Application of L-tryptophanamide hydrochloride and antibacterial composition

Abstract

The present invention belongs to the field of antibiotic adjuvants, and specifically relates to an application of L-tryptophanamide hydrochloride and an antibacterial composition. The present invention discloses an antibacterial application of L-tryptophanamide hydrochloride and its application as a tetracycline antibiotic synergist, and it is found that the antibacterial effect of tetracycline antibiotics can be greatly improved, and the drug resistance of resistant strains can be reversed. At the same time, the present invention also provides an antibacterial composition.

Description

Technical Field

[0001] The invention belongs to the field of antibiotic adjuvants, and specifically relates to an application of L-tryptophanamide hydrochloride and an antibacterial composition. Background Art

[0002] Salmonella typhimurium is an important zoonotic pathogen that not only threatens the development of the aquaculture industry, but can also cause human infection through contaminated food, which has important public health significance. Infection with Salmonella typhimurium often manifests as self-limiting diseases such as vomiting, diarrhea, and abdominal pain, but severe infection can cause death. It is reported that Salmonella typhimurium infection can cause tens of thousands of deaths each year. In addition, Salmonella typhimurium is widely prevalent and is currently the dominant serotype in many countries and regions in the world. Therefore, effective prevention and control measures are urgently needed.

[0003] Tetracycline drugs, including doxycycline, minocycline, tigecycline and other drugs, have been gradually introduced into clinical practice since the 1950s. They enter the cell through passive diffusion and energy-dependent active transport, and can reversibly bind to the A site of the 30S subunit of the bacterial ribosome, preventing aminoacyl tRNA from binding to the receptor site of the mRNA ribosome complex, inhibiting the synthesis of bacterial proteins, thereby inhibiting bacterial growth. At the same time, tetracycline drugs also have a strong inhibitory effect on rickettsia, mycoplasma and chlamydia. However, what is worrying is that with the excessive use of antimicrobial drugs in clinical practice and livestock and poultry farming, the resistance of pathogenic microorganisms to tetracycline drugs is becoming increasingly serious, which poses a serious threat to global public health. Usually, when pathogens develop drug resistance, most clinicians use increased antibiotic dosage or combined with other types of antibiotics to kill resistant bacteria, which will undoubtedly increase the amount or type of antibiotics used, leading to further enhancement of bacterial resistance or further increase in the number of resistant categories, forming a vicious circle, and ultimately leading to the dilemma of no drugs available. At the same time, when new antibiotics are put into use, bacteria will quickly develop resistance to them. Therefore, finding efficient and reliable strategies to reverse antibiotic resistance and restore the effectiveness of existing antibiotics is crucial to solving the increasingly serious problem of antibiotic resistance.

[0004] Many studies have shown that the bacterial response to antimicrobial drugs is a holistic rather than a local reaction. The vast metabolic network of bacteria is closely related to the effectiveness of antibiotics. For resistant strains and sensitive strains, metabolites with significant differences in metabolic flux are potential new candidate drugs or antibiotic adjuvants. By supplementing these differential metabolites, it can be used to restore the differences in the metabolic profiles of resistant bacteria, increase the metabolic activity of resistant strains, and thus make them sensitive to antimicrobial drugs. At present, many studies have shown that exogenous supplementation of bacterial differential metabolites can restore the sensitivity of resistant bacteria to aminoglycosides, tetracyclines, and quinolones. It can be seen that using metabolic reprogramming methods to enhance the effectiveness of existing antibiotics is a promising antibacterial strategy.

[0005] The technical problem to be solved in this case is: how to restore the sensitivity of Gram-negative bacteria to tetracycline antibiotics and effectively improve the antibacterial effect of tetracycline antibiotics. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide an antibacterial application of L-tryptophanamide hydrochloride and its application as a synergist of tetracycline antibiotics to improve the efficacy of tetracycline antibiotics and reverse the drug resistance of resistant strains.

[0007] Meanwhile, the invention also provides an antibacterial composition.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] Firstly, the invention discloses the application of L-tryptophanamide hydrochloride in preparing an antibacterial agent.

[0010] More specifically, the bacterium is one of Shigella sonnei, Escherichia coli, Salmonella, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pasteurella, Enterobacter hallii, Streptococcus suis, and Staphylococcus aureus.

[0011] In addition, the invention discloses L-tryptophanamide hydrochloride as an antibiotic synergist for inhibiting Gram-negative bacteria and application thereof.

