Use of pantothenic acid or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for enhancing the susceptibility of a pathogenic bacterium to daptomycin

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of pantothenic acid or a pharmaceutically acceptable salt thereof in preparation of a medicine for enhancing sensitivity of pathogenic bacteria to daptomycin. It is found for the first time that addition of pantothenic acid or a pharmaceutically acceptable salt thereof can improve the sensitivity of gram-positive bacteria including Staphylococcus aureus, Streptococcus agalactiae and Corynebacterium diphtheriae to daptomycin, significantly improve the bactericidal effect, and further research shows that pantothenic acid or a pharmaceutically acceptable salt thereof can promote the clearance, formation and reduction of pathogenic bacteria biofilm by daptomycin. The finding opens up a new way for overcoming antibiotic resistance and optimizing anti-infection treatment strategies.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to the use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of pathogenic bacteria to daptomycin. BACKGROUND

[0002] With the development and extensive use of antibiotics, the emergence and widespread dissemination of drug-resistant bacteria have become a global concern. This is related to the current misuse or misuse of antibiotics and the slow development of new antibiotics, so there is an urgent need to find new strategies to combat the increase in bacterial drug resistance.

[0003] In this context, developing new antibacterial synergists to restore the sensitivity of drug-resistant bacteria to existing antibacterial drugs through scientific combination therapy has become an important strategy to combat drug resistance. This research direction not only improves the effectiveness of clinical antibacterial therapy, but also effectively delays the evolution of drug-resistant strains, and has important clinical value and practical significance for solving the current antibacterial treatment dilemma. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the defects and deficiencies that many clinical bacteria have developed resistance to existing antibiotic drugs, and the high cost and long development cycle of new antibacterial drugs, and to provide the use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of an antibiotic synergist.

[0005] The purpose of the present application is to provide the use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of pathogenic bacteria to antibiotics.

[0006] Another purpose of the present application is to provide the use of pantothenic acid or a pharmaceutically acceptable salt thereof in combination with an antibiotic in the preparation of an anti-pathogenic bacteria drug.

[0007] Still another purpose of the present application is to provide an anti-pathogenic bacteria drug.

[0008] The above purposes of the present application are achieved by the following technical solutions:

[0009] The present application protects the use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of an antibiotic synergist, and the antibiotic is selected from daptomycin.

[0010] The present application protects the use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of pathogenic bacteria to antibiotics, and the antibiotic is selected from daptomycin, and the pathogenic bacteria are gram-positive bacteria.

[0011] The present application also protects the use of pantothenic acid or a pharmaceutically acceptable salt thereof in combination with an antibiotic in the preparation of an anti-pathogenic bacteria drug, and the antibiotic is selected from daptomycin, and the pathogenic bacteria are gram-positive bacteria.

[0012] Further, the pharmaceutically acceptable salt thereof includes a calcium salt, i.e., the calcium salt is calcium pantothenate.

[0013] Further, the pathogenic bacteria include sensitive bacteria and drug-resistant bacteria.

[0014] Still further, the gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, and Corynebacterium diphtheriae.

[0015] Further, the Staphylococcus aureus includes sensitive bacteria and drug-resistant bacteria, the sensitive bacteria being Methicillin Susceptible Staphylococcus Aureus (MSSA), and the drug-resistant bacteria being Methicillin-Resistant Staphylococcus Aureus (MRSA).

[0016] Further, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof is ≥ 0.25 mM.

[0017] Preferably, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof is ≥ 0.5 mM.

[0018] More preferably, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof is ≥ 1 mM.

[0019] Still further, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof can be 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, etc., or an interval range formed by any of the above values. For example, 0.25-6 mM, 0.5-6 mM, 1-6 mM, etc., but not limited thereto.

[0020] Further, the concentration of the daptomycin is ≥ 50 μg / mL, preferably 125 μg / mL.

[0021] Still further, the concentration of the daptomycin can be 50 μg / mL, 75 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 800 μg / mL, etc., or an interval range formed by any of the above values. For example, 50-800 μg / mL, 100-150 μg / mL, 125-800 μg / mL, etc., but not limited thereto.

[0022] Further, the time for the pantothenic acid or the pharmaceutically acceptable salt thereof to act in combination with the daptomycin is ≥ 1 h, preferably ≥ 3 h.

