Application of valine in preparation of medicine for improving sensitivity of bacteria to antibiotics

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of valine in preparation of a medicine for improving the sensitivity of bacteria to antibiotics. Researches prove that the combination of the valine and the antibiotics can significantly improve the sensitivity of the antibiotics to clinical drug-resistant bacteria and promote the killing of the antibiotics to the drug-resistant bacteria. The in-vivo study on mice further proves that in a systematic infection model, the survival rate of the mice is remarkably increased through combination of valine and antibiotics; in a thigh infection model, the combination effectively reduces the number of bacteria and inflammatory response in an infected area. The action mechanism of the valine is as follows: the valine can promote the uptake of antibiotics by bacteria by improving the proton dynamic potential of the bacteria and the abundance of lauric acid, so that the sterilization efficiency of the valine is improved. In addition, valine is essential amino acid for a human body, so that the valine is high in nutritional value and high in safety. The discovery opens up a new way for overcoming the problem of antibiotic resistance and optimizing an anti-infection treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of valine in the preparation of drugs for improving the sensitivity of bacteria to antibiotics. Background Art

[0002] Staphylococcus aureus Rosenbach, especially methicillin-resistant Staphylococcus aureus (MRSA), is a common clinical pathogen that poses a serious threat to human health. In the "2024 List of Bacterial Priority Pathogens" updated on May 17, 2024, the World Health Organization listed MRSA as a high-priority bacteria, emphasizing its significant harm as one of the drug-resistant bacteria.

[0003] At present, the problem of drug resistance of Gram-positive and Gram-negative bacteria is becoming increasingly serious, and the research and development of new antimicrobial drugs faces many difficulties. In this situation, it is particularly important to find effective antimicrobial synergists and restore the sensitivity of multidrug-resistant bacteria (including MRSA) to existing key antimicrobial drugs through reasonable combination drug strategies. This will not only help improve the clinical efficacy of antimicrobial drugs, but also delay the emergence of drug resistance to a certain extent, which is of great significance for improving the current difficulties faced by antimicrobial treatment. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings that many clinical bacteria have developed resistance to existing antibiotics and that new antibacterial drugs have high research and development costs and long cycles, and to provide the application of valine in the preparation of antibiotic synergists.

[0005] The purpose of the present invention is to provide an application of valine in combination with an antibiotic in the preparation of a drug for improving the sensitivity of bacteria to the antibiotic.

[0006] Another object of the present invention is to provide the use of valine in combination with antibiotics in the preparation of a medicament for preventing and / or treating bacterial infection.

[0007] Another object of the present invention is to provide a drug for improving the sensitivity of bacteria to antibiotics.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The invention discloses an application of protected valine in preparing an antibiotic synergist, wherein the antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or a pharmaceutically acceptable salt thereof.

[0010] Valine (i.e. 2-amino-3-methylbutyric acid) is one of the 20 amino acids that make up protein. It is one of the 8 essential amino acids and glycogenic amino acids for the human body. It can promote normal growth of the body, repair tissues, regulate blood sugar, and provide the required energy. The present invention first discovered that the combination of valine and antibiotics can significantly increase the sensitivity of head antibiotics to clinical drug-resistant bacteria and promote the killing of drug-resistant bacteria by antibiotics. Studies in mice further proved that after valine was combined with antibiotics, the systemic infection model in mice could significantly improve the survival rate, and the thigh model could significantly reduce the amount of bacteria and inflammatory response in the infected area, thereby achieving the treatment of diseases caused by bacterial infection. The mechanism of the above treatment is that valine can promote the uptake of antibiotics by bacteria by increasing the proton motive force and lauric acid abundance of bacteria, thereby improving its bactericidal efficiency. In addition, valine is an essential amino acid for the human body, has high nutritional value, and has been widely used in food and medicine with high safety.

[0011] Furthermore, the cefoperazone sulbactam is usually administered in the form of a salt, preferably in the form of cefoperazone sodium sulbactam sodium. Furthermore, the mass ratio of cefoperazone to sulbactam in the cefoperazone sulbactam is 1:1.

[0012] Furthermore, the amoxicillin-clavulanic acid is usually administered in the form of a salt, preferably in the form of amoxicillin sodium clavulanate potassium. Furthermore, the mass ratio of amoxicillin to clavulanic acid in the amoxicillin-clavulanic acid is (2-7):1.

[0013] Preferably, the antibiotic is used at a concentration of ≥10 μg / mL, more preferably ≥50 μg / mL.

[0014] Preferably, the valine is L-valine.

[0015] Preferably, the valine is used at a concentration of ≥0.1 mM (mmol / L), more preferably ≥1 mM.

[0016] Furthermore, the valine increases the sensitivity of bacteria to antibiotics, and the bacteria are Gram-positive bacteria and Gram-negative bacteria, wherein Gram-negative bacteria do not include Escherichia coli.

[0017] Furthermore, the bacteria include sensitive bacteria and drug-resistant bacteria.

[0018] The invention discloses an application of protected valine in combination with an antibiotic in the preparation of a drug for improving the sensitivity of bacteria to antibiotics. The antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or pharmaceutically acceptable salts thereof; and the bacteria are gram-positive bacteria and gram-negative bacteria, wherein the gram-negative bacteria do not include Escherichia coli.

[0019] Furthermore, the cefoperazone sulbactam is usually administered in the form of a salt, preferably in the form of cefoperazone sodium sulbactam sodium. Furthermore, the mass ratio of cefoperazone to sulbactam in the cefoperazone sulbactam is 1:1.

[0020] Furthermore, the amoxicillin-clavulanic acid is usually administered in the form of a salt, preferably in the form of amoxicillin sodium clavulanate potassium. Furthermore, the mass ratio of amoxicillin to clavulanic acid in the amoxicillin-clavulanic acid is (2-7):1.

[0021] Preferably, the antibiotic is used at a concentration of ≥10 μg / mL, more preferably ≥50 μg / mL.

[0022] Preferably, the valine is L-valine.

[0023] Preferably, the valine is used at a concentration of ≥0.1 mM, more preferably ≥1 mM.

[0024] The present invention also protects the use of valine in combination with antibiotics in the preparation of a drug for preventing or / and treating bacterial infection, wherein the antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or pharmaceutically acceptable salts thereof; and the bacteria are Gram-positive bacteria and Gram-negative bacteria, wherein Gram-negative bacteria do not include Escherichia coli.

[0025] Furthermore, the cefoperazone sulbactam is usually administered in the form of a salt, preferably in the form of cefoperazone sodium sulbactam sodium. Furthermore, the mass ratio of cefoperazone to sulbactam in the cefoperazone sulbactam is 1:1.

