Application of pantothenic acid or pharmaceutically acceptable salt thereof in preparation of medicine for enhancing sensitivity of pathogenic bacteria to daptomycin

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of pantothenic acid or 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 by adding pantothenic acid or pharmaceutically acceptable salt thereof, the sensitivity of gram-positive bacteria including staphylococcus aureus, streptococcus agalactiae and corynebacterium diphtheriae to daptomycin can be improved, and the sterilization effect is remarkably improved; further researches show that pantothenic acid or pharmaceutically acceptable salts thereof can promote removal of pathogenic bacteria biofilms by daptomycin, and form and reduce drug resistance of pathogenic bacteria to daptomycin. The discovery opens up a new way for overcoming the problem of antibiotic resistance and optimizing an anti-infection treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to the use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of pathogens to daptomycin. Background Art

[0002] With the development and widespread use of antibiotics, the emergence and widespread spread of drug-resistant bacteria has become a global concern. This is partly due to the current overuse or misuse of antibiotics and the slow development of new antibiotics. Therefore, there is an urgent need to find new strategies to combat the increase in bacterial resistance.

[0003] In this context, developing novel antimicrobial synergists and restoring the sensitivity of resistant bacteria to existing antimicrobial drugs through scientific combination therapy has become an important strategy for addressing the drug resistance crisis. This research direction not only improves the effectiveness of clinical antimicrobial treatment but also effectively delays the evolution of drug-resistant strains, possessing significant clinical value and practical significance in addressing the current dilemma of antimicrobial therapy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings that many clinical bacteria have developed resistance to existing antibiotics, as well as the high cost and long development cycle of new antibacterial drugs, and to provide the role of pantothenic acid or its pharmaceutically acceptable salts in the preparation of antibiotic synergists.

[0005] The purpose of the present invention 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 pathogens to antibiotics.

[0006] Another object of the present invention is to provide the effect of pantothenic acid or its pharmaceutically acceptable salts combined with antibiotics in the preparation of anti-pathogen drugs.

[0007] Another object of the present invention is to provide an anti-pathogen drug.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

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

[0010] The invention provides an application of protected pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of pathogens to antibiotics. The antibiotic is selected from daptomycin, and the pathogens are Gram-positive bacteria.

[0011] The present invention also protects the effect of pantothenic acid or its pharmaceutically acceptable salt in combination with antibiotics in the preparation of anti-pathogenic bacteria drugs, wherein the antibiotic is selected from daptomycin, and the pathogenic bacteria are Gram-positive bacteria.

[0012] Furthermore, the pharmaceutically acceptable salt thereof includes a calcium salt, that is, the calcium salt is calcium pantothenate.

[0013] Furthermore, the pathogens include sensitive bacteria and drug-resistant bacteria.

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

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

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

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

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

[0019] Furthermore, the concentration of pantothenic acid or a pharmaceutically acceptable salt thereof can be 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, or any range thereof, such as 0.25-6 mM, 0.5-6 mM, 1-6 mM, but is not limited thereto.

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

[0021] Furthermore, the concentration of 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 any range formed by the above values, such as 50-800 μg / mL, 100-150 μg / mL, 125-800 μg / mL, but is not limited thereto.

[0022] Furthermore, the duration of action of the pantothenic acid or a pharmaceutically acceptable salt thereof in combination with daptomycin is ≥1 h, preferably ≥3 h.

[0023] Furthermore, when the pathogen is Staphylococcus aureus, the concentration of pantothenic acid is ≥0.25 mM, and the concentration of daptomycin is ≥50 μg / mL.

[0024] Furthermore, when the pathogen is Staphylococcus aureus, the concentration of the 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 pathogen is Staphylococcus aureus, the concentration of the 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 pathogen is Staphylococcus aureus, the concentration of the pharmaceutically acceptable salt of pantothenic acid (preferably calcium pantothenate) is ≥1 mM, and the concentration of daptomycin is ≥125 μg / mL.

