A method for effectively reducing the dosage of antibiotics and a bactericidal composition

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a method and a bactericidal composition for effectively reducing the dosage of antibiotics. A method for effectively reducing the dosage of antibiotics, comprising: testing the resistance of pathogens to different antibiotics, determining the initial minimum inhibitory concentration of antibiotics on pathogens; combining bacteriophages with different antibiotics, testing the inhibitory effect of the combination of bacteriophages and antibiotics on pathogens; and determining the minimum inhibitory concentration of the combination of bacteriophages and antibiotics through animal experiments. The method of the present invention, by combining bacteriophages with antibiotics, can significantly prolong the lag period of host pathogens, thereby effectively improving the sensitivity of pathogens to antibiotics. On the premise of ensuring the therapeutic effect, the dosage of antibiotics is greatly reduced, the breeding income is increased, the food quality and safety are improved, and the risk of pathogens developing resistance is reduced. The present invention has significant economic benefits and broad application prospects in the prevention and treatment of poultry farming.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a method for effectively reducing the dosage of antibiotics and a bactericidal composition. Background Art

[0002] In livestock and poultry farming, the problem of drug resistance caused by long-term antibiotic use is becoming increasingly serious, not only causing significant losses to the farming industry but also endangering human health. Alternatives to antibiotics are constantly emerging, with antimicrobial peptides, bacteriophages, probiotics, and plant extracts all being hot research targets. However, their effectiveness in actual farming is suboptimal, and none of them currently offer an effective alternative to antibiotics. How to effectively reduce antibiotic use while ensuring the health of farmed animals is an urgent issue in livestock and poultry farming. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for improving the sensitivity of antibiotics and effectively reducing the usage of antibiotics, thereby alleviating the drug resistance of pathogens in the breeding process and improving food quality and safety.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a method for effectively reducing the dosage of antibiotics, comprising:

[0005] Test the resistance of pathogens to different antibiotics and determine the initial minimum inhibitory concentration of antibiotics against pathogens;

[0006] Use phages in combination with different antibiotics to test the inhibitory effect of the combination of phages and antibiotics on pathogens;

[0007] Through animal experiments, the minimum inhibitory concentration of the combination of phage and antibiotics was determined.

[0008] Preferably, the pathogen is Escherichia coli, and the bacteriophage is Escherichia coli phage RDP-EC-16029.

[0009] Preferably, the present invention also provides a bactericidal composition for reducing the amount of antibiotics used; the composition comprises Escherichia coli phage RDP-EC-16029; in the composition, the minimum inhibitory concentration of the antibiotic is reduced to less than 1 / 2 of the original amount.

[0010] Preferably, in the composition, the minimum inhibitory concentration of the antibiotic is reduced to less than 1 / 4 of the original value.

[0011] Preferably, the amount of bacteriophage added to the composition is 10 8 puf / ml.

[0012] The beneficial effects of the present invention are embodied in:

[0013] The method of the present invention, through the combined use of bacteriophage and antibiotics, can significantly prolong the lag phase of host pathogens, thereby effectively increasing the pathogens' sensitivity to antibiotics. While ensuring therapeutic efficacy, it significantly reduces antibiotic usage, increases farming profits, improves food quality and safety, and reduces the risk of pathogens developing drug resistance. This method has significant economic benefits and broad application prospects in the prevention and treatment of poultry farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the minimum inhibitory concentration determination of four antibiotics. DETAILED DESCRIPTION

[0015] To facilitate understanding of the present invention, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0016] The pathogen E10 used in the present invention was isolated from the liver of diseased white-feathered broiler chickens at a farm in Shandong Province. Drug sensitivity tests revealed that the pathogen was resistant to multiple antibiotics. The Escherichia coli E10 is a known strain.

[0017] The bacteriophage RDP-EC-16029 used in this study was isolated from wastewater at a Shandong aquaculture plant. Full genome sequencing of the bacteriophage revealed that the phage lacked virulence and lysogenic genes. RDP-EC-16029 is a known strain (CGMCC No. 18196).

[0018] Example 1 Synergistic Effect of Bacteriophage and Antibiotics on Escherichia coli

[0019] 1. Measurement of Minimum Inhibitory Concentration of Escherichia coli

[0020] 1.1 Prepare 1.024% antibiotic stock solution of four antibiotics: kanamycin, tilmicosin, doxycycline, and chloramphenicol.

