Use of linoleic acid in preparation of drug for improving sensitivity of gram-positive bacteria to antibiotic

Abstract

The present invention belongs to the technical field of biomedicine, and relates to use of linoleic acid in the preparation of a drug for improving the sensitivity of Gram-positive bacteria to an antibiotic. It has been discovered for the first time that the combined use of linoleic acid and an antibiotic can significantly improve the sensitivity of Gram-positive bacteria to the antibiotic. In vivo study data have shown that the combination of linoleic acid and the antibiotic can not only improve the survival rate of mice infected with Gram-positive bacteria, but also enhance the ability of the mice to clear Gram-positive bacteria. Furthermore, formulating linoleic acid and the antibiotic into an anti-infection composition can, in one aspect, achieve a significant clinical anti-infection effect, and in another aspect, prolong the post-antibiotic effect. Moreover, linoleic acid is an essential fatty acid in human and animal nutrition, with high nutritional value, and it has been widely used in food and pharmaceutical products, demonstrating high safety.

Description

Use of linoleic acid in the preparation of a drug for improving the sensitivity of gram-positive bacteria to antibiotics TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to the use of linoleic acid in the preparation of a drug for improving the sensitivity of gram-positive bacteria to antibiotics. BACKGROUND

[0002] Staphyloccocus aureus Rosenbach is a common pathogenic bacterium in clinic, which often causes community and hospital infections, and the infections caused by it rank second, only next to Escherichia coli. The β-lactam drugs represented by penicillin and cephalosporin are one of the important drugs for treating Staphyloccocus aureus infections in clinic. However, due to the wide use of β-lactam drugs in clinic, especially the increase of the detection rate of methicillin-resistant Staphyloccocus aureus (MRSA), the multiple drug resistance characteristics of which to antibacterial drugs, the clinical efficacy of β-lactam drugs is greatly limited.

[0003] Therefore, under the current situation of increasing drug resistance of gram-positive bacteria including MRSA, and increasing difficulty in the development of new antibacterial drugs, it is of great significance to find effective antibacterial drug synergists, and to restore the sensitivity of multiple drug-resistant bacteria including MRSA to the existing key antibacterial drugs through reasonable combination drug strategy, to improve the clinical efficacy of antibacterial drugs and delay the generation of drug resistance. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the defects and deficiencies that gram-positive bacteria have developed resistance to existing antibiotic drugs, and the increasing difficulty in the development of new antibacterial drugs, to provide the use of linoleic acid in the preparation of an antibiotic synergist, which 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.

[0005] The purpose of the present application is to provide the use of linoleic acid in combination with antibiotics in the preparation of a drug for improving the sensitivity of gram-positive bacteria to antibiotics.

[0006] Another purpose of the present application is to provide the use of linoleic acid in combination with antibiotics in the preparation of a drug for preventing or / and treating gram-positive bacterial infections.

[0007] Still another purpose of the present application is to provide a drug for improving the sensitivity of gram-positive bacteria to antibiotics.

[0008] The above-mentioned object of the present application is achieved by the following technical solutions.

[0009] The present application protects the use of linolic acid in the preparation of antibiotic synergists, the antibiotics are 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 above-mentioned antibiotics; and the penicillin antibiotics do not include ampicillin.

[0010] Linolic acid is an important unsaturated fatty acid, which is an essential fatty acid for 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 found that linolic acid can significantly improve the sensitivity of gram-positive bacteria to antibiotics when used in combination with the above-mentioned antibiotics; in vivo research data show that linolic acid combined with the above-mentioned 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 anti-infection composition of linolic acid + antibiotic can not only achieve significant clinical anti-infection effect, but also prolong the post-effect of antibiotic (Postantibiotic Effect, PAE refers to the effect that the inhibition of microorganisms still maintains for a period of time after the serum concentration of antibiotic decreases to below the minimum inhibitory concentration or disappears after contacting the antibiotic); and linolic acid is an essential fatty acid for human and animal nutrition, 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 improving the sensitivity of gram-positive bacteria to antibiotics, the antibiotics are 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 above-mentioned antibiotics; and the penicillin antibiotics do not include ampicillin.

[0013] The present application also protects the use of linolic acid in the preparation of preventing or / and treating gram-positive bacterial infection, the antibiotics are 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 above-mentioned antibiotics; and the penicillin antibiotics do 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 (penicillins + 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, 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 the antibiotic to kill the Gram-positive bacteria.

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

[0023] Further, the linoleic acid as an antibiotic synergist enhances the resistance of the body after infection with gram-positive bacteria.

[0024] Further, the linoleic acid as an antibiotic synergist increases the content of antibiotics into cells. The exogenous addition of linoleic acid can improve the permeability of the bacterial membrane of gram-positive bacteria, thereby increasing the content of antibiotics into cells, thus promoting the sensitivity of bacteria to antibiotics.

[0025] Further, the linoleic acid as an antibiotic synergist delays the aftereffect of gram-positive bacteria on antibiotics.

[0026] Further, the linoleic acid as an antibiotic synergist delays the aftereffect of gram-positive bacteria on cefoperazone sodium and sulbactam sodium.

[0027] The present application also protects a medicine for improving 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, penicillin antibiotics or pharmaceutically acceptable salts thereof or a complex preparation containing one or more of the antibiotics; and the penicillin antibiotics do not include ampicillin.

