Composite bactericidal composition and application thereof in preparation of reagent for killing aeromonas hydrophila oxytetracycline drug-resistant strains

Abstract

The invention belongs to the technical field of prevention and control of drug-resistant bacteria, and particularly discloses a composite bactericidal composition and application thereof in preparation of a reagent for killing aeromonas hydrophila oxytetracycline drug-resistant strains. The minimum inhibitory concentration of the epsilon-polylysine to an aeromonas hydrophila oxytetracycline drug-resistant strain is as low as 3.125 g / mL. Meanwhile, the sterilization mechanism of the epsilon-polylysine on aeromonas hydrophila oxytetracycline drug-resistant strains is discussed through metabonomics research. On the basis, the application finds that various exogenous small molecule metabolites such as 2-phenylacetamide and the like are combined with epsilon-polylysine for use, so that the absorption of drug-resistant bacteria on epsilon-polylysine can be promoted, the sterilization effect of the epsilon-polylysine is effectively enhanced, and the problem of drug resistance of bacteria is solved. Therefore, when the epsilon-polylysine and the exogenous small molecule metabolite are jointly applied, compared with the existing single antibiotic therapy, the epsilon-polylysine has higher activity and better safety and operability in preparation of the reagent for resisting the aeromonas hydrophila oxytetracycline drug-resistant strain.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug-resistant bacteria prevention and control, in particular to a composite bactericidal composition and its use in preparing a reagent for killing drug-resistant strains of Aeromonas hydrophila. BACKGROUND

[0002] Aeromonas hydrophila is a gram-negative opportunistic pathogen widely distributed in water environment, soil and aquatic organisms, which plays a crucial role in the environment-animal-human health system. It can induce red leg syndrome and septicemia in various fish species, leading to diseases such as gastroenteritis and motile Aeromonas hydrophila septicemia (MAS), causing huge economic losses in aquaculture. Aeromonas hydrophila can also cause waterborne and foodborne gastroenteritis, septicemia, necrotizing fasciitis and other complications in humans. Terramycin is a drug commonly used to treat Aeromonas hydrophila, but due to the irrational use of terramycin and horizontal transfer of drug resistance genes, the problem of Aeromonas hydrophila resistance to terramycin is becoming more and more serious. For example, Aeromonas hydrophila from animal, food and human samples in Brazil is mostly resistant to terramycin. The assessment of disease in freshwater fish farming in northern Vietnam showed that the resistance rate of Aeromonas hydrophila to terramycin was between 36.6% and 50.6%, and this resistance was related to the abuse of antibiotics in aquaculture. Overuse of terramycin in aquaculture not only increases the detection frequency and spread of tetracycline resistance genes (tetM) in water samples, but also affects the health of aquatic ecosystems through the discharge of its residues into water bodies. Therefore, it has become an urgent task to find an efficient way to control the problem of Aeromonas hydrophila resistance. tet R The present application relates to the technical field of drug-resistant bacteria prevention and control, in particular to a composite bactericidal composition and its use in preparing a reagent for killing drug-resistant strains of Aeromonas hydrophila.

[0003] ε-polylysine (ε-polylysine) is a natural antibacterial agent with broad-spectrum bactericidal activity, high biological safety, good thermal stability and good water solubility, and is widely used in food preservation and aquaculture. For example, ε-polylysine can significantly change the bacterial community structure of silver carp fillets, inhibit the growth of spoilage bacteria such as Pseudomonas and Shewanella, prolong the shelf life of fish fillets, and slow down chemical changes. However, there is no report on the bactericidal potential of ε-polylysine against terramycin-resistant strains of Aeromonas hydrophila. Pseudomonas Shewanella The present application relates to the technical field of drug-resistant bacteria prevention and control, in particular to a composite bactericidal composition and its use in preparing a reagent for killing drug-resistant strains of Aeromonas hydrophila. SUMMARY

[0004] The present application aims to provide ε-polylysine combined with exogenous small molecule metabolites to promote the absorption of bacteria to ε-polylysine and enhance the bactericidal effect, including drug-resistant bacteria and ordinary bacteria.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows: ​In one aspect, the present application discloses a composite bactericidal composition, which comprises ε-polylysine and a small molecule metabolite.