[0023] Further, when the pathogenic bacteria is Staphylococcus aureus, the concentration of pantothenic acid is > 0.25 mM, and the concentration of daptomycin is > 50 μg / mL.

[0024] Further, when the pathogenic bacteria is Staphylococcus aureus, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 0.25 mM, and the concentration of daptomycin is > 125 μg / mL.

[0025] Preferably, when the pathogenic bacteria is Staphylococcus aureus, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 50 μg / mL.

[0026] More preferably, when the pathogenic bacteria is Staphylococcus aureus, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 125 μg / mL.

[0027] Further, when the pathogenic bacteria is Streptococcus agalactiae, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 100 μg / mL.

[0028] Preferably, when the pathogenic bacteria is Streptococcus agalactiae, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 400 μg / mL.

[0029] More preferably, when the pathogenic bacteria is Streptococcus agalactiae, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 800 μg / mL.

[0030] Preferably, when the pathogenic bacteria is Corynebacterium diphtheriae, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 400 μg / mL.

[0031] More preferably, when the pathogenic bacteria is Corynebacterium diphtheriae, the concentration of pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is > 1 mM, and the concentration of daptomycin is > 500 μg / mL.

[0032] Further, the pantothenic acid or pharmaceutically acceptable salt thereof enhances the ability of daptomycin to kill pathogenic bacteria as an antibiotic potentiator.

[0033] Further, the pantothenic acid or pharmaceutically acceptable salt thereof enhances the ability of daptomycin to clear pathogenic bacterial biofilm as an antibiotic potentiator.

[0034] Further, the pantothenic acid or pharmaceutically acceptable salt thereof enhances the ability of daptomycin to reduce pathogenic bacterial biofilm formation as an antibiotic potentiator.

[0035] Further, the pantothenic acid or the pharmaceutically acceptable salt thereof promotes the pathogenic bacteria to reduce the drug resistance to daptomycin as an antibiotic potentiator.

[0036] The present application protects an anti-pathogenic bacteria medicine, and the effective component of the anti-pathogenic bacteria medicine comprises pantothenic acid or a pharmaceutically acceptable salt thereof and daptomycin.

[0037] Further, the pharmaceutically acceptable salt thereof comprises a calcium salt, that is, the calcium salt is calcium pantothenate.

[0038] Preferably, the effective component of the anti-pathogenic bacteria medicine comprises calcium pantothenate and daptomycin.

[0039] Further, the pathogenic bacteria comprises susceptible bacteria and drug-resistant bacteria.

[0040] Further, the gram-positive bacteria comprises one or more of Staphylococcus aureus, Streptococcus agalactiae and Corynebacterium diphtheriae.

[0041] Further, the Staphylococcus aureus comprises susceptible bacteria and drug-resistant bacteria, the susceptible bacteria is Methicillin Susceptible Staphylococcus Aureus (MSSA), and the drug-resistant bacteria is Methicillin-Resistant Staphylococcus Aureus (MRSA).

[0042] Further, the pathogenic bacteria is gram-positive bacteria.

[0043] Further, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof is ≥0.25 mM.

[0044] Preferably, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof is ≥0.5 mM.

[0045] More preferably, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof is ≥1 mM.

[0046] Further, the concentration of the pantothenic acid or the pharmaceutically acceptable salt thereof can be 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, etc., or an interval range formed by any of the above values. For example, 0.25-6 mM, 0.5-6 mM, 1-6 mM, etc., but not limited thereto.

[0047] Further, the concentration of the daptomycin is ≥50 μg / mL, and preferably 125 μg / mL.

[0048] Further, the concentration of the daptomycin can be 50 μg / mL, 75 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 800 μg / mL, or the like, or an interval range formed by any of the above values. For example, 50-800 μg / mL, 100-150 μg / mL, 125-800 μg / mL, or the like, but not limited thereto.

[0049] Further, the pantothenic acid or pharmaceutically acceptable salt thereof is combined with daptomycin for ≥ 1 h, preferably for ≥ 3 h.

[0050] Further, when the pathogenic bacteria is Staphylococcus aureus, the concentration of the pantothenic acid is ≥ 0.25 mM, and the concentration of the daptomycin is ≥ 50 μg / mL.