[0028] Preferably, the mixing ratio of the linoleic acid and the 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 the 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 the combination of linoleic acid and antibiotics significantly improves the sensitivity of gram-positive bacteria to antibiotics; in vivo study data show that the combination of linoleic acid and antibiotics not only improves the survival rate of mice infected with gram-positive bacteria, but also improves 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 is a graph of the PCR amplification results of the clinical Staphylococcus aureus gene in Example 1.

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

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

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

[0035] Figure 5 is a graph showing the results of the survival rate (A) and clearance ability (B) of mice infected with clinical Staphylococcus aureus that can be improved by linoleic acid in Example 3.

[0036] Figure 6 is a graph showing the results of the sensitivity of clinical Staphylococcus aureus (A) and other gram-positive bacteria (B) to cefoperazone sodium sulbactam sodium that can be improved by linoleic acid, and the sensitivity of Staphylococcus aureus to other antibiotics (C) that can be improved by linoleic acid in Example 4.

[0037] Figure 7 is a graph showing the results of the concentration effect of linoleic acid (A), antibiotic concentration (B), and time gradient (C) on the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium sulbactam sodium that can be improved by linoleic acid in Example 5.

[0038] Figure 8 is a graph showing the results of the survival rate (A) and clearance ability (B) of mice infected with clinical Staphylococcus aureus that can be improved by linoleic acid in combination with cefoperazone sodium sulbactam sodium in Example 6.

[0039] Figure 9 is a graph showing the results of the permeability of clinical Staphylococcus aureus sensitive strain MSSA (A) and drug-resistant strain MRSA (B) that can be improved by adding linoleic acid in Example 7.

[0040] Figure 10 is a graph showing the results of the antibiotic content into clinical Staphylococcus aureus that can be improved by adding linoleic acid in Example 7.

[0041] Figure 11 is a graph showing the results of the post-effect of clinical Staphylococcus aureus on cefoperazone sodium sulbactam sodium that can be delayed by linoleic acid in Example 8. Embodiments of the present application

[0042] The present application is further illustrated by the following description and specific examples, which are not intended to limit the application in any manner. Unless otherwise specified, the reagents, methods, and apparatus employed in the present application are of a type commonly employed by those skilled in the art.

[0043] Example 1 Analysis of antibiotic resistance of clinical Staphylococcus aureus

[0044] Nine strains of sensitive Staphylococcu saureus (MSSA) and 20 strains of Methicillin-resistant Staphylococcu saureus (MRSA) were obtained from clinic, named MSSA 1-9 and MRSA 1-20.

[0045] 1.1 Drug resistance determination of clinical strains

[0046] Twenty-nine strains of Staphylococcu saureus were cultured overnight, then diluted 1:100 to culture the bacteria to OD600 of 0.5, and then diluted 100 times, and the minimum inhibitory concentration (MIC) of these strains to Datomycin (DAP), Vancomycin (VAN), Cefoperazone sodium and sulbactam sodium (SCF) and Oxacillin (OX) was determined according to the Microbial Drug Sensitivity Test Scheme provided by Clinical and Laboratory Standards Institute (CLSI). The results are shown in Table 1.

[0047] Table 1 Minimum inhibitory concentration (MIC) of four antibiotics to 29 strains of Staphylococcu saureus

[0048]

[0049] Note: For OX, MIC≤2 is sensitive bacteria; for DAP: MIC≤1 is sensitive bacteria; for VAN: MIC≤2 is sensitive bacteria.

[0050] As can be seen from Table 1, the above 20 strains of MRSA strains are resistant to the four antibiotics tested, which also shows that the 20 strains of bacteria are not only methicillin-resistant bacteria, but also multiple drug-resistant bacteria (Table 1, Table A), and the 9 strains of MRSA are sensitive to the four antibiotics (Table 1, Table B).

[0051] 1.2 Gene identification of clinical strains

[0052] femB (651bp) is an intrinsic gene of Staphylococcu saureus, mecA (310bp) is a specific gene of methicillin-resistant strains, and PVL is an exotoxin-leukocidin produced by Staphylococcu saureus, which damages the defense barrier and immune response of the body by destroying leukocytes and phagocytes.

[0053] The clinical strains were cultured overnight, and the three genes of each strain were amplified by colony PCR (the primer sequences are shown in Table 2), and the amplification products were detected by gel electrophoresis. The results are shown in Figure 1. As can be seen from the figure, for the MRSA strains, each resistant strain can amplify the three genes, indicating that it is indeed Staphylococcus aureus, and is a methicillin-resistant strain, and all have toxicity. For sensitive strains, each strain can amplify femB, but not mecA, indicating that these strains are indeed Staphylococcus aureus, and are sensitive. However, some strains have exotoxin genes, and some do not.

[0054] Table 2 Primer sequences

[0055]

[0056] Example 2 Linoleic acid can kill Staphylococcus aureus of clinical origin

[0057] 2.1 Preparation of strain samples

[0058] Single colonies of clinical bacteria were picked from solid LB plates and inoculated into 5 mL of LB liquid medium, which was incubated 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 and 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.