[0027] Furthermore, when the pathogen is Streptococcus agalactiae, the concentration of the 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 pathogen is Streptococcus agalactiae, the concentration of the 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 pathogen is Streptococcus agalactiae, the concentration of the 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 pathogen 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 ≥400 μg / mL.

[0031] More preferably, when the pathogen 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.

[0032] Furthermore, the pantothenic acid or a pharmaceutically acceptable salt thereof acts as an antibiotic synergist to enhance the ability of daptomycin to kill pathogens.

[0033] Furthermore, the pantothenic acid or a pharmaceutically acceptable salt thereof acts as an antibiotic synergist to enhance the ability of daptomycin to eliminate pathogenic bacterial biofilms.

[0034] Furthermore, the pantothenic acid or a pharmaceutically acceptable salt thereof acts as an antibiotic synergist to enhance the ability of daptomycin to reduce the formation of biofilms of pathogenic bacteria.

[0035] Furthermore, the pantothenic acid or a pharmaceutically acceptable salt thereof acts as an antibiotic synergist to promote pathogens to reduce their resistance to daptomycin.

[0036] The present invention protects an antipathogenic drug, the active ingredients of which include pantothenic acid or a pharmaceutically acceptable salt thereof and daptomycin.

[0037] Furthermore, the pharmaceutically acceptable salt thereof includes a calcium salt, that is, the calcium salt is calcium pantothenate.

[0038] Preferably, the active ingredients of the anti-pathogen drug include calcium pantothenate and daptomycin.

[0039] Furthermore, the pathogens include sensitive bacteria and drug-resistant bacteria.

[0040] Furthermore, the Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, and Corynebacterium diphtheriae.

[0041] Furthermore, the Staphylococcus aureus includes sensitive bacteria and drug-resistant bacteria, wherein the sensitive bacteria are Methicillin Susceptible Staphylococcus Aureus (MSSA) and the drug-resistant bacteria are Methicillin-Resistant Staphylococcus Aureus (MRSA).

[0042] Furthermore, the pathogen is a Gram-positive bacterium.

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

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

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

[0046] Furthermore, the concentration of pantothenic acid or a pharmaceutically acceptable salt thereof can be 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, or any range thereof, such as 0.25-6 mM, 0.5-6 mM, 1-6 mM, but is not limited thereto.

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

[0048] Furthermore, the concentration of 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 any range formed by the above values, such as 50-800 μg / mL, 100-150 μg / mL, 125-800 μg / mL, but is not limited thereto.

[0049] Furthermore, the duration of action of the pantothenic acid or a pharmaceutically acceptable salt thereof in combination with daptomycin is ≥1 h, preferably ≥3 h.

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

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

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

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

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

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

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

[0057] Preferably, when the pathogen 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 ≥400 μg / mL.

[0058] More preferably, when the pathogen 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 invention has the following beneficial effects:

[0060] This study first discovered that the addition of pantothenic acid or a pharmaceutically acceptable salt thereof can increase the sensitivity of Gram-positive bacteria, including Staphylococcus aureus, Streptococcus agalactiae, and Corynebacterium diphtheriae, to daptomycin, significantly enhancing its bactericidal efficacy. Further research has shown that pantothenic acid or a pharmaceutically acceptable salt thereof can promote the removal and formation of pathogen biofilms by daptomycin and reduce pathogen resistance to daptomycin. This discovery opens up new avenues for overcoming antibiotic resistance and optimizing anti-infective treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a diagram showing the effect of pantothenic acid on improving the sensitivity of MRSA to daptomycin.

[0062] Figure 2 Calcium pantothenate can increase the sensitivity of MRSA to daptomycin. Figures A and B show that this effect is dependent on the concentration of calcium pantothenate and daptomycin, respectively.

[0063] Figure 3 This is a statistical chart showing the correlation between the effect of calcium pantothenate on increasing the sensitivity of MRSA to daptomycin and the duration of action.