[0051] Further, when the pathogenic bacteria is Staphylococcus aureus, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 0.25 mM, and the concentration of the daptomycin is ≥ 125 μg / mL.

[0052] Preferably, when the pathogenic bacteria is Staphylococcus aureus, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of the daptomycin is ≥ 50 μg / mL.

[0053] More preferably, when the pathogenic bacteria is Staphylococcus aureus, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of the daptomycin is ≥ 125 μg / mL.

[0054] Further, when the pathogenic bacteria is Streptococcus agalactiae, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of the daptomycin is ≥ 100 μg / mL.

[0055] Preferably, when the pathogenic bacteria is Streptococcus agalactiae, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of the daptomycin is ≥ 400 μg / mL.

[0056] More preferably, when the pathogenic bacteria is Streptococcus agalactiae, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of the daptomycin is ≥ 800 μg / mL.

[0057] Preferably, when the pathogenic bacteria is Corynebacterium diphtheriae, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of the daptomycin is ≥ 400 μg / mL.

[0058] More preferably, when the pathogenic bacteria is Corynebacterium diphtheriae, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥ 1 mM, and the concentration of daptomycin is ≥ 500 μg / mL.

[0059] Compared with the prior art, the present application has the following beneficial effects:

[0060] The present application first discovers that the addition of pantothenic acid or a pharmaceutically acceptable salt thereof can improve the sensitivity of gram-positive bacteria including Staphylococcus aureus, Streptococcus agalactiae and Corynebacterium diphtheriae to daptomycin, significantly improve the bactericidal effect, and further research shows that pantothenic acid or a pharmaceutically acceptable salt thereof can promote the clearance of daptomycin on pathogenic bacteria biofilm, form and reduce the drug resistance of pathogenic bacteria to daptomycin. This discovery opens up a new way for overcoming antibiotic resistance and optimizing anti-infection treatment strategies. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 Effect of pantothenic acid on improving the sensitivity of MRSA bacteria to daptomycin.

[0062] Figure 2 Calcium pantothenate can improve the sensitivity of MRSA bacteria to daptomycin, wherein the graphs A and B respectively show that this effect has a concentration dependence of calcium pantothenate and a concentration dependence of daptomycin.

[0063] Figure 3 Statistical graph of the correlation data of calcium pantothenate improving the sensitivity of MRSA bacteria to daptomycin and the action time.

[0064] Figure 4 Statistical graph of calcium pantothenate improving the sensitivity of MRSA bacteria biofilm to daptomycin, wherein graph A is a statistical graph of the clearance effect of calcium pantothenate combined with daptomycin on biofilm, and graph B is a statistical graph of the inhibition effect of calcium pantothenate combined with daptomycin on biofilm formation.

[0065] Figure 5 Statistical graph of calcium pantothenate improving the sensitivity of other positive bacteria (Streptococcus agalactiae (A) and Corynebacterium diphtheriae (B)) to daptomycin.

[0066] Figure 6 Statistical graph of the results of calcium pantothenate combined with other antibiotics (gentamicin (A), doxycycline (B), vancomycin (C) and chloramphenicol (D)). DETAILED DESCRIPTION

[0067] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0068] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0069] MRSA: Methicillin-resistant Staphylococcus aureus (MRSA ST239); GBS: Group B Streptococcus; C. diphtheriae: Corynebacterium diphtheriae.

[0070] Figure 2 A represents Figure 2 A in FIG., Figure 2 B represents Figure 2 B in FIG., and the naming of other figures is similarly continued.

[0071] Preparation of test samples in Example 1

[0072] A single colony of MRSA (or GBS, C. diphtheriae) on a plate was picked and inoculated into 30 mL of LB liquid medium (1 L containing 10 g of sodium chloride, 10 g of bacteriological peptone, and 5 g of yeast extract powder) or BHI liquid medium (1 L containing 10 g of tryptone, 5 g of sodium chloride, 2.5 g of disodium hydrogen phosphate, 2 g of glucose, and 500 mL of beef heart infusion) and incubated at 37°C with shaking at 220 rpm for 12 h (or 16 h, 24 h) to reach saturation, and the bacterial cells were collected by centrifugation at 8000 rpm for 3 min, the supernatant was removed, and the cells were washed twice with sterile 0.85% saline. Then, the bacterial cells were resuspended in M9 minimal medium (1 L of A solution containing 17.1 g of dodecahydrate disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 1 g of ammonium chloride, 0.5 g of sodium chloride, and 5 mL of 2M sodium acetate, 2 mL of 1M magnesium sulfate, and 100 μL of 1M calcium chloride were added to 1 L of A solution) or LB liquid medium to adjust the OD 600 value to 0.2, and 3 mL was aliquoted into a test tube for standby use.