[0059] 2.2 Linoleic acid can kill Staphylococcus aureus of clinical origin and has a linoleic acid concentration gradient effect

[0060] According to 2.1 in Example 2, prepare samples of clinical sensitive strain MSSA 2 and clinical resistant strain MRSA 7. Add linoleic acid in the test tubes to make the final concentration 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mM, respectively. Each concentration has 3 biological replicates. Incubate at 37°C and 200 rpm for 10 hours. Detect the viable cell count using plate method, and then calculate the survival rate of bacteria under different linoleic acid concentrations. The calculation formula is: survival rate (%) = (viable cell count after adding linoleic acid / viable cell count without adding linoleic acid) x 100%. The results are shown in Figure 2. As can be seen from the figure, whether it is a sensitive strain or a resistant strain, after the addition of linoleic acid, the survival rate of the bacteria is significantly reduced. And with the increase of linoleic acid concentration, the bactericidal efficiency gradually increases. The specific situation is:

[0061] For the clinically sensitive bacteria MSSA 2 (as shown in Figure 2, A), when 0.01 mM of linoleic acid was added, it played a better killing effect, and the bactericidal effect was increased by 8.07 times (the survival rate decreased 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.

[0062] For the clinically resistant bacteria MRSA 7 (as shown in Figure 2, B), when it was 0.01 mM, the bactericidal effect was also increased by 7.14 times (the survival rate decreased 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.

[0063] Among them, when the concentration of linoleic acid was 1 mM, the bactericidal efficiency of sensitive bacteria and resistant bacteria increased by 1925.93 times and 1257.24 times respectively. Further increasing 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 study.

[0064] This result shows that linoleic acid can kill clinically derived Staphylococcus aureus, including sensitive strains and resistant strains, and has a gradient effect of linoleic acid concentration.

[0065] 2.3 Linoleic acid killing clinically Staphylococcus aureus has time dependence

[0066] MSSA 2 and MRSA 7 samples were prepared according to 2.1 in Example 2, 1 mM of linoleic acid was added in the test tube, and the control was without linoleic acid, and the viable cell count was performed at different times, and the survival rate was calculated to study the relationship between bactericidal efficiency and time.

[0067] The results are shown in Figure 3. As can be seen from the figure, after the addition of linoleic acid, the number of bacteria of clinically MSSA 2 and MRSA 7 did not decrease within 2 hours, and then the number of bacteria decreased obviously with the extension of time, showing time effect. The specific situation is as follows:

[0068] For the drug-resistant bacteria MRSA 7 (Figure 3, panel A), the number of bacteria was reduced by 15 times in 4 hours, and then the number of bacteria was reduced by 137.67-13641.57 times in 6-12 hours. For the sensitive bacteria MSSA 2 (Figure 3, panel B), the number of bacteria was reduced by 12.8 times in 4 hours, and then the number of bacteria was reduced by 216.15-16818.73 times in 6-12 hours.

[0069] The above results show that the killing of clinical Staphylococcus aureus by linoleic acid is time-dependent. 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 in the follow-up test.

[0070] 2.4 Linoleic acid killing of clinical Staphylococcus aureus is universal

[0071] The remaining 8 sensitive bacteria and 19 drug-resistant bacteria samples were prepared according to 2.1 in Example 2. Each strain of bacteria was divided into two groups, M9 control group and 1 mM linoleic acid test group. After incubation at 37°C for 10 hours at 200 rpm, viable cell counting was performed, and the survival rate of each strain after the addition of linoleic acid was calculated. Each strain was biologically repeated three times.

[0072] The results are shown in Figure 4. As can be seen from the figure, linoleic acid can kill all clinical sensitive and drug-resistant bacteria, but the degree of increased sensitivity is different for different strains, which is described as follows:

[0073] For 8 sensitive bacteria (Figure 4, panel A), except for the MSSA1 strain, the bactericidal ratio after the addition of linoleic acid was only 81.97 times, and the bactericidal ratio of the remaining strains was 1925.93-98846.78 times.

[0074] For 19 drug-resistant bacteria (Figure 4, panel B), the bactericidal ratio was 7.39-14918.69 times. Among them, 3 strains with a bactericidal ratio less than 100 were strains 11, 12 and 15; 5 strains with a bactericidal ratio greater than 100 but less than 1000 were strains 8, 13, 14, 16 and 17; and the remaining 11 strains with a bactericidal ratio greater than 1000 were strains 1-6, 9, 10, 18-20.

[0075] Example 3 Linoleic acid can improve the resistance of mice to clinical Staphylococcus aureus infection

[0076] 3.1 Linoleic acid can improve the survival rate of mice to clinical Staphylococcus aureus infection

[0077] The balb / c mice (6-8 weeks, about 20 grams, half male and half female) were divided into 3 groups, 16 in each group, namely the normal saline control group, the antibiotic treatment group and the linoleic acid test group. The challenge strain was drug-resistant MRSA 7, and the infection dose was 1.2x108 CFU, which was injected intraperitoneally. One hour after bacterial infection, the mice in the treatment group were given cefoperazone sodium sulbactam sodium by intravenous injection at a dose of 200 mg / kg; the mice in the experimental group were injected with linoleic acid by intraperitoneal injection at a dose of 100 mg / kg. The death of the mice was observed and recorded every day, 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 x 100%.

[0078] The experimental results are shown in Figure 5A. As can be seen from the figure, in the normal saline control group, 68% died on the first day and all died on the second day; in the antibiotic treatment group, 31% died on the first day and 62% died on the second day, and there was no more death thereafter, with a survival rate of 7%; in the linoleic acid test group, there was no death on the first day, 7% died on the second day, and there was no more death thereafter, with a survival rate of 93%. Compared with the control group, the protection rate of the mice after injection of linoleic acid increased by 93%, and compared with the antibiotic treatment group, the protection rate of the mice after injection of linoleic acid increased by 86%.

[0079] The results show that when the mice are infected with methicillin-resistant bacteria, linoleic acid can significantly improve the resistance of mice to methicillin-resistant bacteria infection under the condition that antibiotic treatment is basically ineffective.

