Application of linoleic acid in preparation of medicine for improving sensitivity of gram-positive bacteria to antibiotics

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of linoleic acid in preparation of a medicine for improving sensitivity of gram-positive bacteria to antibiotics. It is found for the first time that combination of linoleic acid and antibiotics can significantly improve the sensitivity of gram-positive bacteria to antibiotics; in-vivo research data shows that the combination of linoleic acid and antibiotics not only can improve the survival rate of mice infected with gram-positive bacteria, but also can improve the ability of mice to remove gram-positive bacteria; linoleic acid and antibiotics are further prepared into the anti-infection composition, on one hand, the remarkable clinical anti-infection effect can be achieved, and on the other hand, the post-effect of the antibiotics can be prolonged; moreover, the linoleic acid is a necessary fatty acid in nutrition of human bodies and animals, is high in nutritional value, is widely applied to food and medicines, and is high in safety.

Description

[0001] This application is a divisional application of the invention patent application with application date of July 30, 2024, application number 2024110312484, and invention name “Application of linoleic acid in the preparation of drugs for increasing the sensitivity of Gram-positive bacteria to antibiotics”. Technical Field

[0002] The present invention belongs to the field of biomedicine technology and more specifically relates to the use of linoleic acid in the preparation of a drug for increasing the sensitivity of Gram-positive bacteria to antibiotics. Background Art

[0003] Staphylococcus aureus ( Staphyloccocus aureus Rosenbach ) is a common clinical pathogen that often causes community and hospital infections, and the infections it causes are second only to Escherichia coli. Reasonable antibiotic intervention is still an effective means of treating Staphylococcus aureus infections. β-lactam drugs represented by penicillin and cephalosporin are one of the important drugs for the clinical treatment of Staphylococcus aureus infections. However, due to the widespread use of β-lactam drugs in clinical practice, especially methicillin-resistant Staphylococcus aureus ( methicillin-resistant staphylococcus aureus The increase in the detection rate of MRSA and its multidrug-resistant characteristics have greatly limited the clinical efficacy of β-lactam drugs.

[0004] Therefore, in the current situation where the drug resistance of Gram-positive bacteria including MRSA is becoming increasingly serious and the development of new antimicrobial drugs is becoming increasingly difficult, finding effective antimicrobial drug enhancers and restoring the sensitivity of multidrug-resistant bacteria including MRSA to existing key antimicrobial drugs through reasonable combination drug strategies are of great significance for improving the clinical efficacy of antimicrobial drugs and delaying the development of drug resistance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings that Gram-positive bacteria have developed resistance to existing antibiotics and the research and development of new antibacterial drugs is becoming increasingly difficult. The present invention provides the use of linoleic acid in the preparation of antibiotic synergists. The antibiotics can not only improve the survival rate of the body against infected Gram-positive bacteria, but also improve the body's ability to eliminate Gram-positive bacteria.

[0006] The purpose of the present invention is to provide an application of linoleic acid in combination with antibiotics in the preparation of a drug for increasing the sensitivity of Gram-positive bacteria to antibiotics.

[0007] Another object of the present invention is to provide the use of linoleic acid in combination with antibiotics in the preparation of a medicament for preventing and / or treating Gram-positive bacterial infection.

[0008] Another object of the present application is to provide a medicine for improving the sensitivity of gram-positive bacteria to antibiotics.

[0009] The above objects of the present application are achieved by the following technical solutions. The present application protects the use of linolic acid in the preparation of an antibiotic sensitizer, wherein the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a complex preparation containing one or more of the antibiotics; and the penicillin antibiotic does not include ampicillin.

[0010] Linolic acid is an important unsaturated fatty acid, which is an essential fatty acid for the human body, has the effects of lowering cholesterol, softening blood vessels and promoting microcirculation, and can prevent various cardiovascular and cerebrovascular diseases. The inventors' team first discovered that linolic acid can significantly improve the sensitivity of gram-positive bacteria to antibiotics when used in combination with the above antibiotics; in vivo study data show that the combination of linolic acid and the above antibiotics (such as cefoperazone sodium and sulbactam sodium) can not only improve the survival rate of the body infected with gram-positive bacteria, but also improve the clearance ability of the body to gram-positive bacteria. The preparation of an anti-infection composition from linolic acid and antibiotics can not only achieve a significant clinical anti-infection effect, but also prolong the post-effect of antibiotics (Postantibiotic Effect, PAE, is the effect that the inhibition of microorganisms still maintains for a period of time after the serum concentration of antibiotics drops below the minimum inhibitory concentration or disappears after contacting antibiotics); and linolic acid is an essential fatty acid for the human body and animals, has high nutritional value, and has been widely used in food and medicine, and has high safety.

[0011] Further, the linolic acid improves the sensitivity of gram-positive bacteria to antibiotics.

[0012] The present application protects the use of linolic acid in the preparation of an antibiotic sensitizer, wherein the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a complex preparation containing one or more of the antibiotics; and the penicillin antibiotic does not include ampicillin.

[0013] The present application also protects the use of linoleic acid in combination with an antibiotic in the preparation of a medicament for preventing or / and treating Gram-positive bacterial infection, wherein the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a compound preparation containing one or more of the antibiotics; and the penicillin antibiotic does not include ampicillin.

[0014] Preferably, the antibiotic is selected from cefoperazone sodium sulbactam sodium (cephalosporins + β-lactamase inhibitor, i.e. a compound preparation), cefotaxime (cephalosporins), cefoperazone (cephalosporins), amikacin (aminoglycosides), tobramycin (aminoglycosides), gentamicin (aminoglycosides), ofloxacin (quinolones), levofloxacin (quinolones), tetracycline (tetracyclines), meropenem (carbapenems), vancomycin (glycopeptides), azithromycin (macrolides), sulfadiazine (sulfonamides) or amoxicillin sodium clavulanate potassium (penicillin + β-lactamase inhibitor, i.e. a compound preparation).

[0015] Further, the cefoperazone sodium sulbactam sodium is a compound preparation of cefoperazone and sulbactam, wherein both cefoperazone and sulbactam are in the form of sodium salt; further, in the compound preparation, the mass ratio of cefoperazone and sulbactam is 1:1.

[0016] Further, the amoxicillin sodium clavulanate potassium is a compound preparation of amoxicillin and clavulanate, wherein amoxicillin is in the form of sodium salt and clavulanate potassium is in the form of potassium salt; further, in the compound preparation, the mass ratio of amoxicillin and clavulanate is (2~7):1.

