Application of sodium ascorbate in restoring the sensitivity of florfenicol-resistant Streptococcus suis

Abstract

The present invention relates to the application of ascorbic acid in restoring the sensitivity of florfenicol-resistant Streptococcus suis, belongs to the technical field of medical use, and the present invention provides a natural substance salt-sodium ascorbate, wherein the sodium ascorbate can restore the sensitivity of florfenicol-resistant Streptococcus suis and interfere with its ability to form biofilm. It is verified by experiments that when sodium ascorbate and florfenicol are co-incubated with bacteria, the use concentration of antibiotics is significantly reduced, and when sodium ascorbate is used alone, the formation of biofilm of Streptococcus suis can be effectively intervened.

Description

Technical Field

[0001] The invention belongs to the technical field of medical use, relates to a new medical use of ascorbic acid, and specifically relates to the use of ascorbic acid in restoring the sensitivity of florfenicol-resistant Streptococcus suis. Background Art

[0002] Streptococcus suis ( Streptococcus suis, S. suis ) is a pathogen that seriously harms the human and animal bodies. It is generally transmitted through the respiratory and digestive tracts. Animals infected with it can develop pneumonia, meningitis, sepsis and arthritis, and are prone to group outbreaks, bringing economic shocks to the breeding industry. Antimicrobial drugs have always been the main means of treating and preventing Streptococcus suis. However, with the irregular use of a large number of antimicrobial drugs, the strengthening of bacterial defense capabilities, the acceleration of gene mutations and multidrug resistance, many antibiotics are losing their effectiveness at a high rate.

[0003] After years of research, it was found that hemolysin (SLY), capsular polysaccharide (CPS), lysozyme releasing protein (MRP), biofilm (EF) and other substances are the main virulence factors of Streptococcus suis, but they play different roles in its pathogenic process. Bacterial biofilm is a membrane-like substance formed by bacteria in order to adapt to the natural environment by secreting extracellular polymers and wrapping the bacteria in secretions. It is a self-protective growth mode of bacteria. Biofilm can protect bacteria from environmental stress and is resistant or drug-resistant to bacteriophages, various chemical fungicides, host immune system and antibiotics. Therefore, it is easy to cause environmental pollution and human and animal infections, and clinical treatment is difficult. At present, although studies have found that many antibiotics can interfere with the formation of Streptococcus suis biofilm to a certain extent, its removal method is single, and long-term use is prone to cause bacterial multidrug resistance.

[0004] Studies have shown that some natural products have significant antibacterial effects and can inhibit the formation of bacterial biofilms, such as a natural 2-(5H)-furanone - ascorbic acid (vitamin C). Ascorbic acid is a water-soluble vitamin contained in fruits and vegetables. It is responsible for wound healing, scar tissue formation, repair and maintenance of cartilage, bone and tooth growth, and the formation of proteins for skin, tendons, ligaments and blood vessels in the human body. Studies have shown that ascorbic acid has potential antibacterial activity and can reduce the quorum sensing ability and pathogenicity of Clostridium perfringens. Sodium ascorbate is an organic sodium salt produced by replacing the 3-hydroxy proton of ascorbic acid with sodium ions. It is more stable than ascorbic acid. Its molecular structure is as follows Figure 1 shown.

[0005] Sodium ascorbate plays a significant role in antimicrobial therapy and cancer treatment, laying the foundation for the combined use of drugs. However, there has been no report on the combination of sodium ascorbate and antibiotics to restore the sensitivity of drug-resistant Streptococcus suis. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a natural substance salt - sodium ascorbate, which can restore the sensitivity of florfenicol-resistant Streptococcus suis and interfere with its ability to form biofilm. Experiments have verified that when sodium ascorbate and florfenicol are co-incubated with bacteria, the concentration of antibiotics used is significantly reduced, and sodium ascorbate can effectively interfere with the formation of biofilm of Streptococcus suis when used alone.

[0007] The specific scheme adopted by the present invention is:

[0008] First, the application of sodium ascorbate in restoring the sensitivity of florfenicol-resistant Streptococcus suis.

[0009] In a second aspect, a method for restoring the sensitivity of Streptococcus suis is provided, wherein sodium ascorbate is co-incubated with an antibiotic to incubate Streptococcus suis. Preferably, the antibiotic is florfenicol.

[0010] In a third aspect, sodium ascorbate is used in any of the following:

[0011] (a) Inhibit the formation of biofilm of Streptococcus suis;

[0012] (b) Preparation of drugs for inhibiting biofilm formation of Streptococcus suis.

[0013] Preferably, the inhibitory concentration of sodium ascorbate is 10 μg / mL-2560 μg / mL. More preferably, the inhibitory concentration of sodium ascorbate is 160 μg / mL.

[0014] In a fourth aspect, an antimicrobial synergist comprises sodium ascorbate. Preferably, the bacterium is Streptococcus suis; and the synergist is to restore the sensitivity of Streptococcus suis to florfenicol or to interfere with the formation of biofilm of Streptococcus suis by using sodium ascorbate.