[0064] Figure 4 These are statistical graphs showing that calcium pantothenate can increase the sensitivity of MRSA biofilm to daptomycin. Graph A shows the biofilm-clearing effect of calcium pantothenate combined with daptomycin, and Graph B shows the biofilm-inhibiting effect of calcium pantothenate combined with daptomycin.

[0065] Figure 5 The statistical graph shows that calcium pantothenate can improve the sensitivity of other positive bacteria (Streptococcus agalactiae (A) and Corynebacterium diphtheriae (B)) to daptomycin.

[0066] Figure 6 This is a statistical chart showing the results of the combined use of calcium pantothenate with other antibiotics (gentamicin (A), doxycycline (B), vancomycin (C) and chloramphenicol (D)). DETAILED DESCRIPTION

[0067] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

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

[0069] MRSA: methicillin-resistant Staphylococcus aureus (MRSA ST239); GBS: Streptococcus agalactiae; C. diphtheriae: Corynebacterium diphtheriae.

[0070] Figure 2 A means Figure 2 Figure A in Figure 2 B means Figure 2 The naming of other figures is similar.

[0071] Example 1 Preparation of test samples

[0072] A single colony of MRSA (or GBS, C. diphtheriae) on the 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 sodium hydrogen phosphate, 2 g of glucose and 500 mL of bovine heart extract), and cultured at 37 ° C and 220 rpm for 12 h (or 16 h, 24 h) to reach saturation. The bacteria 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, M9 basal medium (1 L of A solution contained 17.1 g of sodium hydrogen phosphate dodecahydrate, 3 g of potassium dihydrogen phosphate, 1 g of ammonium chloride, 0.5 g of sodium chloride, and 5 mL of 2 M sodium acetate, 2 mL of 1 M magnesium sulfate, and 100 μL of 1 L of A solution were added). 1M calcium chloride) or LB liquid medium to adjust the bacterial solution OD 600 When the value reaches 0.2, dispense 3 mL into test tubes for later use.

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

[0074] Daptomycin was added to the samples prepared in the aforementioned M9 basal medium to a final concentration of 125 μg / mL. Pantothenic acid was then added to final concentrations of 0, 0.5, 1, 2, 4, and 6 mM. The cells were incubated at 37°C and 220 rpm for 6 hours. The cells were diluted with 0.85% saline to the appropriate concentration. 10 μL of the dilution was plated and incubated for 12 hours. The cells were counted and the survival rate was calculated. The survival rate formula was (number of viable cells in the groups treated with different daptomycin concentrations / number of viable cells in the blank group) × 100%.

[0075] The results are as follows Figure 1As shown, neither daptomycin treatment alone nor pantothenic acid treatment alone had an effect on bacterial survival rate. When daptomycin and pantothenic acid were used in combination, the bactericidal efficiency of the combination 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 pantothenic acid concentration continued to increase, the survival rate of MRSA remained basically stable between 0.35% and 0.7%. The above results indicate that pantothenic acid can increase the sensitivity of MRSA to daptomycin within a certain concentration range, thereby enhancing the bactericidal effect of daptomycin against MRSA.

[0076] Example 3 Calcium pantothenate increases the sensitivity of MRSA to daptomycin

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

[0078] To the samples prepared in M9 basal medium, daptomycin was first added to a final concentration of 125 μg / mL. Calcium pantothenate was then added to final concentrations of 0, 0.25, 0.5, 1, and 2 mM. The samples were incubated at 37°C and 220 rpm for 6 hours. The samples were then serially diluted with 0.85% saline to the appropriate concentrations. 10 μL of the dilutions were spotted on LB plates and incubated for 12 hours. The viable cells were counted and the survival rate was calculated. The survival rate formula is (viable cell count in the groups treated with different calcium pantothenate concentrations / viable cell count in the blank group) × 100%.

