure 1A), P. aeruginosa MPAO1 (Figure 1B), and A. baumanii KCTC2508 (Figure 1C) and the recovery of the inhibitory activity by a nitrogen monoxide scavenger (C-PTIO). Furanone C-30 (FC), a quorum sensing inhibitor, was used as a control compound. (*P<0.01, **P<0.001, and ***P<0.0001 compared to untreated cells.# P< 0.01, ## P< 0.001, and ### P< 0.0001 vs. C-PTIO-untreated cells.)

[0015] Figure 2 shows the results of the inhibitory activity of tyrosol and 3-hydroxytyrosol on c-di-GMP production in E. coli, P. aeruginosa, and A. baumanii by nitric oxide production. The inhibitory activity of tyrosol and 3-hydroxytyrosol on c-di-GMP production in E. coli BW25113 (Figure 1A), P. aeruginosa MPAO1 (Figure 1B), and A. baumanii KCTC2508 (Figure 1C) and the recovery of the inhibitory activity by a nitric oxide scavenger (C-PTIO) are shown. Furanone C-30 (FC), a quorum sensing inhibitor, was used as a control compound. (*P<0.01, **P<0.001, and ***P<0.0001 compared to untreated cells. # P< 0.01, ## P< 0.001, and ### P< 0.0001 vs. C-PTIO-untreated cells)

[0016] Figure 3 shows the identification of targets of tyrosol compounds for the inhibition of E. coli biofilms. Figure 3A shows the loss of biofilm inhibition activities of tyrosol (i) and 3-hydrosytyrosol (ii) in E. coli BW25113 / pBAD-ybfA by overexpression of ybfA, and the loss of biofilm inhibition activities of furanone C-30 (FC) and (iii), which are Quorum sensing inhibitors, as control compounds. Figure 3B shows the enhancement of biofilm formation by overexpression of ybfA and the reduction by treatment with a nitric oxide scavenger (C-PTIO). (*P<0.01, **P<0.001, and ***P<0.0001 compared to untreated cells. #P< 0.01, ## P< 0.001, and ### P< 0.0001 C-PTIO- vs. untreated cells)

[0017] Figure 4 shows the effects of tyrosol and YbfA on the expression of genes related to the NO production pathway. Figure 4A shows the effects of tyrosol on the expression of genes related to the NO production pathway in an E. coli BW25113 biofilm, and Figure 4B shows the effects of YbfA on the expression of genes related to the NO production pathway in an E. coli BW25113 biofilm. In this case, the completestatin target protein NirC was used as a control.

[0018] Figure 5 shows the effect of YbfA, a tyrosol compound target protein, on the expression of anaerobic metabolism genes, and shows the effect of YbfA on the expression of flagellar synthesis-related genes (fliA, flgC, flgN) and anaerobic metabolism-related genes (hycF, hycl, hyaA) according to the biofilm formation time of E. coli BW25113.

[0019] Figure 6 shows the enhancement of antibiotic sensitivity of E. coli biofilm bacteria to ciprofloxacin and tobramycin by tyrosol. Figure 6A shows the sensitivity of planktonic cells of E. coli BW25113 and YbfA mutant to antibiotics, Figure 6Bi shows the sensitivity of biofilm cells of E. coli BW25113 and YbfA mutant to antibiotics, and Figure 6Bii shows the increase in antibiotic sensitivity of E. coli BW25113 biofilm cells by treatment with tyrosol compounds. At this time, the quorum sensing inhibitor furanone C-30 (FC) and the biofilm inhibitory compound complementatin (com) targeting NirC were used as control compounds. (*P< 0.01, **P< 0.001, and ***P< 0.0001 compared to untreated cells.)

[0020] Figure 7 shows the enhancement of antibiotic sensitivity of E. coli biofilm bacteria to Ceftazidime and Meropenem by tyrosol. Figure 7A shows the sensitivity of planktonic cells of E. coli BW25113 and YbfA mutant to antibiotics, Figure 7Bi shows the sensitivity of biofilm cells of E. coli BW25113 and YbfA mutant to antibiotics, and Figure 7Bii shows the increase in antibiotic sensitivity of E. coli BW25113 biofilm cells by treatment with tyrosol compounds. At this time, the quorum sensing inhibitor furanone C-30 (FC) and the biofilm inhibitory compound complementatin (com) targeting NirC were used as control compounds. (*P< 0.01, **P< 0.001, and ***P< 0.0001 compared to untreated cells.)