[0017] Preferably, in the compound preparation, the mass ratio of amoxicillin and clavulanate is 2:1, 4:1 or 7:1.

[0018] Further, the Gram-positive bacteria are selected from one or more of Staphylococcus aureus, Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecalis.

[0019] Further, the Staphylococcus aureus includes susceptible bacteria and drug-resistant bacteria.

[0020] Further, the susceptible bacteria are Methicillin Susceptible Staphylococcus Aureus (MSSA) and the drug-resistant bacteria are Methicillin-Resistant Staphylococcus Aureus (MRSA).

[0021] Further, the linoleic acid as an antibiotic synergist enhances the ability of antibiotics to kill gram-positive bacteria.

[0022] Further, the linoleic acid as an antibiotic synergist enhances the ability of antibiotics to kill gram-positive bacteria.

[0023] Further, the linoleic acid as an antibiotic synergist enhances the ability of antibiotics to kill gram-positive bacteria.

[0024] Further, the linoleic acid as an antibiotic synergist enhances the ability of antibiotics to kill gram-positive bacteria.

[0025] Further, the linoleic acid as an antibiotic synergist enhances the ability of antibiotics to kill gram-positive bacteria.

[0026] Further, the linoleic acid as an antibiotic synergist enhances the ability of antibiotics to kill gram-positive bacteria.

[0027] The present application also protects a medicine for enhancing the sensitivity of gram-positive bacteria to antibiotics, which contains an effective amount of linoleic acid and antibiotics; the antibiotics are selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof or a compound preparation containing one or more of the antibiotics; and the penicillins do not include ampicillin.

[0028] Preferably, the mixing ratio of the linoleic acid and antibiotics is 1: (2-40000) g / mol. The meaning of the mixing ratio is that 2-40000 g of antibiotics and 1 mol of linoleic acid are used together.

[0029] More preferably, the mixing ratio of the linoleic acid and antibiotics is 1: (50-40000) g / mol.

[0030] Compared with the prior art, the present application has the following beneficial effects: the present application first discovers that linoleic acid is combined with antibiotics, which significantly improves the sensitivity of gram-positive bacteria to antibiotics; in vivo research data show that the combination of linoleic acid and antibiotics can not only improve the survival rate of mice infected with gram-positive bacteria, but also improve the clearance ability of mice to gram-positive bacteria; further, the preparation of linoleic acid and antibiotics into an anti-infection composition can not only achieve a significant clinical anti-infection effect, but also prolong the clinical effect of antibiotics; and linoleic acid is an essential fatty acid in human and animal nutrition, has high nutritional value, and has been widely used in food and medicine, and has high safety. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a clinical Staphylococcus aureus gene PCR amplification result graph in Example 1.

[0032] Figure 2 It is a result statistical graph of the killing effect of linoleic acid on clinical Staphylococcus aureus sensitive bacteria MSSA 2 (A) and drug-resistant bacteria MRSA 7 (B) and the concentration effect of linoleic acid in Example 2.

[0033] Figure 3 It is a result statistical graph of the time effect of linoleic acid on killing clinical Staphylococcus aureus MRSA 7 (A) and MSSA 2 (B) in Example 2.

[0034] Figure 4 It is a result statistical graph of the killing effect of linoleic acid on multiple strains of clinical Staphylococcus aureus sensitive bacteria (A) and drug-resistant bacteria (B) in Example 2.

[0035] Figure 5 It is a result statistical graph of the survival rate (A) and clearance ability (B) of mice infected with clinical Staphylococcus aureus in Example 3.

[0036] Figure 6 It is a result statistical graph of the sensitivity of clinical Staphylococcus aureus (A) and other gram-positive bacteria (B) to cefoperazone sodium sulbactam sodium, and the sensitivity of Staphylococcus aureus to other antibiotics (C) in Example 4.

[0037] Figure 7 It is a result statistical graph of the concentration of linoleic acid (A), the concentration of antibiotics (B) and the time gradient (C) effect of linoleic acid on improving the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium sulbactam sodium in Example 5.

[0038] Figure 8 It is a result statistical graph of the survival rate (A) and clearance ability (B) of mice infected with clinical Staphylococcus aureus in Example 6.

[0039] Figure 9 Statistical chart of the results of the increase in the permeability of the clinical Staphylococcus aureus sensitive strain MSSA (A) and the drug-resistant strain MRSA (B) after the addition of linoleic acid in Example 7.

[0040] Figure 10 Statistical chart of the results of the increase in the amount of antibiotics entering the clinical Staphylococcus aureus after the addition of linoleic acid in Example 7.

[0041] Figure 11 Statistical chart of the results of the delay of the post-effect of cefoperazone sodium and sulbactam sodium on the clinical Staphylococcus aureus by linoleic acid in Example 8. DETAILED DESCRIPTION

[0042] The present application will be further described by the following description of the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Example 1 Analysis of the resistance of clinical Staphylococcus aureus to antibiotics Nine sensitive Staphylococcus aureus strains (MSSA) and 20 methicillin-resistant Staphylococcus aureus strains (MRSA) obtained from the clinic were named MSSA 1-9 and MRSA 1-20. Staphylococcu saureus , MSSA) and 20 methicillin-resistant Staphylococcus aureus strains (MRSA) Methicillin-resistant Staphylococcu saureus , MRSA) were obtained from the clinic and named MSSA 1-9 and MRSA 1-20.

[0045] 1.1 Determination of the resistance of clinical strains The 29 clinical Staphylococcus aureus strains were cultured overnight, then diluted 1:100 to culture the bacteria to an OD600 of 0.5, and then diluted 100-fold, and the minimum inhibitory concentration (MIC) of these strains to daptomycin (DAP), vancomycin (VAN), cefoperazone sodium and sulbactam sodium (SCF) and oxacillin (OX) was determined according to the protocol for microbial susceptibility testing provided by the Clinical and Laboratory Standards Institute (CLSI). The results are shown in Table 1.

[0046] Table 1 Minimum inhibitory concentration (MIC) of four antibiotics to 29 Staphylococcus aureus strains

[0047] Note: For OX, MIC≤2 is sensitive bacteria; for DAP: MIC≤1 is sensitive bacteria; for VAN: MIC≤2 is sensitive bacteria.