[0012] More specifically, the Gram-negative bacteria is Salmonella typhimurium.

[0013] More specifically, the antibiotics that inhibit Gram-negative bacteria are one or more combinations of tetracycline, doxycycline, and minocycline.

[0014] Finally, the invention discloses an antibacterial composition, comprising L-tryptophanamide hydrochloride and an antibiotic for inhibiting gram-negative bacteria.

[0015] More specifically, the antibiotics that inhibit Gram-negative bacteria are one or more combinations of tetracycline, doxycycline, and minocycline.

[0016] More specifically, the concentration of the L-tryptophanamide hydrochloride is not less than 1.1 mg / ml.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention screened for the first time the key metabolite that affects the resistance of Salmonella typhimurium to tetracycline antibiotics, L-tryptophanamide hydrochloride, and confirmed through experiments that L-tryptophanamide hydrochloride has a certain inhibitory effect on a variety of pathogens including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, etc., and the inhibitory concentration is 1.1-4.4 mg / ml. At the same time, L-tryptophanamide hydrochloride can greatly enhance the effects of tetracycline, doxycycline, and minocycline against Salmonella typhimurium. 1.1 mg / ml of L-tryptophanamide hydrochloride can play a synergistic role on the above antibiotics, and 2.2 mg / ml of L-tryptophanamide hydrochloride can enhance the effects of the above antibiotics by more than 2,000 times. At the same time, the synergistic index of L-tryptophanamide hydrochloride and the above three antibiotics is ≤0.5, 1.1mg / ml of L-tryptophanamide hydrochloride can reduce the MIC of the above antibiotics to tetracycline-resistant Salmonella typhimurium by 4 to 16 times, and 2.2mg / ml of L-tryptophanamide hydrochloride can reduce the MIC of the above antibiotics to tetracycline-resistant Salmonella typhimurium by 16 to 32 times, thereby effectively reducing the dosage of the above three tetracyclines. At the same time, the inventors have confirmed through experiments that L-tryptophanamide hydrochloride does not increase the hemolyticity of the drug when used in conjunction with the drug, which is safer. In summary, the present invention effectively solves the prevention and control problem of tetracycline-resistant Salmonella typhimurium, and is widely used to overcome the problems of poor antibacterial effect and large drug dosage caused by drug resistance during clinical application, thereby greatly reducing the cost of medication and reducing the risk of transmission of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A Differential metabolite analysis results of tetracycline-resistant Salmonella typhimurium and tetracycline-sensitive Salmonella typhimurium in negative ion mode;

[0020] Figure 1B Differential metabolite analysis results of tetracycline-resistant Salmonella Typhimurium and tetracycline-susceptible Salmonella Typhimurium in cationic mode;

[0021] Figure 2A The results are the test results of the inhibitory effect of L-tryptophanamide hydrochloride on multi-drug resistant Salmonella typhimurium;

[0022] Figure 2B The results are the test results of the inhibitory effect of L-tryptophanamide hydrochloride on Salmonella typhimurium ATCC14028;

[0023] Figure 2C The results are the test results of the inhibitory effect of L-tryptophanamide hydrochloride on Klebsiella pneumoniae T-YSW-2;

[0024] Figure 2D The results are the test results of the inhibitory effect of L-tryptophanamide hydrochloride on Pseudomonas aeruginosa IPM-4;

[0025] Figure 2E The results are the test results of the inhibitory effect of L-tryptophanamide hydrochloride on Escherichia coli T-YW-2;

[0026] Figure 2F The results are the test results of the inhibitory effect of L-tryptophanamide hydrochloride on Escherichia coli ATCC25922;

[0027] Figure 3A This is a schematic diagram showing the effect of different concentrations of L-tryptophanamide hydrochloride on the growth of tetracycline-resistant Salmonella typhimurium;

[0028] Figure 3B This is a schematic diagram of the killing effect of L-tryptophanamide hydrochloride combined with different antibiotics on Salmonella typhimurium;

[0029] Figure 3C This is a clear picture of the broth after L-tryptophanamide hydrochloride and tetracycline were applied to the LB broth containing bacterial liquid for 6 hours;

[0030] Figure 3D This is a clear picture of the broth after L-tryptophanamide hydrochloride and doxycycline were applied to the LB broth containing bacterial liquid for 6 hours;

