Application of a hinokitiol composition in preparing a Staphylococcus aureus inhibitor

Abstract

The present invention discloses the application of a hinokitiol composition in the preparation of a Staphylococcus aureus inhibitor. After adding hinokitiol, the survival rate of Staphylococcus aureus drug-resistant bacteria significantly decreases when treated with tetracycline, tigecycline, and chlortetracycline, indicating that these two substances can improve the sensitivity of Staphylococcus aureus drug-resistant bacteria to tetracycline, tigecycline, and chlortetracycline and have a synergistic effect. Animal experiments show that the combination of hinokitiol and tetracycline can significantly reduce the Staphylococcus aureus load colonized in the nasal cavity of mice. The hinokitiol provided by the present invention can be combined with tetracycline antibiotics to have a strong synergistic antibacterial effect both in vivo and in vitro, providing a brand-new technical method for the prevention and treatment of diseases in humans and farm animals.

Description

(1) Technical Field

[0001] The present invention relates to the use of a hinokitiol composition in the preparation of a Staphylococcus aureus inhibitor. (2) Background Art

[0002] Staphylococcus aureus is a dangerous human pathogen that asymptomatically colonizes 30% of healthy individuals globally but can cause invasive opportunistic infections. It is a major cause of hospital- and community-associated infections, ranging from mild skin infections to life-threatening diseases such as severe sepsis, necrotizing pneumonia, endocarditis, and bacteremia. With the emergence and rapid spread of multidrug-resistant bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus infections have become increasingly difficult to treat. Currently, MRSA has become one of the most important causes of antibiotic-resistant healthcare-associated infections globally, with high morbidity and mortality rates.

[0003] Tetracycline antibiotics are an old class of broad-spectrum antibiotics. As one of the "big four" antibiotics, tetracycline represents a diverse class of bioactive compounds, from the naturally occurring chlortetracycline to second- and third-generation semi-synthetic derivatives such as minocycline, tigecycline, and the more recent omadacycline. Tetracycline exerts its antibiotic activity by binding to the 30S ribosomal subunit, thereby interfering with bacterial protein synthesis. Due to their strong antibacterial activity against a variety of pathogenic organisms, tetracycline antibiotics have been widely used in animal husbandry and the treatment of clinical diseases, including infections caused by MRSA. However, with the extensive use of tetracycline, the problem of bacterial drug resistance has become increasingly serious, severely limiting its clinical use. Although structural modification to synthesize new tetracycline antibiotics is an important way to overcome bacterial drug resistance, the current R & D investment and output are seriously insufficient, and the efficiency of developing new antibiotics far lags behind the evolutionary speed of bacterial drug resistance. Therefore, there is an urgent need to adopt other approaches to address the increasingly serious problem of drug-resistant bacterial infections. Currently, a recognized effective countermeasure is to develop antibacterial synergists.

[0004] Hinokitiol (β-thujaplicin) is a natural monoterpenoid compound present in the xylem of Cupressaceae trees. It is a tropolone derivative and has been widely used in oral care and therapeutic products due to its high efficiency, broad-spectrum antibacterial, anti-inflammatory, and anti-cancer properties. A recent study showed that Escherichia coli carrying the plasmid-encoded TetA efflux pump gene, after treatment with hinokitiol, resulted in the loss of the tetA resistance gene. Although a large number of studies have been conducted on the antibacterial activity of hinokitiol, there are few reports on the efficacy of the combination of hinokitiol and antibiotics. So far, no one has studied the combination of hinokitiol and tetracycline antibiotics. (3) Summary of the Invention

[0005] The object of the present invention is to provide an application of a hinokitiol composition in the preparation of a Staphylococcus aureus inhibitor. In the present invention, the composition of hinokitiol and tetracycline antibiotics can specifically enhance the synergistic antibacterial activity of tetracycline antibiotics against Staphylococcus aureus, but has no synergistic antibacterial activity against Escherichia coli and Bacillus subtilis. The composition of the present invention can solve the problem of increasingly serious multi-drug resistant Staphylococcus aureus infections.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The present invention provides an application of a hinokitiol composition in the preparation of a Staphylococcus aureus inhibitor. The hinokitiol composition is composed of a combination of hinokitiol and tetracycline antibiotics, and the tetracycline antibiotics include tetracycline, tigecycline, minocycline, and chlortetracycline.