[0048] As shown in Table 1, the above 20 MRSA strains are resistant to the four kinds of antibiotics tested, which also shows that the 20 strains are not only methicillin-resistant bacteria, but also multi-drug resistant bacteria (Table 1, Table A), and 9 MRSA strains are sensitive to the four kinds of antibiotics (Table 1, Table B).

[0049] 1.2 Genetic identification of clinical strains femB (651bp) is an intrinsic gene of Staphylococcus aureus, mecA (310bp) is a unique gene of methicillin-resistant strains, and PVL is an exotoxin-leukocidin produced by Staphylococcus aureus, which damages the defense barrier and immune response of the body by destroying leukocytes and phagocytes.

[0050] The clinical strains were cultured overnight, and the three genes of each strain were amplified by colony PCR (amplification primer sequences are shown in Table 2), and then the amplification products were detected by gel electrophoresis. The results are shown in Figure 1 As shown in the figure, for MRSA strains, each resistant strain can amplify the three genes, which shows that it is indeed Staphylococcus aureus, and is a methicillin-resistant strain, and also has toxicity. For sensitive strains, each strain can amplify femB , but not mecA , which shows that these strains are indeed Staphylococcus aureus, and are sensitive strains. However, some strains have exotoxin genes, and some do not.

[0051] Table 2 Primer sequences

[0052] Example 2 Linoleic acid can kill Staphylococcus aureus from clinical sources 2.1 Preparation of strain samples A single colony of clinical bacteria was picked from a solid LB plate and inoculated into 5 mL of LB liquid medium, which was cultured at 37°C and 200 rpm for 16 hours. The bacterial solution was collected and centrifuged at 8000 rpm for 5 min. The supernatant was removed and the bacterial cells were washed with an equal volume of 0.85% physiological saline. Finally, the bacterial cells were suspended in 1x M9 basic medium (containing 10 mM acetate, 1 M Cacl2). The OD value of the bacterial solution was adjusted to 0.2, and 5 mL was aliquoted into test tubes for subsequent experiments.

[0053] 2.2 Linoleic acid can kill Staphylococcus aureus from clinical sources and has a linoleic acid concentration gradient effect The samples of clinical sensitive strain MSSA 2 and clinical drug-resistant strain MRSA 7 were prepared according to 2.1 in Example 2. Subsequently, linoleic acid was added in the test tubes to make the final concentration of 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mM, respectively. There were 3 biological replicates for each concentration. After incubation at 37℃ for 10 hours at 200 rpm, the viable bacteria were detected by plating, and then the survival rate of the bacteria under different concentrations of linoleic acid was calculated. The calculation formula was: survival rate (%) = (viable bacteria after adding linoleic acid / viable bacteria without adding linoleic acid) x 100%. The results are shown in Figure 2 As can be seen from the figure, the survival rate of the bacteria decreased significantly after the addition of linoleic acid, regardless of sensitive or drug-resistant bacteria. And with the increase of the concentration of linoleic acid, the bactericidal efficiency gradually increased. The specific situation is as follows: For clinical sensitive bacteria MSSA 2 (as shown in A of Figure 2 When 0.01 mM of linoleic acid was added, it could play a good killing effect, and the bactericidal effect was increased by 8.07 times (the survival rate was reduced to 12.39%) compared with the control group. When the concentration of linoleic acid gradually increased from 0.05 mM to 10 mM, the bactericidal efficiency increased by 19.91 times, 129.58 times, 709.17 times, 1925.93 times, 4681.57 times and 9577.32 times (the survival rate also decreased from 5.02% to 0.01%), respectively.

[0054] For clinical drug-resistant bacteria MRSA 7 (as shown in B of Figure 2 When 0.01 mM of linoleic acid was added, the bactericidal effect was also increased by 7.14 times (the survival rate was reduced to 14.00%) compared with the control group. Similarly, when the concentration of linoleic acid gradually increased from 0.05 mM to 10 mM, the bactericidal efficiency increased by 15 times, 39.47 times, 230.77 times, 1257.24 times, 2675.16 times and 4199.48 times (the survival rate also decreased from 6.67% to 0.02%), respectively.

[0055] Among them, when the concentration of linoleic acid was 1 mM, the bactericidal efficiency of sensitive bacteria and drug-resistant bacteria increased by 1925.93 times and 1257.24 times, respectively. With the increase of the concentration of linoleic acid, the bactericidal efficiency also increased. However, since linoleic acid is acidic, when the amount added is too high, it will affect the pH value of the incubation system. Therefore, 1 mM of linoleic acid was selected for the subsequent research.

[0056] This result shows that linoleic acid can kill Staphylococcus aureus of clinical origin, including sensitive strains and drug-resistant strains, and has a concentration gradient effect of linoleic acid.

[0057] 2.3 Linoleic acid killing of clinical Staphylococcus aureus has time dependence MSSA 2 and MRSA 7 samples were prepared according to 2.1 in Example 2, 1 mM linoleic acid was added into each sample in test tube, and no linoleic acid was added as control, and plate viable count was performed at different time, and survival rate was calculated to study the relationship between sterilization efficiency and time.

[0058] The results are shown in Figure 3 The figure shows that the number of MSSA 2 and MRSA 7 clinical bacteria does not decrease within 2 hours after the addition of linoleic acid, and then the number of bacteria decreases obviously with the extension of time, showing time effect. The specific situation is as follows: For drug-resistant bacteria MRSA 7 (A of Figure 3 ), the number of bacteria decreased by 15 times in 4 hours, and then the number of bacteria decreased by 137.67-13641.57 times in 6-12 hours. For sensitive bacteria MSSA 2 (B of Figure 3 ), the number of bacteria decreased by 12.8 times in 4 hours, and then the number of bacteria decreased by 216.15-16818.73 times in 6-12 hours.

[0059] The above results show that linoleic acid has time-dependent killing effect on clinical Staphylococcus aureus. Linoleic acid can achieve good killing effect on clinical Staphylococcus aureus in 10 hours. In order to facilitate operation, 10 hours is taken as the sterilization time of linoleic acid for subsequent research.

[0060] 2.4 Linoleic acid killing of clinical Staphylococcus aureus is universal According to 2.1 in Example 2, the rest 8 sensitive bacteria and 19 drug-resistant bacteria samples were prepared. Each strain was divided into two groups, M9 control group and 1 mM linoleic acid test group. After incubation at 37°C and 200 rpm for 10 hours, viable count was performed, and the survival rate of each strain after the addition of linoleic acid was calculated. Each strain had 3 biological repeats.