[0026] Furthermore, the amoxicillin-clavulanic acid is usually administered in the form of a salt, preferably in the form of amoxicillin sodium clavulanate potassium. Furthermore, the mass ratio of amoxicillin to clavulanic acid in the amoxicillin-clavulanic acid is (2-7):1.

[0027] Preferably, the antibiotic is used at a concentration of ≥10 μg / mL, more preferably ≥50 μg / mL.

[0028] Preferably, the valine is L-valine.

[0029] Preferably, the valine is used at a concentration of ≥0.1 mM, more preferably ≥1 mM.

[0030] Furthermore, the bacteria include sensitive bacteria and drug-resistant bacteria.

[0031] Furthermore, the Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecium.

[0032] Furthermore, the Staphylococcus aureus includes sensitive bacteria and resistant bacteria, wherein the sensitive bacteria are methicillin-sensitive Staphylococcus aureus (MSSA), and the resistant bacteria are methicillin-resistant Staphylococcus aureus (MRSA).

[0033] Furthermore, the Gram-negative bacteria include one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0034] Furthermore, the valine acts as an antibiotic synergist to enhance the ability of antibiotics to kill bacteria.

[0035] Furthermore, the valine acts as an antibiotic synergist to enhance the ability of antibiotics to eliminate bacteria.

[0036] Furthermore, the valine acts as an antibiotic synergist to enhance the body's resistance to bacterial infection.

[0037] Furthermore, the valine acts as an antibiotic synergist to increase the amount of antibiotics entering cells. Exogenous valine can promote the abundance of lauric acid, thereby increasing the permeability of bacterial cell membranes, thereby increasing the amount of antibiotics entering cells, thereby increasing the sensitivity of bacteria to antibiotics.

[0038] Furthermore, the valine acts as an antibiotic synergist to delay the post-effect of Gram-positive bacteria on antibiotics.

[0039] Furthermore, the valine acts as an antibiotic synergist to delay the post-antibiotic effect of bacteria on antibiotics. The post-antibiotic effect (PAE) refers to the effect that after bacteria are exposed to antibiotics, even though the serum concentration of antibiotics drops below the minimum inhibitory concentration or disappears, the inhibitory effect on microorganisms still persists for a period of time.

[0040] The present invention protects a drug for improving the sensitivity of bacteria to antibiotics, comprising an effective amount of valine and an antibiotic; the antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or pharmaceutically acceptable salts thereof; the bacteria are gram-positive bacteria and gram-negative bacteria, wherein the gram-negative bacteria do not include Escherichia coli.

[0041] Furthermore, the cefoperazone sulbactam is usually administered in the form of a salt, preferably in the form of cefoperazone sodium sulbactam sodium. Furthermore, the mass ratio of cefoperazone to sulbactam in the cefoperazone sulbactam is 1:1.

[0042] Furthermore, the amoxicillin-clavulanic acid is usually administered in the form of a salt, preferably in the form of amoxicillin sodium clavulanate potassium. Furthermore, the mass ratio of amoxicillin to clavulanic acid in the amoxicillin-clavulanic acid is (2-7):1.

[0043] Preferably, the antibiotic is used at a concentration of ≥10 μg / mL, more preferably ≥50 μg / mL.

[0044] Preferably, the valine is L-valine.

[0045] Preferably, the valine is used at a concentration of ≥0.1 mM, more preferably ≥1 mM.

[0046] Furthermore, the bacteria include sensitive bacteria and drug-resistant bacteria.

[0047] Furthermore, the Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecium.

[0048] Furthermore, the Gram-negative bacteria include one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0049] Preferably, the mixing ratio of valine to antibiotic is 1: (0.5-4000) mol / g.

[0050] More preferably, the mixing ratio of valine to antibiotic is 1: (2.5-400) mol / g.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention discovered for the first time that the combination of valine and antibiotics can significantly increase the sensitivity of head antibiotics to clinical drug-resistant bacteria and promote the killing of drug-resistant bacteria by antibiotics. In vivo studies in mice further proved that: in the systemic infection model, the combination of valine and antibiotics significantly increased the survival rate of mice; and in the thigh infection model, the combination effectively reduced the number of bacteria and inflammatory response in the infected area. Its mechanism of action is that valine can promote the bacterial uptake of antibiotics by increasing the proton motive force and lauric acid abundance of bacteria, thereby improving its bactericidal efficiency. In addition, valine is an essential amino acid for the human body, which not only has high nutritional value but also high safety. This discovery has opened up a new way to overcome the problem of antibiotic resistance and optimize anti-infection treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a statistical chart showing the results of valine improving the sensitivity of clinical Staphylococcus aureus to multiple antibiotics in Example 2.

[0054] Figure 2 The results of the statistical graphs of the antibiotic concentration (A), valine concentration (B) and time gradient (C) effects of valine on improving the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium in Example 3 are shown.

[0055] Figure 3 The results are statistical graphs showing that the addition of valine in Example 4 increases the sensitivity of various bacteria to cefoperazone sodium and sulbactam sodium; wherein A is a statistical graph showing the results of clinical methicillin-resistant Staphylococcus aureus, B is a statistical graph showing the results of other Gram-positive bacteria, and C is a statistical graph showing the results of clinical Gram-negative bacteria.

[0056] Figure 4 This is a statistical graph showing the results of Example 5 in which valine can delay the post-effects of clinical Staphylococcus aureus on cefoperazone sodium and sulbactam sodium.

[0057] Figure 5 The results of the statistical graph of the muscle infection (A) and the bacterial count (B) of the infected area after the mouse thigh was infected with clinical Staphylococcus aureus in Example 6 and the infected area was treated with antibiotics and valine.

[0058] Figure 6 This is a statistical graph showing the results of Example 6 showing that valine synergistically with cefoperazone sodium and sulbactam sodium can improve the survival rate (A and B) and clearance ability (C) of mice infected with clinical Staphylococcus aureus.

[0059] Figure 7 The results of improving the pyruvate cycle of clinical Staphylococcus aureus after adding valine in Example 7 are statistical graphs; wherein A is a statistical graph of the results of important enzyme activities in the pyruvate cycle; B is a statistical graph of the results of NADH; C is a statistical graph of the results of ATP; D is a statistical graph of the results of proton motive force; and E is a statistical graph of the results of the synergistic effect of valine and SCF on bacterial survival rate after adding CCCP.

[0060] Figure 8 The figures are statistical graphs of the effect of adding lauric acid on MRSA bacteria in Example 7; wherein A is a statistical graph of the lauric acid abundance of MSSA and MRSA and MRSA after adding valine; B is a statistical graph of the results of lauric acid synergistically improving the sensitivity of MRSA to SCF; C is a statistical graph of the cell membrane permeability detection results of MSSA and MRSA, and D and E are statistical graphs of the changes in bacterial permeability after adding lauric acid and valine, respectively.