[0015] A fifth aspect is the use of sodium ascorbate in the preparation of feed or feed additives.

[0016] Beneficial effects: The sodium ascorbate in the present invention is a natural salt, which has the characteristics of a minimum clinically effective dose, less toxic side effects and bacterial tolerance to drugs. The present invention uses this natural salt to restore the sensitivity of drug-resistant strains, reduce the minimum inhibitory concentration of antibiotics and the amount of biofilm formation of Streptococcus suis. The present invention provides a theoretical basis for sodium ascorbate as an antibacterial synergist or feed additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the molecular structure diagram of sodium ascorbate;

[0018] Figure 2 This is a graph showing the effect of different concentrations of sodium ascorbate on biofilm formation of Streptococcus suis. DETAILED DESCRIPTION

[0019] The present invention firstly detects the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of sodium ascorbate and florfenicol on Streptococcus suis, and finds that sodium ascorbate has no bacteriostasis or bactericidal effect and the bacteria are highly resistant to florfenicol; secondly, 1280 μg / mL of florfenicol and sodium ascorbate with different concentration gradients (5-2500 μg / mL) are added to co-culture Streptococcus suis to detect whether the Streptococcus suis recovers its sensitivity to antibiotics; finally, an in vitro biofilm model of Streptococcus suis is established to study the intervention effect of sodium ascorbate on the biofilm formation ability of Streptococcus suis.

[0020] The experiment confirmed that sodium ascorbate can restore the sensitivity of florfenicol-resistant Streptococcus suis and interfere with its ability to form biofilm. When sodium ascorbate is used alone, it interferes with the formation of biofilm of Streptococcus suis, which may be a potential mechanism for restoring the sensitivity of florfenicol-resistant Streptococcus suis.

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0022] Experimental Example 1 Determination of the Minimum Inhibitory Concentration of Sodium Ascorbate and Florfenicol against Streptococcus suis

[0023] 1. Experimental Drugs

[0024] Sodium ascorbate monomer and florfenicol (99.5%) monomer were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0025] Preparation of antibiotic stock solution: Dissolve florfenicol in an appropriate amount of acetonitrile, dilute with water to a final concentration of 1280 μg / mL drug stock solution, filter with a 0.22 μm sterile filter membrane, dispense into sterile 10 mL centrifuge tubes, and store at 4°C. Preparation of sodium ascorbate stock solution: Dissolve the drug in a small amount of water, dilute with water to a final concentration of 10240 μg / mL drug stock solution, filter with a 0.22 μm filter membrane, dispense into sterile 10 mL centrifuge tubes, and store at 4°C in the dark.

[0026] 2. Experimental Methods

[0027] Take the clinical isolate of Streptococcus suis stored in glycerol at -20℃, operate aseptically in the clean bench, use a pipette to inoculate the bacteria into TSB liquid culture medium, and place it in a 37℃ constant temperature incubator for overnight culture. Use an inoculation loop to draw four lines on the TSA plate with the turbid culture liquid for 24 hours, pick out the purified single colony, culture it in TSB overnight, and set aside.

[0028] Add 100 μL of TSB medium to each well of the 96-well plate, add 1280 μg / mL of florfenicol to the second and third columns, and dilute the cultured Streptococcus suis in a gradient dilution method from the third column using a pipette to a final concentration of 10 6 CFU / mL, use a pipette to draw 100 μL and add it to each column except the second column, and incubate at 37°C for 24 hours. The lowest concentration at which no bacteria can be seen growing is the minimum inhibitory concentration of florfenicol. The method for determining the minimum inhibitory concentration of sodium ascorbate against Streptococcus suis is the same as above.

[0029] 3. Experimental Results

[0030] By observing the transparency of the bottom plate in the 96-well plate, it can be seen that the minimum inhibitory concentration of florfenicol against Streptococcus suis is 640 μg / mL, while sodium ascorbate does not inhibit or kill bacteria at the functional drug concentration. The results are shown in Table 1.

[0031] Table 1 Minimum inhibitory concentrations of florfenicol and sodium ascorbate against Streptococcus suis.

[0032] drug Florfenicol Sodium Ascorbate Minimum inhibitory concentration 640 μg / mL >5000μg / mL

[0033] Experimental Example 2 Sodium ascorbate and florfenicol were co-incubated with Streptococcus suis to detect whether the bacteria recovered their sensitivity to antibiotics.

[0034] 1. Experimental Drugs

[0035] 1280 μg / mL of florfenicol and 10240 μg / mL of sodium ascorbate.

[0036] 2. Experimental Methods

[0037] Add 100 μL TSB medium to each well of the 96-well plate, and mix the gradient dilutions of sodium ascorbate and florfenicol in TSB medium. Dilute the revived Streptococcus suis at 1:100 to a final concentration of 10 6 CFU / mL, use a pipette to draw 100 μL into the other columns except the second column, and incubate at 37°C for 24h. To evaluate the antibacterial effect of the combination of antibiotics and sodium ascorbate, the data generated by the checkerboard test were analyzed according to the fractional inhibition concentration index (FICI) using the following equation. FICI=FICIA+FICIB=(CA coMB / MIC A )+(C B coMB / MIC B ), where MIC A and MIC B is the MIC of sodium ascorbate and antibiotics when acting alone, CA COMB and CB COMB It is the MIC when sodium ascorbate and antibiotics are used in combination. Synergy is defined as FICI ≤ 0.5, additive effect is defined as 0.5 < FICI < 4, and antagonism is defined as FICI ≥ 4.

