Use of pyruvate in the preparation of a formulation for increasing the sensitivity of pathogenic bacteria to antibiotics

Abstract

The application belongs to the technical field of medicine, and discloses application of pyruvic acid in preparation of a preparation for improving sensitivity of pathogenic bacteria to antibiotics. Researches of the application show that pyruvic acid can significantly improve the sensitivity of pathogenic bacteria to antibiotics, and pyruvic acid combined with antibiotics can be used to prepare a medicine for treating pathogenic bacterial infection. The medicine has good antibacterial, anti-drug-resistant bacterial and anti-infection effects on pathogenic bacteria such as pseudomonas aeruginosa, acinetobacter baumannii and parahemolytic vibrio, especially drug-resistant bacteria. The medicine has good application value and significance in the production and development of antibacterial drugs and anti-infection drugs in combination with antibiotics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of pyruvic acid in preparation of a preparation for improving sensitivity of pathogenic bacteria to antibiotics. BACKGROUND

[0002] Clinical pathogenic bacteria seriously endanger the health of hospital patients and the healthy development of the animal husbandry industry. Pseudomonas aeruginosa is one of the main pathogenic bacteria of nosocomial infections, and Pseudomonas aeruginosa infection accounts for about 10%-19% of hospital infections, or even higher. Pseudomonas aeruginosa infection can occur in any part or tissue of the human body and can cause respiratory tract infection, blood stream infection, skin infection, central nervous system infection, bone and joint infection, etc. The diseases caused by infection in different parts are different, and the mortality is different: respiratory tract infection can cause pneumonia; blood stream infection has high mortality, can have high fever, and is often accompanied by shock, acute respiratory distress syndrome or disseminated intravascular coagulation; central nervous system infection has a poor prognosis, and the mortality is about 2 / 3 or more. Acinetobacter baumannii usually causes skin, respiratory tract or urinary tract infection, causing symptoms such as skin redness and pus, cough, sputum, shortness of breath, urinary urgency, frequent urination, and urinary pain. If it invades the blood, it can cause severe blood stream infection and can invade the intracranial, causing diseases such as sepsis and meningitis, and showing symptoms such as high fever, chills, stiff neck, projectile vomiting, and even limb dysfunction. Vibrio parahaemolyticus can cause food poisoning and has hemolytic activity, enterotoxin and lethal effect.

[0003] With the extensive use and unreasonable use of antibiotics, the drug resistance of clinical pathogenic bacteria gradually increases and the drug resistance range gradually expands. Acinetobacter baumannii has high drug resistance, so it is difficult to treat patients with severe infection in the clinic, has high mortality, and has a poor prognosis. Pseudomonas aeruginosa is one of the most common multidrug-resistant and pan-resistant pathogenic bacteria in the clinic, and the drug resistance rate has been high for a long time. Over time, drug-resistant bacteria gradually replace sensitive bacteria to become dominant flora, and the massive reproduction of drug-resistant bacteria can further enhance the drug resistance of drug-resistant bacteria. After the clinical pathogenic bacteria develop drug resistance, most clinicians use increased antibiotic doses or combined antibiotic therapy to kill drug-resistant bacteria, which undoubtedly increases the amount or types of antibiotics used, further enhances bacterial drug resistance, or further expands the drug resistance range, which leads to the generation of multidrug-resistant bacteria or pan-resistant bacteria, and forms a vicious cycle, eventually leading to the generation of pan-resistant bacteria, and the situation that there is no available antibiotic, which brings serious challenges to infection treatment. Therefore, it is crucial to develop new technical methods to control clinical pathogenic bacteria and drug-resistant bacteria infection.

