Application of linoleic acid in preparing a medicament for enhancing the antibiotic sensitivity of Gram-positive bacteria

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to the application of linoleic acid in the preparation of a medicament for enhancing the antibiotic sensitivity of Gram-positive bacteria. The present invention discovers for the first time that the combination of linoleic acid and an antibiotic can significantly enhance the antibiotic sensitivity of Gram-positive bacteria; in vivo research data shows that the combination of linoleic acid and an antibiotic can not only improve the survival rate of mice infected with Gram-positive bacteria, but also enhance the ability of mice to eliminate Gram-positive bacteria; further, an anti-infective composition prepared from linoleic acid and an antibiotic can, on the one hand, achieve a significant clinical anti-infective effect, and on the other hand, prolong the post-antibiotic effect; moreover, linoleic acid is an essential fatty acid in human and animal nutrition, has high nutritional value, and has been widely used in foods and drugs, with high safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to the application of linoleic acid in the preparation of a drug for enhancing the sensitivity of Gram-positive bacteria to antibiotics. Background Art

[0002] Staphyloccocus aureus Rosenbach is a common clinical pathogen, often causing community and hospital infections, and the infections it causes rank second, second only to Escherichia coli. Rational antibiotic intervention remains an effective means for treating Staphyloccocus aureus infections. β-lactam drugs represented by penicillin and cephalosporins are one of the important drugs for treating Staphyloccocus aureus infections in clinical practice. However, due to the widespread use of β-lactam drugs in clinical practice, especially the increasing detection rate of methicillin-resistant staphylococcusaureus (MRSA), its characteristic of multi-drug resistance to antibacterial drugs has greatly limited the clinical efficacy of β-lactam drugs.

[0003] Therefore, in the current situation where the drug resistance of Gram-positive bacteria including MRSA is becoming increasingly serious and the research and development of new antibacterial drugs are becoming increasingly difficult, finding effective antibacterial drug synergists and restoring the sensitivity of multi-drug resistant bacteria including MRSA to existing key antibacterial drugs through a reasonable combination drug strategy is of great significance for improving the clinical efficacy of antibacterial drugs and delaying the emergence of drug resistance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies that Gram-positive bacteria have developed drug resistance to existing antibiotic drugs and the research and development of new antibacterial drugs is becoming increasingly difficult, and to provide the application of linoleic acid in the preparation of an antibiotic synergist, wherein the antibiotic can not only improve the survival rate of the body infected with Gram-positive bacteria, but also improve the clearance ability of the body against Gram-positive bacteria.

[0005] The object of the present invention is to provide the application of the combination of linoleic acid and an antibiotic in the preparation of a drug for enhancing the sensitivity of Gram-positive bacteria to antibiotics.

[0006] Another object of the present invention is to provide the application of the combination of linoleic acid and an antibiotic in the preparation of a drug for preventing and / or treating Gram-positive bacterial infections.

[0007] Another object of the present invention is to provide a drug for enhancing the sensitivity of Gram-positive bacteria to antibiotics.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] The present invention protects the use of linoleic acid in the preparation of an antibiotic synergist, wherein the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a combined preparation containing one or more of the above antibiotics; and the penicillins do not include ampicillin.

[0010] Linoleic acid is an important unsaturated fatty acid and an essential fatty acid for the human body. It has the functions of reducing cholesterol, softening blood vessels, and promoting microcirculation, and can prevent the occurrence of various cardiovascular and cerebrovascular diseases. The inventor team first discovered in the present invention that the combination of linoleic acid and the above antibiotics can significantly improve the sensitivity of Gram-positive bacteria to antibiotics; in vivo research data show that the combination of linoleic acid and the above antibiotics (such as cefoperazone sodium and sulbactam sodium) can not only improve the survival rate of the body against Gram-positive bacteria infections, but also improve the ability of the body to clear Gram-positive bacteria. Preparing linoleic acid + antibiotic into an anti-infective composition can, on the one hand, achieve a significant clinical anti-infective effect, and on the other hand, extend the postantibiotic effect (the postantibiotic effect (PAE) of an antibiotic refers to the effect that the inhibitory effect on microorganisms still persists for a period of time after the antibiotic serum concentration drops below the minimum inhibitory concentration or disappears after the bacteria contact the antibiotic); moreover, linoleic acid is an essential fatty acid in human and animal nutrition, has high nutritional value, and has been widely used in food and drugs, with high safety.

[0011] Furthermore, the linoleic acid improves the sensitivity of Gram-positive bacteria to antibiotics.

[0012] The present invention protects the use of the combination of linoleic acid and an antibiotic in the preparation of a drug for improving the sensitivity of Gram-positive bacteria to an antibiotic, wherein the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a combined preparation containing one or more of the above antibiotics; and the penicillins do not include ampicillin.

[0013] The present invention also protects the use of the combination of linoleic acid and an antibiotic in the preparation of a drug for preventing and / or treating Gram-positive bacteria infections, wherein the antibiotic is selected from cephalosporins, aminoglycosides, quinolones, tetracyclines, carbapenems, glycopeptides, macrolides, sulfonamides, penicillins or pharmaceutically acceptable salts thereof, or a combined preparation containing one or more of the above antibiotics; and the penicillins do not include ampicillin.

[0014] Preferably, the antibiotic is selected from cefoperazone sodium and sulbactam sodium (cephalosporin + β-lactamase inhibitor, i.e., a compound preparation), cefotaxime (cephalosporin), cefoperazone (cephalosporin), amikacin (aminoglycoside), tobramycin (aminoglycoside), gentamicin (aminoglycoside), ofloxacin (quinolone), levofloxacin (quinolone), tetracycline (tetracycline), meropenem (carbapenem), vancomycin (glycopeptide), azithromycin (macrolide), sulfadiazine (sulfonamide), or amoxicillin sodium and clavulanate potassium (penicillin + β-lactamase inhibitor, i.e., a compound preparation).