[0021] Figure 8 shows the enhancement of antibiotic sensitivity of Pseudomonas aeruginosa biofilm bacteria by tyrosol. Figure 8A shows the sensitivity of planktonic cells of P. aeruginosa MPAO1 to antibiotics, and Figure 8B shows the result of enhancing antibiotic sensitivity of P. aeruginosa MPAO1 biofilm cells by tyrosol compound treatment. At this time, the Quorum sensing LasR inhibitor furanone C-30 (FC) and the biofilm inhibitory compound completestatin (com) targeting NasA were used as control compounds. (*P<0.01, **P<0.001, and ***P<0.0001 compared to untreated cells.)

[0022] Figure 9 shows the enhancement of antibiotic sensitivity of A. baumannii biofilm bacteria by tyrosol. Figure 9A shows the high sensitivity of A. baumannii planktonic cells to antibiotics, and Figure 9B shows the strong resistance of A. baumannii KCTC2508 biofilm cells to antibiotics. It can be confirmed that the antibiotic sensitivity of A. baumannii biofilm cells by tyrosol compound treatment is enhanced to the level of planktonic cells. At this time, Quorum sensing LasR inhibitor furanone C-30 (FC) and biofilm inhibitory compound completestatin (com) were used as control compounds. (*P< 0.01, **P< 0.001, and ***P< 0.0001 compared to untreated cells.)

[0023] Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0024] A biofilm is a film-like aggregate formed by microorganisms. Microorganisms attach to solid surfaces, forming colonies and slowly growing to form a biofilm covered by extracellular polymeric substances (EPS). This biofilm-removing activity is distinct from general antibacterial activity. Microorganisms in biofilm form are difficult to remove through physical washing and are highly resistant to chemical disinfectants and antibiotics compared to suspended microorganisms. Therefore, removing biofilms or treating biofilm-related infections with disinfectants or antibiotics requires extremely large doses. However, repeated overuse of antibiotics increases resistance, leading to the emergence of resistant bacteria, making it more difficult to control biofilm formation.

[0025] According to the present invention, a novel composition for inhibiting biofilm formation, which can effectively inhibit biofilm formation, is proposed, including a tyrosol compound.

[0026] More specifically, the composition for inhibiting biofilm formation of the present invention contains a compound of the following chemical formula (1) as an active ingredient.

[0027] In the present invention, the term 'tyrosol compound' refers to all compounds of the following chemical formula (1) that contain tyrosol.

[0028]

[0029] In the above chemical formula (1), R3 is a hydroxy group, and R1, R2, R4 and R5 are each independently hydrogen, hydroxyl group, or hydroxyalkyl (C1-C 10 ) group, and the hydroxyalkyl group may be, for example, a hydroxymethyl group, a hydroxyethyl group, or a hydroxypropyl group. For example, R1, R2, R4 and one of R5 may be a hydroxy group, and the others may be hydrogen.

[0030] For example, the compound of the above chemical formula (1) may be at least one selected from the group consisting of tyrosol of the following chemical formula (1-1) and 3-hydroxytyrosol of the following chemical formula (1-2).

[0031]

[0032]

[0033] In the present invention, the expression "comprising as an active ingredient" means comprising a sufficient amount to achieve the efficacy or activity of the tyrosol compound, for example, comprising the compound of the above chemical formula (1) at a concentration of 0.01 to 10 micromolar (μM), for example, comprising it at a concentration of 0.1 to 1 micromolar (μM). Meanwhile, depending on the culture conditions, the effect may be achieved even when comprising it at a concentration of less than 0.1 micromolar (μM). For example, tyrosol (chemical formula 1-1) exhibits excellent activity equivalent to that of furanone C-30 (FC) 100 μM, a well-known QS LaR inhibitor, even at 10 μM, and 3-hydroxytyrosol (chemical formula 1-2) exhibits activity equivalent to that of furanone C-30 (FC) at 1 μM, even at 1 μM.