[0006] As a preferred solution, the small molecule metabolite comprises one or more of 2-phenylacetamide, uridine, L-threonine, L-lysine, citrulline, uracil and L-histidine.

[0007] More preferably, the small molecule metabolite is 2-phenylacetamide.

[0008] As a preferred solution, the concentration of ε-polylysine is 12.5 µg / mL, and the concentration of the small molecule metabolite is 2.5 mM-40 mM.

[0009] As a preferred solution, the composition promotes the absorption of ε-polylysine by bacteria.

[0010] As a preferred solution, the bacteria comprise Aeromonas hydrophila gentamicin-resistant bacteria, Vibrio parahaemolyticus, Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Staphylococcus aureus or Candida albicans. These bacteria are common pathogenic bacteria in humans and farmed animals, including drug-resistant and non-drug-resistant bacteria, and Staphylococcus aureus is a gram-positive bacterium, and Aeromonas hydrophila is a model bacterium for studying drug resistance of aquatic pathogens, so these bacteria are good representative bacteria of drug-resistant and non-drug-resistant bacteria.

[0011] In another aspect, the present application discloses the use of the above-mentioned composite bactericidal composition in the preparation of a reagent for killing Aeromonas hydrophila terramycin-resistant strains.

[0012] Experiments show that the MIC of ε-polylysine for Aeromonas hydrophila terramycin-resistant bacteria is 3.125 µg / mL, and after adding ε-polylysine, the survival rate of the strain is found to be significantly reduced, indicating that ε-polylysine can efficiently kill bacteria. The present application also finds that ε-polylysine can be used in combination with exogenous small molecule metabolites to improve the bactericidal effect of ε-polylysine on the strain. These results show that the method of improving the sensitivity of the strain to ε-polylysine by combining ε-polylysine with exogenous small molecule metabolites can be used to achieve the purpose of efficiently killing drug-resistant bacteria and ordinary bacteria, especially the combination of ε-polylysine and 2-phenylacetamide, which exhibits good broad-spectrum synergistic bactericidal effect. The present application proves through experiments that the addition of exogenous small molecule metabolites to ε-polylysine can significantly improve the sensitivity of Aeromonas hydrophila terramycin-resistant bacteria to ε-polylysine, providing a new technical method for the treatment of drug-resistant bacteria and ordinary bacteria.

[0013] The present application also discloses a new bactericide containing epsilon-polylysine and small molecule metabolites, or a preparation for improving the bactericidal effect of epsilon-polylysine on drug-resistant bacteria, which mainly comprises epsilon-polylysine and small molecule metabolites.

[0014] Although the embodiments only select the Aeromonas hydrophila gentamicin-resistant strain, Vibrio parahaemolyticus, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Candida albicans, but it does not constitute a limitation on the scope of protection, the reasons are as follows: first, Aeromonas hydrophila is a model organism for studying bacterial drug resistance mechanism, and its conclusion can be extrapolated to the closely related species in Aeromonas; second, the selected strains cover gram-negative (Escherichia coli, Vibrio parahaemolyticus, Salmonella typhimurium), gram-positive (Staphylococcus aureus) and fungi (Candida albicans) three categories, basically covering the main pathogenic groups of humans and farmed animals, and having typical representative significance; third, bacteria can have drug-resistant and non-drug-resistant states, that is, drug-resistant and non-drug-resistant strains of the same bacteria.