[0008] The concentration ratio of the tetracycline antibiotics to hinokitiol is 1:0.01 to 320.

[0009] The Staphylococcus aureus can be an antibiotic-sensitive or drug-resistant strain, and the drug-resistant bacteria can be selected from one of methicillin-resistant Staphylococcus aureus (MRSA), tetracycline-resistant Staphylococcus aureus, or multi-drug resistant bacteria, etc.

[0010] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0011] After adding hinokitiol in the present invention, the survival rate of Staphylococcus aureus drug-resistant bacteria treated with tetracycline, tigecycline, and chlortetracycline decreased significantly, indicating that these two substances can improve the sensitivity of Staphylococcus aureus drug-resistant bacteria to tetracycline, tigecycline, and chlortetracycline and have a synergistic effect. Animal experiments show that the combination of hinokitiol and tetracycline can significantly reduce the load of Staphylococcus aureus colonized in the nasal cavity of mice.

[0012] The hinokitiol provided by the present invention can be combined with tetracycline antibiotics to have a strong synergistic antibacterial effect both in vivo and in vitro, providing a new technical method for the prevention and treatment of diseases of humans and farmed animals. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shows the bactericidal effect of the combination of hinokitiol and tetracycline against methicillin-resistant Staphylococcus aureus ATCC 43300.

[0014] Figure 2 Shows the bactericidal effect of the combination of hinokitiol and tigecycline against tetracycline-resistant Staphylococcus aureus ATCC 51153.

[0015] Figure 3 The bactericidal effect of hinokitiol combined with chlortetracycline against methicillin-resistant Staphylococcus aureus CGMCC 1.12409.

[0016] Figure 4 The decolonization effect of hinokitiol combined with tetracycline on Staphylococcus aureus in the nasal cavity of mice. (V) Specific implementation manners

[0017] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0018] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. DMSO represents dimethyl sulfoxide.

[0019] Composition of MH broth (g / L): beef powder 2.0, soluble starch 1.5, acid-hydrolyzed casein 17.5, solvent is water, pH value 7.3 ± 0.2.

[0020] MH plate: (g / L): beef powder 2.0, soluble starch 1.5, acid-hydrolyzed casein 17.5, agar 20, solvent is water, pH value 7.3 ± 0.2.

[0021] Composition of protease soy agar (TSA) plate (g / L): tryptone 15g, soy peptone 5g, sodium chloride 5g, agar 15g, solvent is water, pH 7.3 ± 0.2.

[0022] Example 1. Drug sensitivity experiment by microdilution method

[0023] The minimum inhibitory concentration (MIC) of antibacterial drugs against bacteria was determined by the microdilution method using a 96-well plate. According to the experimental guidance protocol of the Clinical and Laboratory Standards Institute (CLSI) of the United States, the minimum inhibitory concentration (MIC) of antibacterial drugs against bacteria was determined by the broth microdilution method. The specific experimental operations are as follows:

[0024] (1) Inoculate each of the test bacteria in Table 1 onto an MH plate and culture at 37°C for 24 h. Select a single colony from each plate and inoculate it onto an MH slant, then culture at 37°C for 24 h for standby. Pick a loop of the test bacteria from the activated slant into an MH liquid medium, culture overnight at 37°C and 160 rpm, and dilute it with the MH liquid medium to a bacterial concentration of 10 6 cfu / ml to obtain the test bacterial solution.

[0025] (2) The minimum inhibitory concentration (MIC) was determined by the double dilution method, and all operations were carried out in a laminar flow hood. The test compound was diluted with MH broth to a final concentration of 128 μg / mL. 100 μL of each dilution was added to a 96-well plate, and then 100 μL of the test bacterial solution was added to each well. The mixture was incubated at 37 °C for 20 hours, and the OD 600 value was measured. At the same time, visual observation was used, and the lowest concentration that completely inhibited the growth of the indicator bacteria was defined as the MIC. The results are shown in Table 1.