[0073] Example 2 Pantothenic acid improves the sensitivity of MRSA to daptomycin

[0074] Daptomycin was added to the above prepared sample in M9 minimal medium to a final concentration of 125 μg / mL, and pantothenic acid was added to a final concentration of 0, 0.5, 1, 2, 4, and 6 mM, and incubated at 37°C with shaking at 220 rpm for 6 h. Then, the sample was diluted with 0.85% saline to an appropriate concentration, and 10 μL of the diluted sample was plated and incubated for 12 h, and the number of viable cells was counted and the survival rate was calculated. The formula for the survival rate was (the number of viable cells in the different daptomycin concentration treatment groups / the number of viable cells in the blank group) x 100%.

[0075] The results are shown in Table 1. Figure 1As shown, neither daptomycin treatment alone nor pantothenic acid treatment alone could affect bacterial survival. However, when daptomycin and pantothenic acid were used in combination, the bactericidal efficiency of 125 μg / mL daptomycin and 1 mM pantothenic acid was nearly 129 times higher than that of 125 μg / mL daptomycin alone. However, as the concentration of pantothenic acid continued to increase, the survival rate of MRSA remained relatively stable between 0.35% and 0.7%. These results indicate that pantothenic acid can improve the sensitivity of MRSA to daptomycin within a certain concentration range, thereby enhancing the bactericidal effect of daptomycin on MRSA.

[0076] Example 3: Calcium pantothenate improves the sensitivity of MRSA to daptomycin.

[0077] 1. The sensitivity of MRSA to daptomycin increases with increasing calcium pantothenate concentration.

[0078] In the samples prepared using M9 basal medium, daptomycin was first added to a final concentration of 125 μg / mL, followed by the addition of calcium pantothenate to achieve final concentrations of 0, 0.25, 0.5, 1, and 2 mM. The samples were incubated at 37℃ and 220 rpm for 6 h. Afterward, the samples were serially diluted with 0.85% physiological saline to appropriate concentrations. 10 μL of each diluted solution was spotted onto LB agar plates and incubated for 12 h. The viable cell count was then recorded, and the survival rate was calculated. The survival rate was calculated as (viable cell count in different calcium pantothenate treatment groups / viable cell count in the control group) × 100%.

[0079] The results are as follows Figure 2 As shown in Figure A, without daptomycin, treatment with calcium pantothenate alone had no effect on bacterial survival. However, with the addition of daptomycin, even at low concentrations (0.25 mM) of calcium pantothenate, MRSA survival decreased significantly. As the concentration of calcium pantothenate increased, MRSA became increasingly sensitive to antibiotics. Treatment with 125 μg / mL daptomycin alone had little effect on bacterial survival, while the combination of 125 μg / mL daptomycin and 2 mM calcium pantothenate resulted in a bactericidal effect of 872 times. These results indicate that calcium pantothenate can enhance the sensitivity of MRSA to daptomycin, and a concentration gradient effect exists. Therefore, both pantothenic acid and its salts can enhance the sensitivity of MRSA to daptomycin.

[0080] 2. The addition of calcium pantothenate makes the sensitivity of MRSA to daptomycin concentration-dependent.

[0081] First, add calcium pantothenate to the sample prepared with M9 basic medium to a final concentration of 1 mM, then add daptomycin to a final concentration of 0, 50, 75, 100, 125, 150 μg / mL, incubate at 37°C, 220 rpm for 6 h, take 10 μL of the dilution and plate for 12 h, then count and calculate the survival rate. The formula for the survival rate is (the number of viable bacteria in the different daptomycin concentration treatment groups / the number of viable bacteria in the blank group) x 100%.