[0061] The results are shown in Figure 4 The figure shows that linoleic acid can kill all clinical sensitive bacteria and drug-resistant bacteria, but the degree of increasing sensitivity is different for different strains, which is described as follows: For 8 sensitive bacteria (A of Figure 4 ), except MSSA1 strain, the sterilization multiple after the addition of linoleic acid was only 81.97 times, and the sterilization multiple of the rest strains was 1925.93-98846.78 times.

[0062] For 19 drug-resistant bacteria (B of Figure 4(Figure B in the figure) The sterilization multiples ranged from 7.39 to 14918.69. Three strains (11, 12, and 15) had sterilization multiples below 100 times; five strains (8, 13, 14, 16, and 17) had sterilization multiples greater than 100 times; and 11 strains (1-6, 9, 10, and 18-20) had sterilization multiples greater than 1000 times.

[0063] Example 3 Linoleic acid can improve the resistance of mice to clinical Staphylococcus aureus infection 3.1 Linoleic acid can improve the survival rate of mice against clinical Staphylococcus aureus infection Balb / c mice (6-8 weeks, approximately 20 g, half male and half female) were divided into three groups, 16 in each group: a normal saline control group, an antibiotic treatment group, and a linoleic acid test group. The challenge strain was the drug-resistant MRSA 7, and the infection dose was 1.2 × 10 8 CFU were measured by intraperitoneal injection. One hour after bacterial infection, mice in the treatment group were intravenously administered cefoperazone sodium and sulbactam sodium at a dose of 200 mg / kg; mice in the experimental group were intraperitoneally administered linoleic acid at a dose of 100 mg / kg. Mouse mortality was observed and recorded daily, and the survival rate of each group was calculated daily for a total of 7 days. The calculation formula was: Survival rate = number of surviving mice per group / total number of mice per group × 100%.

[0064] The experimental results are shown in Figure 5 As shown in Figure A, in the saline control group, 68% died on the first day, and all died on the second day. In the antibiotic-treated group, 31% died on the first day, 62% died on the second day, and no further deaths occurred, with a survival rate of 7%. In the linoleic acid-treated group, no deaths occurred on the first day, 7% died on the second day, and then stabilized, with no further deaths, for a survival rate of 93%. Compared with the control group, the protection rate of mice injected with linoleic acid increased by 93%, and compared with the antibiotic-treated group, the protection rate of mice injected with linoleic acid increased by 86%.

[0065] The results show that when mice are infected with methicillin-resistant bacteria, linoleic acid can significantly improve the mice's resistance to methicillin-resistant bacteria infection when antibiotic treatment is basically ineffective.

[0066] 3.2 Linoleic acid can improve the clearance of clinical Staphylococcus aureus infection in mice Balb / c mice (6-8 weeks old, approximately 20 g, half male and half female) were divided into two groups, 6 in each group, a control group and a linoleic acid test group. The challenge strain was methicillin-resistant Staphylococcus aureus MRSA 7, and the infection dose was 1.4×10 6CFU, intraperitoneal injection. One hour after bacterial infection, linoleic acid was given intraperitoneally at a dose of 100 mg / kg. After 6 hours, the same weight of internal organs (liver, spleen, kidney) were taken for thorough grinding, then dilution plating was performed, and the number of bacteria in the organs was counted.

[0067] The results are shown in Figure 5 B of FIG. 1, it can be seen that, after the mice were infected with drug-resistant bacteria, if linoleic acid was given, the bacterial load in the spleen, kidney and liver of the mice decreased by 1563 times, 1009 times and 4419 times, respectively. The results show that linoleic acid can significantly improve the clearance of mice from clinical Staphylococcus aureus infection.

[0068] Example 4: Linoleic acid can improve the sensitivity of gram-positive bacteria to antibiotics 4.1 Linoleic acid can improve the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium The results of 2.4 in Example 2 found that linoleic acid had a killing effect on clinically derived Staphylococcus aureus. However, it was also found that different strains had different bactericidal effects, and the bactericidal effect of some strains was less than 1000 times. Since bacterial infection is still treated with antibiotics at present, can linoleic acid synergize with antibiotics to improve the sensitivity of the strain to antibiotics? Therefore, all strains MRSA 8, 11-17 and MSSA 1 in 2.4 in Example 2 with a linoleic acid bactericidal effect less than 1000 times were selected, and strains MSSA2, MSSA8, MRSA4, 9 and 18 with a linoleic acid bactericidal effect greater than 1000 times were also selected, a total of 14 strains of bacteria were used as objects for linoleic acid synergized with antibiotics for bactericidal test.

[0069] The above strain samples were prepared according to 2.1 in Example 2, and each strain was divided into 4 groups: M9 control group, antibiotic group, linoleic acid group, linoleic acid + antibiotic group. The antibiotic was cefoperazone sodium and sulbactam sodium, and the concentration used for each strain was shown in Table 3, and the concentration of linoleic acid was 1 mM. After incubation at 37°C for 10 hours at 200 rpm, the viable bacteria were detected by plating, and then the survival rate of bacteria in different treatment groups was calculated. The calculation formula was: survival rate (%) = (viable bacteria after adding linoleic acid and / or antibiotics / viable bacteria in the control group) x 100%.

[0070] The results are shown in Figure 6As shown in Figure A in the above table, these strains have no bactericidal effect or weak bactericidal effect when single-antibiotic is added, and the bactericidal multiples are only between 1.07 and 2.57. However, if linoleic acid is added on the basis of the antibiotic, the sensitivity of all strains to the antibiotic is significantly improved. For the strains with a single-antibiotic linoleic acid bactericidal effect of less than 1000 times, the bactericidal multiples are further increased by 7.88-282.44 times on the basis of the original single-antibiotic linoleic acid. For the strains with a single-antibiotic linoleic acid bactericidal effect of more than 1000 times, the bactericidal multiples are further increased by 1.35-4 times on the basis of the original single-antibiotic linoleic acid.