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

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

[0017] Preferably, in the compound preparation, the mass ratio of amoxicillin to clavulanic acid 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 sensitive bacteria and drug-resistant bacteria.

[0020] Further, the sensitive 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 Gram-positive bacteria.

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

[0023] Furthermore, the linoleic acid, as an antibiotic synergist, enhances the resistance of the body after being infected with Gram-positive bacteria.

[0024] Furthermore, the linoleic acid, as an antibiotic synergist, increases the content of antibiotics entering the cells. Exogenous addition of linoleic acid can improve the permeability of the bacterial membrane of Gram-positive bacteria, thereby increasing the content of antibiotics entering the cells, and thus promoting the sensitivity of bacteria to antibiotics.

[0025] Furthermore, the linoleic acid, as an antibiotic synergist, delays the post-antibiotic effect of Gram-positive bacteria.

[0026] Furthermore, the linoleic acid, as an antibiotic synergist, delays the post-antibiotic effect of Gram-positive bacteria on cefoperazone sodium and sulbactam sodium.

[0027] The present invention also protects a drug for increasing the sensitivity of Gram-positive bacteria to antibiotics, which contains an effective amount of linoleic acid and an antibiotic; the antibiotic is 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 above antibiotics; and the penicillins do not include ampicillin.

[0028] Preferably, the mixing ratio of linoleic acid and the antibiotic is 1:(2 - 40000) g / mol. The meaning represented by this mixing ratio is that 2 - 40000 g of the antibiotic and 1 mol of linoleic acid are used in combination.

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

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention first discovers that the combination of linoleic acid and an antibiotic significantly increases the sensitivity of Gram-positive bacteria to the antibiotic; in vivo research data shows 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 improve the ability of mice to clear Gram-positive bacteria; further preparing linoleic acid and the antibiotic into an anti-infective composition can, on the one hand, achieve a significant clinical anti-infective effect, and on the other hand, extend the clinical effect of the antibiotic; moreover, linoleic acid is an essential fatty acid in human and animal nutrition, has high nutritional value, and has been widely used in food and drugs, with high safety. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0032] Figure 2Statistical chart of the results of linoleic acid killing clinical Staphylococcus aureus sensitive bacteria MSSA 2(A) and resistant bacteria MRSA7(B) and the bactericidal effect having a linoleic acid concentration effect in Example 2.

[0033] Figure 3 Statistical chart of the results of the time effect of linoleic acid killing clinical Staphylococcus aureus MRSA 7(A) and MSSA 2(B) in Example 2.

[0034] Figure 4 Statistical chart of the results of linoleic acid killing multiple strains of clinical Staphylococcus aureus sensitive bacteria (A) and resistant bacteria (B) in Example 2.

[0035] Figure 5 Statistical chart of the results of linoleic acid improving the survival rate (A) and clearance ability (B) of mice against clinical Staphylococcus aureus infection in Example 3.

[0036] Figure 6 Statistical chart of the results of linoleic acid improving the sensitivity of clinical Staphylococcus aureus (A) and other Gram-positive bacteria (B) to cefoperazone sodium and sulbactam sodium, and improving the sensitivity of Staphylococcus aureus to other antibiotics (C) in Example 4.

[0037] Figure 7 Statistical chart of the results of the linoleic acid concentration (A), antibiotic concentration (B), and time gradient (C) effects of linoleic acid improving the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium in Example 5.

[0038] Figure 8 Statistical chart of the results of linoleic acid synergistically with cefoperazone sodium and sulbactam sodium improving the survival rate (A) and clearance ability (B) of mice against clinical Staphylococcus aureus infection in Example 6.

[0039] Figure 9 Statistical chart of the results of improving the permeability of clinical Staphylococcus aureus sensitive bacteria MSSA (A) and resistant bacteria MRSA (B) after adding linoleic acid in Example 7.

[0040] Figure 10 Statistical chart of the results of increasing the antibiotic content entering clinical Staphylococcus aureus after adding linoleic acid in Example 7.

[0041] Figure 11 Statistical chart of the results of linoleic acid delaying the post-antibiotic effect of cefoperazone sodium and sulbactam sodium on clinical Staphylococcus aureus in Example 8. Detailed implementation manner

[0042] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

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

[0044] Example 1 Analysis of Antibiotic Resistance of Clinical Staphylococcus aureus

[0045] Nine clinically obtained sensitive Staphylococcus aureus (MSSA) and 20 Methicillin-resistant Staphylococcus aureus (MRSA) were named MSSA 1-9 and MRSA 1-20.

[0046] 1.1 Determination of Drug Resistance of Clinical Strains

[0047] Twenty-nine clinical Staphylococcus aureus strains were cultured overnight. After subculturing at a ratio of 1:100 and culturing the bacteria until the OD600 reached 0.5, they were then diluted 100-fold. According to the microbial susceptibility test protocol provided by the Clinical and Laboratory Standards Institute (CLSI), the minimum inhibitory concentrations (MICs) of these strains against daptomycin (DAP), vancomycin (VAN), cefoperazone sodium and sulbactam sodium (SCF), and oxacillin (OX) were determined. The results are shown in Table 1.

[0048] Table 1 Determination of the Minimum Inhibitory Concentration (MIC) of Four Antibiotics against 29 Strains of Staphylococcus aureus

[0049]

[0050] Note: For OX, MIC ≤ 2 indicates sensitive bacteria; for DAP: MIC ≤ 1 indicates sensitive bacteria; for VAN: MIC ≤ 2 indicates sensitive bacteria.

[0051] As can be seen from Table 1, the above 20 MRSA strains were all resistant to the four tested antibiotics, indicating that these 20 strains were not only methicillin-resistant bacteria but also multi-drug resistant bacteria (Table A in Table 1), while the 9 MSSA strains were sensitive to the four antibiotics (Table B in Table 1).