[0082] From Figure 2 B can be seen that when no calcium pantothenate is added, even if the daptomycin reaches 150 μg / mL, it has no effect on the survival rate of the bacteria, but when calcium pantothenate is added, 50 μg / mL of daptomycin can kill nearly 90% of the bacteria, and as the concentration of the antibiotic increases, the survival rate of MRSA decreases from 110.1% to 0.01%. The bactericidal efficiency of the combination of 150 μg / mL daptomycin and 1 mM calcium pantothenate is about ten times higher than that of the antibiotic alone. The results of this experiment show that calcium pantothenate can increase the sensitivity of MRSA to daptomycin, and the bactericidal effect is related to the concentration of the antibiotic, the higher the concentration of the antibiotic, the better the bactericidal effect.

[0083] Example 4 Effect of the action time on the degree to which calcium pantothenate increases the sensitivity of MRSA to daptomycin

[0084] Four experimental groups were set up, including a blank group, a group with 125 μg / mL daptomycin alone, a group with 1 mM calcium pantothenate alone, and a combination group, which were incubated at 37°C, 220 rpm for 8 h, 100 μL of bacterial solution was taken out every hour, diluted step by step, and the number of viable bacteria was detected by plating, and the survival rate at different times was calculated after 12 h of overnight culture. The formula for the survival rate is (the number of viable bacteria in each treatment group at a certain time point / the number of viable bacteria in the blank group at the corresponding time point) x 100%.

[0085] The results are shown in Figure 3 Compared with the antibiotic alone group, the survival rate of the bacteria in the combination group decreased to 18.37% after 1 hour of treatment, and the survival rate continued to decrease as the action time of calcium pantothenate increased. After 6 h of treatment, the survival rate of the bacteria tended to be stable, and the optimal bactericidal time was 6 h, at which the bactericidal fold was about 50. The above results show that calcium pantothenate can increase the sensitivity of MRSA to daptomycin, and within a certain time range, the longer the action time, the more obvious the effect.

[0086] Example 5 Correlation between the increase in the sensitivity of MRSA to daptomycin by calcium pantothenate and bacterial biofilm

[0087] 1. Calcium pantothenate can promote the removal of daptomycin from bacterial biofilm

[0088] Biofilm, also known as biofilm, refers to a group of organized bacteria wrapped by bacterial extracellular macromolecules attached to the surface of living or non-living objects. The production of bacterial biofilm can improve its resistance to harsh environment, thereby leading to bacterial drug resistance. MRSA is used to prepare biofilm to study the removal effect of calcium pantothenate combined with daptomycin on biofilm. MRSA single colony was picked up in 30 mL LB liquid medium, and was cultured at 37℃, 220 rpm for 12 h to reach saturation state. Sterilized 6 mm PE50 catheter (0.58 mm x 0.96 mm) was dispensed into 15 mL centrifuge tube, 5 mL fresh LB liquid medium was added, and 1:100 saturated bacterial liquid was transferred, and was cultured at 37℃. Fresh LB liquid medium was replaced every 24 h, and was continuously cultured for 3 days. The prepared PE50 catheter was washed with physiological saline for 3 times, and was placed in 0.5 mL M9 basic medium containing different drugs (blank group, single 125 μg / mL daptomycin group, single 1 mM calcium pantothenate group and combined group), and was incubated at 37℃, 220 rpm for 6 h. After water bath ultrasonic for 10 min, dilution plate was carried out, and survival rate was calculated after 12 h of overnight culture.

[0089] The results are shown in Figure 4 As shown in A, the bactericidal ratio of the single 125 μg / mL daptomycin group compared with the blank group was 3.48 times, and the bactericidal ratio of the combined group compared with the blank group was 11.43 times. It can be seen that the addition of calcium pantothenate can improve the removal efficiency of daptomycin on biofilm by 3.29 times. The above results show that calcium pantothenate can promote the removal of daptomycin on biofilm, thereby improving the bactericidal effect.

[0090] 2. Calcium pantothenate combined with daptomycin can reduce the formation of bacterial biofilm

[0091] Since the existence form of pathogenic MRSA is mainly in the form of biofilm, crystal violet staining method is used to detect the biofilm formation of different treatment groups. As described before, the sample was prepared, and four experimental groups were set, including blank group, single 125 μg / mL daptomycin group, single 1 mM calcium pantothenate group and combined group, and 200 μL was taken in each group and incubated at 37℃ for 6 h. Then M9 was poured out, and 0.85% physiological saline was used to wash twice, and was naturally air dried at room temperature. 150 μL 0.1% crystal violet was added to each well, and was stained for 20 min, and then 0.85% physiological saline was used to wash to remove excess crystal violet, and was air dried, and then 150 μL 95% ethanol was added to dissolve crystal violet, and was shaken uniformly. Finally, the optical density of each well was detected by enzyme label instrument at 570 nm wavelength.