[0034] The bacteria and related biofilms to which the composition for inhibiting biofilm formation and the composition for assisting antibacterial activity of the present invention can be applied are not particularly limited, and can be included without limitation as long as they are related to gram-negative bacteria with an outer membrane.

[0035] Specifically, it may be a pathogenic gram-negative bacterium, for example, an Escherichia sp. bacterium, an Acinetobacter sp. bacterium, a Pseudomonas sp. bacterium, or a Klebsiella sp. bacterium. Specifically, the bacteria of the genus Escherichia include, but are not limited to, Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, and Escherichia vulneris. The bacteria of the genus Acinetobacter include, but are not limited to, Acinetobacter baumannii, Acinetobacter junii, Acinetobacter boissieri, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, and Acinetobacter. These include, but are not limited to, Acinetobacter nosocomialis, Acinetobacter schindleri, and Acinetobacter ursingii.The above-mentioned bacteria of the genus Pseudomonas include, but are not limited to, Pseudomonas aeruginosa; Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas chlororaphis, Pseudomonas pertucinogena, Pseudomonas stutzeri, Pseudomonas syringae, etc. The above-mentioned Klebsiella bacteria include Klebsiella pneumonia, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella terrigena, etc., and may be an antibacterial auxiliary composition for one or more bacteria selected from the group consisting of, for example, E. coli, P. aeruginosa, and Acinetobacter baumannii, or an agent for inhibiting biofilm formation formed by these bacteria.

[0036] Furthermore, the bacteria may exhibit resistance to antibiotics, particularly at least one of the antibiotics mentioned herein. Such “antibiotic-resistant bacteria” refer to bacteria that are no longer affected or barely affected by at least one antibiotic that was previously effective, and that have developed the ability to withstand antibiotics that previously exhibited effective antibacterial activity. Antibiotic-resistant bacteria can transmit the resistance ability to their offspring. The mechanisms of antibiotic resistance are diverse. For example, resistance may arise through non-permeability mechanisms that physically prevent the antibiotic from reaching the site of action inside or on the bacteria, efflux mechanisms that rapidly remove the antibiotic from the bacteria, thereby preventing an effective amount of the antibiotic from reaching the site of action inside or on the bacteria, metabolic mechanisms that destroy the antibiotic, convert the antibiotic to a harmless or less harmful compound, or allow the compound to be excreted more easily, bypass mechanisms in which the bacteria utilize a different pathway than the one inhibited by the antibiotic, or through bacteria that are less sensitive to the antibiotic target form or do not have the target.

[0037] Additionally, the antibiotic-resistant bacteria may be resistant to two or more antibiotics, and are also called “multidrug-resistant microorganisms.”

[0038] The compound of the above chemical formula (1) of the present invention can inhibit the formation of biofilms by controlling the production of nitric oxide based on the NO-mediated biofilm formation mechanism, and more specifically, it is based on a new mechanism that can inhibit biofilm formation by inhibiting the formation of c-di-GMP by producing an excessive amount of nitric oxide.

[0039] The biofilm inhibition target protein of the tyrosol compound of the present invention is ybfA (Putative periplasmic protein), and ybfA inhibits the expression of the anaerobic transcription factor fnr.

[0040] According to another aspect of the present invention, an antimicrobial auxiliary composition is provided, which may also be referred to as an "antimicrobial auxiliary" or an "antibiotic auxiliary", and which means that even if it does not directly kill the bacteria, it increases the susceptibility of the bacteria to antibiotics by suppressing the bacteria's resistance to antibiotics or inducing intracellular accumulation of antibiotics.

[0041] More specifically, the antimicrobial auxiliary composition of the present invention comprises a compound of the following chemical formula (1) as an active ingredient and may be used in combination with an antibiotic to enhance antimicrobial activity against biofilms. In addition, the antimicrobial auxiliary composition of the present invention may enhance biofilm antibiotic sensitivity.

[0042]

[0043] In the above chemical formula (1), R3 is a hydroxy group, and R1, R2, R4 and R5 are each independently hydrogen, hydroxyl group, or hydroxyalkyl (C1-C 10 ) group, and the hydroxyalkyl group may be, for example, a hydroxymethyl group, a hydroxyethyl group, or a hydroxypropyl group. For example, R1, R2, R4 and one of R5 may be a hydroxy group, and the others may be hydrogen.