[0026] According to the CLSI drug susceptibility breakpoint criteria, when the MIC of Staphylococcus aureus to tetracycline is ≥ 16 μg / mL, it can be judged that the Staphylococcus aureus is a tetracycline-resistant strain. As shown in Table 1, the MICs of Staphylococcus aureus ATCC 43300, ATCC 29213, and ATCC 25923 to tetracycline were all 0.25 μg / mL, which were tetracycline-sensitive strains; while the MICs of Staphylococcus aureus ATCC 51153 and CGMCC 1.12409 to tetracycline were 16 - 32 μg / mL, so they were tetracycline-resistant strains. It is worth mentioning that the MICs of these drug-resistant Staphylococcus aureus to hinokitiol were similar to those of the tetracycline-sensitive strains.

[0027] Table 1. MICs of hinokitiol and tetracycline against different strains

[0028]

[0029]

[0030] Example 2. Hinokitiol can selectively enhance the synergistic antibacterial effect of tetracycline against Staphylococcus aureus

[0031] The checkerboard broth dilution method was used to determine the synergistic antibacterial effect between hinokitiol and different antibiotics. Escherichia coli, Bacillus subtilis, and Staphylococcus aureus were used as the test bacteria. The MICs when used alone and in combination were recorded, and the FICI index was used to calculate and judge the combined use effect of the two drugs. The specific operations are as follows:

[0032] 1. Selectivity of hinokitiol

[0033] The checkerboard broth dilution method was used to determine the synergistic antibacterial effect between hinokitiol and tetracycline. The concentration ranges of the combined drugs included hinokitiol at 1 / 64 MIC to 2 MIC concentrations (0.5 - 64 μg / mL), and tetracycline at 1 / 32 MIC to 2 MIC concentrations (0.015 - 64 μg / mL). Using fresh MH broth as the culture medium, 50 μL of hinokitiol with 8 concentration gradients was added to rows A - H of a 96 - well plate. 50 μL of tetracycline with 8 concentration gradients was added to columns 1 - 7, and column 8 served as the growth control. A bacterial suspension prepared from fresh test bacteria was adjusted to a concentration of 1×10 6 CFU / mL using MH broth. 100 μL of the bacterial suspension was added to each well, mixed evenly, and after incubation at 37 °C for 20 h, the MIC results were read using the method of Example 1. The fractional inhibitory concentration index (FICI) was used to judge the combined effect. FICI = MIC A drug combination / MIC A drug alone + MIC B drug combination / MIC B drug alone. When FICI ≤ 0.5, the two drugs were determined to have a synergistic effect; when 0.5 < FICI ≤ 1, the two drugs were determined to have an additive effect; when 1 < FICI ≤ 2, the two drugs were determined to have an irrelevant effect; when FICI > 2, the two drugs were determined to have an antagonistic effect.

[0034] The results are shown in Table 2. Hinokitiol greatly enhanced the antibacterial activity of tetracycline against Staphylococcus aureus. Hinokitiol at 1 - 2 μg / ml could selectively enhance the antibacterial activity of tetracycline against Staphylococcus aureus, reducing the MIC of tetracycline against Staphylococcus aureus by 4 - 64 times. The FICI of the combined use of the two drugs against all tested Staphylococcus aureus was less than 0.5, but there was no synergistic enhancement effect on Bacillus subtilis and Escherichia coli, indicating that hinokitiol could specifically enhance the antibacterial activity of tetracycline against Staphylococcus aureus.

[0035] Table 2. Synergistic antibacterial results of hinokitiol in selectively enhancing tetracycline against Staphylococcus aureus

[0036]

[0037]