[0031] Figure 3E This is a clear picture of the broth after L-tryptophanamide hydrochloride and minocycline were applied to the LB broth containing bacterial liquid for 6 hours;

[0032] Figure 4A The antibacterial effect of the combination of different concentrations of L-tryptophanamide hydrochloride and 1 / 2MIC of TET (tetracycline);

[0033] Figure 4B The antibacterial effect of the combination of different concentrations of L-tryptophanamide hydrochloride and 1 / 2MIC of DOX (doxycycline);

[0034] Figure 4C The antibacterial effect of the combination of different concentrations of L-tryptophanamide hydrochloride and 1 / 2MIC of MH (minocycline);

[0035] Figure 5A The results of the chessboard analysis of the interaction between L-tryptophanamide hydrochloride and TET;

[0036] Figure 5B The results of the chessboard analysis of the combined effects of L-tryptophanamide hydrochloride and DOX;

[0037] Figure 5C The results of the chessboard analysis of the interaction between L-tryptophanamide hydrochloride and MH;

[0038] Figure 5D The bactericidal time curve of L-tryptophanamide hydrochloride and TET acting alone or in combination;

[0039] Figure 5E The bactericidal time curve of L-tryptophanamide hydrochloride and DOX acting alone or in combination;

[0040] Fig. 5F The bactericidal time curve of L-tryptophanamide hydrochloride and MH acting alone or in combination;

[0041] Fig. 6A The results are the validation results of the antibacterial activity of L-tryptophanamide hydrochloride in combination with tetracycline, doxycycline and minocycline against Klebsiella pneumoniae;

[0042] Figure 6B The results are the validation results of the antibacterial activity of L-tryptophanamide hydrochloride in combination with tetracycline, doxycycline and minocycline against Pseudomonas aeruginosa;

[0043] Figure 6C The results are the verification results of the antibacterial activity of L-tryptophanamide hydrochloride against Escherichia coli after being combined with tetracycline, doxycycline and minocycline respectively;

[0044] Fig.6D The antibacterial results of L-tryptophanamide hydrochloride combined with tetracycline, doxycycline and minocycline when inhibiting different bacteria;

[0045] Fig. 7A This is a result diagram showing the effect of the combination of L-tryptophanamide hydrochloride and TET on the hemolysis rate of red blood cells;

[0046] Figure 7B This is a result diagram showing the effect of the combination of L-tryptophanamide hydrochloride and DOX on the hemolysis rate of red blood cells;

[0047] Figure 7C This is a graph showing the effect of the combination of L-tryptophanamide hydrochloride and MH on the hemolysis rate of red blood cells. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0049] Example 1 Screening of Differential Metabolites of Tetracycline-Resistant Salmonella and Tetracycline-Sensitive Salmonella

[0050] Salmonella typhimurium R1 and Salmonella typhimurium R41 were isolated and preserved in our laboratory;

[0051] The drug sensitivity results of Salmonella typhimurium R1 and Salmonella typhimurium R41 are shown in Table 1.

[0052] Table 1 Results of drug sensitivity test of Salmonella typhimurium R1 and Salmonella typhimurium R41 (unit: μg / ml)

[0053]

[0054] Non-targeted metabolomics analysis of differential metabolites between Salmonella Typhimurium R1 and Salmonella Typhimurium R41:

[0055] (1) Place the bacterial sample in an EP tube, add 300 μL of 80% methanol aqueous solution, and freeze in liquid nitrogen for 5 minutes; thaw on ice, vortex for 30 seconds, sonicate for 6 minutes, and centrifuge at 5000 rpm and 4°C for 1 minute. Take the supernatant into a new centrifuge tube and freeze-dry it into dry powder; add the corresponding 10% methanol solution according to the sample volume to dissolve it, and inject it into LC-MS for analysis.

[0056] (2) The offline data (.raw) files were imported into CD 3.1 library search software for processing. The retention time, mass-to-charge ratio and other parameters of each metabolite were simply screened. The retention time deviation was set to 0.2 min and the mass deviation was set to 5 ppm to align the peaks of different samples for more accurate identification. The mass deviation was then set to 5 ppm, the signal intensity deviation was set to 30%, the signal-to-noise ratio was set to 3, the minimum signal intensity, and the summed ion information were set to extract the peaks. The peak area was quantified and the target ions were integrated. The molecular formula was then predicted based on the molecular ion peaks and fragment ions and compared with the mzCloud (https: / / www.mzcloud.org / ), mzVault and Masslist databases. The blank sample was used to remove the background ions and the original quantitative results were standardized. Finally, the identification and relative quantitative results of the metabolites were obtained.