[0061] Fig. 9This is a statistical chart showing the effect of adding valine and / or CCCP on the antibiotic content of clinical Staphylococcus aureus in Example 7. DETAILED DESCRIPTION

[0062] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0063] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0064] Figure 2 A means Figure 2 Figure A in Figure 3 A stands for Figure 3 This is Figure A in the figure. The meanings of the other figures are similar.

[0065] Valine is commercially available L-valine.

[0066] Example 1 Preparation of clinical bacterial samples

[0067] Nine strains of sensitive Staphylococcus aureus (MSSA) and 19 strains of methicillin-resistant Staphylococcus aureus (MRSA) obtained from clinical trials were named MSSA 1 to 9 and MRSA 1 to 9, 11 to 20. For details of the strains, see patent application 202411031248.4.

[0068] Pick a single bacterial colony and inoculate it in 5 mL LB medium, and culture it at 37 ° C and 200 rpm for 16 hours; take an appropriate amount of bacterial solution and centrifuge it at 8000 rpm for 5 min to collect the bacteria, remove the supernatant and wash the bacteria three times with an equal volume of 0.85% saline, suspend it in 1×M9 medium (containing 10 mM acetate, 1 mM CaCl2), and use M9 medium to adjust the bacterial solution concentration to OD 600 The concentration was 0.2 and divided into 5 mL test tubes for later use.

[0069] Example 2 Valine can improve the sensitivity of clinical methicillin-resistant Staphylococcus aureus to antibiotics

[0070] The purpose of this study was to study whether valine could increase the sensitivity of bacteria to commonly used antibiotics in clinical practice using clinical methicillin-resistant Staphylococcus aureus as the target.

[0071] To this end, MRSA 2 bacterial samples were prepared according to Example 1. The samples were divided into 3 groups according to each antibiotic: M9 control group, single antibiotic group, antibiotic + valine group. 3 biological replicates. The experimental antibiotics and dosages are shown in Table 1, and the valine concentration is 5mM. After culturing for 10 hours at 37°C and 200rpm, the viable bacteria were counted by plate method, and then the survival rate of bacteria after adding valine and not adding valine was calculated based on the addition of different antibiotics. The calculation formula is: survival rate (%) = number of bacteria after adding antibiotics or antibiotics + valine / number of bacteria in the M9 control group × 100%.

[0072] There are 20 kinds of antibiotics used in the experiment, see Table 1 for details. The results show that 10 antibiotics (cefoperazone sodium and sulbactam sodium (the mass ratio of cefoperazone to sulbactam is 1:1, and both are used in this ratio unless otherwise specified), ceftriaxone sodium, ceftazidime, amoxicillin sodium and clavulanate potassium (the mass ratio of amoxicillin to clavulanic acid is 5:1, and both are used in this ratio unless otherwise specified), moxifloxacin, levofloxacin, erythromycin, roxithromycin, and amikacin) have weak or no bactericidal effects when used alone (at the currently selected antibiotic concentration). However, when valine is added, the sensitivity of bacteria to these antibiotics is significantly increased ( Figure 1 ). Among them, the best effect was achieved when valine was used in combination with cefoperazone sodium and sulbactam sodium, with the bactericidal multiple increased by 250.7 times. In addition, cephradine, cefazolin, amoxicillin, carbenicillin, metronidazole, gentamicin, kanamycin, tobramycin, tetracycline and tigecycline were used in combination with valine, and the combined bactericidal effect was not significantly improved compared with the effect of using antibiotics alone.

[0073] Table 1 Antibiotics used and their concentrations

[0074]

[0075]

[0076] Example 3 Study on the effect of valine on the sensitivity of clinical methicillin-resistant Staphylococcus aureus to cefoperazone sodium and sulbactam sodium

[0077] In order to confirm that valine improves the sensitivity of clinical methicillin-resistant Staphylococcus aureus to cefoperazone sodium and sulbactam sodium SCF, MRSA 2 was selected for in-depth study on different concentrations of antibiotics, different concentrations of valine and different action times.

[0078] 3.1 Valine improves the sensitivity of clinical bacteria to antibiotics and has an antibiotic concentration gradient effect

[0079] MRSA 2 bacterial samples were prepared according to Example 1. The samples were divided into an M9 control group and an experimental group. The experimental group was a group with different concentrations of cefoperazone sodium and sulbactam sodium (i.e., SCF) (0, 10, 25, 50, 100, 200, and 400 μg / mL), and a group with the corresponding concentration of SCF+5mM valine. Three biological replicates were performed. The culture was cultured at 37°C and 200rpm for 10 hours, and the viable bacteria were counted by the plate method. Then, the survival rate of the bacteria after adding SCF or SCF and valine treatment based on the addition of different concentrations of antibiotics was calculated, and the calculation formula was: survival rate (%) = the number of bacteria after adding SCF or SCF+valine treatment / the number of M9 control bacteria × 100%. Valine synergistically improves the bactericidal efficiency of cefoperazone sodium and sulbactam sodium, and is evaluated according to the bactericidal multiple. The bactericidal multiple calculation formula is: bactericidal multiple = survival rate of SCF group / survival rate of SCF+valine group.

[0080] Results Figure 2 A. As can be seen from the figure, after adding valine, as the concentration of cefoperazone sodium and sulbactam sodium increases, the bactericidal efficiency against clinical bacteria increases significantly. The specific situation is:

[0081] On the basis of 5mM valine, the addition of low concentrations of cefoperazone sodium and sulbactam sodium (10 and 25μg / mL) improved the bactericidal efficiency (the bactericidal multiples were 1.11 and 1.42 times, respectively), but the degree of improvement was small; when the concentration of cefoperazone sodium and sulbactam sodium was 50-400μg / mL, the bactericidal efficiency of clinical bacteria increased significantly with the increase of antibiotic concentration, and the bactericidal multiples increased by 8.93 times, 105.53 times, 259.41 times and 388.82 times, respectively (the survival rate decreased from 94.42%, 84.42%, 70.04% and 42.77% without valine to 10.57%, 0.80%, 0.27% and 0.11% after adding valine, respectively).

[0082] 3.2 Valine improves the sensitivity of clinical bacteria to antibiotics with a valine concentration gradient effect

[0083] According to Example 1, MRSA 2 bacterial samples were prepared. The bacterial samples were divided into an M9 control group and an experimental group. The experimental group was treated with different concentrations of valine (0.1-20 mM) alone, and the corresponding concentrations of valine + 200 μg / mL SCF. Three biological replicates were performed. The cells were cultured at 37°C and 200 rpm for 10 hours, and the viable bacteria were counted using a plate method. The survival rate of the bacteria after adding SCF and not adding SCF was then calculated based on the addition of different concentrations of valine. The calculation formula was: survival rate (%) = the number of bacteria after adding valine or SCF + valine / the number of M9 control bacteria × 100%. Valine synergistically improves the bactericidal efficiency of cefoperazone sodium and sulbactam sodium, and is evaluated based on the bactericidal multiple. The bactericidal multiple calculation formula is: bactericidal multiple = survival rate of the valine group / survival rate of the antibiotic + valine group.