[0004] It is known that there is a general dose-dependent relationship between the level of drug resistance of clinical pathogenic bacteria and the use of antibiotics. The irrational use of antibiotics is an important inducement for the generation of multi-drug resistant bacteria, and is an important problem and urgent scientific problem faced by health departments around the world. Therefore, at the G20 Summit in Hangzhou, China in 2016, the problem of antibiotic resistance was listed as one of the themes of the conference, and at the same time, China issued the “China Bacterial Resistance Containment Action Plan (2016-2020) ”, with the joint efforts of 14 departments at the national level to effectively contain bacterial resistance. The speed of the generation of drug-resistant bacteria accelerates with the development and update of antibiotics, and the corresponding drug-resistant strains are generated soon after the new antibiotics are put into use. In the face of the long development cycle and high development cost of antibiotics, the development speed of new antibiotics lags behind the generation speed of drug-resistant bacteria, which brings great pressure to the clinical treatment of patients infected with pathogenic bacteria and the prevention of bacterial infection.

[0005] Therefore, the development of new antibiotics and antibacterial methods has very important practical significance for the prevention and treatment of pathogenic bacterial infections, the protection of human health and the health of ecological environment in the breeding industry. SUMMARY

[0006] The technical problem to be solved by the present application is to provide pyruvic acid as a small molecule substance for enhancing the bactericidal effect of antibiotics, so as to prevent and treat Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus bacterial infections, and to be used for the preparation of anti-infection drugs.

[0007] The present application provides the use of pyruvic acid in the preparation of a drug for preventing or treating pathogenic bacterial infections.

[0008] Another object of the present application is to provide a drug for preventing or treating infections containing pyruvic acid.

[0009] The present application achieves the above-mentioned application purposes through the following technical solutions:

[0010] After incubating drug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus with exogenous pyruvic acid, the research results show that pyruvic acid can improve the sensitivity of drug-resistant bacteria to antibiotics and can improve the bactericidal efficiency of antibiotics on drug-resistant bacteria. Therefore, the present application provides the following application schemes:

[0011] The use of pyruvic acid in the preparation of a drug for preventing or treating pathogenic bacterial infections.

[0012] The use of pyruvic acid in the preparation of a drug for preventing or treating pathogenic bacterial infections.

[0013] The pyruvic acid can improve the sensitivity of pathogenic bacteria to antibiotics, and can be used for combined preparation of antibacterial drugs to further enhance the sensitivity of pathogenic bacteria to the antibacterial drugs. When the sensitivity of pathogenic bacteria to the antibacterial drugs is enhanced by using the scheme of the present application, the reference use administration dose range is 10-200 mg / time.

[0014] Preferably, the pathogenic bacteria are Pseudomonas aeruginosa, Acinetobacter baumannii or Vibrio parahaemolyticus.

[0015] In addition, further, the pyruvic acid and the antibiotic are used together in the above scheme.

[0016] Preferably, the mass ratio of pyruvic acid to antibiotic is 1:(0.001-1000).

[0017] Preferably, the mass ratio of pyruvic acid to antibiotic is 1:(0.01-100).

[0018] Specifically, the pyruvic acid is used as an antibiotic enhancer to enhance the ability of the antibiotic to kill pathogenic bacteria. The pyruvic acid is used as an antibiotic enhancer to enhance the ability of the antibiotic to clear pathogenic bacteria in vivo. The pyruvic acid is used as an antibiotic enhancer to promote the ability of a host to clear pathogenic bacteria in vivo after the host is infected with the pathogenic bacteria.

[0019] Preferably, the antibiotic includes but is not limited to lipopeptides, cephem, fluoroquinolones, carbapenems, aminoglycosides.

[0020] More preferably, the antibiotic includes but is not limited to colistin, polymyxin B, ceftazidime, levofloxacin, meropenem, and gentamicin.

[0021] According to the disclosed content, a new antibacterial agent for improving the bacteriostatic or bactericidal effect of an antibacterial drug on drug-resistant pathogenic bacteria can be developed, i.e. a drug for preventing or treating infection, which mainly contains pyruvic acid. Preferably, it also contains the above-mentioned antibiotic.

[0022] Preferably, the mass ratio of pyruvic acid to antibiotic is 1:(0.001-1000).

[0023] More preferably, the mass ratio of pyruvic acid to antibiotic is 1:(0.01-100).

[0024] As an alternative embodiment, the drug is a solid powder or a liquid preparation.