[0080] 3.2 Linoleic acid can improve the clearance of mice to clinical Staphylococcus aureus infection

[0081] The balb / c mice (6-8 weeks, about 20 grams, half male and half female) were divided into 2 groups, 6 in each group, namely the control group and the linoleic acid test group. The challenge strain was methicillin-resistant Staphylococcus aureus MRSA 7, and the infection dose was 1.4x106 CFU, which was injected intraperitoneally. One hour after bacterial infection, linoleic acid was administered intraperitoneally at a dose of 100 mg / kg. After 6 hours, equal weights of internal organs (liver, spleen, kidney) were ground thoroughly, then diluted and plated, and the number of bacteria in the organs was counted.

[0082] The results are shown in Figure 5B. As can be seen from the figure, when the mice are infected with drug-resistant bacteria, if linoleic acid is given, the bacterial load in the spleen, kidney and liver of the mice decreases by 1563 times, 1009 times and 4419 times, respectively. The results show that linoleic acid can significantly improve the clearance of mice to clinical Staphylococcus aureus infection.

[0083] Example 4 Linoleic acid can improve the sensitivity of gram-positive bacteria to antibiotics

[0084] 4.1 Linoleic acid can improve the susceptibility of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium

[0085] The results of 2.4 in Example 2 found that linoleic acid had a killing effect on Staphylococcus aureus of clinical origin. However, it was also found that different strains had different bactericidal effects, and some strains had a bactericidal effect less than 1000 times. Since bacterial infections are still treated with antibiotics, can linoleic acid synergize with antibiotics to improve the susceptibility of the strain to antibiotics? For this purpose, all strains MRSA 8, 11-17 and MSSA 1 in 2.4 of Example 2 with a linoleic acid bactericidal effect less than 1000 times were selected, and strains MSSA2, MSSA8, MRSA4, 9, 18 with a linoleic acid bactericidal effect greater than 1000 times were also selected, a total of 14 bacteria as objects, to conduct a bactericidal test of linoleic acid synergized with antibiotics.

[0086] 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, and linoleic acid + antibiotic group. The antibiotic was cefoperazone sodium and sulbactam sodium, and the concentration used for each strain was shown in Table 3. The concentration of linoleic acid was 1 mM. After incubation at 37°C for 10 hours at 200 rpm, the viable cell count was detected by plate method, 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 antibiotics / viable cell count of the control group) x 100%.

[0087] The results are shown in Figure 6A. As can be seen from the figure, when these strains were added with antibiotics alone, there was no bactericidal effect or the bactericidal effect was very weak, with a bactericidal ratio of only 1.07-2.57 times. However, if linoleic acid was added on the basis of antibiotics, the susceptibility of all strains to antibiotics was significantly improved. For strains with a bactericidal effect less than 1000 times when linoleic acid was added alone, the bactericidal ratio was further increased by 7.88-282.44 times on the basis of the original addition of linoleic acid alone. For strains with a bactericidal effect greater than 1000 times when linoleic acid was added alone, the bactericidal ratio was further increased by 1.35-4 times on the basis of the original addition of linoleic acid alone.

[0088] Table 3 Concentration of cefoperazone sodium and sulbactam sodium used in the test

[0089]

[0090] 4.2 Linoleic acid can improve the susceptibility of other gram-positive bacteria to cefoperazone sodium and sulbactam sodium

[0091] 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 determination, of these bacteria is shown in Table 4. Each strain was divided into 4 groups: M9 control group, antibiotic group, linoleic acid group, linoleic acid + antibiotic group. The antibiotic was cefoperazone sodium 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 for 10 hours at 200 rpm, the viable cell count was determined by plating, and then the survival rate of the 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%.

[0092] Table 4. Bacterial drug resistance detection

[0093]

[0094] Note: Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes: penicillin ≤0.25 is sensitive, and there is no standard for resistance and intermediates; cephalosporins ≤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.

[0095] Enterococcus faecalis: penicillin: ≤8 is sensitive, no intermediate has been reported, and ≥16 is resistant; tetracycline: ≤4 is sensitive, 8 is intermediate, and ≥16 is resistant; levofloxacin: ≤2 is sensitive, 4 is intermediate, and ≥8 is resistant; vancomycin: ≤4 is sensitive, 8-16 is intermediate, and ≥32 is resistant.

[0096] Bacillus subtilis: penicillin ≤8 is sensitive, 8-16 is intermediate, and ≥32 is resistant; cephalosporins ≤4 is sensitive, 8 is intermediate, and ≥16 is resistant; tobramycin ≤4 is sensitive, 8 is intermediate, and ≥16 is resistant; tetracycline: ≤4 is sensitive, 8 is intermediate, and ≥16 is resistant; levofloxacin: ≤0.5 is sensitive, 1 is intermediate, and ≥2 is resistant.

[0097] R: resistant; I: intermediate; S: sensitive.

[0098] As can be seen from Table 4, the above-mentioned gram-positive bacteria are resistant to most of the antibiotics tested, and are multi-drug resistant bacteria.

[0099] As shown in B of FIG. 6, these strains have no bactericidal effect or weak bactericidal effect when single antibiotic is added, and the bactericidal multiples are only between 1.1-1.69. However, if linoleic acid is added on the basis of the antibiotic, the sensitivity of all strains to the antibiotic is significantly improved, and the bactericidal multiples of Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecalis are increased by 315.58 times, 3.71 times, 2254.49 times, 77.45 times and 284.59 times, respectively.