[0044] Preferably, the compound of the above chemical formula (1) may be at least one selected from the group consisting of tyrosol of the above chemical formula (1-1) and 3-hydroxytyrosol of the above chemical formula (1-2).

[0045] Here, the above "antibiotic" generally means a substance having antibacterial activity, for example, penicillins, cephalosporins, monobactams and carbapenems, which are beta-lactam antibiotics with a beta-lactam ring as their basic structure; polymyxins, which exhibit an antibacterial effect by changing the cell membrane permeability of bacteria; aminoglycosides, which exhibit an antibacterial effect by moving into the periplasmic space through porins in the outer membrane of Gram-negative bacteria and then moving into the cell; macrolides, which exhibit an antibacterial effect by binding to 50S of bacterial ribosomes and inhibiting protein synthesis; tetracyclines, which exhibit an antibacterial effect on various types of bacteria and are known as broad-spectrum antibiotics; glycopeptides, which exhibit an antibacterial effect by inhibiting bacterial cell wall biosynthesis; and antibacterial agents isolated from Streptomyces lincolnensis and having antibacterial activity against anaerobic bacteria. It may be at least one selected from the group consisting of good lincomycins, quinolones that exhibit antibacterial effects by inhibiting the replication of bacterial DNA, combinations thereof, and derivatives thereof, and in particular, it may be an aminoglycoside or quinolone antibiotic.

[0046] For example, the antibiotic may be selected from the group consisting of Meropenem, Imipenem / Cilastatin, Ertapenem, Doripenem, Ceftazidime, Cephalexin, Cefotaxime, Cefixime, Cefepime, levofloxacin, moxifloxacin, norfloxacin, gentamicin, amikacin, streptomycin, neomycin, gentamicin, ciprofloxacin, and tobramycin, for example. It may be at least one selected from the group consisting of ciprofloxacin, tobramycin, Meropenem and Ceftazidime.

[0047] For example, the compound of the above chemical formula (1) is included at a concentration of 0.01 to 10 micromolar (μM), and can be included at a concentration of 0.1 to 1 micromolar (μM). Meanwhile, depending on the culture conditions, an effect may be achieved even when included at a concentration of less than 0.1 micromolar (μM). For example, tyrosol (chemical formula 1-1) exhibits excellent activity equivalent to that of furanone C-30 (FC) 100 μM, a well-known QS LaR inhibitor, even at 10 μM, and 3-hydroxytyrosol (chemical formula 1-2) exhibits excellent activity equivalent to that of furanone C-30 (FC) 100 μM, even at 1 μM, respectively. The antibacterial auxiliary composition of the present invention can be administered together with an antibiotic, before or after antibiotic treatment, when administering an antibiotic, and can be used as an auxiliary agent to enhance the antibacterial activity of an antibiotic.

[0048] According to another aspect of the present invention, an antibacterial composition comprising the tyrosol compound of the present invention and an antibiotic is provided. The tyrosol compound of the present invention is a compound of the above-described chemical formula (1), and all of the above-described contents are applied as is.

[0049] More specifically, the antibacterial composition of the present invention comprises an antibiotic; and a compound of the above chemical formula (1) as an active ingredient. Preferably, the compound of the above chemical formula (1) may be at least one selected from the group consisting of tyrosol of the above chemical formula (1-1) and 3-hydroxytyrosol of the above chemical formula (1-2).

[0050] As described above, the tyrosol compound of the present invention has the effect of enhancing the antibiotic susceptibility of antibiotic-resistant bacteria. Therefore, when used in combination with antibiotics, the antibacterial activity of conventional antibiotics can be restored. The antibiotics applicable in this case are as described above.

[0051] In particular, the tyrosol compound of the present invention can reduce the strong antibiotic resistance of biofilms by inhibiting ybfA (Putative periplasmic protein), a biofilm inhibition target protein, thereby suppressing the expression of the anaerobic transcription factor fnr.

[0052] The antibacterial composition of the present invention may contain an antibiotic at a concentration of 1 to 500 ppm, and even when containing an antibiotic at a concentration of, for example, 2 to 100 ppm or 3 to 50 ppm, the combined use of the tyrosol compound may enhance the sensitivity of target biofilm bacteria to antibiotics, thereby allowing them to be killed even with a small amount of antibiotics.