[0052] 1.2 Gene Identification of Clinical Strains

[0053] femB (651 bp) is an intrinsic gene of Staphylococcus aureus, mecA (310 bp) is a specific gene of methicillin-resistant strains, and PVL is an exotoxin - leukocidin produced by Staphylococcus aureus, which damages the body's defense barrier and immune response by destroying white blood cells and phagocytes.

[0054] Overnight culture of clinical strains, and these three genes of each strain were amplified by colony PCR respectively (the primer sequences for amplification are shown in Table 2), and then the amplification products were detected by gel electrophoresis. The results are shown in Figure 1 , as can be seen from the figure, for MRSA strains, these three genes can be amplified from each drug-resistant strain, indicating that it is indeed Staphylococcus aureus and is a methicillin-resistant strain, and all are toxic. For sensitive strains, femB can be amplified from each strain, but mecA cannot be amplified, indicating that these strains are indeed Staphylococcus aureus and are sensitive bacteria. However, some strains have the exotoxin gene while some do not.

[0055] Table 2 Primer sequences

[0056]

[0057] Example 2 Linoleic acid can kill Staphylococcus aureus from clinical sources

[0058] 2.1 Strain sample preparation

[0059] Single colonies of clinical bacteria were picked from solid LB plates and inoculated into 5 mL of LB liquid medium, and cultured at 37 °C and 200 rpm for 16 hours; the bacterial liquid was collected, centrifuged at 8000 rpm for 5 min, the supernatant was removed, and the cells were washed with an equal volume of 0.85% physiological saline, and finally the cells were suspended in 1×M9 minimal medium (containing 10 mM acetate and 1 M CaCl2); the OD value of the bacterial liquid was adjusted to 0.2, and 5 mL was aliquoted into test tubes for subsequent experiments.

[0060] 2.2 Linoleic acid can kill Staphylococcus aureus from clinical sources and has a linoleic acid concentration gradient effect

[0061] Samples of clinical sensitive strain MSSA 2 and clinical drug-resistant strain MRSA 7 were prepared according to 2.1 in Example 2. Linoleic acid was added to test tubes respectively to make the final concentrations 0, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mM. There were 3 biological replicates for each concentration. Cultured at 37 °C and 200 rpm for 10 hours, the viable cell count was detected by plating, and then the survival rate of bacteria at different linoleic acid concentrations was calculated. The calculation formula is: Survival rate (%) = (viable cell count after adding linoleic acid / viable cell count without adding linoleic acid) × 100%. The results are shown in Figure 2A. As can be seen from the figure, whether it is sensitive bacteria or drug-resistant bacteria, after adding linoleic acid, the survival rate of the bacteria decreases significantly. Moreover, as the concentration of linoleic acid increases, the bactericidal efficiency gradually increases. Specifically:

[0062] For the clinically sensitive bacterium MSSA 2 (as shown in Figure A of Figure 2 ), when 0.01 mM of linoleic acid is added, it can play a good bactericidal role, and the bactericidal effect is increased by 8.07 times compared with the control group (the survival rate drops to 12.39%); when the concentration of linoleic acid gradually increases from 0.05 mM to 10 mM, the increased multiples of bactericidal efficiency are 19.91 times, 129.58 times, 709.17 times, 1925.93 times, 4681.57 times, and 9577.32 times respectively (the survival rate also drops from 5.02% to 0.01%).

[0063] For the clinically drug-resistant bacterium MRSA 7 (as shown in Figure B of Figure 2 ), when it is 0.01 mM, the bactericidal effect is also increased by 7.14 times compared with the control group (the survival rate drops to 14.00%); similarly, when the concentration of linoleic acid gradually increases from 0.05 mM to 10 mM, the increased multiples of bactericidal efficiency are 15 times, 39.47 times, 230.77 times, 1257.24 times, 2675.16 times, and 4199.48 times respectively (the survival rate also drops from 6.67% to 0.02%).

[0064] Among them, when the concentration of linoleic acid is 1 mM, the bactericidal efficiencies of sensitive bacteria and drug-resistant bacteria are increased by 1925.93 times and 1257.24 times respectively. Further increasing the concentration of linoleic acid, the bactericidal multiple also increases. However, since linoleic acid is acidic, when the added amount is too high, it will affect the pH value of the incubation system. Therefore, 1 mM of linoleic acid was selected for the subsequent experiments.

[0065] This result shows that linoleic acid can kill Staphylococcus aureus of clinical origin, including sensitive strains and drug-resistant strains, and has a linoleic acid concentration gradient effect.

[0066] 2.3 The killing of clinical Staphylococcus aureus by linoleic acid is time-dependent

[0067] Prepare MSSA 2 and MRSA 7 samples according to 2.1 in Example 2. Add 1 mM of linoleic acid to test tubes respectively, use no addition of linoleic acid as a control, perform plate viable count at different times, and calculate the survival rate to study the relationship between bactericidal efficiency and time.

[0068] The results are as shown in Figure 3As shown, it can be seen from the figure that for clinical bacteria MSSA 2 and MRSA 7, after adding linoleic acid, the number of bacteria did not decrease within 2 hours, and then the number of bacteria decreased significantly with the extension of time, showing a time effect. The specific situation is as follows:

[0069] For the drug-resistant bacterium MRSA 7 ( Figure 3 Figure A), the number of bacteria decreased by 15 times in 4 hours, and then the reduction multiple of the number of bacteria was 137.67 - 13641.57 times from 6 to 12 hours. For the sensitive bacterium MSSA2 ( Figure 3 Figure B), the number of bacteria decreased by 12.8 times in 4 hours, and then the reduction multiple of the number of bacteria was 216.15 - 16818.73 times from 6 to 12 hours.