[0084] The results are as follows Figure 2 As shown in B, after adding valine to the clinical bacteria on the basis of adding cefoperazone sodium and sulbactam sodium, as the concentration of valine increases, its bactericidal efficiency against clinical bacteria increases more significantly, and remains unchanged after reaching 5mM. The specific results are described as follows:

[0085] When valine was added alone at any concentration, bacterial survival remained basically unchanged. On the basis of adding SCF, when the concentration of valine was 0.1 and 0.5 mM, the bactericidal efficiency was improved, but the degree of improvement was small, and the bactericidal multiples were 1.10 and 1.14 times respectively; when the concentration of valine increased from 1 mM to 5 mM, the bactericidal efficiency increased by 4.07 times, 63.59 times and 382.59 times respectively (the survival rate decreased from 87.46%, 101.11%, 103.3% to 21.51%, 1.59%, 0.27%), and when the concentration of valine reached 10 mM and above, its bactericidal multiple no longer increased.

[0086] 3.3 Valine has a time effect in improving the sensitivity of clinical bacteria to antibiotics

[0087] MRSA 2 bacterial samples were prepared according to Example 1, and the bacterial samples were divided into 4 groups: M9 control group, 5mM valine group, 200μg / mL SCF group and valine+SCF synergistic group. 3 biological replicates. Cultured at 37°C and 200rpm, samples were taken every 2 hours, and viable bacteria were counted by plate method. The survival rate of bacteria in each of the 4 groups was calculated at each time point, and the calculation formula was: survival rate (%) = number of bacteria in each group at different time points / initial number of bacteria × 100%. The synergistic improvement of the bactericidal efficiency of cefoperazone sodium and sulbactam sodium at different time points was evaluated according to the bactericidal multiples at different time points in each group. The calculation formula for the bactericidal multiples at the same time point is: bactericidal multiples = survival rate of the SCF group at the same time point / survival rate of the SCF+valine group.

[0088] The results are as follows Figure 2 As shown in C, after adding valine to the clinical bacteria on the basis of adding cefoperazone sodium and sulbactam sodium, the bactericidal efficiency of clinical bacteria increased significantly as time prolonged. The specific results are described as follows: MRSA2 at 2 hours, valine did not increase the bactericidal efficiency of cefoperazone sodium and sulbactam sodium; when the bactericidal time increased to 4 hours, the bactericidal efficiency began to appear, and the bactericidal multiple increased by 1.85 times; when the bactericidal time increased from 6 hours to 10 hours, the bactericidal efficiency was significantly improved, and the bactericidal multiple increased from 33.88 times to 287.54 times (the survival rate decreased from 80.64% and 80.51% to 2.38% and 0.28%, respectively). When the bactericidal time was extended to 12 hours or even 24 hours, the bactericidal efficiency remained unchanged at first, and then gradually decreased.

[0089] Example 4: Valine increases the sensitivity of various bacteria to cefoperazone sodium and sulbactam sodium

[0090] 4.1 It is common that valine increases the sensitivity of MRSA to cefoperazone sodium and sulbactam sodium in clinical practice

[0091] According to Example 1, 18 clinical MRSA bacterial samples were prepared, and each strain was divided into 4 groups: M9 control group, single antibiotic SCF group, single valine group, and antibiotic SCF + valine group. Valine was 5mM, and the concentrations of cefoperazone sodium and sulbactam sodium were: 300μg / mL for MRSA 11, 400μg / mL for MRSA13 and 19, and 200μg / mL for other bacteria. After 10h of action, the number of viable bacteria was counted, and the plate method was used for viable bacteria counting. Then the survival rate of bacteria after adding antibiotics or / and valine treatment was calculated, and the calculation formula was: survival rate (%) = number of bacteria after adding or not adding antibiotics or / and valine treatment / number of M9 control bacteria × 100%. 3 biological replicates.

[0092] The results are as follows Figure 3 As shown in A, valine can increase the sensitivity of all clinical MRSA bacteria to cefoperazone sodium and sulbactam sodium. The degree of increased sensitivity varies, ranging from 9 to 250 times.

[0093] 4.2 Valine can increase the sensitivity of other Gram-positive bacteria to cefoperazone sodium and sulbactam sodium

[0094] Gram-positive bacterial samples such as Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecium were prepared according to Example 1, and each strain was divided into 3 groups: M9 control group, antibiotic SCF group, antibiotic SCF + valine synergistic group. Valine was 5mM, and the concentrations of cefoperazone sodium and sulbactam sodium were: 50μg / mL for Streptococcus agalactiae, 400μg / mL for Streptococcus pyogenes, 200μg / mL for Bacillus subtilis, and 400μg / mL for Enterococcus faecium. After 10 hours of action, the number of live bacteria was counted, and the plate method was used for live bacteria counting. Then the survival rate of bacteria after treatment in each group was calculated, and the calculation formula was: survival rate (%) = the number of bacteria after treatment with or without antibiotics or / and valine / the number of M9 control bacteria × 100%. 3 biological replicates.

[0095] Results Figure 3 B. As can be seen from the figure, valine can increase the sensitivity of Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecium to cefoperazone sodium and sulbactam sodium. The degree of increased sensitivity varies, ranging from 5 to 7 times.

[0096] 4.3 Valine can increase the sensitivity of other Gram-negative bacteria to cefoperazone sodium and sulbactam sodium

[0097] According to Example 1, Gram-negative bacteria samples (clinically resistant Pseudomonas aeruginosa (PA) strains No. 1, No. 2, No. 204 and No. 205, clinically resistant Acinetobacter baumannii (Aba) strains No. 25, No. 48, No. 252 and No. 303, clinically resistant Klebsiella pneumoniae (Kpn) strains No. 10, No. 18, No. 116 and No. 118, clinically resistant Escherichia coli (Eco) strains No. 56, No. 57, No. 116 and No. 117) were prepared (the drug resistance information of the above strains is shown in Tables 2 to 5. It can be seen from the table that the above listed The bacteria were resistant to most of the antibiotics tested and were multi-drug resistant. Each strain was divided into 3 groups: M9 control group, antibiotics alone group, antibiotics + valine group. Valine was 5mM, and the antibiotic concentration was 400μg / mL. The number of live bacteria was counted after 10 hours of action, and the plate method was used to count the live bacteria. Then the survival rate of bacteria after treatment in each group was calculated, and the calculation formula was: survival rate (%) = number of bacteria after treatment with or without antibiotics and / or valine / number of M9 control bacteria × 100%. 3 biological replicates.