[0015] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects: The present application specifically discloses a natural antibacterial peptide, epsilon-polylysine, which can efficiently kill Aeromonas hydrophila terramycin-resistant strains. Research shows that the MIC of epsilon-polylysine to the resistant strain is only 3.125 µg / mL, which can independently and quickly kill bacteria. Through metabolomics analysis, the bactericidal metabolic mechanism of epsilon-polylysine on Aeromonas hydrophila terramycin-resistant strains is explored, and further through metabolic reprogramming strategy, exogenous small molecule metabolites are used to synergize with epsilon-polylysine to kill Aeromonas hydrophila terramycin-resistant bacteria, and finally it is found that a plurality of exogenous small molecule metabolites including 2-phenylacetamide have a significant synergistic bactericidal effect with epsilon-polylysine. Therefore, the bactericidal reagent prepared by epsilon-polylysine alone or in combination with exogenous small molecule metabolites has higher activity, better safety and operability in efficiently killing Aeromonas hydrophila terramycin-resistant bacteria than traditional single antibiotics, and provides a new perspective and idea for the prevention and control of Aeromonas hydrophila drug resistance. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0017] Figure 1Survival rate of strains under the action of different concentrations of ε-polylysine in Example 1 of the present application; data are expressed as mean ± SEM (n = 3 biological replicates); Figure 2 Metabolic differences revealed by OPLS-DA and pathway enrichment analysis results in Example 2 of the present application, wherein Figure 2 a S-plot generated by OPLS-DA; Figure 2 b Bubble chart of differential metabolic pathways based on MetaboAnalyst analysis, showing metabolic pathways between ε-polylysine treatment group and control group; Figure 3 Figure of bactericidal effect of exogenous small molecule metabolites in combination with ε-polylysine on strains in Example 3 of the present application; data are expressed as mean ± SEM (n = 3 biological replicates), and significance was analyzed by T test, p < 0.05, p <0.01, p < 0.001, p < 0.0001; Figure 4 Figure of determination of intracellular ε-polylysine content of strains after the action of exogenous small molecule metabolites in combination with ε-polylysine in Example 4 of the present application; data are expressed as mean ± SEM (n = 3 biological replicates), and significance was analyzed by T test, p < 0.0001; Figure 5 Figure of the universality of the bactericidal effect of 2-phenylacetamide in combination with ε-polylysine in Example 5 of the present application, wherein Figure 5 a MIC determination of ε-polylysine on 7 different pathogenic bacteria; Figure 5 b Effect of 2-phenylacetamide in combination with ε-polylysine on the bactericidal effect on 7 different pathogenic bacteria; data are expressed as mean ± SEM (n = 3 biological replicates), and significance was analyzed by T test, p < 0.0001. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If specific conditions are not indicated in the embodiments, conventional conditions or manufacturer recommended conditions are used. If the reagents or instruments used are not indicated by the manufacturer, they are all conventional products that can be purchased on the market.

[0019] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to specific embodiments.

[0020] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0021] Example 1 This embodiment investigates the bactericidal effect of different concentrations of ε-polylysine (ε-PL) on the oxytetracycline-resistant strain of Aeromonas hydrophila (OTC-A01).

[0022] OTC-A01 was cultured in Muller-Hinton (MH) medium at 30°C and 220 rpm until the logarithmic growth phase. The optical density (OD) was measured at 600 nm using MH medium after dilution. 600 The concentration of ε-PL was diluted to 0.02 using a two-fold dilution method in 96-well plates to achieve concentrations of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.56 μg / mL, 0.78 μg / mL, and 0.39 μg / mL. 100 µL of the diluted bacterial culture was added to 100 µL of MH medium containing the corresponding antibiotic and incubated at 37°C for 16 h. A 200 μL MH medium was used as a negative control. Each sample was tested three times. The concentration of the antimicrobial substance at which bacterial growth was not visible to the naked eye was used as the MIC value. The MIC of ε-polylysine against OTC-A01 was 3.125 µg / mL, while that of oxytetracycline was 50 µg / mL, according to the two-fold dilution method. This indicates that ε-polylysine can kill OTC-A01 more efficiently than oxytetracycline and is an alternative antibacterial substance for controlling this strain.