[0070] The above results indicate that the killing of clinical Staphylococcus aureus by linoleic acid is time-dependent. Linoleic acid can achieve a good killing effect on clinical Staphylococcus aureus in 10 hours. For the convenience of operation, the subsequent experiments were carried out with 10 hours as the bactericidal time of linoleic acid.

[0071] 2.4 The killing of clinical Staphylococcus aureus by linoleic acid is universal

[0072] Prepare the samples of the remaining 8 sensitive bacteria and 19 drug-resistant bacteria according to 2.1 in Example 2. Each strain of bacteria was divided into 2 groups, namely the M9 control group and the 1 mM linoleic acid test group. After incubation at 37 °C and 200 rpm for 10 hours, viable cell counting was carried out, and the survival rate of each strain of bacteria after adding linoleic acid was calculated. There were 3 biological replicates for each strain.

[0073] The results are shown in Figure 4 , and it can be seen from the figure that linoleic acid can kill all clinical sensitive bacteria and drug-resistant bacteria. However, for different strains, the degree of improving sensitivity is different, and the specific description is as follows:

[0074] For the 8 sensitive bacteria ( Figure 4 Figure A), except for the MSSA1 strain, the bactericidal multiple after adding linoleic acid was only 81.97 times, and the bactericidal multiples of the remaining strains were 1925.93 - 98846.78 times.

[0075] For the 19 drug-resistant bacteria ( Figure 4 Figure B), the bactericidal multiples were 7.39 - 14918.69 times. Among them, there were 3 strains with a bactericidal effect lower than 100 times, namely strains 11, 12, and 15; there were 5 strains greater than 100 and less than 1000 times, namely strains 8, 13, 14, 16, and 17; the remaining 11 strains were greater than 1000 times, namely strains 1 - 6, 9, 10, 18 - 20.

[0076] 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 infected with clinical Staphylococcus aureus

[0077] Balb / c mice (6 - 8 weeks old, about 20 g, half male and half female) were divided into 3 groups, with 16 mice in each group, namely the normal saline control group, the antibiotic treatment group, and the linoleic acid test group. The virulent strain was the drug-resistant strain MRSA7, and the infection dose was 1.2×10 8 CFU, administered by intraperitoneal injection. One hour after bacterial infection of the mice, the mice in the treatment group were administered cefoperazone sodium and 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 daily, and the survival rate of each group was calculated every day for 7 days. The calculation formula is: survival rate = number of surviving mice in each group / total number of mice in each group × 100%.

[0078] The experimental results are shown in Figure 5 Figure A in. 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, 62% died on the second day, and no more died thereafter, with a survival rate of 7%; in the linoleic acid experimental group, no deaths occurred on the first day, 7% died on the second day, and then stabilized and no more died, with a survival rate of 93%. Compared with the control group, the protection rate of the mice increased by 93% after injection of linoleic acid. Compared with the antibiotic treatment group, the protection rate of the mice increased by 86% after injection of linoleic acid.

[0079] This result indicates that when mice are infected with methicillin-resistant bacteria, linoleic acid can significantly improve the resistance of mice to methicillin-resistant bacteria infection when antibiotic treatment is basically ineffective.

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

[0081] Balb / c mice (6 - 8 weeks old, about 20 g, half male and half female) were divided into 2 groups, with 6 mice in each group, namely the control group and the linoleic acid test group. The virulent strain was methicillin-resistant Staphylococcus aureus MRSA7, and the infection dose was 1.4×10 6 CFU, administered by intraperitoneal injection. One hour after bacterial infection, linoleic acid was administered by intraperitoneal injection at a dose of 100 mg / kg. Six hours later, an equal weight of internal organs (liver, spleen, kidneys) was taken and thoroughly ground, then diluted and plated, and the number of bacteria in the organs was counted.

[0082] The results are shown in Figure 5In Figure B, it can be seen from the figure that when mice were infected with drug-resistant bacteria and treated with linoleic acid, the bacterial loads in the spleen, kidney, and liver of the mice decreased by 1563-fold, 1009-fold, and 4419-fold, respectively. The results indicate that linoleic acid can significantly enhance the clearance of clinical Staphylococcus aureus infection in mice.

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

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

[0085] In the 2.4 of Example 2, it was found that linoleic acid had a bactericidal effect on clinical Staphylococcus aureus. However, it was also found that the bactericidal effects on different strains were different, and the bactericidal effects on some strains were less than 1000-fold. Given that bacterial infections are still treated with antibiotics at present, can linoleic acid combined with antibiotics improve the sensitivity of strains to antibiotics? Therefore, all strains MRSA8, 11-17, and MSSA1 with a bactericidal effect of linoleic acid less than 1000-fold in 2.4 of Example 2 were selected, and at the same time, strains MSSA2, MSSA8, MRSA4, 9, and 18 with a bactericidal multiple of linoleic acid greater than 1000-fold were selected. A total of 14 strains of bacteria were used as the objects for the bactericidal test of linoleic acid combined with antibiotics.

[0086] Prepare the above strain samples according to 2.1 in Example 2. 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 is shown in Table 3. The concentration of linoleic acid was 1 mM. After incubation at 37 °C and 200 rpm for 10 hours, the viable cell count was detected by plate, 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) × 100%.

[0087] The results are shown in Figure 6 Figure A. It can be seen from the figure that these strains had no bactericidal effect or a very weak bactericidal effect when treated with antibiotics alone, and the bactericidal multiple was only between 1.07 - 2.57-fold. However, if linoleic acid was added on the basis of antibiotics, the sensitivity of all strains to antibiotics was significantly improved. For the strains with a bactericidal effect of linoleic acid alone less than 1000-fold, the bactericidal multiple was further increased by 7.88 - 282.44-fold on the basis of the original single addition of linoleic acid. For the strains with a bactericidal multiple of linoleic acid alone greater than 1000-fold, the bactericidal multiple was further increased by 1.35 - 4-fold on the basis of the original single addition of linoleic acid.