[0057] The analysis results are shown in Figure 1A and Figure 1B ,in, Figure 1A The results of differential metabolite analysis in negative ion mode between tetracycline-resistant Salmonella typhimurium and tetracycline-sensitive Salmonella typhimurium; Figure 1B Results of differential metabolite analysis in cationic mode between tetracycline-resistant and tetracycline-sensitive Salmonella Typhimurium.

[0058] pass Figure 1A and Figure 1B It can be seen that there are significant differences in the contents of 94 substances between the strongly resistant and weakly resistant Salmonella typhimurium of tetracycline drugs, of which 24 substances are significantly down-regulated and 70 substances are significantly up-regulated. L-tryptophanamide hydrochloride ( Figure 1A The most down-regulated substance was 23 times lower in the resistant strains than in the sensitive strains, suggesting that it may be the key substance causing the difference in tetracycline resistance between the two strains. At the same time, this substance is also the first in VIP (variable weight importance) among all differential metabolites.

[0059] Example 2 Antibacterial spectrum and minimum inhibitory concentration determination of L-tryptophanamide hydrochloride

[0060] The antibacterial activity of L-tryptophanamide hydrochloride against bacteria was determined by the broth microdilution method. The test strains and the determination results are shown in Table 1. It has certain antibacterial activity against a variety of common pathogens in clinical and livestock and poultry breeding industries, including Escherichia coli, Salmonella, Klebsiella pneumoniae, etc.

[0061] The specific steps of the broth microdilution method are as follows:

[0062] (1) Suspend the strain to be tested in MH broth medium to obtain a bacterial concentration of 1×10 6 CFU / mL of bacterial suspension.

[0063] (2) Take a 96-well plate, add 100 μL of MH broth culture medium to each well, add 100 μL of antibacterial drug solution to each well in the first column, and dilute it in multiples from the first column to the tenth column; then add 100 μL of the bacterial suspension prepared in step (1) to each well, incubate at 37°C for 16 h to 18 h, and observe the lowest concentration of L-tryptophanamide hydrochloride that inhibits bacterial growth. Set up positive and negative control wells, add 100 μL of MH broth culture medium and 100 μL of the bacterial suspension prepared in step (1) to each positive control well, and only add 200 μL of MH broth culture medium to the negative control well.

[0064] The experimental results are shown in Table 2;

[0065] Table 2 MIC of L-tryptophanamide hydrochloride against various Gram-negative and Gram-positive bacteria

[0066]

[0067] Table 2 shows that the MIC of L-tryptophanamide hydrochloride against the eight Gram-negative bacteria tested is 2.2-4.4 mg / ml, and the MIC against the two Gram-positive bacteria tested is 1.1-2.2 mg / ml. Figure 2A to Figure 2F The results of visual observation of the inhibitory effect of L-tryptophanamide hydrochloride on some Gram-negative bacteria are shown.

[0068] Figure 2A This is a diagram showing the results of visual observation of the inhibitory effect of L-tryptophanamide hydrochloride on multi-drug resistant Salmonella typhimurium R1;

[0069] Figure 2B This is a diagram showing the results of visual observation of the inhibitory effect of L-tryptophanamide hydrochloride on Salmonella typhimurium ATCC14028;

[0070] Figure 2C This is a diagram showing the results of visual observation of the inhibitory effect of L-tryptophanamide hydrochloride on Klebsiella pneumoniae T-YSW-2;

[0071] Figure 2D This is a visual observation result of the inhibitory effect of L-tryptophanamide hydrochloride on Pseudomonas aeruginosa IPM-4.

[0072] Figure 2E This is a diagram showing the visual observation result of the inhibitory effect of L-tryptophanamide hydrochloride on Escherichia coli T-YW-2;

[0073] Figure 2F This is a diagram showing the results of visual observation of the inhibitory effect of L-tryptophanamide hydrochloride on Escherichia coli ATCC25922.