[0098] Results Figure 3 C. As can be seen from the figure, except for clinically resistant Escherichia coli, valine can increase the sensitivity of other bacteria to cefoperazone sodium and sulbactam sodium. The degree of increased sensitivity varies, ranging from 2 to 13 times.

[0099] Table 2 Drug resistance information of clinical Klebsiella pneumoniae

[0100]

[0101]

[0102] Note: The mass ratio of piperacillin to tazobactam in piperacillin sodium and tazobactam sodium is 8:1. Unless otherwise specified, this ratio applies to both of them. The mass ratio of ceftazidime to avibactam in ceftazidime avibactam sodium is usually 4:1. Unless otherwise specified, this ratio applies to both of them.

[0103] Table 3 Drug resistance information of clinical Escherichia coli

[0104]

[0105]

[0106] Table 4 Drug resistance information of clinical Pseudomonas aeruginosa

[0107]

[0108] Note: X means not detected; the mass ratio of ticarcillin to clavulanic acid in ticarcillin-clavulanate potassium is 15:1.

[0109] Table 5 Drug resistance information of clinical Acinetobacter baumannii

[0110]

[0111]

[0112] Note: X means not detected; the mass ratio of ampicillin to sulbactam in ampicillin sodium and sulbactam sodium is 2:1.

[0113] Example 5: Valine can delay the aftereffect of cefoperazone sodium and sulbactam sodium on clinical methicillin-resistant Staphylococcus aureus

[0114] Using Staphylococcus aureus as a representative of Gram-positive bacteria MRSA 2, the effect of valine combined with cefoperazone sodium and sulbactam sodium was further explored.

[0115] Pick a single bacterial colony into a 250 mL conical flask containing 50 mL LB, culture it overnight at 37 °C and 200 rpm, then transfer the bacteria to a 5 mL LB tube at a ratio of 1:100. When the bacteria grow to an OD of 600When the pH is 0.2, collect the bacteria by centrifugation and wash the bacteria three times with physiological saline. Resuspend the bacteria with 5mL MHB, take 500μL of the above bacterial solution and add it to a test tube containing 4.5mL MHB. Each strain is divided into 7 groups: MHB control group, 1 / 2MIC SCF and 1 / 2MIC SCF+5mM valine, 1MIC SCF and 1MIC SCF+5mM valine, 2MIC SCF and 2MIC SCF+5mM valine. There are 3 biological replicates for each treatment. Incubate at 37℃200rpm for 2 hours. Take 100μL of the above bacterial solution and dilute it 1000 times, then take 100μL and add it to a 4.9mL MHB test tube. Incubate the diluted bacterial solution at 37℃ and 200rpm, and take 100μL of the bacterial solution at 0, 1, 2, 4, 6, 8, and 12 hours respectively. After dilution, use a plate to detect the number of viable bacteria. All strains have 3 biological replicates. Then, the bacterial growth curve was constructed by taking the time point as the horizontal axis and the average value of the logarithm of the corresponding colony number as the vertical axis.

[0116] PAE was then calculated from the bacterial growth curve using the following formula: PAE = TC, where T is the time required for the viable bacterial count in the test culture to increase by 1 Log10 CFU from the count observed immediately after dilution, and C is the corresponding time for the control group not exposed to the antibiotic. For this test, PAE (i.e., PAE) was calculated when SCF was added alone. SCF ), PAE with SCF+valine (PAE SCF+缬氨酸 ), by comparing PAE SCF and PAE SCF+缬氨酸 , to determine whether the exogenous addition of valine would prolong the PAE of the antibiotic SCF. PAE is the post-antibiotic effect, which refers to the effect that after the bacteria are exposed to antibiotics, although the serum concentration of antibiotics drops below the minimum inhibitory concentration (MIC) or disappears, the inhibitory effect on microorganisms still persists for a period of time.

[0117] The growth curve results were plotted as Figure 4. Through further calculation, the PAE of the strain after treatment with cefoperazone sodium sulbactam sodium alone, cefoperazone sodium sulbactam sodium + valine was obtained under the three conditions of 1 / 2MIC, 1MIC and 2MIC. The results are summarized in Table 6. The PAE of the cefoperazone sodium sulbactam sodium antibiotic group, i.e., the SCF group, in the table is the result of the comparison between the addition of antibiotics and the control group; the PAE of cefoperazone sodium sulbactam sodium + valine, i.e., SCF + valine, in the table is the result of the comparison between the addition of cefoperazone sodium sulbactam sodium + valine and the addition of antibiotics alone. It can be seen from the table that the PAE of cefoperazone sodium sulbactam sodium at 1 / 2MIC, 1MIC and 2MIC were 1.36, 1.6 and 1.94 hours, respectively. The PAE of SCF + valine was prolonged compared with the PAE of the antibiotic group, which was prolonged by 1.69, 2.17 and 2.66 hours, respectively.

[0118] In summary, the addition of valine can not only promote the bactericidal effect of cefoperazone sodium and sulbactam sodium, but also delay its after-effects, thereby prolonging the bactericidal effect of cefoperazone sodium and sulbactam sodium.

[0119] Table 6 PAE of valine against MRSA Staphylococcus aureus (hours)

[0120]

[0121] Example 6 In vivo efficacy study of valine in improving the sensitivity of clinical methicillin-resistant Staphylococcus aureus

[0122] Balb / c mice (6-8 weeks, male, about 20 grams) were used as experimental subjects. Through thigh muscle infection experiments and systemic infection experiments (including survival rate experiments and bacterial counts of visceral organs after infection), an in vivo efficacy study on the effect of exogenous valine on improving the sensitivity of clinical methicillin-resistant Staphylococcus aureus to cefoperazone sodium and sulbactam sodium was carried out.

[0123] 6.1 Mouse thigh muscle infection model

[0124] The experiment was conducted with MRSA2 strain. Male balb / c mice were divided into 3 groups: control group, antibiotics alone group (200 mg / kg), antibiotics (200 mg / kg) and valine (200 mg / kg) combined group. There were 4 mice in each group. Each mouse was treated with cyclophosphamide by intraperitoneal injection, resulting in a decrease in neutrophils in the mice. The treatment time and dose were 4 days and 1 day before infection, respectively, and the doses were 150 mg / kg and 100 mg / kg, respectively.

[0125] Each mouse was infected with MRSA2 strain cultured overnight for 16 hours. The infection dose was 2.8×10 5CFU was injected intramuscularly into the right thigh of mice. Two hours after infection, each mouse was given the drug by tail vein injection according to the above grouping, and the control group was injected with the same volume of normal saline. 24 hours after administration, the mice were euthanized by CO2 inhalation.