[0100] 4.3 Linoleic acid can improve the sensitivity of clinical Staphylococcus aureus to other antibiotics

[0101] The clinical drug-resistant bacteria MRSA 9 sample was prepared according to 2.1 in Example 2, and was divided into 4 groups: M9 control group, linoleic acid group, antibiotic group, linoleic acid + antibiotic group. The concentrations of antibiotics in the test were: vancomycin was 100 μg / mL, and the concentrations of the other antibiotics were all 100 μg / mL; the concentration of linoleic acid was 0.01 mM. After incubation at 37°C for 10 hours at 200 rpm, the viable cell count was detected by plate method, 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%.

[0102] The results are shown in C of FIG. 6. As shown in the figure, when single antibiotic is added, the survival rate of bacteria is 44.82% for vancomycin and 67.24-98.27% for the rest. When single linoleic acid is added, the survival rate of bacteria is 70%. However, if both are used, that is, linoleic acid is added on the basis of the added antibiotic, the sensitivity of the strains to the antibiotic is improved, and the multiples are between 2.82-2826, except for polymyxin, clindamycin and ampicillin.

[0103] These results show that after linoleic acid and cefoperazone sodium sulbactam sodium, or other antibiotics such as tetracycline, meropenem, amikacin, levofloxacin, tobramycin, gentamicin, vancomycin, cefotaxime, azithromycin, amoxicillin sodium clavulanate potassium and sulfadiazine are used together, the bactericidal ability of the antibiotics to these bacteria is enhanced, which indicates that linoleic acid can not only significantly improve the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium sulbactam sodium, but also can improve the sensitivity of these gram-positive bacteria such as Streptococcus, Bacillus subtilis and Enterococcus to cefoperazone sodium sulbactam sodium and other antibiotics including tetracycline, meropenem, amikacin, levofloxacin, tobramycin, gentamicin, vancomycin, cefotaxime, azithromycin, amoxicillin sodium clavulanate potassium and sulfadiazine.

[0104] Example 5 Linoleic acid improves the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium sulbactam sodium

[0105] Staphylococcus aureus as a representative of gram-positive bacteria, cefoperazone sodium sulbactam sodium as a representative of antibiotics, further explore the effect of linoleic acid combined with antibiotics.

[0106] 5.1 With linoleic acid concentration effect

[0107] Prepare the clinical drug-resistant strain MRSA 12 sample according to 2.1 in Example 2, and divide it into 7 groups: an antibiotic control group (200 μg / mL cefoperazone sodium sulbactam sodium), and 6 test groups: on the basis of adding 200 μg / mL cefoperazone sodium sulbactam sodium, add linoleic acid to make its final concentration be 0.01, 0.05, 0.1, 0.5, 1 and 5 mM respectively. Each concentration has 3 biological repeats. Culture at 37°C 200 rpm for 10 hours, detect the viable bacteria number by plate, and then convert it into Log10 value. A decrease of 1 Log10 indicates a 10-fold decrease in bacteria number, and a decrease of 2 Log10 indicates a 100-fold decrease in bacteria number. The results are shown in Figure 7A. As can be seen from the figure, after adding linoleic acid on the basis of adding antibiotics, the number of surviving bacteria decreases. And with the increase of the concentration of linoleic acid added, the number of surviving bacteria decreases more significantly.

[0108] Specifically, when 0.01 mM linoleic acid is added on the basis of adding antibiotics, it can play a good role in improving the sensitivity of antibiotics. Compared with the control group, i.e. adding antibiotics alone, the number of surviving bacteria decreases by nearly 0.5 Log, i.e. 0.5 order of magnitude, which means that the number of bacteria decreases by about 5 times. When the concentration of linoleic acid increases from 0.05 mM to 5 mM, the number of surviving bacteria decreases by 1.32, 1.97, 2.86, 3.28 and 3.73 orders of magnitude, respectively, which means that the number of bacteria decreases from tens of times to thousands of times.

[0109] 5.2 With antibiotic concentration effect

[0110] Prepare the clinical drug-resistant strain MRSA 12 sample according to 2.1 in Example 2, and divide it into 6 groups: a linoleic acid control group (1 mM), and 5 test groups: on the basis of adding 1 mM linoleic acid, add cefoperazone sodium sulbactam sodium to make its final concentration be 10, 50, 100, 200 and 400 μg / mL respectively. Each concentration has 3 biological repeats. Culture at 37°C 200 rpm for 10 hours, detect the viable bacteria number by plate, and then convert it into Log10 value. The results are shown in Figure 7B. As can be seen from the figure, after adding antibiotics on the basis of adding linoleic acid, the number of surviving bacteria decreases. And with the increase of the concentration of antibiotics added, the number of surviving bacteria decreases more significantly.

[0111] Specifically, when 10 μg / mL of antibiotics was added to linoleic acid, the antibiotic sensitivity was improved, and the number of surviving bacteria was reduced by 0.59 Log, i.e., 0.59 orders of magnitude, or about 6 times, compared with the control group, i.e., 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, i.e., the number of bacteria was reduced by nearly 200 times, to more than 10,000 times.