[0053] The composition for inhibiting biofilm formation and the composition for antibacterial assistance of the present invention are not particularly limited in their formulations, and can be administered orally or parenterally during clinical administration.

[0054] For example, it can be used in the form of a general pharmaceutical preparation. In this case, parenteral administration can mean administration via routes other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular, and subcutaneous.

[0055] More specifically, when the composition of the present invention is used as a pharmaceutical, it can be administered in various dosage forms. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include Witepsol, macrogol, Tween 61, cacao butter, liurin butter, and glycerogelatin.

[0056] In addition, the composition of the present invention can be used by mixing with various carriers acceptable as pharmaceuticals, such as saline solution or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers can be used as pharmaceuticals to increase stability or absorbability.

[0057] When the composition of the present invention is used as a food additive, the biofilm formation-inhibiting composition, antibacterial composition, and antibacterial auxiliary composition may be added as is or used together with other food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredients may be appropriately determined depending on the intended use.

[0058] There are no special restrictions on the types of the above foods, and examples of foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense are included.

[0059] Meanwhile, when the composition of the present invention is used as an over-the-counter drug additive, the biofilm formation-inhibiting composition, antibacterial composition, and antibacterial auxiliary composition of the present invention may be added as is or used together with other over-the-counter drugs or over-the-counter drug ingredients, and may be used appropriately according to conventional methods. The mixing amount of the effective ingredients may be appropriately determined depending on the intended use.

[0060] The pharmaceutical composition of the present invention is not limited thereto, but may preferably be a disinfectant, shower foam, garglin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

[0061] Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely illustrative examples to aid understanding of the present invention and are not intended to limit the scope of the present invention.

[0062] Example

[0063] 1. Confirmation of the anti-biofilm activity of tyrosol compounds

[0064] To confirm the anti-biofilm activity of tyrosol compounds against E. coli, P. aeruginosa, and Acinetobacter baumannii, E. coli BW25113 (Fig. 1A), P. aeruginosa MPAO1 (Fig. 1B), and Acinetobacter baumannii KCTC2508 (Fig. 1C) were treated with tyrosol represented by chemical formula (1-1) and 3-hydroxytyrosol represented by chemical formula (1-2) at different concentrations, respectively.

[0065]

[0066]

[0067] As a result, as can be confirmed in Fig. 1, the tyrosol compound inhibited E. coli biofilm formation by 8.7, 23.8, and 39.9% at concentrations of 0.1, 1, and 10 μM without affecting bacterial growth (Fig. 1A). This was confirmed to be 10 times more potent than furanone C-30 (FC), a well-known QS LaR inhibitor. In addition, the 3-hydroxytyrosol compound inhibited E. coli biofilm formation by 13.1, 25.7, 32.1, 44.5, and 49.9% at concentrations of 0.1, 0.3, 1, 3, and 10 μM without affecting bacterial growth, showing activity that is at least 3 times stronger than tyrosol.

[0068] Meanwhile, it was confirmed that the biofilm inhibition activity of these tyrosol compounds was eliminated by treatment with C-PTIO, a NO (nitric oxide) scavenger, and thus it was found that the tyrosol compound inhibited biofilm formation by producing excessive amounts of NO. In Fig. 1, the control represents before C-PTIO treatment, and '+C-PTIO' represents after C-PTIO treatment.

[0069] That is, it was found that the tyrosol compound inhibits the mechanism of NO-mediated biofilm formation, and it is a compound with a new mechanism different from existing QS inhibitors.

[0070] In particular, the tyrosol compound also reduced biofilm formation of clinically refractory Gram-negative bacteria, P. aeruginosa and Acinetobacter baumannii, by inhibiting NO formation (Fig. 1B and Fig. 1C), and it was found that the 3-hydroxytyrosol compound showed activity at least three times stronger than tyrosol.

[0071] Referring to Figure 1, it can be confirmed that biofilm formation of E. coli, P. aeruginosa, and Acinetobacter baumannii is inhibited in a concentration-dependent manner by the tyrosol compound.