[0088] Table 3 Concentration of cefoperazone sodium and sulbactam sodium used for strains in the experiment

[0089]

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

[0091] Prepare samples of Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis according to 2.1 in Example 2. The drug resistance, i.e., MIC determination of these bacteria is shown in Table 4. Each strain is divided into 4 groups: M9 control group, antibiotic group, linoleic acid group, and linoleic acid + antibiotic group. The antibiotic is cefoperazone sodium and sulbactam sodium, and the concentration used for each strain is: 300 μg / mL for Streptococcus iniae, 50 μg / mL for Streptococcus agalactiae, 400 μg / mL for Streptococcus pyogenes, 200 μg / mL for Bacillus subtilis, and 400 μg / mL for Enterococcus faecalis; the concentration of linoleic acid is 0.01 mM. After incubating at 37°C and 200 rpm for 10 hours, the viable cell count is detected by plate, and then the survival rate of bacteria in different treatment groups is calculated. The calculation formula is: Survival rate (%) = (number of viable cells after adding linoleic acid and / or antibiotic / number of viable cells in the control group) × 100%.

[0092] Table 4 Detection of bacterial drug resistance

[0093]

[0094] Note: For Streptococcus iniae, Streptococcus agalactiae, and Streptococcus pyogenes: for penicillins, ≤0.25 is sensitive, and there are no standards for resistance and intermediate; for cephalosporins, ≤0.25 is sensitive, and there are no standards for resistance and intermediate; for carbapenems, ≤0.5 is sensitive, and there are no standards for resistance and intermediate; for tetracycline: ≤2 is sensitive, 4 is intermediate, ≥8 is resistant, for levofloxacin: ≤2 is sensitive, 4 is intermediate, ≥8 is resistant; for vancomycin, ≤1 is sensitive, and there are no standards for resistance and intermediate.

[0095] For Enterococcus faecalis: for penicillins: ≤8 is sensitive, no report on intermediate, ≥16 is resistant; for tetracycline: ≤4 is sensitive, 8 is intermediate, ≥16 is resistant; for levofloxacin: ≤2 is sensitive, 4 is intermediate, ≥8 is resistant; for vancomycin: ≤4 is sensitive, 8 - 16 is intermediate, ≥32 is resistant.

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

[0097] R: Resistant; I: Intermediate; S: Sensitive.

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

[0099] As can be seen from Figure 6 Figure B in , when these strains were treated with antibiotics alone, there was no bactericidal effect or the bactericidal effect was very weak, and the bactericidal multiple was only between 1.1 and 1.69 times. However, if linoleic acid was added to the antibiotics, the sensitivity of all strains to the antibiotics was significantly improved. The bactericidal multiples of Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis and Enterococcus faecalis 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] Prepare the clinical drug-resistant bacteria MRSA9 sample according to 2.1 in Example 2 and divide it into 4 groups: M9 control group, linoleic acid group, antibiotic group, linoleic acid + antibiotic group. The antibiotic concentrations in the experiment were as follows: vancomycin was 100 μg / mL, and the other antibiotics were all 100 μg / mL; the linoleic acid concentration was 0.01 mM. After incubating at 37 °C and 200 rpm for 10 hours, the viable bacteria count was detected by plate, and then the survival rate of bacteria in different treatment groups was calculated. The calculation formula was: survival rate (%) = (number of viable bacteria after adding linoleic acid and / or antibiotics / number of viable bacteria in the control group) × 100%.

[0102] The results are shown in Figure 6 Figure C in . As can be seen from the figure, when the bacteria were treated with antibiotics alone, the survival rate was 44.82% for vancomycin and 67.24 - 98.27% for the others. When treated with linoleic acid alone, the survival rate of the bacteria was 70%. However, if the two were used in combination, that is, linoleic acid was added on the basis of adding antibiotics, the sensitivity of the strains to the antibiotics was improved except for polymyxin, clindamycin and ampicillin, and the multiple was between 2.82 and 2826 times.

[0103] These results indicate that after linoleic acid is combined with 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, the bactericidal ability of the antibiotics against these bacteria is enhanced, indicating that linoleic acid can not only significantly improve the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium, but also improve the sensitivity of Gram-positive bacteria such as streptococcus, bacillus subtilis and fecal coccus 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.

[0104] Example 5 Linoleic acid enhances the sensitivity of clinical Staphylococcus aureus to cefoperazone sodium and sulbactam sodium, showing concentration effects of linoleic acid, antibiotic concentration, and time

[0105] Using Staphylococcus aureus as a representative of Gram-positive bacteria and cefoperazone sodium and sulbactam sodium as representatives of antibiotics, the combined effect of linoleic acid and antibiotics was further explored.

[0106] 5.1 Concentration effect of linoleic acid

[0107] Prepare 12 samples of clinical drug-resistant strain MRSA according to 2.1 in Example 2 and divide them into 7 groups: an antibiotic control group (200 μg / mL cefoperazone sodium and sulbactam sodium), and 6 experimental groups: on the basis of adding 200 μg / mL cefoperazone sodium and sulbactam sodium, add linoleic acid respectively to make its final concentration 0.01, 0.05, 0.1, 0.5, 1, and 5 mM. There are 3 biological replicates for each concentration. Incubate at 37 °C and 200 rpm for 10 hours, and use a plate to detect the number of viable bacteria, and then convert it into a Log10 value. A decrease of 1 Log10 means a decrease of 1 order of magnitude, indicating a 10-fold decrease in the number of bacteria, and a decrease of 2 Log10 means a decrease of 2 orders of magnitude, indicating a 100-fold decrease in the number of bacteria. The results are shown in Figure 7 Figure A in

[0108] The specific situation is as follows: When adding 0.01 mM linoleic acid on the basis of adding antibiotics, it can play a good role in improving the sensitivity of antibiotics. Compared with the control group with only antibiotics added, the number of surviving bacteria decreased by nearly 0.5 Log, that is, 0.5 order of magnitude, which means the number of bacteria decreased by about 5 times. When the concentration of linoleic acid gradually increased from 0.05 mM to 5 mM, the number of surviving bacteria decreased by 1.32, 1.97, 2.86, 3.28, and 3.73 orders of magnitude respectively, which means the number of bacteria decreased from more than ten times to thousands of times.