[0126] Then we observed the muscle condition of the bacterial infection site on the right thigh of the mouse and took photos. Figure 5 As shown in A, it can be seen from the figure that the infected part of the control group was severely ulcerated, the infected part of the antibiotic treatment group was significantly healed compared with the control group, and there was basically no ulceration in the antibiotic plus valine group.

[0127] Then, 50 mg of the right thigh muscle of each mouse was removed under sterile conditions, added with 500 μl of normal saline, homogenized, serially diluted, and counted on an LB plate. Figure 5 As shown in B, the bactericidal efficiency of the leg muscles of mice administered with valine in combination with cefoperazone sodium and sulbactam sodium increased by nearly 2 orders of magnitude (i.e., 100 times) compared with the administration of antibiotics alone.

[0128] 6.2 Valine synergizes with cefoperazone sodium and sulbactam sodium to improve the survival rate of mice infected with methicillin-resistant Staphylococcus aureus

[0129] Balb / c mice (6-8 weeks, about 20 g, half male and half female) were divided into 4 groups, 10 mice in each group, and treated with different concentrations of cefoperazone sodium and sulbactam sodium antibiotics. The challenge strain was methicillin-resistant MRSA 12, and the infection dose was 1.0×10 8 CFU, by intraperitoneal injection. After 1 hour of bacterial infection in each mouse, cefoperazone sodium and sulbactam sodium were administered by tail vein injection, with doses of 50 mg / kg, 100 mg / kg, 200 mg / kg and 400 mg / kg, respectively. The death of mice was observed and recorded daily, and the survival rate of each group was calculated every day for a total of 7 days. The calculation formula is: survival rate (%) = number of surviving mice in each group / total number of mice in each group × 100%.

[0130] The experimental results are shown in Figure 6 A, as shown in the figure, after mice were infected with Staphylococcus aureus, mice treated with 4 doses of antibiotics all died to varying degrees on the first day, and the highest dose, 400 mg / kg, had the lowest mortality rate of 40%. However, on the second day, except for the mice with this dose, which still had a survival rate of 10%, all the mice with the other doses died.

[0131] Furthermore, a treatment experiment of Staphylococcus aureus infection was conducted by adding valine exogenously on the basis of adding 200 mg / kg of antibiotics. Balb / c mice (6-8 weeks, about 20 grams, half male and half female) were divided into 5 groups, 10 mice in each group, including antibiotic control group (dose of 200 mg / kg), antibiotic + different doses of valine test group. The doses of valine were 50 mg / kg, 100 mg / kg, 200 mg / kg and 400 mg / kg respectively. The challenge strain was methicillin-resistant MRSA 12, and the infection dose was 1.0×10 8 CFU, by intraperitoneal injection. One hour after bacterial infection of mice, antibiotics were administered by tail vein injection; valine was injected by intraperitoneal injection. The death of mice was observed and recorded daily, and the survival rate of each group was calculated every day for a total of 7 days. The calculation formula was: survival rate (%) = number of surviving mice in each group / total number of mice in each group × 100%.

[0132] The experimental results are shown in Figure 6 B. As can be seen from the figure, in the antibiotic control group, 50% of the mice died on the first day, and all died on the second day; in the valine synergistic antibiotic test group, 30% of the mice died on the first day and 50% died on the second day at four doses. There was a difference on the third day. The mice at 200mg / kg and 400mg / kg doses of valine tended to be stable, and no mice died until the 7th day, with a survival rate of 50%. The mice at 100mg / kg dose of valine died 70%, and then tended to be stable, no longer died, and the survival rate was 30%. The mice at 50mg / kg dose of valine died 80%, and then tended to be stable, no longer died, and the survival rate was 20%.

[0133] The results showed that when mice were infected with methicillin-resistant bacteria, all mice died when only antibiotics were used for treatment, i.e., antibiotics were ineffective, but as long as valine was added exogenously, the mice's resistance to methicillin-resistant bacteria infection was improved. Moreover, the resistance to infection was dose-dependent on valine, and the higher the dose, the higher the resistance of mice to methicillin-resistant bacteria infection.

[0134] 6.3 Valine synergizes with cefoperazone sodium and sulbactam sodium to improve the clearance of clinical Staphylococcus aureus infection in mice

[0135] Balb / c mice (6-8 weeks old, about 20 g, half male and half female) were divided into 4 groups, 6 mice in each group, namely, saline control group, valine control group, antibiotic treatment group, and antibiotic + valine test group. The challenge strain was methicillin-resistant Staphylococcus aureus MRSA 12, and the infection dose was 1.2×10 6CFU was measured by intraperitoneal injection. One hour after bacterial infection, mice in the antibiotic treatment group were intravenously injected with cefoperazone sodium and sulbactam sodium at a dose of 200 mg / kg; mice in the valine group were intraperitoneally injected with valine at a dose of 200 mg / kg; mice in the antibiotic + valine test group were treated with antibiotics (all at a dose of 200 mg / kg) and valine (doses of 50, 100, 200 and 400 mg / kg, respectively) according to their respective injection methods. After 6 hours, equal weight of viscera (liver, spleen, kidney) were taken and fully ground, then diluted and plated, and the number of counts was counted.

[0136] Results Figure 6 C. As can be seen from the figure, when mice are infected with drug-resistant bacteria, if valine is given, the bacterial load of the mouse spleen, kidney and liver remains basically unchanged. If antibiotics are given, the bacterial load of the infected mouse liver, spleen and kidney decreases by 3.82 times, 62.18 times and 6.92 times, respectively; if valine and antibiotics are given, the bacterial load of the mouse liver, spleen and kidney decreases by 364.52 times, 3628.25 times and 724.74 times, respectively. Compared with the antibiotic treatment group, the bacterial load of the liver, spleen and kidney of mice treated with valine and antibiotics decreased by 95.33 times, 58.35 times and 104.74 times, respectively. This result shows that the combination of valine and antibiotics can significantly improve the clearance of clinical Staphylococcus aureus infection in mice.

[0137] Example 7 Mechanism of valine in improving the sensitivity of methicillin-resistant Staphylococcus aureus to cefoperazone sodium and sulbactam sodium

[0138] To study how valine improves the antibiotic sensitivity of clinical methicillin-resistant Staphylococcus aureus, the following experiment was conducted.

[0139] 7.1 Exogenous valine activates the bacterial pyruvate cycle, increases bacterial NADH, and thus enables the bacteria to produce more proton motive force.