[0092] From Figure 4B shows that, compared with the daptomycin-only group, the biofilm formation in the combined group was significantly reduced. This indicates that the addition of calcium pantothenate can increase the sensitivity of MRSA to daptomycin, thereby reducing biofilm formation and achieving good bactericidal and pathogenic effects.

[0093] Example 6: Calcium pantothenate can improve the susceptibility of other positive bacteria to daptomycin.

[0094] 1. Calcium pantothenate can increase the susceptibility of Streptococcus agalactiae (GBS) to daptomycin.

[0095] Single colonies of GBS were picked from BHI solid plates and incubated overnight at 37°C and 220 rpm for 16 hours until saturation. The bacterial cells were collected by centrifugation and the OD of the bacterial culture was adjusted with M9 basal medium. 600 The concentration was adjusted to 0.2, and each sample was packaged into 3 mL containers for later use. First, calcium pantothenate was added to the sample to a final concentration of 1 mM. Then, daptomycin was added to achieve final concentrations of 0, 100, 200, 400, and 800 μg / mL. After incubation for 6 hours, the samples were diluted and spotted onto a plate. After overnight incubation, the survival rate was calculated. The survival rate was calculated as (number of viable bacteria in different daptomycin treatment groups / number of viable bacteria in the control group) × 100%.

[0096] The results are as follows Figure 5 As shown in Figure A, neither daptomycin alone nor calcium pantothenate alone had a bactericidal effect on GBS. However, when daptomycin was combined with calcium pantothenate, the survival rate of GBS decreased with the increase of daptomycin concentration. When daptomycin reached 800 μg / mL, the bactericidal efficiency increased by nearly 12 times, indicating that calcium pantothenate can also improve the sensitivity of GBS to antibiotics and that there is an antibiotic concentration effect.

[0097] 2. Calcium pantothenate can increase the susceptibility of Corynebacterium diphtheriae to daptomycin.

[0098] Single colonies of Corynebacterium diphtheriae were picked from LB solid plates and incubated in 30 mL of LB liquid medium overnight at 37°C and 220 rpm for 24 h until saturation. The bacterial cells were collected by centrifugation and the OD of the bacterial culture was adjusted with M9 basal medium. 600 The concentration was adjusted to 0.2, and each sample was packaged into 3 mL containers for later use. First, calcium pantothenate was added to the sample to a final concentration of 1 mM, then daptomycin was added to achieve final concentrations of 0, 400, and 500 μg / mL. After incubation for 6 hours, the samples were diluted and spotted onto a plate. After overnight incubation, the survival rate was calculated. The survival rate was calculated as (number of viable bacteria in different daptomycin treatment groups / number of viable bacteria in the control group) × 100%.

[0099] The results are as follows Figure 5As shown in Figure 6A, 6B, 6C and 6D, daptomycin alone and calcium pantothenate alone had no effect on the survival of C. diphtheriae, while the addition of calcium pantothenate to daptomycin caused the survival of C. diphtheriae to decrease with increasing concentrations of daptomycin, with a nearly 18-fold increase in killing efficiency at 500 μg / mL daptomycin, indicating that calcium pantothenate also increases the sensitivity of C. diphtheriae to antibiotics and that there is a concentration effect of the antibiotic.

[0100] Example 7 Combination of calcium pantothenate with other classes of antibiotics

[0101] To investigate whether calcium pantothenate also increases the sensitivity of MRSA to other classes of antibiotics, gentamicin (0, 200, 400, 800, 1600, 3200 μg / mL), doxycycline (0, 62.5, 125, 250, 500, 1000 μg / mL), vancomycin (0, 250, 500, 1000, 2000, 4000 μg / mL) and chloramphenicol (0, 500, 1000, 2000, 4000 μg / mL) were combined with 1 mM calcium pantothenate, and the results are shown in Figures 6A, 6B, 6C and 6D. Figure 6 As can be seen from Figures 6A, 6B, 6C and 6D, in addition to the gradient killing effect of doxycycline alone on MRSA, MRSA has strong resistance to the other three antibiotics, and the addition of calcium pantothenate also cannot make MRSA sensitive to gentamicin (6A), doxycycline (6B), vancomycin (6C) and chloramphenicol (6D), indicating that calcium pantothenate has a unique effect of increasing the sensitivity of MRSA to daptomycin.