[0109] 5.2 Antibiotic concentration effect

[0110] Prepare 12 samples of clinical drug-resistant strain MRSA according to 2.1 in Example 2 and divide them into 6 groups: a linoleic acid control group (1 mM), and 5 experimental groups: on the basis of adding 1 mM linoleic acid, add cefoperazone sodium and sulbactam sodium respectively to make its final concentration 10, 50, 100, 200, and 400 μg / mL. There are 3 biological replicates for each concentration. Incubate at 37 °C and 200 rpm for 10 hours, and use a plate to detect the number of viable bacteria, and then convert it into a Log10 value. The results are shown in Figure 7In Figure B, as can be seen from the figure, after adding antibiotics on the basis of adding linoleic acid, the number of surviving bacteria decreased. Moreover, as the concentration of the added antibiotics increased, the number of surviving bacteria decreased more significantly.

[0111] The specific situation is as follows: When adding 10 μg / mL of antibiotics on the basis of adding linoleic acid, it can play a better role in improving the sensitivity to antibiotics. Compared with the control group (adding only linoleic acid), the number of surviving bacteria decreased by 0.59 Log, that is, 0.59 orders of magnitude, which means the number of bacteria decreased by about 6 times. When the concentration of antibiotics gradually increased from 50 μg / mL to 400 μg / mL, the decreases in the number of surviving bacteria were 1.76, 3.24, 4.15, and 4.61 orders of magnitude respectively, which means the number of bacteria decreased from nearly 200 times to tens of thousands of times.

[0112] 5.3 Has a time effect

[0113] Prepare clinical drug-resistant strain MRSA 12 samples according to 2.1 in Example 2, and divide them into 3 large groups: M9 control group, antibiotic control group (200 μg / mL cefoperazone sodium sulbactam), linoleic acid (1 mM) + antibiotic (200 μg / mL cefoperazone sodium sulbactam) test group. Incubate at 37 °C with 200 rpm, sample every 2 hours within 12 hours, detect the viable bacteria count by using a plate, and then convert it into a Log10 value. Each test sample has 3 biological replicates. The results are shown in Figure 7 Figure C. As can be seen from the figure, the number of bacteria in the M9 control group remained basically stable within 12 hours; as time prolonged, the number of bacteria in the antibiotic group and the linoleic acid + antibiotic group gradually decreased; particularly importantly, at the same time point, compared with the number of surviving bacteria in the antibiotic group, the number of surviving bacteria in the linoleic acid + antibiotic group was significantly reduced. Moreover, as time prolonged, the number of surviving bacteria decreased more significantly.

[0114] The specific situation is as follows: At 2 hours, compared with the number of surviving bacteria in the antibiotic group, the number of surviving bacteria in the linoleic acid + antibiotic group decreased by 0.67 orders of magnitude, which means the number of bacteria decreased by about 7 times. From 4 to 12 hours, the number of surviving bacteria in the linoleic acid + antibiotic group decreased by 1.21 - 2.96 orders of magnitude, which means the number of bacteria decreased by about a dozen times - a thousand times.

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

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

[0117] 6.1 Linoleic acid in combination with cefoperazone sodium and sulbactam sodium can improve the survival rate of mice infected with clinical Staphylococcus aureus

[0118] Taking Staphylococcus aureus as the representative of Gram-positive bacteria and cefoperazone sodium and sulbactam sodium as the representative of antibiotics, the effect of the combination of linoleic acid and antibiotics was further explored.

[0119] Balb / c mice (6 - 8 weeks old, about 20 g, half male and half female) were divided into 4 groups, with 16 mice in each group, namely the normal saline control group, the antibiotic treatment group, the linoleic acid test group, and the antibiotic + linoleic acid test group. The challenge strain was methicillin-resistant Staphylococcus aureus (MRSA12), and the infection dose was 1.2×10 8 CFU, and the intraperitoneal injection method was used. One hour after the mice were infected with bacteria, the mice in the antibiotic treatment group were given cefoperazone sodium and sulbactam sodium by intravenous injection, with a dose of 200 mg / kg; the mice in the linoleic acid experimental group were injected with linoleic acid by intraperitoneal injection, with a dose of 200 mg / kg; the mice in the antibiotic + linoleic acid test group were treated with antibiotics and linoleic acid according to their respective injection methods and doses. The death of the mice was observed and recorded daily, and the survival rate of each group was calculated every day for 7 days. The calculation formula was: survival rate (%) = the number of surviving mice in each group / the total number of mice in each group × 100%.

[0120] The experimental results are shown in Figure 8 Figure A in it. It can be seen from the figure that in the normal saline control group, 81% died on the first day and all died on the second day; in the antibiotic treatment group, 31% died on the first day, 62% died on the second day, and no more deaths occurred thereafter, with a survival rate of 7%; in the linoleic acid test group, 7% died on the first day, 62% died on the second day, 7% died on the third day, and then it stabilized and no more deaths occurred, with a survival rate of 24%. In the antibiotic + linoleic acid experimental group, no deaths occurred on the first day, 13% died on the second day, and then it stabilized and no more deaths occurred, with a survival rate of 87%.