[0074] Example 3 Detection of the synergistic effect of L-tryptophanamide hydrochloride on antibiotics

[0075] Effects of different concentrations of L-tryptophanamide hydrochloride on strain growth:

[0076] (1) Tetracycline-resistant Salmonella typhimurium R1 monoclonal was selected and cultured in LB broth at 37°C and 180 rpm overnight. The cells were then washed three times with PBS solution, and the cell pellet was resuspended in LB broth and the OD600 reading of the bacterial solution was adjusted to about 1.0.

[0077] (2) Take 20 ml of fresh LB broth medium, add L-tryptophanamide hydrochloride solution with a final concentration of 0-4.4 mg / ml, then add an equal amount of the bacterial solution prepared in step (1) to each group of broth, shake and mix, and incubate at 37°C, 180 rpm for 24 h. Take 1 ml of the broth containing the bacterial solution in PBS at 0, 6, 12, 18, and 24 h, respectively, and dilute it continuously. Use XLT-4 agar medium to count the cells by plate pouring method.

[0078] The results are as follows Figure 3A As shown, the results showed that 4.4 mg / ml of L-tryptophanamide hydrochloride had a significant inhibitory effect on the growth of tetracycline-resistant Salmonella typhimurium R1, while 0.55-2.2 mg / ml of L-tryptophanamide hydrochloride alone had little effect on the growth of tetracycline-resistant Salmonella typhimurium R1.

[0079] Figure 3A This is a graph showing the inhibitory effect of different concentrations of L-tryptophanamide hydrochloride on the growth of tetracycline-resistant Salmonella typhimurium R1;

[0080] Detection of the synergistic effect of L-tryptophanamide hydrochloride on antibiotics:

[0081] (1) Tetracycline-resistant Salmonella typhimurium R1 monoclonal was selected and cultured in LB broth at 37°C and 180 rpm overnight. The culture was then washed three times with PBS, and the bacterial pellet was resuspended in LB broth and the OD600 reading was adjusted to about 1.0.

[0082] (2) Take 20 ml of fresh LB broth medium, add 16 antibiotic solutions with a final concentration of 1 / 2 MIC respectively, then add 0 or 2.2 mg / ml of L-tryptophanamide hydrochloride to each group of broth, shake and mix, add an equal amount of the bacterial solution prepared in step (1) to each group of broth, shake and mix again, incubate at 37°C, 180 rpm for 6 h, take 1 ml of the above broth containing the bacterial solution in PBS and dilute it continuously, and use XLT-4 agar medium to count by plate pouring method.

[0083] The results are as follows Figure 3B As shown, the results showed that 2.2 mg / ml of L-tryptophanamide hydrochloride significantly enhanced the killing effect of tetracycline drugs tetracycline, doxycycline, and minocycline on Salmonella typhimurium R1, and the survival rate of Salmonella typhimurium R1 decreased by more than 2000 times compared with the effect of antibiotics alone. Figure 3C to 2E The changes in turbidity of the broth of each group after different treatments are shown. The clearer the broth, the lower the survival rate of the bacterial colonies in the broth.

[0084] Figure 3B The killing effect of L-tryptophanamide hydrochloride combined with different antibiotics on Salmonella typhimurium R1 is shown in the table;

[0085] Figure 3C This is a picture of the clarity of the broth after L-tryptophanamide hydrochloride and tetracycline acted on the broth;

[0086] Figure 3D This is a picture of the clarity of the broth after L-tryptophanamide hydrochloride and doxycycline acted on the broth;

[0087] Figure 3E This is a picture of the clarity of the broth after L-tryptophanamide hydrochloride and minocycline were added to the broth.

[0088] Example 4 Concentration dependence test of the synergistic effect of L-tryptophanamide hydrochloride on tetracycline antibiotics

[0089] (1) Tetracycline-resistant Salmonella typhimurium R1 monoclonal was selected and cultured in LB broth at 37°C and 180 rpm overnight. The cells were then washed three times with PBS solution, and the cell pellet was resuspended in LB broth and the OD600 reading of the bacterial solution was adjusted to about 1.0.

[0090] (2) Take 20 ml of fresh LB broth medium, add L-tryptophanamide hydrochloride solution with a final concentration of 0-4.4 mg / ml, then add an equal amount of tetracycline drugs with a final concentration of 1 / 2 MIC to the broth of each group, and finally add an equal amount of the bacterial solution prepared in step (1) to each group. After shaking and mixing, incubate at 37°C, 180 rpm for 6 h. After 6 h, take 1 ml of the broth containing the bacterial solution in PBS and dilute it continuously. Use XLT-4 agar medium to count by plate pouring method.