[0025] The technical scheme of the present application has the following beneficial effects:

[0026] The present application provides a kind of drug for treating infection.The present application research shows that, with pyruvic acid preparation treatment of infection drug, pathogenic bacteria such as pseudomonas aeruginosa, baumanii or parahemolytic vibrio, especially drug-resistant bacteria, have good antibacterial, anti-drug-resistant bacteria, anti-infection effect, and have good application value and significance in the production and development of antibacterial drugs, anti-infective drugs.

[0027] Pyruvic acid can improve the sensitivity of pathogenic bacteria to antibiotics, can be combined with colistin and other antibiotics for antibacterial drug preparation, has stronger antibacterial activity, thereby overcoming the difficulty problem of preventing and treating drug-resistant bacterial infection in clinic, while effectively reducing the amount and type of antibiotics used in clinic, reducing the drug resistance rate of pathogenic bacteria to antibiotics due to large amount of antibiotics used in clinic, overcoming the problem of drug residues in ecological environment due to large amount of antibiotics used, promoting the sustainable use of antibacterial drugs and reducing the residues of antibiotics in ecological environment.

[0028] In addition, based on the present application, a new technical method of combined use of pyruvic acid and colistin and other antibiotics for preventing and treating bacterial infection is provided for clinic. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Pyruvic acid concentration-dependent effect of pyruvic acid on improving the sensitivity of pseudomonas aeruginosa to colistin.

[0030] Figure 2 Pyruvic acid concentration-dependent effect of pyruvic acid on improving the sensitivity of baumanii to colistin.

[0031] Figure 3 Pyruvic acid concentration-dependent effect of pyruvic acid on improving the sensitivity of parahemolytic vibrio to colistin.

[0032] Figure 4 Colistin concentration-dependent effect of pyruvic acid on improving the sensitivity of pseudomonas aeruginosa to colistin.

[0033] Figure 5 Colistin concentration-dependent effect of pyruvic acid on improving the sensitivity of baumanii to colistin.

[0034] Figure 6 Colistin concentration-dependent effect of pyruvic acid on improving the sensitivity of parahemolytic vibrio to colistin.

[0035] Figure 7 Time-dependent effect of pyruvic acid on improving the sensitivity of pseudomonas aeruginosa to colistin.

[0036] Figure 8 Time-dependent effect of pyruvic acid on improving the sensitivity of baumanii to colistin.

[0037] Figure 9Pyruvate can improve the sensitivity of multiple drug resistant Pseudomonas aeruginosa to various antibiotics (ceftazidime, meropenem, levofloxacin, gentamicin).

[0038] Figure 10 Pyruvate can improve the sensitivity of multiple drug resistant Pseudomonas aeruginosa to various antibiotics (ceftazidime, meropenem, levofloxacin, gentamicin).

[0039] Figure 11 Pyruvate has no significant effect on the sensitivity of multiple drug resistant Pseudomonas aeruginosa to penicillin and amikacin.

[0040] Figure 12 Pyruvate can improve the sensitivity of multiple drug resistant Acinetobacter baumannii to various antibiotics (ceftazidime, meropenem, levofloxacin, gentamicin).

[0041] Figure 13 Pyruvate has no significant effect on the sensitivity of multiple drug resistant Acinetobacter baumannii to penicillin and amikacin.

[0042] Figure 14 Pyruvate can improve the sensitivity of multiple drug resistant Vibrio parahaemolyticus to various antibiotics (ceftazidime, meropenem, levofloxacin, gentamicin).

[0043] Figure 15 Pyruvate has no significant effect on the sensitivity of multiple drug resistant Vibrio parahaemolyticus to penicillin and amikacin. DETAILED DESCRIPTION

[0044] The present application is further illustrated by the following specific examples and the accompanying drawings of the specification, but the examples do not limit the present application in any form.

[0045] Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0046] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0047] Example 1 Screening of antibiotic-resistant strains of clinically pathogenic bacteria

[0048] The strains of Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus isolated from the clinic were detected for the minimum inhibitory concentration (MIC) of 13 kinds of antibiotics by the drug sensitivity identification card test method. Multiple drug resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus of clinical origin were screened by drug sensitivity test.