[0121] The results showed that in the linoleic acid test group of mice, the protection rate increased by 24% compared with the normal saline control group and increased by 17% compared with the antibiotic treatment group. In the antibiotic + linoleic acid test group of mice, the protection rate increased by 63% compared with the linoleic acid test group and increased by 80% compared with the antibiotic treatment group. It shows that when mice are infected with methicillin-resistant bacteria, in the case where antibiotic treatment is basically ineffective, linoleic acid can somewhat improve the resistance of mice to methicillin-resistant bacteria infection, but if linoleic acid and antibiotics are used in combination, the resistance of mice to methicillin-resistant bacteria infection has been significantly improved.

[0122] 6.2 Linoleic acid in combination with cefoperazone sodium and sulbactam sodium can improve the clearance of mice infected with clinical Staphylococcus aureus

[0123] Balb / c mice (6 - 8 weeks old, approximately 20 g, half male and half female) were divided into 4 groups, with 6 mice in each group, namely the normal saline control group, the antibiotic treatment group, the linoleic acid test group, and the antibiotic + linoleic acid test group. The virulent strain was methicillin-resistant Staphylococcus aureus MRSA 12, and the infection dose was 1.4×10 6 CFU, and the injection method was intraperitoneal injection. One hour after bacterial infection, mice in the antibiotic treatment group were administered cefoperazone sodium and sulbactam sodium by intravenous injection, with a dose of 200 mg / kg; mice in the linoleic acid test group were injected with linoleic acid by intraperitoneal injection, with a dose of 200 mg / kg; mice in the antibiotic + linoleic acid test group were treated with antibiotics and linoleic acid according to their respective injection methods and doses. After 6 hours, visceral organs (liver, spleen, kidneys) of equal weight were taken, thoroughly ground, then diluted and plated, and the number of colonies was counted.

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

[0125] Example 7 Linoleic acid increases the entry of antibiotics into the cell by enhancing the permeability of the bacterial membrane, thereby achieving sensitivity to antibiotics

[0126] Taking Staphylococcus aureus as a representative of Gram-positive bacteria and cefoperazone sodium and sulbactam sodium as representatives of antibiotics, the mechanism of the combined use of linoleic acid and antibiotics to improve the drug efficacy was further explored.

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

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

[0129] 7.2 Exogenous linoleic acid can promote the content of antibiotics entering bacterial cells

[0130] The method of detecting the inhibitory effect of antibiotics on microorganisms and calculating the antibiotic activity (titer) was used to determine the concentration of antibiotics entering bacterial cells.

[0131] Sample preparation for determining the concentration of antibiotics in bacterial cells: Prepare the MRSA 7 strain samples according to Example 2.1 and divide them into 3 groups, namely the control group without substances and antibiotics, the cefoperazone sodium sulbactam group, and the cefoperazone sodium sulbactam + linoleic acid group. There are 3 biological replicates in each group. Incubate at 37°C and 200 rpm for 10 hours, then centrifuge to collect the bacterial cells and wash them multiple times to remove the residual antibiotics in the culture medium. Suspend the bacterial cells in 1×M9 basic medium (containing 10 mM acetate and 1 M CaCl2); adjust the OD value of the bacterial suspension to 1.0 and centrifuge to collect 10 mL of the bacterial suspension. After adding 350 μL of the ultra-crushing solution containing 2% SDS and performing ultrasonic crushing (ultrasonic crushing on ice, 35% of the ultrasonic power, ultrasonic for 2 s and stop for 3 s, and the ultrasonic crushing time is 25 min), centrifuge to take the supernatant and filter to remove the possible residual bacterial cells in the supernatant to obtain the ultra-crushing solutions of the control group, the cefoperazone sodium sulbactam group, and the cefoperazone sodium sulbactam + linoleic acid group bacteria respectively.

[0132] Preparation of the test strain: Cultivate Staphylococcus aureus ATCC17978 overnight until saturated, dilute it with 1×M9 basic medium (containing 10 mM acetate and 1 M CaCl2) to an OD600 of 0.2, and then dilute it 10,000 times.

[0133] Standard curve drawing: Take 100 μL of the prepared diluted test strain solution and add 20 μL of cefoperazone sodium sulbactam respectively to make the final concentrations 0, 10, 20, 40, 60, and 80 ng / mL. After mixing the above samples, incubate them in a constant temperature shaker at 37°C and 200 rpm for 10 h, and detect the viable cell count by plate. Draw a standard curve with the number of bacterial cells as the ordinate and the antibiotic concentration as the abscissa.

[0134] Determination of the concentration of antibiotics in bacterial cells: Take 100 μL of the prepared diluted test strain solution and add 20 μL of the prepared samples of antibiotics in bacterial cells, namely the ultra-crushing solution of the control group, the ultra-crushing solution of the cefoperazone sodium sulbactam group, and the ultra-crushing solution of the cefoperazone sodium sulbactam + linoleic acid group. After mixing, incubate at 37°C and 200 rpm for 10 h, and detect the viable cell count by plate. Subsequently, calculate the concentration of antibiotics in the samples according to the standard curve formula of cefoperazone sodium sulbactam 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 amount of antibiotics entering the cells increased significantly. Compared with adding antibiotics alone, after adding linoleic acid, the amount of antibiotics entering the cells increased by 9.8 times. The results indicate that linoleic acid can promote the content of antibiotics entering bacterial cells.

[0136] The above experimental results show that after exogenous addition of linoleic acid, the cell membrane permeability of bacteria increases, and at the same time, the amount of antibiotics entering bacterial cells increases. These indicate that linoleic acid increases the content of antibiotics entering the cells by improving the permeability of the bacterial membrane, thereby promoting the sensitivity of bacteria to antibiotics.

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

[0138] Taking Staphylococcus aureus as a representative of Gram - positive bacteria and cefoperazone sodium and sulbactam sodium as representatives of antibiotics, the effects of combined use of linoleic acid and antibiotics were further explored.