[0112] 5.3 Time effect

[0113] The clinical drug-resistant strain MRSA 12 sample was prepared according to 2.1 in Example 2 and was divided into three 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. Culturing was performed at 37°C and 200 rpm, and sampling was performed every 2 hours within 12 hours, and the number of viable bacteria was detected by plating, and then converted into Log10 values. Each test sample had three biological replicates. The results are shown in FIG. 7, C, which shows that the number of bacteria in the M9 control group remained stable within 12 hours; the number of bacteria in the antibiotic group and the linoleic acid + antibiotic group gradually decreased as time increased; and, importantly, the number of surviving bacteria in the linoleic acid + antibiotic group was significantly reduced compared with the number of surviving bacteria in the antibiotic group at the same time point. Moreover, the number of surviving bacteria decreased more significantly as time increased.

[0114] Specifically, at 2 hours, the number of surviving bacteria in the linoleic acid + antibiotic group was reduced by 0.67 orders of magnitude compared with the number of surviving bacteria in the antibiotic group, i.e., the number of bacteria was reduced by about 7 times. From 4 to 12 hours, the number of surviving bacteria in the linoleic acid + antibiotic group was reduced by 1.21-2.96 orders of magnitude, i.e., the number of bacteria was reduced by about 10-1,000 times.

[0115] The above test results show that linoleic acid can synergize with cefoperazone sodium sulbactam sodium to improve the sensitivity of drug-resistant Staphylococcus aureus to cefoperazone sodium sulbactam sodium, and has an antibiotic concentration and linoleic acid concentration effect, and also has a time effect.

[0116] Example 6 Linoleic acid synergizes with cefoperazone sodium sulbactam sodium to improve the resistance of mice to clinical Staphylococcus aureus infection

[0117] 6.1 Linoleic acid synergizes with cefoperazone sodium sulbactam sodium to improve the survival rate of mice to clinical Staphylococcus aureus infection

[0118] Staphylococcus aureus as a representative of gram-positive bacteria, cefoperazone sodium sulbactam sodium as a representative of antibiotics, further explore the role of linoleic acid combined with antibiotics.

[0119] The balb / c mice (6-8 weeks, about 20 grams, half male and half female) were divided into 4 groups, 16 in each group, respectively, saline control group, antibiotic treatment group, linoleic acid test group, antibiotic + linoleic acid test group. The challenge strain is MRSA 12, the infection dose is 1.2x108 CFU, and the abdominal injection method is used. One hour after bacterial infection, the antibiotic treatment group of mice was given intravenous injection of cefoperazone sodium sulbactam sodium, the dose was 200mg / kg; the linoleic acid test group of mice was given intraperitoneal injection of linoleic acid, 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. The death of mice was observed and recorded daily, and the survival rate of each group was calculated every day, a total of 7 days. The calculation formula is: survival rate (%) = the number of surviving mice in each group / the total number of mice in each group x 100%.

[0120] The experimental results are shown in Figure 8A, from which it can be seen that the saline control group, the first day 81% died, the second day all died; the antibiotic treatment group died 31% on the first day, 62% on the second day, and no longer died thereafter, with a survival rate of 7%; the linoleic acid test group died 7% on the first day, 62% on the second day, 7% on the third day, and thereafter tended to be stable, no longer died, with a survival rate of 24%. The antibiotic + linoleic acid test group had no deaths on the first day, 13% died on the second day, and thereafter tended to be stable, no longer died, with a survival rate of 87%.

[0121] The results show that the linoleic acid test group of mice, if compared with the saline control group, the protection rate increased by 24%, and 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%, and compared with the antibiotic treatment group, the protection rate increased by 80%. It shows that when the mice are infected with methicillin-resistant bacteria, the resistance of the mice to methicillin-resistant bacteria infection is improved to a certain extent under the condition that antibiotic treatment is basically ineffective, but if linoleic acid and antibiotics are used together, the resistance of the mice to methicillin-resistant bacteria infection is significantly improved.

[0122] 6.2 Linoleic acid synergizes with cefoperazone sodium sulbactam sodium to improve the clearance of mice from clinical Staphylococcus aureus infection

[0123] The balb / c mice (6-8 weeks, about 20 grams, half male and half female) were divided into 4 groups, 6 in each group, which were 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 1.4x106 CFU, which was injected intraperitoneally. One hour after bacterial infection, the antibiotic treatment group of mice was given cefoperazone sodium sulbactam sodium by intravenous injection, the dose was 200mg / kg; the linoleic acid test group of mice was injected with 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, the same weight of internal organs (liver, spleen, kidney) were ground thoroughly, then diluted and plated, and the number was counted.

[0124] The results are shown in Figure 8B. As can be seen from the figure, when the mice were infected with drug-resistant bacteria, if treated with antibiotics, the bacterial load in the spleen, kidney and liver of the infected mice decreased by 55 times, 40 times and 33 times respectively; if treated with linoleic acid, the bacterial load in the spleen, kidney and liver of the mice decreased by 20 times, 8 times and 17 times respectively. If treated with linoleic acid and antibiotics, the bacterial load in the spleen, kidney and liver of the mice decreased by 136 times, 111 times and 97 times respectively compared with the antibiotic treatment group; decreased by 51 times, 22 times and 50 times respectively compared with the linoleic acid test group. The results showed that the combination of linoleic acid and antibiotics can significantly improve the clearance of mice against clinical Staphylococcus aureus infection.

[0125] Example 7 Linoleic acid increases the permeability of bacterial membrane to increase the entry of antibiotics into the cell and achieve sensitivity to antibiotics

[0126] Staphylococcus aureus was used as a representative of gram-positive bacteria, and cefoperazone sodium sulbactam sodium was used as a representative of antibiotics to further explore the mechanism of the combination of linoleic acid and antibiotics to improve efficacy.