[0079] The results are as follows Figure 2 As shown in A, when daptomycin was not added, treatment with calcium pantothenate alone had no effect on bacterial survival rate. However, when daptomycin was added, the MRSA survival rate dropped significantly even under low concentration (0.25mM) calcium pantothenate treatment. As the concentration of calcium pantothenate increased, MRSA became more and more sensitive to antibiotics. Treatment with 125μg / mL of daptomycin alone had little effect on bacterial survival rate, while the combination of 125μg / mL daptomycin and 2mM calcium pantothenate had a bactericidal multiple of up to 872 times. The above results show that calcium pantothenate can increase the sensitivity of MRSA to daptomycin, and there is a concentration gradient effect. It can be seen that pantothenic acid and its salts can enhance the sensitivity of MRSA to daptomycin.

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

[0081] Calcium pantothenate was added to samples prepared in M9 basal medium to a final concentration of 1 mM. Daptomycin was then added to final concentrations of 0, 50, 75, 100, 125, and 150 μg / mL. The samples were incubated at 37°C and 220 rpm for 6 hours. 10 μL of the dilution was plated and incubated for 12 hours. The cells were counted and the survival rate was calculated. The survival rate formula is (number of viable cells in the daptomycin-treated group at each concentration / number of viable cells in the blank group) × 100%.

[0082] Depend on Figure 2 B showed that when calcium pantothenate was not added, daptomycin had no effect on bacterial survival even at a concentration of 150 μg / mL. However, when calcium pantothenate was added, 50 μg / mL of daptomycin killed nearly 90% of the bacteria, and as the antibiotic concentration increased, the survival rate of MRSA decreased from 110.1% to 0.01%. The combination of 150 μg / mL daptomycin and 1 mM calcium pantothenate increased the bactericidal efficiency by approximately 10,000 times compared to the antibiotic alone. These results indicate that calcium pantothenate can increase MRSA's sensitivity to daptomycin, and its bactericidal effect is related to the antibiotic concentration: the higher the antibiotic concentration, the better the bactericidal effect.

[0083] Example 4 Effect of Action Time on the Degree of Sensitivity of MRSA to Daptomycin Enhanced by Calcium Pantothenate

[0084] Four experimental groups were set up, including a blank control, a group treated with 125 μg / mL daptomycin alone, a group treated with 1 mM calcium pantothenate alone, and a combination group. The cells were incubated at 37°C and 220 rpm for 8 hours. Every hour, 100 μL of the bacterial solution was aspirated and serially diluted. The viable bacterial count was determined on a plate. After 12 hours of overnight incubation, the survival rate at different time points was calculated. The survival rate formula was (viable bacterial count at a given time point in each treatment group / viable bacterial count at the corresponding time point in the blank group) × 100%.

[0085] The results are as follows Figure 3 As shown, compared with the antibiotic-only group, the bacterial survival rate in the combination group dropped to 18.37% after just one hour of treatment. This rate continued to decline as the duration of calcium pantothenate exposure increased. After six hours of treatment, the bacterial survival rate stabilized, indicating that the optimal bactericidal duration is six hours, with a multiplier of approximately 50. These results demonstrate that calcium pantothenate can increase MRSA's susceptibility to daptomycin, and within a certain timeframe, the longer the exposure, the more pronounced the effect.

[0086] Example 5 Correlation between the effect of calcium pantothenate on the susceptibility of MRSA to daptomycin and bacterial biofilm

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

[0088] Biofilms, also known as biofilms, are organized bacterial communities encapsulated by extracellular macromolecules and attached to the surfaces of living or inanimate objects. The formation of bacterial biofilms increases resistance to hostile environments, leading to drug resistance. MRSA biofilms were prepared to investigate the efficacy of calcium pantothenate in combination with daptomycin in removing biofilms. A single MRSA colony was placed in 30 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm for 12 hours to saturation. Sterilized 6 mm PE50 catheters (0.58 mm × 0.96 mm) were then dispensed into 15 mL centrifuge tubes, 5 mL of fresh LB liquid medium was added, and the saturated bacterial suspension was transferred at a 1:100 ratio. The tubes were then incubated at 37°C. Fresh LB liquid medium was replaced every 24 hours for 3 consecutive days. The prepared PE50 catheter was washed thoroughly three times with normal saline and placed in 0.5 mL of M9 basal culture medium containing different drugs (blank group, group with 125 μg / mL daptomycin alone, group with 1 mM calcium pantothenate alone, and combination group). The tubes were incubated at 37°C and 220 rpm for 6 h. After water bath sonication for 10 min, the tubes were diluted and plated, and the survival rate was calculated after overnight culture for 12 h.