[0140] Several studies have shown that exogenous addition of reduced metabolites after bacterial resistance will promote the pyruvate cycle of resistant bacteria, thereby increasing the sensitivity of resistant bacteria to antibiotics. If the pyruvate cycle is enhanced, the activity of important enzymes in the cycle, such as pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), will increase, and more NADH, ATP and proton motive force will also increase. To this end, MSSA 5 and MRSA 2 were cultured overnight, and after collecting these two bacteria, MRSA 2 was treated with 5mM valine for 10 hours, while MSSA 5 and MRSA 2 were treated with only M9 as controls. Then the following were measured:

[0141] Key enzyme activities of bacterial pyruvate cycle: Take 30 mL of the above-prepared bacterial sample and centrifuge at 8000 rpm for 5 min. Resuspend the cells in PBS, ultrasonically disrupt for 15 minutes (650 W total power, 35% output, pulse for 2 seconds, pause for 3 seconds, the whole process is carried out on ice water), and then centrifuge at 12,000 rpm for 10 minutes. The protein concentration was determined using the BCA protein assay kit (Beyotime Biotechnology, P0009). When measuring PDH and KGDH, 0.15 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrabromoazole (MTT), 2.5 mM MgCl2, 6.5 mM methoxysulfate phenazine (PMS), 0.2 mM thiamine PPi (TPP) and 80 mM sodium pyruvate / α-ketoglutarate potassium salt were added to the reaction solution, and the amount of water added was 200 μL. For SDH and MDH assays, 0.15 mM MTT, 2.5 mM MgCl2, 13 mM PMS, 80 mM sodium succinate / malate were added to the reaction solution, and the amount of water added was 200 μL. All reaction mixtures were incubated at 37 °C, MDH / PDH / KGDH for 5 minutes, and SDH for 10 minutes. Finally, colorimetric measurements were performed at 562 nm. All reactions were protected from light. At least three biological replicates were performed.

[0142] Results Figure 7 A. As can be seen from the figure, the enzyme activities of PDH, α-KGDH, SDH and MDH of MRSA2 decreased. However, after the addition of valine, the enzyme activities of PDH, α-KGDH, SDH and MDH increased significantly, approaching the enzyme activities of these corresponding enzymes of MSSA5. This shows that after the bacteria become resistant, the pyruvate cycle decreases, and the activities of these important enzymes decrease. After the exogenous addition of valine, the pyruvate cycle is activated, thereby increasing the activities of these important enzymes.

[0143] NADH: EnzyChrom TM NADH detection kit (BioAssay Systems) was used for detection. After collecting bacteria and adjusting OD600 to 1.0, 2 mL of bacteria were centrifuged and washed three times with sterile saline. Resuspended in NADH extraction buffer for 15 seconds, and then incubated in 60°C water for 5 minutes. Add assay buffer and back extraction buffer, vortex for 10 seconds, centrifuge at 12000×g for 5 minutes, and take the supernatant for determination.

[0144] Results Figure 7 B. As can be seen from the figure, NADH decreases after bacterial resistance, while NADH increases after exogenous addition of valine.

[0145] ATP: Using BacTiter-Glo TMMicrobial Cell Viability Assay (Promega, USA) was used to measure the viability of microbial cells. Bacteria were collected and resuspended in sterile saline. The sample was diluted to a bacterial count of 10 7 CFU / mL, 50 μL was dropped into a 96-well plate. The sample was then mixed with 50 μL of pre-equilibrated BacTiter-Glo™ reagent (Promega, USA) for 5 min, and luminescence was measured using a Victor X5 multimode plate reader.

[0146] Results Figure 7 C. As can be seen from the figure, ATP decreases after bacterial resistance, while ATP increases after exogenous addition of valine.

[0147] Proton motive force: 8 strains of MSSA (1-8) and 8 strains of MRSA (1-8) were selected from clinical strains for the experiment. Bacterial samples were prepared according to Example 1. Each MRSA strain was treated with 5 mM valine for 10 hours, while MSSA and MRSA were treated with only M9 as controls. The treated bacteria were then adjusted to a concentration of 10 6 CFU / mL, 1 mL was transferred to a 1.5 mL EP tube, 10 μL 3 mM DiOC2 (3,3'-diethyloxa-carbocyanine iodide) was added, the tube was thoroughly mixed and incubated at 37°C for 30 min. Before loading, the tube was transferred to a flow cytometry analysis tube and detected using a flow cytometer FACSCalibur flowcytometer (Becton Dickinson, San Jose, CA, USA). The parameters were set according to the instrument operating procedures. The green fluorescence excitation wavelength of the dye DiOC2 (3) was 488 nm and the emission wavelength was 530 nm. The red fluorescence excitation wavelength was 488 nm and the emission wavelength was 610 nm. The ratio of red light intensity to green light intensity represents the strength of membrane potential. The proton motive force (PMF) value was calculated using the formula LOG (10 3 / 2×Y mean / X mean), where Y mean and X mean represent the red light intensity and green light intensity, respectively. The determination was performed according to the instructions of the BacLight bacterial membrane potential kit (Invitrogen).

[0148] The results are as follows Figure 7 As shown in Figure D, it can be seen that the proton motive force of MRSA is significantly reduced compared with MSSA. After adding valine, the proton motive force of MRSA is significantly increased.

[0149] CCCP is a membrane potential disruptor that can inhibit the generation of proton motive force. To confirm whether exogenous valine increases the sensitivity of bacteria to antibiotics by promoting the generation of proton motive force, MRSA2 bacterial samples were prepared and divided into 8 groups: 2 control groups (valine alone, SCF alone), 6 antibiotics + valine groups (gradient concentrations of CCCP: 0, 0.01, 0.05, 0.1, 0.5, 1 μM), and OD 600 The bacterial solution was diluted to 0.2 in M9 medium and incubated on a shaker at 37°C and 200 rpm for 10 h before counting on a dilution plate.

[0150] Results Figure 7 E. As can be seen from the figure, the bacterial growth was not affected by the addition of valine or SCF alone, while the bacterial survival rate in the antibiotic + valine group decreased significantly. However, after adding different concentrations of CCCP, the bactericidal effect of valine synergistically with cefoperazone and sulbactam gradually weakened and was negatively correlated with the CCCP concentration, indicating that the entry of antibiotics may depend on the proton motive force.

[0151] The proton motive force measurement and the experiment on the effect of adding CCCP on bacterial survival rate showed that the addition of valine promoted the generation of bacterial proton motive force, thereby increasing the sensitivity of bacteria to antibiotics.

[0152] 7.2 Exogenous valine increases the permeability of bacterial cell membranes by promoting the abundance of lauric acid;

[0153] Studies have shown that lauric acid is related to membrane permeability. To this end, MRSA 2 was used as a control and the abundance of lauric acid in MRSA 2 was determined by GC / MS. The results showed that compared with sensitive bacteria, the abundance of lauric acid in MRSA 2, i.e., the drug-resistant bacteria, was significantly reduced. However, after MRSA2 was treated with valine, its abundance of lauric acid increased ( Figure 8 A).

[0154] Then, the sensitivity test of antibiotics to resistant bacteria with exogenous addition of lauric acid was carried out. The exogenous addition of lauric acid was carried out in the same way as the above-mentioned addition of valine. The results are shown in Figure 8 B. As can be seen from the figure, after adding lauric acid exogenously to SCF, the sensitivity of MRSA2 to antibiotics increased significantly.