[0049] Example 2 Pyruvate enhances the sensitivity of colistin-resistant multiple drug resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus to colistin

[0050] 1. Preparation of bacterial samples:

[0051] Single colony was picked and cultured in 50 mL LB liquid medium at 37 °C, 200 rpm for 14 hours to reach saturation state. After collecting the bacterial solution, the supernatant was removed by centrifugation at 10,000 rpm for 10 minutes. An equal volume of 0.85% physiological saline was added to resuspend the bacterial cells, which were then centrifuged at 10,000 rpm for 10 minutes to remove the supernatant. The bacterial cells were resuspended in M9 medium (containing 10 mM acetate) and adjusted to an OD 600 of 0.6. Then, 5 mL was taken and dispensed into a sterilized test tube for later use.

[0052] 2. Pyruvate enhances the sensitivity of colistin-resistant multiple-drug-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Vibrio parahaemolyticus to colistin in a concentration-dependent manner:

[0053] After adding 40 μg / mL colistin to the prepared bacterial suspension and adding different concentrations of pyruvate solution (0-20 mM), the mixture was incubated at 37 °C, 200 rpm for 6 hours. Then, the viable bacterial count was determined by plating, and the bacterial survival rate with different concentrations of pyruvate in combination with colistin was calculated.

[0054] The calculation formula is:

[0055] It was found that in Pseudomonas aeruginosa ( Figure 1 ), the bacterial survival rate without adding pyruvate was 59.56%. However, after adding pyruvate, the bacterial survival rate decreased from 42.14% to 13.11% with increasing concentration, and the bactericidal effect increased from 1.43 times to 4.54 times. Considering the concentration of pyruvate, 5 mM pyruvate could enhance the sensitivity by about 2.75 times, so this concentration was used in the subsequent experiments. In Acinetobacter baumannii ( Figure 2 ), the bacterial survival rate without adding pyruvate was 65.44%. However, after adding pyruvate, the bacterial survival rate decreased from 52.09% to 10.20% with increasing concentration, and the bactericidal effect increased from 1.26 times to 6.42 times. Considering the concentration of pyruvate, 5 mM pyruvate could enhance the sensitivity by about 4.23 times, so this concentration was used in the subsequent experiments. In Vibrio parahaemolyticus ( Figure 3 ), the bacterial survival rate without adding pyruvate was 75.61%. However, after adding pyruvate, the bacterial survival rate decreased from 61.38% to 32.02% with increasing concentration, and the bactericidal effect increased from 1.23 times to 2.36 times. Considering the concentration of pyruvate, 5 mM pyruvate could enhance the sensitivity by about 1.76 times, so this concentration was used in the subsequent experiments.

[0056] 3. Pyruvate enhances the sensitivity of polymyxin-resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus to colistin in a colistin concentration-dependent manner:

[0057] At a fixed pyruvate concentration of 5 mM, the effect of pyruvate on the sensitivity of polymyxin-resistant Pseudomonas aeruginosa to colistin was investigated at different colistin concentrations. The bacteria were incubated at 37°C and 200 rpm for 6 hours, and then the viable bacteria were counted by plating. The results showed that the survival rate of Pseudomonas aeruginosa decreased from 41.07% to 15.01% with increasing colistin concentration, and the corresponding enhancement of colistin bactericidal effect was from 2.28-fold to 3.36-fold, i.e., the bactericidal effect increased significantly with increasing colistin concentration. Figure 4 Figure 5 The survival rate of Acinetobacter baumannii decreased from 24.91% to 7.11% with increasing colistin concentration, and the corresponding enhancement of colistin bactericidal effect was from 2.98-fold to 4.63-fold, i.e., the bactericidal effect increased significantly with increasing colistin concentration. Figure 6 The survival rate of Vibrio parahaemolyticus decreased from 46.32% to 8.06% with increasing colistin concentration, and the corresponding enhancement of colistin bactericidal effect was from 1.95-fold to 4.09-fold, i.e., the bactericidal effect increased significantly with increasing colistin concentration.