[0089] The results are as follows Figure 4 As shown in A, the bactericidal multiple of the group with 125 μg / mL daptomycin alone was 3.48 times that of the blank group, while the bactericidal multiple of the combined group was 11.43 times that of the blank group. It can be seen that the addition of calcium pantothenate increased the efficiency of daptomycin in clearing biofilms by 3.29 times. The above results indicate that calcium pantothenate can promote the clearance of biofilms by daptomycin, thereby improving the bactericidal effect.

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

[0091] Because pathogenic MRSA primarily exists as a biofilm, crystal violet staining was used to assess biofilm formation in the different treatment groups. Samples were prepared as described above, and four experimental groups were set up: a blank control, a group treated with 125 μg / mL daptomycin alone, a group treated with 1 mM calcium pantothenate alone, and a combination group. 200 μL of each solution was incubated statically at 37°C for 6 hours. The M9 was then removed, rinsed twice with 0.85% saline, and air-dried at room temperature. Each well was stained with 150 μL of 0.1% crystal violet for 20 minutes, then rinsed with 0.85% saline to remove excess violet. After air-drying, 150 μL of 95% ethanol was added to dissolve the crystal violet and shaken thoroughly. Finally, the optical density of each well was measured at 570 nm using a microplate reader.

[0092] Depend on Figure 4B showed that compared with the daptomycin group alone, the biofilm formation in the combination group was significantly reduced. This shows that the addition of calcium pantothenate can increase the sensitivity of MRSA to daptomycin, thereby reducing biofilm formation and achieving a good bactericidal and pathogenicity inhibition effect.

[0093] Example 6 Calcium pantothenate can increase the sensitivity of other positive bacteria to daptomycin

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

[0095] A single GBS colony was picked from the BHI solid plate and placed in 30 mL of BHI liquid medium. The culture was cultured overnight at 37°C and 220 rpm for 16 h until saturation. The bacteria were collected by centrifugation and the OD value of the culture solution was adjusted using M9 minimal medium. 600 The value was adjusted to 0.2, and 3 mL of each solution was dispensed for later use. Calcium pantothenate was first added to the sample to a final concentration of 1 mM, followed by daptomycin at final concentrations of 0, 100, 200, 400, and 800 μg / mL. After incubation for 6 hours, the samples were diluted and plated. After overnight culture, the survival rate was calculated. The survival rate formula is (number of viable cells in the groups treated with different daptomycin concentrations / number of viable cells in the blank group) × 100%.

[0096] The results are as follows Figure 5 As shown in A, it can be seen that daptomycin alone and calcium pantothenate alone have no bactericidal effect on GBS. When daptomycin is combined with calcium pantothenate, the survival rate of GBS decreases with the increase of daptomycin concentration. When daptomycin reaches 800 μg / mL, the bactericidal efficiency increases by nearly 12 times, indicating that calcium pantothenate can also increase the sensitivity of GBS to antibiotics and there is an antibiotic concentration effect.

[0097] 2. Calcium pantothenate can increase the sensitivity of C. diphtheriae to daptomycin

[0098] A single colony of Corynebacterium diphtheriae was picked from the LB solid plate and placed in 30 mL of LB liquid medium. The culture was cultured overnight at 37°C and 220 rpm for 24 h until saturation. The bacteria were collected by centrifugation and the OD value of the culture solution was adjusted using M9 minimal medium. 600 The value was adjusted to 0.2, and 3 mL of each group was dispensed for later use. Calcium pantothenate was first added to the sample to a final concentration of 1 mM, followed by daptomycin at final concentrations of 0, 400, and 500 μg / mL. After incubation for 6 hours, the samples were diluted and plated. After overnight culture, the survival rate was calculated. The survival rate formula is (number of viable cells in the groups treated with different daptomycin concentrations / number of viable cells in the blank group) × 100%.