[0038] 2. Detection of the combination of hinokitiol and other antibiotics

[0039] The checkerboard broth dilution method was used to determine the synergistic antibacterial effect between hinokitiol and different antibiotics. The test bacteria used were Staphylococcus aureus ATCC 51153 and ATCC 43300. The concentration ranges of the combined drugs included hinokitiol at 1 / 64 MIC to 2 MIC concentrations (0.5 - 64 μg / mL), and different antibiotics at 1 / 64 MIC to 2 MIC concentrations (0.015 - 64 μg / mL). Using fresh MH broth as the culture medium, 50 μL of hinokitiol at 8 concentration gradients was added to rows A - H of a 96 - well plate. 50 μL of antibiotics at 8 concentration gradients was added to columns 1 - 7, and column 9 was used as the growth control. A bacterial suspension prepared from fresh test bacteria was adjusted to a concentration of 1×10 6 CFU / mL using MH broth, and 100 μL of the bacterial suspension was added to each well. After mixing evenly and incubating at 37°C for 20 h, the MIC results were judged using the method of Example 1. The fractional inhibitory concentration index (FICI) was used to judge the combined effect.

[0040] Except for tetracycline, the combination of hinokitiol and other tetracycline antibiotics such as tigecycline, minocycline, and chlortetracycline also showed good synergistic antibacterial activity against Staphylococcus aureus (FICI < 0.5) (Table 3). However, there was no synergistic effect when combined with other types of antibiotics such as ciprofloxacin and vancomycin (FICI > 0.5), indicating that hinokitiol can specifically enhance the antibacterial activity of tetracycline antibiotics against Staphylococcus aureus.

[0041] Table 3. Hinokitiol can selectively improve the synergistic antibacterial results of tigecycline and chlortetracycline against Staphylococcus aureus

[0042]

[0043]

[0044] Example 3. Adding hinokitiol can improve the bactericidal effect of tetracycline antibiotics against clinically drug - resistant Staphylococcus aureus

[0045] The time - kill experiment was used to further evaluate the synergistic antibacterial activity of the combination of hinokitiol and tetracycline antibiotics against Staphylococcus aureus. The specific operations were as follows:

[0046] 1. Hinokitiol combined with tetracycline

[0047] (1) One loop of the test Staphylococcus aureus ATCC 43300 was picked from the slant and inoculated into a test tube containing 4 mL of MH broth, and cultured with shaking at 37°C and 120 rpm for 16 h. The bacterial suspension was aspirated into a test tube containing 4 mL of MH broth and mixed well to make the bacterial suspension concentration 10 8 CFU / mL.

[0048] (2) Another 4 test tubes were taken and divided into 4 groups: Group 1 was the control group, adding 4 mL of MH broth; Group 2 was the hinokitiol group, adding 4 mL of MH broth containing hinokitiol with a final concentration of 1 μg / mL; Group 3 was the tetracycline group, adding 4 mL of MH broth containing tetracycline with a final concentration of 4 μg / mL; Group 4 was the hinokitiol + tetracycline group, adding 4 mL of MH broth containing hinokitiol with a final concentration of 1 μg / mL and tetracycline with a final concentration of 4 μg / mL.

[0049] (3) The bacterial suspensions in step (1) were respectively added to the test tubes of each group in step (2), 50 μL was added to each group, mixed evenly, and cultured with shaking at 37 °C and 120 rpm; 100 μL of the bacterial suspension was taken at different times (0, 1, 2, 4, 8, 24 h), ten-fold gradient diluted with sterile normal saline to about 10 4 CFU / mL, and then spread on MH agar plates, and cultured statically at 37 °C for 24 h, and then colony counting was carried out.

[0050] The results are shown in Figure 1 , after 24 h of treatment of drug-resistant Staphylococcus aureus, the number of surviving bacteria in the combined use group (combined use of 1 μg / mL hinokitiol and 4 μg / mL tetracycline) was 1000 times lower than that in the group using hinokitiol or tetracycline alone, indicating that the combined use of the two has a synergistic bactericidal effect on drug-resistant Staphylococcus aureus.

[0051] 2. Hinokitiol combined with tigecycline

[0052] The Staphylococcus aureus in step 1 was changed to Staphylococcus aureus ATCC 51153, and tetracycline was replaced with 0.03 μg / mL tigecycline, and other operations were the same. The results are shown in Figure 2 .

[0053] As Figure 2 shown, after 24 h of treatment of drug-resistant Staphylococcus aureus, the number of surviving bacteria in the combined use group (combined use of 1 μg / mL hinokitiol and 0.03 μg / mL tigecycline) was 10 8 times lower than that in the group using hinokitiol or tigecycline alone, indicating that the combined use of the two has a synergistic bactericidal effect on drug-resistant Staphylococcus aureus.