[0021] 1.2. Minimum inhibitory concentration determination

[0022] Antibiotics can inhibit bacterial growth in vitro. The minimum inhibitory concentration (MOI) of an antibiotic is the minimum concentration of the drug that inhibits the growth of pathogens or kills them. It is used to measure the ability of anti-infective drugs (including antibiotics, antibacterial drugs, and chemically synthesized drugs) to combat pathogens. From a microbiological perspective, this is achieved by achieving and maintaining a certain concentration of the anti-infective drug. Inhibition of pathogen growth must occur at or above the MOI in vivo or in vitro. The lower the concentration, the greater the anti-pathogen activity. Because antibiotics are highly dose-dependent, antibiotics below the MOI will not effectively inhibit bacterial growth.

[0023] like Figure 1 As shown, 100 μL of sterile saline was added to a 96-well plate. 100 μL of each of the four antibiotic stock solutions was added to the first column of wells. The plates were then serially diluted to the 11th column, discarded, and the last column was used as a negative control. 100 μL of a purified bacterial suspension (100-fold dilution of the growth medium) was then added to each well. The plates were incubated overnight at 37°C, and the results were recorded after 12-16 hours.

[0024] 1.3 Experimental Results

[0025] Table 1. Minimum inhibitory concentrations of four antibiotics against Escherichia coli

[0026] antibiotic Kanamycin Tilmicosin doxycycline Diflucan Minimum dilution <![CDATA[1 / 2 7 ]]> <![CDATA[1 / 2 7 ]]> <![CDATA[1 / 2 11 ]]> <![CDATA[1 / 2 8 ]]>

[0027] 2. Inhibition of pathogen E10 by combination of four antibiotics and bacteriophage

[0028] 2.1 Based on the minimum inhibitory concentration measured in the above experiments, a synergistic experiment between phage and antibiotics was designed.

[0029] E. coli E10 was diluted 100-fold with LB medium and divided into two 100-mL aliquots. Add 1 mL of phage to one aliquot and 1 mL of culture medium to the other. In a sterilized colorimetric tube, add 2 mL of antibiotics at varying concentrations according to the susceptibility ratios shown in Table 1. Then, add 2 mL of E. coli and the phage dilution. Add 2 mL of the E. coli dilution to the other aliquot, bringing the total volume to 4 mL. The tubes were then placed in an automated microbial growth instrument at 37°C, 200 rpm, and measured every 5 minutes for 72 hours. The results are shown below.

[0030] Table 2. Inhibition results of kanamycin and phage combination on pathogen E10

[0031]

[0032] As shown in Table 2, at the minimum inhibitory concentration (MIC) of kanamycin, the lag period of E10 was 1500 minutes. Adding phage at the same concentration extended the lag period to over 4500 minutes, significantly prolonging the lag period and inhibiting the growth and reproduction of pathogens for a long time. A comparison of lag periods reveals that the lag period of kanamycin at the MIC is 1500 minutes, while the combination with phage reduces the MIC to one-quarter of the original concentration, resulting in a lag period of 2800 minutes.

[0033] Table 3. Inhibitory effects of doxycycline and phage combination on pathogen E10

[0034]

[0035] As shown in Table 3, at the minimum inhibitory concentration (MIC) of doxetine, the lag period of E10 was 1800 minutes. Adding phage at the same concentration extended the lag period to over 4500 minutes, significantly prolonging the lag period and inhibiting the growth and reproduction of pathogens for a long time. A comparison of lag periods reveals that the lag period of doxetine at the MIC is 1800 minutes, while the combination with phage reduces the MIC to 1 / 4 of the original concentration, resulting in a lag period of 2000 minutes.

[0036] Table 4. Inhibitory effects of tilmicosin and phage combination on pathogen E10

[0037]

[0038]

[0039] As shown in Table 4, at the minimum inhibitory concentration (MIC) of tilmicosin, the lag phase of E10 was 1000 minutes. Adding phage at the same concentration extended the lag phase to over 4500 minutes, significantly prolonging the lag phase and inhibiting the growth and reproduction of pathogens for a long time. A comparison of lag phases reveals that the lag phase of tilmicosin at the MIC is 1000 minutes, while the combination with phage reduces the MIC to 1 / 8 of the original concentration, resulting in a lag phase of 1200 minutes.