[0072] 2. Confirmation of c-di-GMP production inhibitory activity of tyrosol compounds

[0073] Since the mechanism of NO-mediated biofilm formation is known to be that small amounts of NO produced enhance the activity of the DGC (diguanylate cyclase) enzyme to produce c-di-GMP, the inhibitory activity of tyrosol compounds on c-di-GMP production in E. coli, P. aeruginosa, and Acinetobacter baumannii was investigated.

[0074] As a result, similar to the biofilm inhibitory activity, it was confirmed that the 3-hydroxytyrosol compound inhibited the c-di-GMP production of the strain with an activity at least three times stronger than tyrosol (Fig. 2). In addition, the inhibitory activity of this tyrosol compound disappeared by C-PTIO treatment, indicating that the tyrosol compound inhibited c-di-GMP formation by excessively producing NO. In contrast, FC of a different mechanism as a control group rather increased c-di-GMP production. In Fig. 2, the control represents before C-PTIO treatment, and '+C-PTIO' represents after C-PTIO treatment.

[0075] Therefore, it can be confirmed that tyrosol compounds inhibit biofilm formation by inhibiting the production of c-di-GMP by excessively producing NO.

[0076] 3. Identification of target proteins for biofilm inhibition by tyrosol compounds

[0077] (1) Identification of YbfA, a target protein for E. coli biofilm inhibition

[0078] In the same way as the previously reported method for identifying the target protein NirC of the biofilm-inhibiting compound completestatin (mBio 11: e00878, 2020), the anti-biofilm target of tyrosol and 3-hydroxytyrosol was confirmed to be YbfA using a KeioEscherichia coli knockout library, and was verified through an overexpression assay.

[0079] In more detail, ybfA was cloned into the pBAD plasmid containing an arabinose-inducible promoter to construct an overexpressing E. coli (E. coli BW25113 / pBAD-ybfA), and the biofilm inhibitory activity of the tyrosol compound was compared with that of the wild type (E. coli BW25113) and the strain containing only the vector (E. coli BW25113 / pBAD) to verify the target.

[0080] As a result, tyrosol and 3-hydroxytyrosol compounds showed biofilm inhibitory activity in wild-type bacteria and vector-containing strains, but biofilm inhibitory activity disappeared in the ybfA overexpressing strain (Fig. 3Ai, ii).

[0081] In contrast, the QS inhibitor FC, as a control, exhibited biofilm-inhibitory activity even in the ybfA-overexpressing strain, as expected (Fig. 3Aiii). These results demonstrate that tyrosol compounds inhibit biofilm formation by inhibiting the YbfA protein.

[0082] The result that biofilm formation is inhibited when YbfA protein is inhibited suggests that YbfA is a protein that promotes biofilm formation, and this was proven in the ybfA overexpressing strain through the result that biofilm formation increased compared to the wild type and vector-only strains when ybfA was overexpressed (Fig. 3B).

[0083] Furthermore, this increased biofilm formation was reverted to its original state by C-PTIO, indicating that YbfA promotes biofilm formation by producing small amounts of NO. This indicates a novel mechanism of NO-mediated biofilm formation by YbfA, while previously only NO-mediated biofilm formation by NirC (nitrite transporter) had been reported.

[0084] 4. Effect of tyrosol on the expression of the anaerobic transcription factor FNR and the NO-generating gene napA.

[0085] To investigate the mechanism by which tyrosol compounds produce excessive amounts of NO, we investigated the expression of genes involved in the denitrification pathway, a pathway that produces NO in bacteria.

[0086] As a result, tyrosol increased the expression of the anaerobic transcription factor fnr and the NO-generating gene napA (Fig. 4A). When the expression of fnr and napA genes in the tyrosol compound target protein YbfA mutant was examined, the expression of fnr and napA genes was increased, and napA gene expression was increased in the fnr mutant (Fig. 4B). These results indicate that YbfA suppresses fnr expression, thereby suppressing napA gene expression, resulting in the production of a small amount of NO.

[0087] 5. Confirmation of Tyrosol's inhibition of anaerobic biofilm formation and multidrug resistance gene expression.

[0088] (1) Confirmation of inhibition of anaerobic biofilm formation

[0089] Since YbfA represses the expression of the anaerobic transcription factor fnr, and tyrosol increases fnr expression through YbfA inhibition, we investigated whether YbfA-mediated biofilm formation occurs under anaerobic conditions.