[0091] The results are as follows Figures 4A to 4C As shown, 1.1-4.4 mg / ml of L-tryptophanamide hydrochloride significantly enhanced the antibacterial effect of each tetracycline drug, indicating that the synergistic effect of L-tryptophanamide hydrochloride on tetracycline antibiotics has a certain concentration dependence.

[0092] Figure 4A The antibacterial effect diagram of the combination of different concentrations of L-tryptophanamide hydrochloride and 1 / 2MIC TET (tetracycline);

[0093] Figure 4B The diagram is the antibacterial effect of the combination of different concentrations of L-tryptophanamide hydrochloride and 1 / 2MIC DOX (doxycycline);

[0094] Figure 4C The graph shows the antibacterial effect of different concentrations of L-tryptophanamide hydrochloride and 1 / 2MIC MH (minocycline).

[0095] Example 5 Checkerboard analysis to detect the synergistic antibacterial activity of L-tryptophanamide hydrochloride and three tetracycline drugs against Salmonella typhimurium

[0096] (1) A single colony of tetracycline-resistant Salmonella typhimurium R1 was selected and cultured in MH broth at 37°C for 4 to 6 hours. The culture was then diluted 1:100 with MH broth.

[0097] (2) Three tetracycline drugs were separately taken, dissolved in sterile water and diluted with MH broth medium to obtain antibiotic solutions with concentrations of 32 μg / mL respectively.

[0098] (3) L-tryptophan amide hydrochloride was taken, dissolved in sterile water and diluted with MH broth medium to obtain an antibacterial drug solution with a concentration of 4.4 mg / mL.

[0099] (4) A 96-well flat-bottom plate was taken. Tetracycline (0 - 512 μg / mL), doxycycline (0 - 256 μg / mL), and minocycline (0 - 512 μg / mL) were diluted along the abscissa respectively, and L-tryptophan amide hydrochloride was diluted along the ordinate (0 - 8.8 mg / mL). 100 μL of the bacterial solution obtained in step (1) was evenly added into the wells of the 96-well plate. After incubation at 37 °C for 18 h, the optical density of each well at 600 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0100] The fractional inhibitory concentration (FIC) index was calculated according to the following formula:

[0101] FIC = MIC of tetracycline drugs (used in combination) / MIC of tetracycline drugs (used alone) + MIC of L-tryptophan amide hydrochloride (used in combination) / MIC of L-tryptophan amide hydrochloride (used alone)

[0102] When FIC ≤ 0.5, the interaction between the two drugs was determined to be a synergistic effect, and the smaller the FIC index, the stronger the synergistic effect; when 0.5 < FIC ≤ 4, it was considered that there was no interaction between the two drugs; when FIC > 4, the two drugs produced an antagonistic effect.

[0103] The results of the checkerboard analysis were as Figures 5A to 5C shown. The results showed that L-tryptophan amide hydrochloride could significantly enhance the antibacterial activities of tetracycline, doxycycline, and minocycline against tetracycline-resistant Salmonella typhimurium, and the synergistic fold was 16 to 32 times. The fractional inhibitory concentration (FIC) index of the combination was less than or equal to 0.5, indicating a significant synergistic effect of the combination.

[0104] Figure 5A were the experimental results of the checkerboard analysis of the co-action of L-tryptophan amide hydrochloride and TET;

[0105] Figure 5B were the experimental results of the checkerboard analysis of the co-action of L-tryptophan amide hydrochloride and DOX;

[0106] Figure 5C were the experimental results of the checkerboard analysis of the co-action of L-tryptophan amide hydrochloride and MH.

[0107] Example 6 Time-kill curves of L-tryptophan amide hydrochloride and three tetracycline drugs

[0108] (1) Tetracycline-resistant Typhimurium R1 monoclonal clones were selected and cultured in MH broth at 37°C overnight. The next day, the cells were washed three times with PBS, and then the cell pellet was resuspended in MH broth and the concentration of the cell solution was adjusted to 10 6 CFU / mL or so.

[0109] (2) Prepare three tetracycline drugs and L-tryptophanamide hydrochloride solutions respectively, calculate the final concentrations, and then add them individually or together into fresh MH broth medium.