[0054] 3. Hinokitiol combined with chlortetracycline

[0055] The Staphylococcus aureus in step 1 was changed to Staphylococcus aureus CGMCC1.12409, and tetracycline was replaced with 1 μg / mL chlortetracycline, and other operations were the same. The results are shown in Figure 3 .

[0056] As Figure 3 shown, after 24 h of treatment of drug-resistant Staphylococcus aureus, the number of surviving bacteria in the combined use group (combined use of 1 μg / mL hinokitiol and 1 μg / mL chlortetracycline) was 104 times, indicating that the combination of the two has a synergistic bactericidal effect on drug-resistant Staphylococcus aureus.

[0057] Overall Figures 1-3 , hinokitiol and tetracycline ( Figure 1 ), tigecycline ( Figure 2 ), and chlortetracycline ( Figure 3 ) combination significantly enhanced the bactericidal effect on clinically isolated Staphylococcus aureus.

[0058] Example 4. Hinokitiol enhances the decolonization effect of tetracycline on Staphylococcus aureus in the nasal cavity of mice

[0059] Staphylococcus aureus ATCC 43300 was spread on tryptic soy agar (TSA) plates containing 500 μg / mL streptomycin to screen for mutant strain 43300 with streptomycin resistance R , and the MIC of antibacterial drugs against bacteria was determined by the microdilution method in 96-well plates to confirm that 43300 R was a streptomycin-resistant strain with an MIC of streptomycin greater than 500 μg / mL.

[0060] The mutant strain was inoculated into MH broth medium and cultured overnight at 37 °C with shaking at 160 rpm. After centrifugation at 6000 rpm for 10 minutes, the supernatant was removed, and the bacterial cells were washed twice with the same volume of sterile saline. Then the bacterial cells were resuspended in sterile saline, and the bacterial concentration was adjusted to 10 9 CFU / mL. 10 μL of the bacterial suspension was dropped into the nasal cavity of ICR mice (weighing 18 - 26 g) to colonize in the nasal cavity of the mice. After the mice were raised for 24 h, 10 μL of the drug at the specified concentration (sterile saline, 0.5 mg / kg tetracycline, 160 mg / kg hinokitiol, 0.5 mg / kg tetracycline + 160 mg / kg hinokitiol) was dropped into the nasal cavity. After continuing to raise for 48 h, the animals were sacrificed by cervical dislocation, and the nasal carriage of Staphylococcus aureus was evaluated. The nose was wiped with 75% ethanol externally, dissected with sterile scissors, and the nasal tissue was excised. Then the sample was vigorously vortexed in 200 μl of saline for 15 seconds. 50 μl of the nasal suspension from each nose was spread on TSA medium plates containing streptomycin (500 μg / ml), and after culturing at 37 °C for 24 h, the colonies were counted by visual observation.

[0061] As Figure 4 shown, compared with the sterile saline control treatment group, both hinokitiol (160 mg / kg) and tetracycline (0.5 mg / kg) could reduce the number of bacteria in the nasal cavity of mice; however, when hinokitiol (160 mg / kg) was combined with tetracycline (0.5 mg / kg), its antibacterial effect was stronger than that of the two used alone, indicating that the combined application of hinokitiol and tetracycline also showed a significant synergistic antibacterial effect in vivo.

Claims

1. Application of hinokitiol combined with tetracycline antibiotics in the preparation of a drug for inhibiting Staphylococcus aureus, Characterized in that, The tetracycline antibiotics are tetracycline, tigecycline, minocycline or chlortetracycline.

2. The application according to claim 1, Characterized in that, The concentration ratio of the tetracycline antibiotics to hinokitiol is 1:0.01 to 320.

3. The application according to claim 1, Characterized in that, The Staphylococcus aureus is an antibiotic-resistant strain.

4. The application according to claim 3, Characterized in that, The strain is one of methicillin-resistant Staphylococcus aureus, tetracycline-resistant Staphylococcus aureus or multi-drug resistant Staphylococcus aureus.

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