[0090] When we examined genes related to flagellar synthesis (fliA, flgC, flgN) and anaerobic metabolism (hycF, hycl, hyaA) in planktonic and biofilm cells, we found that in the wild strain, anaerobic genes increased from 3 hours of biofilm culture and reached a maximum at 12:00, indicating that anaerobic biofilm formation began from 3 hours of culture. However, in the ybfA mutant, the expression of anaerobic metabolism genes was significantly reduced, confirming that YbfA promotes anaerobic biofilm formation (Fig. 5A). This suggests that YbfA is involved in the late stage of biofilm development.

[0091] In contrast, the nirC mutant showed no change in anaerobic metabolism gene expression, confirming that it is not related to anaerobic biofilm formation.

[0092] 6. Confirmation of increased sensitivity to biofilm antibiotics

[0093] (1) Enhancement of antibiotic sensitivity of E. coli biofilm bacteria by inhibition of YbfA by tyrosol

[0094] We investigated whether inhibition of YbfA by treatment with tyrosol would reduce the strong antibiotic resistance of biofilms.

[0095] 1) Investigation of sensitivity to antibiotics ciprofloxacin and tobramycin

[0096] First, the sensitivity of planktonic and biofilm cells of wild E. coli to bacteriocidal antibiotics such as ciprofloxacin and tobramycin was investigated. As a result, planktonic cells began to die at 3 and 30 ppm of ciprofloxacin and tobramycin, respectively (Fig. 6A), but biofilm cells showed strong resistance, surviving even at 1000 ppm (Fig. 6Bi).

[0097] However, biofilm cells from the ybfA mutant were as sensitive to antibiotics as floating cells. As expected, biofilm cells from the nirC mutant, used as a control, exhibited strong antibiotic resistance. These results can be interpreted as evidence of the ybfA mutant's reduced anaerobic biofilm and increased antibiotic sensitivity due to the reduction of multidrug resistance genes.

[0098] To investigate whether inhibition of YbfA by tyrosol treatment reduces the strong antibiotic resistance of biofilms, antibiotic sensitivity of biofilms treated with tyrosol was examined (Fig. 6Bii). When treated with 10 μM tyrosol, biofilm bacteria began to be killed by the antibiotics ciprofloxacin and tobramycin at 3 and 10 ppm, respectively, showing sensitivity at the level of floating cells. In contrast, the QS inhibitor furanone C-30 (FC) and the NirC inhibitor completestatin (com) had no effect.

[0099] Meanwhile, 3-hydroxytyrosol showed an effect almost similar to that of tyrosol at 10 μM when treated with 1 μM, indicating that 3-hydroxytyrosol increased the sensitivity of biofilms to antibiotics 10 times more strongly than tyrosol.

[0100] 2) Sensitivity test for antibiotics ceftazidime and meropenem

[0101] Sensitivity was investigated under the same conditions as in 6.(1) 1) above, except that ceftazidime and meropenem were used as antibiotics.

[0102] As a result, the antibiotics Meropenem and Ceftazidime showed antibacterial activity against suspended cells starting from 1 ppm, but biofilm cells showed resistance even at 1000 ppm (Fig. 7A). In addition, biofilm cells of the ybfA mutant were sensitive to antibiotics like suspended cells (Fig. 7Bi). To investigate whether treatment with tyrosol reduces the strong antibiotic resistance of biofilms, the antibiotic sensitivity of biofilms treated with tyrosol was examined (Fig. 7Bii). When treated with 10 μM tyrosol, biofilm bacteria started to be killed by the antibiotics Meropenem and Ceftazidime at 1 ppm, showing sensitivity at the level of suspended cells. Meanwhile, 3-hydroxytyrosol showed an effect almost similar to that of 10 μM tyrosol when treated with 1 μM, indicating that 3-hydroxytyrosol increased the sensitivity of biofilms to antibiotics 10 times more strongly than tyrosol.