[0040] Table 5. Inhibition results of the combination of fusin and bacteriophage against pathogen E10

[0041]

[0042] As shown in Table 5, at the minimum inhibitory concentration (MIC) of fufenazine, the lag period of E10 was 510 minutes. Adding bacteriophage at the same concentration extended the lag period to over 4500 minutes, significantly prolonging the lag period and inhibiting the growth and reproduction of pathogens for a long time. A comparison of lag periods shows that the lag period of fufenazine at the MIC is 510 minutes, while the combination with bacteriophage can reduce the MIC to 1 / 4 of the original concentration.

[0043] In summary, the combined use of bacteriophages with kanamycin, doxycycline, tilmicosin, and sulfaquinoxaline can reduce their drug sensitivity concentrations to 1 / 4 to 1 / 8 of their original levels. In inhibiting the growth and reproduction of pathogens, antibiotics and bacteriophages complement each other, creating a highly effective synergistic effect, effectively increasing antibiotic sensitivity, reducing usage, and mitigating the emergence of drug resistance.

[0044] 2.2 Mechanism analysis

[0045] The microbial growth curve includes four periods: lag phase (bacteria cannot divide normally and the total number of bacteria does not increase), logarithmic growth phase, stable phase, and decay phase.

[0046] Characteristics of the lag phase: the growth rate constant is zero, the bacteria are coarse, the RNA content increases, the metabolic activity is strong, and the resistance to adverse environments decreases.

[0047] Characteristics of the logarithmic phase: the fastest growth rate, vigorous metabolism, active enzyme system, the number of live bacteria and the total number of bacteria are roughly the same, and the chemical composition, morphology and physical and chemical properties of the cells are basically consistent.

[0048] Characteristics of the stable period: the number of live bacteria remains relatively stable, the total number of bacteria reaches the highest level, and the accumulation of cell metabolic products reaches the highest peak.

[0049] Characteristics of the decay stage: the bacterial death rate is greater than the rate of new generation, the entire group shows negative growth, cells begin to become deformed, and cell death occurs through autolysis.

[0050] In phage-antibiotic synergy experiments, OD600 was continuously monitored, and data was collected to calculate the corresponding inhibition time (see Tables 2-5). In the positive control group, bacteria grew normally in the culture medium, with the OD600 gradually increasing and the reaction system becoming turbid. The higher the OD600, the more turbid the reaction system, and the higher the microbial content.

[0051] The synergistic effect of bacteriophages and antibiotics can significantly extend the lag period, indicating that the combination of the two can effectively inhibit the growth of bacteria and achieve the effect of "1+1>2". This has high application value in actual production, can significantly reduce the use of antibiotics, increase breeding efficiency, ensure food health and safety, and meet people's demand for green and healthy food.

[0052] In vitro lysis experiments using multidrug-resistant Escherichia coli E10 as a test subject demonstrated that bacteriophage RDP-EC-16029 could lyse E. coli E10. Furthermore, bacteriophage RDP-EC-16029 altered the growth curve of E. coli and prolonged its lag phase, which plays a crucial role in increasing E. coli's sensitivity to antibiotics.

[0053] The experimental results show that the sensitivity of Escherichia coli E10 to the four antibiotics is different, in the order of sulfaquinoxaline > doxycycline > kanamycin = tilmicosin.

[0054] 3. The synergistic effect of antibiotics and bacteriophages can effectively prolong the antibacterial time, enhance the bactericidal effect, increase the sensitivity of antibiotics, and reduce the use of antibiotics.

[0055] (1) Kanamycin has a significant synergistic effect with bacteriophage, which can effectively reduce its usage to 1 / 4 of the original amount.

[0056] (2) The synergistic effect between doxycycline and bacteriophage is quite obvious, which can effectively reduce its usage to 1 / 4 of the original amount.

[0057] (3) The synergistic effect of sulfamethoxazole and bacteriophage is quite obvious, which can effectively reduce its usage to 1 / 4 of the original amount.

[0058] (4) Tilmicosin has a significant synergistic effect with bacteriophage, which can effectively reduce its usage to 1 / 8 of the original amount.

[0059] Example 2 Animal Experiment

[0060] Highly pathogenic and multidrug-resistant Escherichia coli E10 was selected and injected into an animal model. Initially, 500 chickens were used. After successful modeling, 340 of the remaining 360 chickens were selected and grouped as shown in Table 6.

[0061] The combination of antibiotics and bacteriophages was selected as the experimental object; negative and positive controls were set up to record the growth status of the chickens. At the beginning of the experiment, the broilers were 15 days old. The amount of bacteriophage added was 10 8 puf / ml.