[0038] 3. Experimental Results

[0039] The drug dosage was counted by observing the transparency of the bottom plate in the 96-well plate, and the FICI mean value when sodium ascorbate and florfenicol were used in combination was calculated according to the FICI calculation formula. The results are as follows:

[0040] FICI=(40μg / mL / 640μg / mL)+(5μg / mL / >5000μg / mL)≈0.0625.

[0041] According to the results, FICI≤0.5, which means that the two drugs are synergistic when used together. The antibiotic concentration before adding sodium ascorbate was 640μg / mL, and the antibiotic concentration after adding sodium ascorbate was 40μg / mL. The concentration at which the antibiotic works was greatly reduced, indicating that sodium ascorbate can restore the sensitivity of florfenicol-resistant Streptococcus suis.

[0042] Experimental Example 3 An in vitro biofilm model of Streptococcus suis was established to study the intervention effect of sodium ascorbate on the biofilm formation ability of Streptococcus suis.

[0043] 1. Experimental Drugs

[0044] 10240 μg / mL sodium ascorbate.

[0045] 2. Experimental Methods

[0046] Inoculate the glycerol-preserved Streptococcus suis on TSA agar plates and culture at 37°C for 24 hours. Pick a single colony and inoculate it in TSB medium and culture it at 37°C overnight. Dilute the cultured bacteria to about 1×10 6 CFU / mL, 200 μL was inoculated into a 96-well polystyrene microfiber plate and cultured at 37 °C for 24 h.

[0047] Take out the 96-well plate, gently aspirate the free bacteria with a pipette, and wash twice with sterile PBS to ensure complete removal. Add 240μl of 95% ethanol to each well, fix for 15 minutes, wash with sterile PBS, and dry. Add 240μl of 1% crystal violet to each well and stain for 20 minutes, then rinse the staining solution with sterile PBS and dry. Dissolve the adherent bacteria bound to the dye with 240μl of 95% ethanol for 30 minutes. Use an enzyme reader (595nm) to measure the OD value, and repeat each group of experiments three times.

[0048] When studying the intervention effect of sodium ascorbate on the biofilm formation ability of Streptococcus suis, different concentrations (5-2560 μg / mL) of sodium ascorbate were added in advance, and then the bacterial biofilm was cultured for analysis. The experimental method was the same as above.

[0049] 3. Experimental Results

[0050] like Figure 2 As shown, the results showed that sodium ascorbate at a concentration of 10μg / mL-2560μg / mL had varying degrees of interference with the formation of biofilm of Streptococcus suis, but the use of sodium ascorbate at 160μg / mL could significantly inhibit the formation of biofilm of Streptococcus suis, with an inhibition rate of up to 29.5%.

[0051] In summary, the present invention adopts drug sensitivity analysis and biofilm in vitro model, and for the first time proposes that sodium ascorbate can restore the sensitivity of drug-resistant strains to antibiotics and has an in vitro intervention effect on the biofilm of Streptococcus suis. Before using sodium ascorbate, florfenicol can only play an antibacterial effect at a concentration of 640μg / mL. After adding sodium ascorbate and co-incubating, 40μg / mL can inhibit bacteria, indicating that sodium ascorbate can restore the sensitivity of drug-resistant strains to antibiotics. In addition, we detected the intervention effect of sodium ascorbate on the formation of biofilm of Streptococcus suis. We found that sodium ascorbate at a concentration of 10μg / mL-2560μg / mL interfered with the formation of biofilm to varying degrees, but this interference reached a peak at a concentration of 160μg / mL, and the inhibition rate of biofilm could reach 29.5%. This research finding may be a potential mechanism for sodium ascorbate to restore the sensitivity of drug-resistant strains to antibiotics. Sodium ascorbate is a salt of ascorbic acid, which means that while sodium ascorbate can restore drug sensitivity and inhibit biofilm formation, it can also protect body tissues and organs, enhance body immunity, and help reduce drug toxic side effects.

[0052] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. Application of sodium ascorbate in the preparation of a drug for restoring the sensitivity of florfenicol-resistant Streptococcus suis.

2. Use of sodium ascorbate and antibiotics in the preparation of a drug for restoring the sensitivity of Streptococcus suis, characterized in that: Sodium ascorbate and antibiotics are used to co-incubate Streptococcus suis; the antibiotic is florfenicol.

3. The use of sodium ascorbate in the preparation of a drug for inhibiting biofilm formation of Streptococcus suis, characterized in that: The inhibitory concentration of the sodium ascorbate is 40 μg / mL-640 μg / mL.

4. The use according to claim 3, characterized in that: The inhibitory concentration of sodium ascorbate was 160 μg / mL.

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