[0071] Table 3. Cefoperazone sodium and sulbactam sodium concentration used in the test

[0072] 4.2 Linoleic acid can improve the sensitivity of other gram-positive bacteria to cefoperazone sodium and sulbactam sodium The Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecalis samples were prepared according to 2.1 in Example 2. The drug resistance, i.e. MIC, of these bacteria is shown in Table 4. Each strain was divided into four groups: M9 control group, antibiotic group, linoleic acid group, linoleic acid + antibiotic group. The antibiotic was cefoperazone sodium and sulbactam sodium, and the concentration used for each strain was: 300 ug / mL for Streptococcus iniae, 50 ug / mL for Streptococcus agalactiae, 400 ug / mL for Streptococcus pyogenes, 200 ug / mL for Bacillus subtilis, and 400 ug / mL for Enterococcus faecalis; the concentration of linoleic acid was 0.01 mM. After incubation at 37°C and 200 rpm for 10 hours, the viable cell count was detected by plate counting, and then the survival rate of bacteria in different treatment groups was calculated. The calculation formula was: survival rate (%) = (viable cell count after adding linoleic acid and / or antibiotic / viable cell count of the control group) x 100%.

[0073] Table 4. Bacterial drug resistance test

[0074] Note: For Streptococcus iniae, Streptococcus agalactiae and Streptococcus pyogenes, penicillin ≤0.25 is sensitive, and there is no standard for resistance and intermediates; cefalosporins ≤0.25 is sensitive, and there is no standard for resistance and intermediates; carbapenems ≤0.5 is sensitive, and there is no standard for resistance and intermediates; tetracycline: ≤2 is sensitive, 4 is intermediate, and ≥8 is resistant; levofloxacin: ≤2 is sensitive, 4 is intermediate, and ≥8 is resistant; vancomycin ≤1 is sensitive, and there is no standard for resistance and intermediates.

[0075] Enterococcus faecalis: Penicillins: ≤8 is sensitive, no intermediate values ​​have been reported, and ≥16 is resistant; Tetracyclines: ≤4 is sensitive, 8 is intermediate, and ≥16 is resistant; Levofloxacins: ≤2 is sensitive, 4 is intermediate, and ≥8 is resistant; Vancomycin: ≤4 is sensitive, 8-16 is intermediate, and ≥32 is resistant.

[0076] Bacillus subtilis: Penicillins ≤8 are sensitive, 8-16 are intermediate, and ≥32 are resistant; cephalosporins ≤4 are sensitive, 8 are intermediate, and ≥16 are resistant; Tobramycin ≤4 are sensitive, 8 are intermediate, and ≥16 are resistant; Tetracycline: ≤4 are sensitive, 8 are intermediate, and ≥16 are resistant; Levofloxacin: ≤0.5 are sensitive, 1 is intermediate, and ≥2 are resistant.

[0077] R: resistant; I: intermediate; S: sensitive.

[0078] As shown in Table 4, the Gram-positive bacteria listed above are resistant to most of the antibiotics tested and are multidrug-resistant bacteria.

[0079] Depend on Figure 6 As shown in Figure B, antibiotics alone had no or very weak bactericidal effect on these strains, with kill rates ranging from only 1.1 to 1.69. However, the addition of linoleic acid significantly improved the sensitivity of all strains to the antibiotics, with kill rates increasing by 315.58, 3.71, 2254.49, 77.45, and 284.59 for Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis, respectively.

[0080] 4.3 Linoleic acid can increase the sensitivity of clinical Staphylococcus aureus to other antibiotics Clinically resistant MRSA 9 samples were prepared according to Section 2.1 of Example 2 and divided into four groups: an M9 control group, a linoleic acid group, an antibiotic group, and a linoleic acid + antibiotic group. The antibiotic concentrations used in the experiment were: vancomycin at 100 μg / mL, and all other antibiotics at 100 μg / mL; the linoleic acid concentration was 0.01 mM. After incubation at 37°C and 200 rpm for 10 hours, the viable bacterial count was determined using a plate, and the survival rate of the bacteria in each treatment group was calculated. The calculation formula was: Survival rate (%) = (viable bacterial count after addition of linoleic acid and / or antibiotics / viable bacterial count in the control group) × 100%.

[0081] See the results Figure 6The survival rate of bacteria was 44.82% when vancomycin was added alone, and the survival rate of bacteria was 67.24-98.27% when other antibiotics were added alone. The survival rate of bacteria was 70% when linoleic acid was added alone. However, if linoleic acid was added in combination with antibiotics, that is, linoleic acid was added on the basis of adding antibiotics, the sensitivity of the strains to the antibiotics was improved, and the fold was 2.82-2826, except for polymyxin, clindamycin and ampicillin.

[0082] These results show that the bactericidal ability of antibiotics on these bacteria is enhanced after linoleic acid and cefoperazone sodium and sulbactam sodium, or other antibiotics such as tetracycline, meropenem, amikacin, levofloxacin, tobramycin, gentamicin, vancomycin, cefotaxime, azithromycin, amoxicillin sodium clavulanate potassium and sulfadiazine are used in combination, indicating that linoleic acid not only can significantly improve the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium, but also can improve the sensitivity of these gram-positive bacteria such as streptococcus, bacillus subtilis and coprococcus to cefoperazone sodium and sulbactam sodium and other antibiotics including tetracycline, meropenem, amikacin, levofloxacin, tobramycin, gentamicin, vancomycin, cefotaxime, azithromycin, amoxicillin sodium clavulanate potassium and sulfadiazine.

[0083] Example 5 Linoleic acid improves the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium Staphylococcus aureus is taken as a representative of gram-positive bacteria, and cefoperazone sodium and sulbactam sodium is taken as a representative of antibiotics, to further explore the effect of linoleic acid combined with antibiotics.

[0084] 5.1 Concentration effect of linoleic acid The clinical drug-resistant strain MRSA 12 sample was prepared according to 2.1 in Example 2, and was divided into 7 groups: an antibiotic control group (200 μg / mL cefoperazone sodium and sulbactam sodium), and 6 test groups: on the basis of adding 200 μg / mL cefoperazone sodium and sulbactam sodium, linoleic acid was added to make the final concentration 0.01, 0.05, 0.1, 0.5, 1 and 5 mM, respectively. Each concentration had 3 biological repeats. After being cultured at 37°C and 200 rpm for 10 hours, the viable bacteria count was detected by plate detection, and then the Log10 value was converted. A decrease of 1 Log10 indicates a 10-fold decrease in the number of bacteria, and a decrease of 2 Log10 indicates a 100-fold decrease in the number of bacteria. The results are shown in Figure 7 As shown in the A graph in FIG. 1, after adding linoleic acid on the basis of adding antibiotics, the number of surviving bacteria decreased. And with the increase of the concentration of added linoleic acid, the number of surviving bacteria decreased more significantly.