[0139] Taking three clinical strains MSSA 2, MRSA 7 and MRSA12 as representatives. Pick a single bacterial colony and inoculate it into a 250 mL conical flask containing 50 mL of LB, and culture it overnight at 37 °C with 200 rpm. Then transfer the bacteria at a ratio of 1:100 to a 5 mL LB test tube. When the bacteria grow to OD 600 is 0.2, centrifuge to collect the bacterial cells and wash the bacteria three times with physiological saline. Resuspend the bacterial cells with 5 mL of MHB, and take 500 μL of the above bacterial solution and add it to a test tube containing 4.5 mL of MHB. Each strain is 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 has 3 biological replicates. Incubate at 37 °C with 200 rpm for 2 hours. Take 100 μL of the above bacterial solution, dilute it 1000 times, and then take 100 μL and add it to a test tube containing 4.9 mL of MHB. Incubate the diluted bacterial solution at 37 °C with 200 rpm, and respectively take 100 μL of the bacterial solution at 0, 1, 2, 4, 6, 8, 12 hours, dilute it and detect the viable cell count by plating. All strains have 3 biological replicates. Then, with the time point as the abscissa and the average value of the logarithm of the corresponding colony count as the ordinate, construct the bacterial growth curve.

[0140] Then, the PAE was calculated through the bacterial growth curve, and the calculation formula was as follows: PAE = T - C, where T was the time required for the viable bacteria count in the test culture to increase by 1 Log10 CFU from the count immediately observed after dilution, and C was the corresponding time of the control group not exposed to the antibiotic. For this experiment, the PAE when adding SCF alone (i.e., PAE SCF ), the PAE when adding SCF + linoleic acid (PAE SCF+亚油酸 ) were calculated. By comparing PAE SCF and PAE SCF+亚油酸 , it was determined whether the exogenous addition of linoleic acid would prolong the PAE of the antibiotic SCF.

[0141] The growth curve results were plotted as Figure 11 . Through further calculations, the PAEs of the three strains were obtained under two conditions of 1×MIC and 2×MIC, when adding cefoperazone sodium and sulbactam antibiotic alone, and after treatment with linoleic acid + cefoperazone sodium and sulbactam. In the table, the PAE of the cefoperazone sodium and sulbactam antibiotic group, i.e., the SCF group, was the result of comparing the antibiotic with the control group; the PAE of linoleic acid + cefoperazone sodium and sulbactam in the table, i.e., after treatment with SCF + linoleic acid, was the result of comparing linoleic acid + cefoperazone sodium and sulbactam with adding the antibiotic alone. The results are presented in Table 5. The specific details are as follows:

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

[0143]

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

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

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

[0147] Overall, linoleic acid could prolong the PAE of SCF, and there were differences in the prolongation time among different strains.

[0148] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. The use of linoleic acid in the preparation of an antibiotic synergist, characterized in that: The antibiotic is selected from cephalosporin antibiotics or pharmaceutically acceptable salts thereof or a compound preparation containing one or more of the antibiotics; The linoleic acid increases the sensitivity of Gram-positive bacteria to antibiotics; The antibiotic is selected from cefoperazone sodium, sulbactam sodium, ceftriaxone or cefoperazone; When the antibiotic is selected from cefoperazone sodium and sulbactam sodium, the Gram-positive bacteria is selected from one or more of Staphylococcus aureus, Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis; When the antibiotic is selected from cefotaxime or cefoperazone, the Gram-positive bacteria is selected from Staphylococcus aureus.

2. 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 cephalosporin antibiotics or pharmaceutically acceptable salts thereof or a compound preparation containing one or more of the antibiotics; The antibiotic is selected from cefoperazone sodium, sulbactam sodium, ceftriaxone or cefoperazone; When the antibiotic is selected from cefoperazone sodium and sulbactam sodium, the Gram-positive bacteria is selected from one or more of Staphylococcus aureus, Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis; When the antibiotic is selected from cefotaxime or cefoperazone, the Gram-positive bacteria is selected from Staphylococcus aureus.

3. Use of linoleic acid in combination with antibiotics in the preparation of a drug for preventing and / or treating Gram-positive bacterial infection, characterized in that: The antibiotic is selected from cephalosporin antibiotics or pharmaceutically acceptable salts thereof or a compound preparation containing one or more of the antibiotics; The antibiotic is selected from cefoperazone sodium, sulbactam sodium, ceftriaxone or cefoperazone; When the antibiotic is selected from cefoperazone sodium and sulbactam sodium, the Gram-positive bacteria is selected from one or more of Staphylococcus aureus, Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis; When the antibiotic is selected from cefotaxime or cefoperazone, the Gram-positive bacteria is selected from Staphylococcus aureus.

4. A drug for increasing the sensitivity of Gram-positive bacteria to antibiotics, characterized in that: Containing an effective amount of linoleic acid and an antibiotic; the antibiotic is selected from cephalosporin antibiotics or pharmaceutically acceptable salts thereof or a compound preparation containing one or more of the antibiotics; The antibiotic is selected from cefoperazone sodium, sulbactam sodium, ceftriaxone or cefoperazone; When the antibiotic is selected from cefoperazone sodium and sulbactam sodium, the Gram-positive bacteria is selected from one or more of Staphylococcus aureus, Streptococcus iniae, Streptococcus agalactiae, Streptococcus pyogenes, Bacillus subtilis, and Enterococcus faecalis; When the antibiotic is selected from cefotaxime or cefoperazone, the Gram-positive bacteria is selected from Staphylococcus aureus.

5. The drug according to claim 4, characterized in that: The mixing ratio of the linoleic acid and the antibiotic is 1: (2-40000) g / mol.

6. The drug according to claim 5, characterized in that: The mixing ratio of the linoleic acid and the antibiotic is 1: (50-40000) g / mol.

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