[0062] Table 6. Experimental groups

[0063]

[0064]

[0065] Ten days after administration, broiler chickens from different experimental groups were dissected to observe the therapeutic effects on air sac inflammation. The negative control group showed obvious pericardial and hepatic encapsulation, with numerous cheesy deposits in the air sac. The positive control group (kanamycin, chloramphenicol, doxycycline, and tilmicosin) showed clear air sacs after feeding, with no turbidity or pericardial encapsulation. The phage group (RDP-EC-16029) showed no obvious pericardial and hepatic encapsulation, with significantly reduced air sac symptoms, and the air sacs were generally bright and free of turbidity. The 1 / 2 kanamycin + phage RDP-EC-16029, 1 / 4 kanamycin + phage RDP-EC-16029, and 1 / 2 kanamycin + phage RDP-EC-16029 groups showed clear air sacs after feeding, with no turbidity or pericardial encapsulation. The air sacs of the 1 / 2 doxycycline + phage RDP-EC-16029 and 1 / 4 doxycycline + phage RDP-EC-16029 groups were clear, with no turbidity or pericardial hepatic folds. The air sacs of the 1 / 2 pyrethroid + phage RDP-EC-16029, 1 / 4 pyrethroid + phage RDP-EC-16029, and 1 / 8 pyrethroid + phage RDP-EC-16029 groups were clear, with no turbidity or pericardial hepatic folds. The air sacs of the 1 / 2 tilmicosin + phage RDP-EC-16029 and 1 / 4 tilmicosin + phage RDP-EC-16029 groups were clear, with no turbidity or pericardial hepatic folds. The number of survivors and treatment efficacy after the experiment are shown in Table 7.

[0066] Table 7. Survivor numbers and treatment effects

[0067]

[0068]

[0069] The above results indicate that the combination of antibiotics and bacteriophages provided by the present invention can effectively reduce the amount of antibiotics used. In actual production, while ensuring therapeutic efficacy, the dosage of lincomycin, chloramphenicol, doxycycline, and tilmicosin can be reduced to 1 / 8-1 / 4 of the normal dosage, thereby reducing the amount of antibiotics used, increasing breeding income, and ensuring food quality and safety.

[0070] In summary, in vitro antibacterial experiments and animal experiments have confirmed that the combination of antibiotics and bacteriophages can effectively inhibit the growth and reproduction of multidrug-resistant E. coli and significantly reduce the use of antibiotics. This has broad application prospects in actual production. In the treatment of multidrug-resistant E. coli, by combining with bacteriophages, more effective treatment can be achieved and the use of antibiotics can be reduced. Reducing the use of antibiotics can effectively reduce the occurrence of drug-resistant bacteria and ensure food quality and safety.

Claims

1. A method for effectively reducing the dosage of antibiotics, characterized in that: include: Test the resistance of pathogens to different antibiotics and determine the initial minimum inhibitory concentration of antibiotics against pathogens; Use bacteriophages in combination with different antibiotics to test the inhibitory effect of the combination of bacteriophages and antibiotics on pathogens; Through animal experiments, the minimum inhibitory concentration of the combination of phage and antibiotics was determined; The bacteriophage is Escherichia coli phage RDP-EC-16029, with a deposit number of CGMCC NO.18196; the pathogen is Escherichia coli; the antibiotic is any one of kanamycin, tilmicosin, doxycycline, and chloramphenicol; the combination of bacteriophage and antibiotic prolongs the lag phase of the pathogen and increases the sensitivity of the pathogen to the antibiotic, thereby reducing the dosage of the antibiotic while ensuring efficacy.

2. A bactericidal composition for reducing the amount of antibiotics used, characterized in that: The composition comprises Escherichia coli phage RDP-EC-16029, with a deposit number of CGMCC NO.18196. In the composition, the antibiotic is any one of kanamycin, tilmicosin, doxycycline, and chloramphenicol. The combination of the phage and the antibiotic prolongs the lag phase of the pathogen and increases the sensitivity of the pathogen to the antibiotic, thereby reducing the minimum inhibitory concentration of the antibiotic to less than half of the original value while ensuring efficacy.

3. The bactericidal composition according to claim 2, characterized in that: In the composition, the minimum inhibitory concentration of the antibiotic is reduced to less than 1 / 4 of the original level.

4. The bactericidal composition according to claim 2, wherein: In the composition, the amount of bacteriophage added is 10 8 puf / ml.

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