[0110] (3) Take an equal amount of the bacterial solution obtained in step (1) and add it to the broth of each group mentioned above. Take 1 ml of the broth containing the bacterial solution in PBS at 0 h, 2 h, 4 h, 6 h, 8 h, 16 h, and 24 h, respectively, and dilute it continuously. Use XLT-4 agar medium to count by plate pouring method.

[0111] The experimental results are shown in Figures 5D to 5F The results showed that L-tryptophanamide hydrochloride can significantly enhance the antibacterial activity of tetracycline, doxycycline and minocycline against tetracycline-resistant Salmonella typhimurium.

[0112] Figure 5D The bactericidal time curves of L-tryptophanamide hydrochloride and TET used alone and together;

[0113] Figure 5E The bactericidal time curves of L-tryptophanamide hydrochloride and DOX used alone and together;

[0114] Fig. 5F This is the bactericidal time curve of L-tryptophanamide hydrochloride and MH used alone and together.

[0115] Example 7 Broad-spectrum detection of the synergistic and synergistic effects of L-tryptophanamide hydrochloride on tetracycline drugs

[0116] Broad-spectrum detection of the synergistic effect of L-tryptophanamide hydrochloride on tetracycline drugs:

[0117] (1) Klebsiella pneumoniae T-YSW-2, Pseudomonas aeruginosa IPM-4, and Escherichia coli T-YW-2 monoclonal clones were selected and cultured in LB broth at 37°C overnight. The next day, the cells were washed three times with PBS, and then the cell pellet was resuspended in LB broth medium and the bacterial solution concentration was adjusted to 10 6 CFU / mL or so.

[0118] (2) Prepare three tetracycline drugs and L-tryptophanamide hydrochloride solutions respectively, calculate the final concentrations, and then add them individually or together into fresh LB broth medium.

[0119] (3) Take an equal amount of the bacterial solution obtained in step (1) and add it to the above-mentioned broths of each group, incubate in a constant temperature shaker at 37°C for 6 hours, take 1 ml of the above-mentioned broth containing the bacterial solution in PBS after 6 hours and dilute it continuously, and use LB agar medium to count by plate pouring method.

[0120] The experimental results are shown in Figures 6A to 6C The results showed that L-tryptophanamide hydrochloride can also enhance the antibacterial activity of tetracycline, doxycycline, and minocycline against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli.

[0121] Fig. 6A The results are the validation results of the antibacterial activity of L-tryptophanamide hydrochloride in combination with tetracycline, doxycycline and minocycline against Klebsiella pneumoniae;

[0122] Figure 6B The results are the validation results of the antibacterial activity of L-tryptophanamide hydrochloride in combination with tetracycline, doxycycline and minocycline against Pseudomonas aeruginosa;

[0123] Figure 6C The results are the validation results of the antibacterial activity of L-tryptophanamide hydrochloride in combination with tetracycline, doxycycline and minocycline against Escherichia coli;

[0124] Broad-spectrum detection of the synergistic effect of L-tryptophanamide hydrochloride on tetracycline drugs:

[0125] (1) Monoclonal clones of Klebsiella pneumoniae T-YSW-2, Pseudomonas aeruginosa IPM-4, and Escherichia coli T-YW-2 were selected and cultured in MH broth at 37°C for 4 to 6 hours. The bacterial suspension was then diluted 1:100 with MH broth.

[0126] (2) Subsequent operations and judgment criteria are the same as those in Example 2.

[0127] The experimental results are shown in Fig.6D The results showed that L-tryptophanamide hydrochloride also had a synergistic effect with three tetracycline drugs, tetracycline, doxycycline, and minocycline, in inhibiting the growth of Klebsiella pneumoniae T-YSW-2, Pseudomonas aeruginosa IPM-4, and Escherichia coli T-YW-2.

[0128] Fig.6D The antibacterial results of L-tryptophanamide hydrochloride and three tetracycline drugs, tetracycline, doxycycline and minocycline, when inhibiting different bacteria.

[0129] Example 7 Red blood cell hemolysis experiment

[0130] (1) Dissolve L-tryptophanamide hydrochloride in PBS solution and dilute to obtain L-tryptophanamide hydrochloride solutions with final concentrations of 4MIC, 2MIC, MIC, 1 / 2MIC, and 0. Take a 96-well cell culture plate, add 50 μl of the above solution to the first 11 columns of the 96-well flat-bottom plate, and add water to the 12th column as a positive control.