[0103] (2) Enhanced antibiotic sensitivity of Pseudomonas aeruginosa biofilms to tyrosol

[0104] The antibiotic sensitivity of biofilm bacteria of P. aeruginosa to tyrosol was investigated in a similar manner to that of E. coli. As with E. coli, wild-type P. aeruginosa cells were killed by ciprofloxacin and tobramycin at 3–30 ppm (Fig. 8A), whereas biofilm cells exhibited strong resistance, surviving even at 1,000 ppm.

[0105] However, when treated with 10 μM tyrosol, biofilm cells began to die at 3 and 10 ppm of the antibiotics ciprofloxacin and tobramycin, respectively, showing strong antibiotic sensitivity (Fig. 8B). Meanwhile, 3-hydroxytyrosol increased the antibiotic sensitivity of biofilms 10-fold more strongly than tyrosol. Controls such as furanone C-30 (FC) and the NirC inhibitor completestatin (com) had no effect.

[0106] (3) Enhancement of antibiotic sensitivity of Acinetobacter baumannii biofilm bacteria by tyrosol

[0107] The antibiotic sensitivity of Acinetobacter baumannii biofilms to tyrosol was investigated in the same manner as for E. coli. As in E. coli, in wild-type A. baumannii, suspended cells were killed by ciprofloxacin and tobramycin at 3 ppm, but biofilm cells survived even at 1,000 ppm, showing strong resistance (Fig. 9). However, when treated with 10 μM tyrosol, biofilm cells were killed by ciprofloxacin and tobramycin at 3 and 10 ppm, respectively, showing strong antibiotic sensitivity. 3-Hydroxytyrosol increased the antibiotic sensitivity of biofilms 10-fold more strongly than tyrosol. As expected, furanone C-30 (FC) and completestatin, as controls, had no effect.

[0108] Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations are possible within a scope that does not depart from the technical spirit of the present invention described in the claims.

Claims

1. Contains the compound of the following chemical formula (1) as an active ingredient, In the above chemical formula (1), R3 is a hydroxy group, R1, R2, R4 and R5 are each independently hydrogen, hydroxyl group, or hydroxyalkyl (C1-C 10 ) A composition for inhibiting biofilm formation.

2. A composition for inhibiting biofilm formation, wherein the compound of formula (1) in paragraph 1 is at least one selected from the group consisting of tyrosol of formula (1-1) below and 3-hydroxytyrosol of formula (1-2) below.

3. In the first paragraph, the composition for inhibiting biofilm formation is a composition for inhibiting biofilm formation formed by one or more bacteria selected from the group consisting of E. coli, P. aeruginosa, and Acinetobacter baumannii.

4. A composition for inhibiting biofilm formation, comprising the compound of chemical formula (1) in a concentration of 0.01 to 10 micromolar (μM) in the first paragraph.

5. In the first paragraph, the compound of the chemical formula (1) is a composition for inhibiting biofilm formation, which inhibits biofilm formation by regulating the production of nitrogen monoxide based on the NO-mediated biofilm formation mechanism.

6. A composition for inhibiting biofilm formation, wherein the target protein of the compound of chemical formula (1) in paragraph 1 is ybfA (Putative periplasmic protein).

7. An antibacterial auxiliary composition containing a compound of the following chemical formula (1) as an active ingredient and used in combination with an antibiotic to enhance antibacterial activity against a biofilm: In the above chemical formula (1), R3 is a hydroxy group, R1, R2, R4 and R5 are each independently hydrogen, hydroxyl group, or hydroxyalkyl (C1-C 10 )crawl.

8. In the 7th paragraph, the compound of formula (1) is at least one selected from the group consisting of tyrosol of the following formula (1-1) and 3-hydroxytyrosol of the following formula (1-2), an antibacterial auxiliary composition.

9. In the 7th paragraph, the antibiotic is an antibacterial auxiliary composition selected from the group consisting of levofloxacin, moxifloxacin, norfloxacin, gentamicin, amikacin, streptomycin, neomycin, gentamicin, ciprofloxacin, tobramycin, meropenem, and ceftazidime.

10. An antibacterial auxiliary composition comprising the compound of formula (1) in a concentration of 0.01 to 10 micromolar (μM) in accordance with paragraph 7.

11. An antibacterial auxiliary composition according to claim 7, wherein the target protein of the compound of chemical formula (1) is ybfA (Putative periplasmic protein).

Images