[0102] Example 8 Calcium pantothenate makes MRSA less likely to develop resistance to daptomycin

[0103] To investigate whether the addition of calcium pantothenate changes the resistance of MRSA to daptomycin, resistance frequency determination was performed. Based on the standard of the Clinical and Laboratory Standards Institute (CLSI), the minimum inhibitory concentration (MIC) of daptomycin for MRSA was determined to be 10 μg / mL, and LB solid plates containing different concentrations of daptomycin (2 MIC-32 MIC) and 1 mM calcium pantothenate were prepared. Single colonies of MRSA were picked and inoculated in 30 mL of LB liquid medium, which was incubated at 37°C, 220 rpm for 12 h to reach saturation, and the bacterial cells were collected by centrifugation and adjusted to an OD value of 1.0 with M9 basic medium. 100 μL of the bacterial solution was spread on the above plates, and after overnight incubation, the number of resistant mutant colonies was recorded. 600

[0104] Table 1 Results of calcium pantothenate making MRSA bacteria less likely to develop resistance to daptomycin

[0105]

[0106] ​The experimental results are shown in Table 1, and it can be seen that there are 3 resistant single colonies on the LB plate containing 2MIC daptomycin (without adding calcium pantothenate), and the resistant frequency is 3 / 10 10 , while there are no colonies at other concentrations. It can be seen that MRSA can develop a certain resistance to daptomycin, and the addition of calcium pantothenate can promote MRSA to reduce the resistance to daptomycin.

[0107] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. The application of pantothenic acid in the preparation of drugs that enhance the sensitivity of pathogenic bacteria to antibiotics, characterized in that, The antibiotic is selected from daptomycin, the pathogen is a Gram-positive bacterium, and the Gram-positive bacterium is methicillin-resistant Staphylococcus aureus.

2. The application of pantothenic acid combined with antibiotics in the preparation of antimicrobial drugs, characterized in that, The antibiotic is selected from daptomycin, the pathogen is a Gram-positive bacterium, and the Gram-positive bacterium is methicillin-resistant Staphylococcus aureus.

3. The application according to claim 1 or 2, characterized in that, The concentration of pantothenic acid is ≥0.25 mM.

4. The application according to claim 1 or 2, characterized in that, The concentration of daptomycin is ≥50 μg / mL.

5. An antimicrobial drug, characterized in that, The active ingredients of the antimicrobial drug include pantothenic acid and daptomycin, and the pathogen is a Gram-positive bacterium, specifically methicillin-resistant Staphylococcus aureus.

6. The antimicrobial drug according to claim 5, characterized in that, The concentration of pantothenic acid is ≥0.25 mM.

7. The application of calcium pantothenate in the preparation of drugs that enhance the sensitivity of pathogenic bacteria to antibiotics, characterized in that, The antibiotic is selected from daptomycin, and the pathogen is a Gram-positive bacterium, including one or more of methicillin-resistant Staphylococcus aureus, agalactiae, and Corynebacterium diphtheriae.

8. The application of calcium pantothenate in combination with antibiotics in the preparation of antimicrobial drugs, characterized in that, The antibiotic is selected from daptomycin, and the pathogen is a Gram-positive bacterium, including one or more of methicillin-resistant Staphylococcus aureus, agalactiae, and Corynebacterium diphtheriae.

9. The application according to claim 7 or 8, wherein the concentration of calcium pantothenate is ≥0.25 mM.

10. The application according to claim 7 or 8, wherein the concentration of daptomycin is ≥50 μg / mL.

11. An antimicrobial drug, characterized in that, The active ingredients of the antimicrobial drug include calcium pantothenate and daptomycin. The pathogens are Gram-positive bacteria, including one or more of methicillin-resistant Staphylococcus aureus, agalactolytica, and Corynebacterium diphtheriae.

12. The antimicrobial drug according to claim 11, characterized in that, The concentration of calcium pantothenate is ≥0.25 mM.

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