[0127] 7.1 Exogenous linoleic acid can improve the permeability of bacterial membrane

[0128] Bacteria were prepared according to 2.1 in Example 2. A total of 9 strains of MSSA and 10 strains of MRSA were prepared. Each strain of bacteria was divided into two groups, one group was a control group and the other group was a linoleic acid (1 mM) experimental group. Incubation was performed at 37°C for 10 hours in a 200 rpm shaking incubator. Then 100 μL was taken into 900 μL of M9, and 2 μL of 2.5 mM SYTO9 dye was added. Incubation was performed at 37°C for 45 minutes at 200 rpm, and fluorescence was detected by flow cytometry. The change in membrane permeability was determined by comparing the fluorescence intensity of the test group and the control group. The results are shown in Figure 9. Regardless of whether it was MSSA or MRSA, the membrane permeability of the bacteria was enhanced after the addition of exogenous linoleic acid. This indicates that linoleic acid can increase the membrane permeability of bacteria.

[0129] 7.2 Exogenous linoleic acid can promote the entry of antibiotics into the intracellular content of bacteria

[0130] The concentration of antibiotics entering the intracellular bacteria was determined by detecting the inhibitory effect of antibiotics on microorganisms and calculating the activity (titer) of the antibiotics.

[0131] Sample preparation for determining the intracellular antibiotic concentration of bacteria: MRSA 7 strain samples were prepared according to Example 2.1, and were divided into three groups, namely the control group without adding substances and antibiotics, the cefoperazone sodium and sulbactam sodium group, and the cefoperazone sodium and sulbactam sodium + linoleic acid group. Each group had three biological replicates. Incubation was performed at 37°C for 10 hours at 200 rpm, and then the bacteria were collected by centrifugation and washed multiple times to remove residual antibiotics in the culture medium. The bacteria were suspended in 1x M9 basic medium (containing 10 mM acetate, 1 M CaCl2); the OD value of the bacterial solution was adjusted to 1.0, and 10 mL of the bacterial solution was collected by centrifugation. After adding 350 μL of ultrasonic breaking solution containing 2% SDS (ultrasonic breaking was performed on ice, 35% ultrasonic breaking power, ultrasonic breaking for 2 s and stopping for 3 s, ultrasonic breaking time for 25 min), the supernatant was collected by centrifugation, and the bacteria in the supernatant were removed by filtration. The ultrasonic breaking solution of the control group, the cefoperazone sodium and sulbactam sodium group, and the cefoperazone sodium and sulbactam sodium + linoleic acid group was obtained.

[0132] Preparation of test strains: Staphylococcus aureus ATCC17978 was incubated overnight to saturation, and then diluted with 1x M9 basic medium (containing 10 mM acetate, 1 M CaCl2) to an OD600 of 0.2, and then diluted 10000 times.

[0133] Standard curve preparation: 100 μL of the prepared test strain diluent was taken, and 20 μL of cefoperazone sodium and sulbactam sodium was added to make the final concentration 0, 10, 20, 40, 60, and 80 ng / mL, respectively. After mixing the above samples, incubation was performed at 37°C for 10 hours in a 200 rpm constant temperature shaking incubator, and the number of viable bacteria was detected by plating. The standard curve was drawn with the number of bacterial cells as the vertical coordinate and the concentration of antibiotics as the horizontal coordinate.

[0134] Bacterial intracellular antibiotic concentration determination: 100 μL of prepared test strain diluent was taken, and 20 μL of prepared bacterial intracellular antibiotic sample, i.e. control group ultrabreaking liquid, cefoperazone sodium and sulbactam sodium group ultrabreaking liquid, cefoperazone sodium and sulbactam sodium + linoleic acid group ultrabreaking liquid, was added respectively. After mixing, it was incubated at 37°C, 200 rpm for 10 h, and then the viable cell count was detected by plate method. Subsequently, the concentration of antibiotics in the sample was calculated according to the standard curve formula of cefoperazone sodium and sulbactam sodium and the number of bacteria.

[0135] The results are shown in Figure 10. As can be seen from the figure, after adding linoleic acid on the basis of adding antibiotics, the intracellular antibiotic content increased significantly. Compared with adding antibiotics alone, the amount of intracellular antibiotic increased by 9.8 times after adding linoleic acid. The results show that linoleic acid can promote the content of antibiotics entering the bacterial intracellular.

[0136] The above test results show that after adding exogenous linoleic acid, the cell membrane permeability of bacteria increases, and the amount of antibiotics entering the bacterial intracellular also increases. This shows that linoleic acid promotes the sensitivity of bacteria to antibiotics by increasing the permeability of bacterial membrane and thus increasing the intracellular content of antibiotics.

[0137] Example 8 Linoleic acid can delay the post-antibiotic effect (PAE) of cefoperazone sodium and sulbactam sodium

[0138] Staphylococcus aureus was used as a representative of gram-positive bacteria, and cefoperazone sodium and sulbactam sodium were used as a representative of antibiotics to further explore the effect of linoleic acid combined with antibiotics.