[0099] The results are as follows Figure 5As shown in Figure B, daptomycin alone and calcium pantothenate alone had no effect on the survival rate of Corynebacterium diphtheriae. However, when daptomycin and calcium pantothenate were used simultaneously, the addition of calcium pantothenate caused the survival rate of Corynebacterium diphtheriae to decrease with the increase of daptomycin concentration. When the daptomycin concentration reached 500 μg / mL, the bactericidal efficiency increased by nearly 18 times, indicating that calcium pantothenate can also increase the sensitivity of Corynebacterium diphtheriae to antibiotics, and there is an antibiotic concentration effect.

[0100] Example 7 Combined use of calcium pantothenate and other antibiotics

[0101] To investigate whether calcium pantothenate can also increase the sensitivity of MRSA to other 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 screened in combination with 1 mM calcium pantothenate. The results are as follows: Figure 6 As shown in the data, it can be seen that except for the gradient bactericidal effect of doxycycline alone on MRSA, MRSA has strong resistance to the other three antibiotics. At the same time, the addition of calcium pantothenate cannot make MRSA sensitive to gentamicin (6A), doxycycline (6B), vancomycin (6C) and chloramphenicol (6D). It can be seen that calcium pantothenate is unique in 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 would alter MRSA's resistance to daptomycin, a resistance frequency assay was performed. Based on the Clinical and Laboratory Standards Institute (CLSI) standard, the minimum inhibitory concentration (MIC) of MRSA to daptomycin was determined to be 10 μg / mL. LB solid plates containing different concentrations of daptomycin (2 MIC to 32 MIC) and 1 mM calcium pantothenate were prepared. A single MRSA colony was picked and placed in 30 mL of LB liquid medium. The plates were cultured overnight at 37°C and 220 rpm for 12 hours to saturation. The cells were then collected by centrifugation and the OD value of the culture medium was adjusted using M9 minimal medium. 600 When the value reaches 1.0, 100 μL of bacterial solution was spread on the above plate, and the number of drug-resistant mutant colonies was recorded after overnight culture.

[0104] Table 1 Results of the effect of calcium pantothenate on the resistance of MRSA to daptomycin

[0105]

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

[0107] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The role of pantothenic acid or its pharmaceutically acceptable salt in the preparation of an antibiotic synergist, characterized in that: The antibiotic is selected from daptomycin.

2. Use of pantothenic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the sensitivity of pathogens to antibiotics, characterized in that: The antibiotic is selected from daptomycin, and the pathogen is Gram-positive bacteria.

3. The role of pantothenic acid or its pharmaceutically acceptable salts in combination with antibiotics in the preparation of anti-pathogen drugs, characterized in that: The antibiotic is selected from daptomycin, and the pathogen is Gram-positive bacteria.

4. The use according to claim 2 or 3, characterized in that: The pharmaceutically acceptable salts thereof include calcium salts.

5. The use according to claim 2 or 3, characterized in that: The Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, and Corynebacterium diphtheriae.

6. The use according to any one of claims 1 to 5, wherein the concentration of pantothenic acid or a pharmaceutically acceptable salt thereof is ≥ 0.25 mM.

7. The use according to any one of claims 1 to 5, wherein the concentration of daptomycin is ≥50 μg / mL.

8. An anti-pathogen drug, characterized in that: The active ingredients of the anti-pathogen drug include pantothenic acid or a pharmaceutically acceptable salt thereof and daptomycin.

9. The anti-pathogen drug according to claim 8, characterized in that: The Gram-positive bacteria include one or more of Staphylococcus aureus, Streptococcus agalactiae, and Corynebacterium diphtheriae.

10. The anti-pathogen drug according to claim 8, characterized in that: The concentration of pantothenic acid or a pharmaceutically acceptable salt thereof is ≥0.25 mM.

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