[0155] Then, the permeability of the MRSA bacterial cell membrane was measured. Bacteria were prepared according to Example 1, with 9 strains of MSSA (1-9) and 10 strains of MRSA (1-9, 11). Incubated on a shaker at 37°C and 200rpm for 10 hours. Then 100μL was added to 900μL M9 and 2μL 2.5mM SYTO9 dye was added. Incubated at 37°C and 200rpm for 45 minutes, fluorescence was detected by flow cytometry, and changes in membrane permeability were determined by comparing fluorescence intensity. The results are shown in Figure 8 C. As can be seen from the figure, the permeability of MRSA cell membrane is lower than that of MSSA, which means that the permeability of bacterial resistant cell membrane is reduced.

[0156] Finally, MRSA2 bacterial samples were prepared according to Example 1. The bacteria were then divided into 3 groups, one group was a control group, one group was a test group with valine (5mM) added, and one group was a test group with lauric acid (5mM) added. The cells were incubated on a shaker at 37°C and 200rpm for 10 hours. Then 100 μL was taken into 900 μL M9 and 2 μL 2.5mM SYTO9 dye was added. The cells were incubated at 37°C and 200rpm for 45 minutes, and fluorescence was detected by flow cytometry. The changes in membrane permeability were determined by comparing the fluorescence intensity of the test group with valine or lauric acid added with that of the control group.

[0157] Results Figure 8 D and 8E. As can be seen from the figure, whether adding lauric acid ( Figure 8 D) or add valine ( Figure 8 E), the membrane permeability of MRSA2 was enhanced, indicating that valine and lauric acid increase the sensitivity of bacteria to antibiotics by increasing the membrane permeability of bacteria.

[0158] 7.3 Increased proton motive force (PMF) and membrane permeability increase the amount of antibiotics entering the cell

[0159] Bacterial death is related to the amount of antibiotics that enter the bacteria, and bacterial resistance is due to the fact that the concentration of antibiotics that enter the bacteria is lower than the concentration that causes their death. In order to study whether valine improves the antibiotic sensitivity of clinical methicillin-resistant Staphylococcus aureus by increasing the concentration of antibiotics that enter the bacteria. The present invention prepared bacterial samples according to Example 1, and the experiment was divided into 2 groups: a control group (only cefoperazone sodium and sulbactam sodium were added) and an experimental group (cefoperazone sodium and sulbactam sodium were added) and incubated in a shaking incubator at 37°C and 200 rpm for 10 hours. The cultured bacteria were washed and resuspended in physiological saline, and the OD was adjusted. 600The value was set to 1.0, 1 mL of bacteria was ultrasonically broken, and the supernatant was taken after centrifugation for the determination of antibiotic content. The cefoperazone ELISA rapid diagnostic kit (purchased from Shenzhen Green Poetry Source Biotechnology Co., Ltd.) was used for detection. The specific steps were as follows: 50 μL of sample (or standard) and 100 μL of cefoperazone capture protein were added to a 96-well plate, gently mixed and incubated at 25°C in the dark for 30 minutes, then 250 μL of washing solution was added to wash the plate 3 times, and the washing solution was completely dried for the last time; 150 μL of antibody 2 was added, gently mixed and incubated at 25°C in the dark for 30 minutes, and the plate was washed 3 times (the method is the same as above); 100 μL of reaction solution TMB was added, gently mixed and incubated at 25°C in the dark for 10-15 minutes; finally, 100 μL of reaction stop solution was added to terminate the reaction, and the absorbance of the reaction product was determined by setting the microplate reader at 450 nm. The standard curve was drawn with the percent absorbance of the standard as the ordinate and the semi-logarithm of the concentration of the cefoperazone standard (ng / mL) as the abscissa. The actual concentration of cefoperazone = sample detection concentration × dilution factor.

[0160] The results are as follows Fig. 9 As shown in the figure, the amount of antibiotics in the bacteria in the experimental group with the addition of cefoperazone sodium sulbactam sodium and valine increased by 3.18 times compared with the control group with only cefoperazone sodium sulbactam sodium. If the proton motive force inhibitor CCCP is added, the intracellular antibiotic content will decrease significantly. The results show that valine can indeed increase the number of antibiotics entering the bacteria, and it is related to the generation of proton motive force. The above results show that exogenous valine increases the sensitivity of bacteria to antibiotics by promoting the pyruvate cycle and generating more proton motive force; at the same time, exogenous valine promotes the increase in the abundance of bacterial lauric acid, thereby increasing the permeability of bacterial membranes. The combined effect of the two increases the concentration of antibiotic drugs entering the bacterial cells, thereby increasing the sensitivity of bacteria to antibiotics.

[0161] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The use of valine in the preparation of an antibiotic synergist, characterized in that: The antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or a pharmaceutically acceptable salt thereof.

2. The application according to claim 1, characterized in that: The valine increases the sensitivity of bacteria to antibiotics, and the bacteria are Gram-positive bacteria and Gram-negative bacteria, wherein the Gram-negative bacteria do not include Escherichia coli.

3. The use of valine in combination with antibiotics in the preparation of a drug for increasing the sensitivity of bacteria to antibiotics, characterized in that: The antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or pharmaceutically acceptable salts thereof; the bacteria are Gram-positive bacteria and Gram-negative bacteria, wherein Gram-negative bacteria do not include Escherichia coli.

4. Use of valine in combination with an antibiotic in the preparation of a medicament for preventing and / or treating bacterial infection, characterized in that: The antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or pharmaceutically acceptable salts thereof; the bacteria are Gram-positive bacteria and Gram-negative bacteria, wherein Gram-negative bacteria do not include Escherichia coli.

5. The use according to any one of claims 2 to 4, characterized in that: The Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecium.

6. The use according to any one of claims 2 to 4, characterized in that: The Gram-negative bacteria include one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

7. A drug for increasing the sensitivity of bacteria to antibiotics, characterized in that: It contains an effective amount of valine and an antibiotic; the antibiotic is selected from cefoperazone sulbactam, ceftriaxone, ceftazidime, amoxicillin-clavulanic acid, moxidectin, levofloxacin, erythromycin, roxithromycin, amikacin or a pharmaceutically acceptable salt thereof; the bacteria are gram-positive bacteria and gram-negative bacteria, wherein gram-negative bacteria do not include Escherichia coli.

8. The drug according to claim 7, characterized in that: The Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecium.

9. The drug according to claim 7, characterized in that: The Gram-negative bacteria include one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

10. The drug according to any one of claims 7 to 9, characterized in that: The mixing ratio of valine to antibiotic is 1: (0.5-4000) mol / g.

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