[0139] Three strains of MSSA 2, MRSA 7 and MRSA 12 were used as representatives. Single colony of bacteria was picked and inoculated into 50 mL LB in a 250 mL conical flask, incubated at 37°C, 200 rpm overnight, then 1:100 transferred into 5 mL LB test tube, when the bacteria grew to OD600 of 0.2, the bacteria were collected by centrifugation and washed with normal saline for three times. The bacteria were resuspended with 5 mL MHB, and 500 μL of the bacteria solution was added into 4.5 mL MHB test tube. 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. Incubated at 37°C, 200 rpm for 2 hours. 100 μL of the above bacteria solution was diluted 1000 times, and then 100 μL was added into 4.9 mL MHB test tube. The diluted bacteria solution was incubated at 37°C, 200 rpm, and 100 μL of the bacteria solution was taken at 0, 1, 2, 4, 6, 8, 12 hours, respectively, and then diluted and detected by plate counting. All strains had 3 biological replicates. Then the time point was taken as the horizontal coordinate, and the average value of the logarithm of the number of bacteria was taken as the vertical coordinate, to construct the bacterial growth curve.

[0140] Then the PAE was calculated by the bacterial growth curve, and the formula was as follows: PAE = T-C, wherein T was the time required for the count of viable bacteria in the test culture to increase by 1 Log10 CFU from the count observed immediately after dilution, and C was the corresponding time of the control group without exposure to antibiotics. For this test, the PAE of SCF alone (PAESCF) and the PAE of SCF + linoleic acid (PAESCF + linoleic acid) were calculated, and whether the exogenous addition of linoleic acid would prolong the PAE of SCF, the antibiotic, was determined by comparing PAESCF and PAESCF + linoleic acid.

[0141] The growth curve results were plotted in FIG. 11. Through further calculation, the PAE of the three strains under the conditions of 1 × MIC and 2 × MIC of cefoperazone sodium and sulbactam sodium alone and linoleic acid + cefoperazone sodium and sulbactam sodium was obtained. The PAE of the cefoperazone sodium and sulbactam sodium group in the table, i.e. the SCF group, was the result of adding the antibiotic compared with the control group; the PAE of the linoleic acid + cefoperazone sodium and sulbactam sodium in the table, i.e. the SCF + linoleic acid treatment, was the result of adding linoleic acid + cefoperazone sodium and sulbactam sodium compared with the antibiotic alone. The results are shown in Table 5. The specific conditions are described as follows:

[0142] Table 5 PAE (hours) of SCF, SCF + linoleic acid exposure for 2 hours on three strains of Staphylococcus aureus

[0143]

[0144] For MSSA 2 strain, the PAE of cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC were 0.15 and 2.11 hours, respectively. The PAE of SCF+linoleic acid was prolonged compared with the PAE of antibiotic group, which were prolonged by 2.01 and 2.82 hours, respectively.

[0145] For MRSA 7 strain, the PAE of cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC were 1.71 and 3.51 hours, respectively. The PAE of SCF+linoleic acid was prolonged compared with the PAE of antibiotic group, which were prolonged by 3.44 and 4.06 hours, respectively.

[0146] For MRSA 12 strain, the PAE of cefoperazone sodium and sulbactam sodium at 1xMIC and 2xMIC were 0.96 and 1.04 hours, respectively. The PAE of SCF+linoleic acid was prolonged compared with the PAE of antibiotic group, which were prolonged by 1.52 and 2.37 hours, respectively.

[0147] Overall, linoleic acid could prolong the PAE of SCF, and the prolonging time was different for different strains.

[0148] The foregoing examples are illustrative only and are not intended to limit the scope of the methods described herein. The appended claims are intended to claim as broad a range as is allowed under the rules. The examples presented herein are presented to demonstrate the application of the application. Accordingly, the applicant intends that the appended claims not be limited to the choice of examples presented herein. Some numerical ranges are presented herein in a range format. It should be understood that this manner of presentation is merely intended to clarify and not to limit the scope of the range. The description of a range format is intended to cover each and every number within the range, including the endpoints.

Claims

1. Use of linoleic acid for the preparation of an antibiotic potentiator, characterized in that, The antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a combination preparation containing one or more of said antibiotics; and the penicillin antibiotic does not include ampicillin.

2. Use according to claim 1, characterized in that, The linoleic acid increases the sensitivity of the gram-positive bacteria to the antibiotic.

3. Use of linoleic acid in combination with an antibiotic for the preparation of a medicament for increasing the sensitivity of Gram-positive bacteria to an antibiotic, characterized in that, The antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a combination preparation containing one or more of said antibiotics; and the penicillin antibiotic does not include ampicillin.

4. Use of linoleic acid in combination with an antibiotic for the preparation of a medicament for the prevention or / and treatment of a Gram-positive bacterial infection, characterized in that, The antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a combination preparation containing one or more of said antibiotics; and the penicillin antibiotic does not include ampicillin.

5. The use according to any one of claims 1 to 4, characterized in that, The antibiotic is selected from cefoperazone sodium sulbactam sodium, cefotaxime, cefoperazone, amikacin, tobramycin, gentamicin, ofloxacin, levofloxacin, tetracycline, meropenem, vancomycin, azithromycin, sulfadiazine or amoxicillin sodium clavulanate potassium.

6. 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, Enterococcus faecalis.

7. A medicament for increasing the sensitivity of Gram-positive bacteria to antibiotics, characterized in that, An effective amount of linoleic acid and an antibiotic; the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillin antibiotics or pharmaceutically acceptable salts thereof or a combination preparation containing one or more of said antibiotics; and the penicillin antibiotic does not include ampicillin.

8. The medicament according to claim 7, characterized in that, The antibiotic is selected from cefoperazone sodium sulbactam sodium, cefotaxime, cefoperazone, amikacin, tobramycin, gentamicin, ofloxacin, levofloxacin, tetracycline, meropenem, vancomycin, azithromycin, sulfadiazine or amoxicillin sodium clavulanate potassium.

9. The medicament 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 medicament 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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