[0131] (2) Dissolve tetracyclines in PBS solution, add 50 μl of tetracycline solution to the first column of a 96-well flat-bottom plate, dilute to the right in multiples until the end of the tenth column and discard.

[0132] (3) Prepare fresh defibrinated sheep blood. Centrifuge 10 mL of sheep blood at 4°C, 3000 x g for 10 min and discard the supernatant. Wash the precipitate at the bottom of the tube twice with PBS and resuspend it in PBS. Take 3.2 mL of the above suspension and add it to 36.8 mL of PBS to prepare an 8% red blood cell suspension. Take 100 microliters of red blood cell solution and add it to the above 96-well plate. Mix well and place in a 37°C incubator for 1 hour. After 1 hour, take 120 microliters of supernatant and add it to a new 2 mL centrifuge tube. After centrifugation at 3000 x g for 3-5 minutes, take 100 microliters of supernatant and add it to a new 96-well plate. Use an M200 microplate reader to measure OD576.

[0133] Hemolysis rate (%) = [(OD576 sample - OD576 blank) / (OD576 water - OD576 blank)] × 100%.

[0134] If the hemolysis rate is lower than 4%, it proves that the substance does not significantly enhance the hemolytic effect of the drug.

[0135] The experimental results are shown in Figures 7A to 7C It can be seen that all concentrations of L-tryptophanamide hydrochloride did not significantly increase the hemolysis rate of the three tetracycline drugs, and the hemolysis rate of each drug combination was below 4%, indicating that L-tryptophanamide hydrochloride at the effective concentration would not significantly increase the hemolysis rate of red blood cells and was relatively safe.

[0136] Fig. 7A This is a graph showing the effect of the combination of L-tryptophanamide hydrochloride and TET on the hemolysis rate;

[0137] Figure 7B This is a graph showing the effect of the combination of L-tryptophanamide hydrochloride and DOX on the hemolysis rate;

[0138] Figure 7C This is a graph showing the effect of the combination of L-tryptophanamide hydrochloride and MH on the hemolysis rate.

[0139] Summarize:

[0140] Based on the results of Examples 1 to 7 above, the following conclusions can be drawn:

[0141] 1. The metabolic analysis of the strongly resistant Salmonella typhimurium R1 and the weakly resistant Salmonella typhimurium R41 showed that L-tryptophanamide hydrochloride was the key substance that caused the difference in their resistance to tetracyclines.

[0142] 2. L-tryptophanamide hydrochloride has certain antibacterial properties, but its MIC concentration is relatively high;

[0143] 3. L-tryptophanamide hydrochloride significantly enhanced the killing effect of tetracycline drugs on Salmonella typhimurium;

[0144] 4. The lowest concentration dependence of various tetracycline drugs on L-tryptophanamide hydrochloride is 1.1 mg / ml. When the concentration is lower than 1.1 mg / ml, the two drugs can also show obvious synergistic performance;

[0145] 5. The graded antibacterial concentration index of various tetracycline drugs combined with L-tryptophanamide hydrochloride was less than or equal to 0.5, indicating that the combination of the two drugs had a significant synergistic effect;

[0146] 6. L-tryptophanamide hydrochloride can significantly enhance the antibacterial activity of tetracycline, doxycycline and minocycline against tetracycline-resistant Salmonella typhimurium;

[0147] 7. L-tryptophanamide hydrochloride has a broad spectrum of synergistic effects on tetracycline drugs, and this synergistic effect exists for Salmonella typhimurium and other Gram-negative bacteria in different states;

[0148] 8. L-tryptophanamide hydrochloride at all concentrations will not significantly increase the hemolysis rate of red blood cells and is relatively safe.

[0149] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, and it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. Application of L-tryptophanamide hydrochloride in the preparation of a synergist of an antibiotic for inhibiting Gram-negative bacteria, wherein the antibiotic for inhibiting Gram-negative bacteria is one or more combinations of tetracycline, doxycycline, and minocycline, and the Gram-negative bacteria is Salmonella typhimurium.

2. Use of a combination of L-tryptophanamide hydrochloride and an antibiotic that inhibits Gram-negative bacteria in the preparation of an antibacterial composition for inhibiting Gram-negative bacteria; the antibiotic is a combination of one or more of tetracycline, doxycycline, and minocycline; and the Gram-negative bacteria is Salmonella typhimurium.

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