[0085] Specifically, when 0.01 mM linoleic acid was added to the antibiotics, the antibiotic sensitivity was improved, and the number of surviving bacteria was reduced by nearly 0.5 Log (0.5 order of magnitude), that is, the number of bacteria was reduced by about 5 times, compared with the control group (antibiotics alone). When the concentration of linoleic acid was gradually increased from 0.05 mM to 5 mM, the number of surviving bacteria was reduced by 1.32, 1.97, 2.86, 3.28, and 3.73 orders of magnitude, respectively, that is, the number of bacteria was reduced by tens of times to thousands of times.

[0086] 5.2 Antibiotic concentration effect The clinical drug-resistant strain MRSA 12 sample was prepared according to 2.1 in Example 2 and divided into 6 groups: a linoleic acid control group (1 mM), and 5 test groups: 1 mM linoleic acid was added, and then cefoperazone sodium sulbactam sodium was added to make the final concentration 10, 50, 100, 200, and 400 μg / mL, respectively. Each concentration had 3 biological repeats. The culture was incubated at 37°C and 200 rpm for 10 hours, and then the number of viable bacteria was detected by plating, and then converted into Log10 value. The results are shown in FIG. B of Figure 7 As can be seen from the figure, after adding antibiotics on the basis of linoleic acid, the number of surviving bacteria was reduced. And with the increase of the concentration of added antibiotics, the number of surviving bacteria was reduced more significantly.

[0087] Specifically, when 10 μg / mL antibiotics were added on the basis of linoleic acid, the antibiotic sensitivity was improved, and the number of surviving bacteria was reduced by 0.59 Log (0.59 order of magnitude), that is, the number of bacteria was reduced by about 6 times, compared with the control group (linoleic acid alone). When the concentration of antibiotics was gradually increased from 50 μg / mL to 400 μg / mL, the number of surviving bacteria was reduced by 1.76, 3.24, 4.15, and 4.61 orders of magnitude, respectively, that is, the number of bacteria was reduced by nearly 200 times to tens of thousands of times.

[0088] 5.3 Time effect The clinical drug-resistant strain MRSA 12 sample was prepared according to 2.1 in Example 2 and divided into 3 groups: an M9 control group, an antibiotic control group (200 μg / mL cefoperazone sodium sulbactam sodium), and a linoleic acid (1 mM) + antibiotic (200 μg / mL cefoperazone sodium sulbactam sodium) test group. The culture was incubated at 37°C and 200 rpm, and samples were taken every 2 hours within 12 hours, and then the number of viable bacteria was detected by plating, and then converted into Log10 value. Each detection sample had 3 biological repeats. The results are shown in FIG. B of Figure 7As shown in Figure C, the bacterial count in the M9 control group remained essentially stable over 12 hours. Over time, the bacterial counts in the antibiotic and linoleic acid + antibiotic groups gradually decreased. Importantly, at the same time point, the number of surviving bacteria in the linoleic acid + antibiotic group was significantly lower than that in the antibiotic group. Furthermore, the decrease in the number of surviving bacteria became even more pronounced over time.

[0089] The specific results are as follows: After 2 hours, the number of surviving bacteria in the linoleic acid + antibiotic group decreased by 0.67 orders of magnitude, or about 7 times, compared to the surviving bacteria in the antibiotic group. From 4 to 12 hours, the number of surviving bacteria in the linoleic acid + antibiotic group decreased by 1.21 to 2.96 orders of magnitude, or about 10 to 1,000 times.

[0090] The above test results show that linoleic acid can cooperate with cefoperazone sodium and sulbactam sodium to improve the sensitivity of resistant Staphylococcus aureus to cefoperazone sodium and sulbactam sodium, and has antibiotic concentration and linoleic acid concentration effects, as well as time effects.

[0091] Example 6 Linoleic acid synergistically combines cefoperazone sodium and sulbactam sodium to improve the resistance of mice to clinical Staphylococcus aureus infection 6.1 Linoleic acid synergistically combines cefoperazone sodium and sulbactam sodium to improve the survival rate of mice infected with Staphylococcus aureus Taking Staphylococcus aureus as the representative of Gram-positive bacteria and cefoperazone sodium and sulbactam sodium as the representatives of antibiotics, the effect of combined use of linoleic acid and antibiotics was further explored.

[0092] Balb / c mice (6-8 weeks, approximately 20 g, half male and half female) were divided into four groups, 16 in each group: a normal saline control group, an antibiotic treatment group, a linoleic acid test group, and an antibiotic + linoleic acid test group. The challenge strain was methicillin-resistant MRSA 12, and the infection dose was 1.2 × 10 8 CFU were measured by intraperitoneal injection. One hour after bacterial infection, mice in the antibiotic treatment group received 200 mg / kg of cefoperazone sodium and sulbactam sodium via intravenous injection. Mice in the linoleic acid experimental group received 200 mg / kg of linoleic acid via intraperitoneal injection. Mice in the antibiotic + linoleic acid experimental group received antibiotics and linoleic acid according to their respective injection methods and dosages. Mice were observed and recorded daily for mortality, and the survival rate of each group was calculated daily for a total of 7 days. The calculation formula was: Survival rate (%) = number of surviving mice per group / total number of mice per group × 100%.

[0093] The experimental results are shown in Figure 8Figure A in the figure, the physiological saline control group, the first day of 81% death, the second day of all death; antibiotic treatment group of the first day of 31% death, the second day of 62% death, after no longer death, survival rate of 7%; linoleic acid test group in the first day of 7% death, the second day of 62% death, the third day of 7% death, after tend to be stable, no longer death, survival rate of 24%. Antibiotic + linoleic acid experimental group in the first day of no death, the second day of 13% death, after tend to be stable, no longer death, survival rate of 87%.

[0094] The results show that the linoleic acid test group of mice, if compared with the physiological saline control group, the protection rate increased by 24%, compared with the antibiotic treatment group, the protection rate increased by 17%. And the antibiotic + linoleic acid test group of mice, if compared with the linoleic acid test group, the protection rate increased by 63%, compared with the antibiotic treatment group, the protection rate increased by 80%. It is shown that when the mice are infected with methicillin-resistant bacteria, the resistance of the mice to methicillin-resistant bacteria infection is improved by linoleic acid, but if linoleic acid and antibiotics are used together, the resistance of the mice to methicillin-resistant bacteria infection is significantly improved.