[0058] 4. Pyruvate enhances the sensitivity of polymyxin-resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus to colistin in a time-dependent manner:

[0059] The prepared bacterial solution was added with 5 mM pyruvate and 40 μg / mL colistin, and incubated at 37°C and 200 rpm for 12 hours. The viable bacteria were counted by plating every 2 hours, and the survival rate at different time points after the synergistic effect of pyruvate and colistin was calculated.

[0060] The calculation formula was:

[0061] The results showed that the survival rate of Pseudomonas aeruginosa was 57.31% at 2 hours after adding colistin and pyruvate, and decreased significantly with time, with a survival rate of 15.01% at 6 hours, i.e., the bactericidal efficiency was enhanced by 1.58-4.22-fold. The survival rate gradually decreased with time, but the change trend was slow. Figure 7 The survival rate of Acinetobacter baumannii decreased from 24.91% to 7.11% with increasing colistin concentration, and the corresponding enhancement of colistin bactericidal effect was from 2.98-fold to 4.63-fold, i.e., the bactericidal effect increased significantly with increasing colistin concentration. Figure 8 ​), after adding colistin and pyruvic acid, its survival rate was 47.2% in 2 hours, and its survival rate decreased significantly with time, and its survival rate was 17.02% in 6 hours, that is, the bactericidal efficiency can be enhanced by 1.88-3.72 times, and then the survival rate gradually decreases with time, but the change trend is slow. In Vibrio parahaemolyticus ( Figure 9 ), after adding colistin and pyruvic acid, its survival rate was 31.24% in 2 hours, and its survival rate decreased significantly with time, and its survival rate was 14.51% in 6 hours, that is, the bactericidal efficiency can be enhanced by 2.97-3.67 times, and then the survival rate gradually decreases with time, but the change trend is slow.

[0062] Example 3 Pyruvic acid can enhance the sensitivity of multi-drug resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Vibrio parahaemolyticus to various antibacterial drugs

[0063] The prepared Pseudomonas aeruginosa bacterial liquid sample was added with 5mM pyruvic acid and 6 kinds of antibiotics (antibiotic concentrations were: 50, 100, 200 μg / mL of ceftazidime, 12.5, 25, 50 μg / mL of meropenem, 4, 8, 16 μg / mL of levofloxacin, 16, 32, 64 μg / mL of gentamicin, 50, 100, 200 μg / mL of penicillin, 50, 100, 200 μg / mL of amikacin), and placed in 37℃, 200rpm for 6 hours, and the bacterial survival rate under different antibiotic conditions was calculated. It was found that ( Figure 10 ) under the condition of adding 5mM pyruvic acid, ceftazidime was enhanced by 2.25, 2.58, 2.29 times respectively, meropenem was enhanced by 1.66, 2.74, 3.47 times respectively, levofloxacin was enhanced by 1.41, 2.65, 3.17 times respectively, gentamicin was enhanced by 1.58, 1.95, 2.75 times respectively, penicillin was enhanced by 1.04, 1.11, 1.01 times respectively, and amikacin was 0.94, 1.06, 1.09 times. This shows that pyruvic acid can enhance the sensitivity of colistin-resistant multi-drug resistant Pseudomonas aeruginosa to various antibacterial drugs, but there is no significant enhancement for penicillin and amikacin ( Figure 11 ).