[0095] 6.2 Linoleic acid synergizes with cefoperazone sodium and sulbactam sodium to improve the clearance of mice from clinical staphylococcus aureus infection Balb / c mice (6-8 weeks, about 20g, half male and half female) were divided into 4 groups, 6 in each group, namely physiological saline control group, antibiotic treatment group, linoleic acid test group, and antibiotic + linoleic acid test group. The challenge strain was methicillin-resistant staphylococcus aureus MRSA 12, and the infection dose was 1.4x10 6 CFU, using intraperitoneal injection. After 1 hour of bacterial infection, the antibiotic treatment group of mice was given cefoperazone sodium and sulbactam sodium by intravenous injection, the dose was 200mg / kg; the linoleic acid test group of mice was given linoleic acid by intraperitoneal injection, the dose was 200mg / kg; the antibiotic + linoleic acid test group of mice was treated with antibiotics and linoleic acid according to their respective injection methods and doses. After 6 hours, equal weight of internal organs (liver, spleen, kidney) were taken and ground thoroughly, then diluted and plated, and the number was counted.

[0096] The results are shown in Figure 8Figure B in the drawings, it can be seen that, after the mice were infected with drug-resistant bacteria, if given antibiotic treatment, the bacterial load in the spleen, kidney and liver of the infected mice decreased by 55 times, 40 times and 33 times, respectively; if given linoleic acid treatment, the bacterial load in the spleen, kidney and liver of the mice decreased by 20 times, 8 times and 17 times, respectively. If given linoleic acid and antibiotic treatment, the bacterial load in the spleen, kidney and liver of the mice decreased by 136 times, 111 times and 97 times, respectively, as compared with the antibiotic treatment group; and decreased by 51 times, 22 times and 50 times, respectively, as compared with the linoleic acid test group. The results show that, after linoleic acid and antibiotics are used in combination, the clearance of the mice to clinical S. aureus infection can be significantly improved.

[0097] Example 7 Linoleic acid increases the intracellular entry of antibiotics by increasing the membrane permeability of bacteria to achieve sensitivity to antibiotics Taking S. aureus as a representative of gram-positive bacteria and cefoperazone sodium sulbactam sodium as a representative of antibiotics, the mechanism of the combination of linoleic acid and antibiotics to improve the efficacy was further explored.

[0098] 7.1 Exogenous linoleic acid can increase the membrane permeability of bacteria According to 2.1 in Example 2, bacteria were prepared, 9 strains of MSSA and 10 strains of MRSA. Each strain of bacteria was divided into two groups, one group was the control group and the other group was the linoleic acid (1 mM) test group. Incubation was performed at 37°C for 10 hours on a 200 rpm shaking table. Then 100 μL to 900 μL M9 was taken, and 2 μL of 2.5 mM SYTO9 dye was added. Incubation was performed at 37°C for 45 minutes at 200 rpm, and the fluorescence was detected by flow cytometry. The change in membrane permeability was judged by comparing the fluorescence intensity of the test group and the control group. The results are shown in Figure 9 Regardless of MSSA or MRSA, the membrane permeability of bacteria is enhanced after the addition of exogenous linoleic acid. This shows that linoleic acid can increase the membrane permeability of bacteria.

[0099] 7.2 Exogenous linoleic acid can promote the entry of antibiotics into the intracellular content of bacteria The method of detecting the inhibition of antibiotics on microorganisms was used to calculate the activity (titer) of antibiotics to determine the concentration of antibiotics entering the intracellular content of bacteria.

[0100] Sample preparation for determination of intracellular antibiotic concentration of bacteria: MRSA 7 strain samples were prepared according to Example 2.1 and divided into three groups, namely a control group without the addition of substances and antibiotics, a cefoperazone sodium and sulbactam sodium group, and a cefoperazone sodium and sulbactam sodium + linoleic acid group. Each group had three biological replicates. The samples were incubated at 37°C and 200 rpm for 10 hours, and then the bacterial cells were collected by centrifugation and washed multiple times to remove residual antibiotics in the culture medium. The bacterial cells were suspended in 1x M9 basic medium (containing 10 mM acetate, 1 M CaCl2), and the OD value of the bacterial suspension was adjusted to 1.0. 10 mL of the bacterial suspension was collected by centrifugation. After adding 350 μL of ultrasonic cracking solution containing 2% SDS (ultrasonic cracking was performed on ice, with an ultrasonic cracking power of 35%, ultrasonic cracking for 2 s and stopping for 3 s, and the ultrasonic cracking time was 25 min), the supernatant was collected by centrifugation, and the bacterial cells that might remain in the supernatant were removed by filtration, thereby obtaining the ultrasonic cracking solution of the control group, the cefoperazone sodium and sulbactam sodium group, and the cefoperazone sodium and sulbactam sodium + linoleic acid group.

[0101] Preparation of test strains: Staphylococcus aureus ATCC17978 was incubated overnight to saturation, diluted with 1x M9 basic medium (containing 10 mM acetate, 1 M CaCl2) to an OD600 of 0.2, and then diluted 10,000 times.

[0102] Preparation of standard curve: 100 μL of the prepared test strain diluent was added with 20 μL of cefoperazone sodium and sulbactam sodium to obtain a final concentration of 0, 10, 20, 40, 60, and 80 ng / mL, respectively. After mixing the samples, they were incubated at 37°C and 200 rpm for 10 hours on a constant temperature shaker, and the viable cell count was determined by plating. The standard curve was plotted with the bacterial cell count as the vertical coordinate and the antibiotic concentration as the horizontal coordinate.

[0103] Determination of intracellular antibiotic concentration of bacteria: 100 μL of the prepared test strain diluent was added with 20 μL of the prepared intracellular antibiotic sample, namely the control group ultrasonic cracking solution, the cefoperazone sodium and sulbactam sodium group ultrasonic cracking solution, and the cefoperazone sodium and sulbactam sodium + linoleic acid group ultrasonic cracking solution. After mixing, the samples were incubated at 37°C and 200 rpm for 10 hours, and the viable cell count was determined by plating. Subsequently, the concentration of antibiotics in the samples was calculated according to the standard curve formula of cefoperazone sodium and sulbactam sodium and the bacterial count.

[0104] The results are shown in Figure 10 As shown in the figure, after adding linoleic acid based on the addition of antibiotics, the intracellular antibiotic content increased significantly. Compared with the addition of antibiotics alone, the amount of intracellular antibiotics increased by 9.8 times after the addition of linoleic acid. The results show that linoleic acid can promote the content of antibiotics entering the bacterial cells.

[0105] The above test results show that after adding linoleic acid exogenously, the cell membrane permeability of the bacteria increases, and the amount of antibiotics entering the intracellular bacteria increases. This indicates that linoleic acid increases the permeability of the bacterial membrane, thereby increasing the intracellular content of antibiotics, thereby promoting the sensitivity of bacteria to antibiotics.

[0106] Example 8 Linoleic acid can delay the post-antibiotic effect (PAE) of cefoperazone sodium and sulbactam sodium Taking Staphylococcus aureus as a representative of gram-positive bacteria and cefoperazone sodium and sulbactam sodium as a representative of antibiotics, the effect of linoleic acid combined with antibiotics was further explored.

[0107] Taking three clinical strains MSSA 2, MRSA 7 and MRSA 12 as representatives. Single bacterial colony was picked and inoculated in a 250 mL conical flask containing 50 mL LB and incubated at 37°C, 200 rpm overnight. Then the bacteria were transferred to 5 mL LB test tubes at a ratio of 1:100. When the bacteria grew to OD 600 0.2, the bacteria were collected by centrifugation and washed with physiological saline three times. The bacteria were resuspended with 5 mL MHB, and 500 μL of the above bacterial solution was added to a test tube containing 4.5 mL MHB. Each strain was divided into 5 groups: MHB control group, 1 × MIC SCF, 1 × MIC SCF + 1 mM linoleic acid, 2 × MIC SCF, 2 × MIC SCF + 1 mM linoleic acid. Each treatment had 3 biological replicates. Incubate at 37°C, 200 rpm for 2 hours. Take 100 μL of the above bacterial solution and dilute it 1000 times, then take 100 μL and add it to a 4.9 mL MHB test tube. The diluted bacterial solution was incubated at 37°C, 200 rpm. At 0, 1, 2, 4, 6, 8, 12 hours, 100 μL of the bacterial solution was taken and diluted, and then the viable bacterial count was detected by plating. All strains had 3 biological replicates. Then take the time point as the horizontal coordinate, and the average value of the logarithm of the number of colonies corresponding to it as the vertical coordinate, to construct the bacterial growth curve.

[0108] Then calculate the PAE by the bacterial growth curve, the formula is as follows: PAE = T-C, where T is the time required for the viable bacterial count in the test culture to increase by 1 Log10 CFU observed immediately after dilution, and C is the corresponding time of the control group without exposure to antibiotics. For this test, the PAE of SCF alone (i.e. PAE SCF ), the PAE of SCF + linoleic acid (PAE SCF+亚油酸 ), by comparing PAE SCF and PAE SCF+亚油酸 , to determine whether the exogenous addition of linoleic acid will prolong the PAE of the antibiotic SCF.

[0109] The growth curve results were plotted as Figure 11 The PAE of the three strains under the treatment of cefoperazone sodium and sulbactam sodium alone and linoleic acid + cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC were obtained through further calculation. The PAE of the cefoperazone sodium and sulbactam sodium group in the table, namely the SCF group, is the result of comparison between the addition of the antibiotic and the control group; the PAE of the linoleic acid + cefoperazone sodium and sulbactam sodium in the table, namely the SCF + linoleic acid group, is the result of comparison between the addition of linoleic acid + cefoperazone sodium and sulbactam sodium and the addition of the antibiotic alone. The results are shown in Table 5. The specific conditions are described as follows: Table 5 PAE (hours) of three strains of Staphylococcus aureus under the treatment of SCF and SCF + linoleic acid for 2 hours

[0110] For the MSSA 2 strain, the PAE of cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC was 0.15 and 2.11 hours, respectively. The PAE of the SCF + linoleic acid group was prolonged by 2.01 and 2.82 hours, respectively, compared with the PAE of the antibiotic group.

[0111] For the MRSA 7 strain, the PAE of cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC was 1.71 and 3.51 hours, respectively. The PAE of the SCF + linoleic acid group was prolonged by 3.44 and 4.06 hours, respectively, compared with the PAE of the antibiotic group.

[0112] For the MRSA 12 strain, the PAE of cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC was 0.96 and 1.04 hours, respectively. The PAE of the SCF + linoleic acid group was prolonged by 1.52 and 2.37 hours, respectively, compared with the PAE of the antibiotic group.

[0113] Overall, linoleic acid can prolong the PAE of SCF, and the prolongation time varies among different strains.

[0114] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. The application of linoleic acid in the preparation of antibiotic synergists, characterized in that: The antibiotic is selected from tetracyclines, carbapenems, glycopeptides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a compound preparation containing one or more of the antibiotics; and the penicillins do not include ampicillin.

2. The application according to claim 1, characterized in that The linoleic acid increases the sensitivity of Gram-positive bacteria to antibiotics.

3. The use of linoleic acid in combination with antibiotics in the preparation of a drug for increasing the sensitivity of Gram-positive bacteria to antibiotics, characterized in that: The antibiotic is selected from tetracyclines, carbapenems, glycopeptides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a compound preparation containing one or more of the antibiotics; and the penicillins do not include ampicillin.

4. Use of linoleic acid in combination with antibiotics in the preparation of a medicament for preventing and / or treating Gram-positive bacterial infection, characterized in that: The antibiotic is selected from tetracyclines, carbapenems, glycopeptides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a compound preparation containing one or more of the antibiotics; and the penicillins do not include ampicillin.

5. The use according to any one of claims 1 to 4, characterized in that: The antibiotic is selected from tetracycline, meropenem, vancomycin, sulfadiazine or amoxicillin sodium and clavulanate potassium.

6. The use according to any one of claims 2 to 4, characterized in that: The Gram-positive bacteria are selected from one or more of Staphylococcus aureus, Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis.

7. A drug for increasing the sensitivity of Gram-positive bacteria to antibiotics, characterized in that: Contains an effective amount of linoleic acid and an antibiotic; the antibiotic is selected from tetracyclines, carbapenems, glycopeptides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a compound preparation containing one or more of the antibiotics; and the penicillins do not include ampicillin.

8. The medicine according to claim 7, characterized in that The antibiotic is selected from tetracycline, meropenem, vancomycin, sulfadiazine or amoxicillin sodium and clavulanate potassium.

9. The drug according to claim 7 or 8, characterized in that The mixing ratio of the linoleic acid and the antibiotic is 1: (2-40000) g / mol.

10. The drug according to claim 9, characterized in that The mixing ratio of the linoleic acid and the antibiotic is 1: (50-40000) g / mol.

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