[0064] The prepared Acinetobacter baumannii bacterial liquid sample was added with 5mM pyruvic acid and 6 kinds of antibiotics (antibiotic concentrations were: 50, 100, 200 μg / mL of ceftazidime, 20, 40, 80 μg / mL of meropenem, 20, 40, 80 μg / mL of levofloxacin, 40, 80, 160 μg / mL of gentamicin, 50, 100, 200 μg / mL of penicillin, 20, 40, 80 μg / mL of amikacin), and placed in 37℃, 200rpm for 6 hours, and the bacterial survival rate under different antibiotic conditions was calculated. It was found that ( Figure 12) : under the condition of adding 5mM pyruvic acid, ceftazidime was enhanced by 1.73, 2.89, 3.49 times, meropenem was enhanced by 1.20, 1.64, 3.35 times, levofloxacin was enhanced by 1.29, 2.42, 2.29 times, gentamicin was enhanced by 2.52, 3.86, 4.09 times, penicillin was enhanced by 1.06, 1.02, 1.12 times, and amikacin was enhanced by 1.04, 0.98, 1.02 times. This shows that pyruvic acid can enhance the sensitivity of polymyxin multi-drug resistant P. aeruginosa to various antibacterial drugs, but has no significant enhancement on penicillin and amikacin. Figure 13

[0065] The prepared V. parahaemolyticus bacterial liquid sample was added with 5mM pyruvic acid and 6 kinds of antibiotics (antibiotic concentrations were: ceftazidime 50, 100, 200 μg / mL, meropenem 32, 64, 128 μg / mL, levofloxacin 16, 32, 64 μg / mL, gentamicin 32, 64, 128 μg / mL, penicillin 50, 100, 200 μg / mL, and amikacin 32, 64, 128 μg / mL), and was placed at 37°C, 200 rpm for 6 hours, and the bacterial survival rate under different antibiotic conditions was calculated. It was found that Figure 14 ) : under the condition of adding 5mM pyruvic acid, ceftazidime was enhanced by 2.33, 2.88, 4.40 times, meropenem was enhanced by 2.64, 4.88, 13.67 times, levofloxacin was enhanced by 1.64, 2.42, 2.62 times, gentamicin was enhanced by 2.25, 2.97, 3.18 times, penicillin was enhanced by 1.00, 0.88, 0.89 times, and amikacin was enhanced by 1.04, 0.98, 1.02 times. This shows that pyruvic acid can enhance the sensitivity of polymyxin multi-drug resistant P. aeruginosa to various antibacterial drugs, but has no significant enhancement on penicillin and amikacin. Figure 15

[0066] Example 4 Pyruvic acid can enhance the sensitivity of various pathogenic bacteria to polymyxin

[0067] 1. Determination of the minimum inhibitory concentration of polymyxin to various pathogenic bacteria:

[0068] The minimum inhibitory concentration (MIC) of polymyxin to P. aeruginosa, A. baumannii, and V. parahaemolyticus was detected by using a drug sensitivity identification card, and the results are shown in Table 1 below.

[0069] Table 1

[0070]

[0071] ​​The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. Use of pyruvate for the preparation of a preparation for increasing the sensitivity of pathogenic bacteria to antibiotics, characterized in that, The pathogenic bacteria are Acinetobacter baumannii or Vibrio parahaemolyticus; the antibiotics include colistin, levofloxacin, meropenem, and gentamicin.

2. Use of pyruvate for the preparation of an antibiotic potentiator, characterized in that, The pathogenic bacteria are Acinetobacter baumannii or Vibrio parahaemolyticus; the antibiotics include colistin, levofloxacin, meropenem, and gentamicin.

3. Use of pyruvate in combination with an antibiotic for the manufacture of a medicament for the prevention or treatment of an infection with pathogenic bacteria, characterized in that, The pathogenic bacteria are Acinetobacter baumannii or Vibrio parahaemolyticus; the antibiotics include colistin, levofloxacin, meropenem, and gentamicin.

4. Use according to claim 3, characterized in that, The pyruvic acid in the medicine is used as an antibiotic enhancer to enhance the ability of the antibiotic to kill pathogenic bacteria.

5. Use according to claim 3, characterized in that, The pyruvic acid in the medicine is used as an antibiotic enhancer to enhance the ability of the antibiotic to eliminate pathogenic bacteria in vivo.

6. Use according to claim 3, characterized in that, The pyruvic acid in the medicine is used as an antibiotic enhancer to promote the ability of the host to eliminate pathogenic bacteria in vivo after being infected with pathogenic bacteria.

Images