[0023] Bactericidal assays were performed using cultures of the OTC-A01 strain in the logarithmic growth phase. Bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 5 min, washed three times with sterile 0.85% (w / v) physiological saline, and then resuspended in M9 medium supplemented with 2 mM anhydrous sodium acetate, 1 mM MgSO4, and 1 mM CaCl2. The cell density of the resuspended culture was adjusted to OD using sterile M9 medium. 600The value was 1.0. Then the bacterial suspension was aliquoted into sterile test tubes (5 μL per tube), and ε-poly-L-lysine was added to a final concentration of 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively. The bacterial suspension without the addition of ε-poly-L-lysine was set up in parallel as an untreated control group to monitor the natural survival rate of the bacteria under the same culture conditions. After 6 h of culture at 30°C with 220 rpm shaking, ten-fold gradient dilutions were made for each bacterial culture using sterile 0.85% physiological saline. 10 μL of each dilution was spotted on TSA plates, with three replicates for each group to ensure reproducibility of the results. The survival rate of the bacteria was calculated based on the colony-forming units (CFU) counted on each plate after 12-16 h of culture at 30°C, with the untreated control group as the 100% survival rate benchmark. The results are shown in Figure 1 Figure 1, the survival rates of OTC-A01 were 27.53%, 3.23%, 0.49%, 0.38%, 0.18% and 0.10%, respectively. With the increase of the concentration of ε-poly-L-lysine, the survival rate of OTC-A01 decreased in a dose-dependent manner. Under the action of 100 μg / mL ε-poly-L-lysine, the survival rate of OTC-A01 was 0.101%, indicating that ε-poly-L-lysine can significantly control the growth of OTC-A01.

[0024] Example 2 This example studies the potential biomarkers of OTC-A01 after ε-poly-L-lysine treatment.

[0025] The results of S-plot analysis based on the OPLS-DA model are shown in Figure 2 Figure 2a, which is used to identify the intragroup differential metabolites, and each dot represents a single metabolite, with the potential biomarkers highlighted in blue, the absolute values of the covariance p and the correlation p (corr) are greater than or equal to 0.05 and 0.5, respectively. With the covariance threshold |p(1)|≥0.05 and the correlation coefficient |p(corr)|≥0.5 as the screening criteria, 7 key down-regulated differential metabolites were identified between the ε-poly-L-lysine group and the Ctrl group, represented by blue dots in the lower left, which were 2-phenylacetamide, L-lysine, L-histidine, uridine, citrulline, uracil and L-threonine. Among them, the contribution degree p (1) = 0.51) and the correlation pThe most significant finding was at (corr = 0.94), indicating that this substance is a key component of the phenylalanine metabolic pathway. Notably, the abundance of 2-phenylacetamide in the ε-polylysine-treated group showed a significant downward trend, suggesting that this metabolite may play a crucial role in the inactivation of OTC-A01 by ε-polylysine.

[0026] Further analysis of metabolic pathway enrichment was conducted using the Metaboanalyst platform, such as... Figure 2 b. The results showed that ε-polylysine treatment significantly affected six core metabolic pathways ( P < 0.05. Among these, the biosynthetic pathways of valine, leucine, and isoleucine were upregulated; these branched-chain amino acids play important roles in bacterial growth and energy metabolism. Conversely, the biosynthetic pathways of phenylalanine, pyrimidine, arginine, phenylalanine, tyrosine, and tryptophan, as well as purine metabolism, were all significantly downregulated. Notably, the phenylalanine metabolic pathway showed the highest pathway impact value (impact value = 1.0) and statistical significance (-log10-0.05) in the bubble plot. p The value of 4.46 indicates that this pathway may be a key target for the bactericidal effect of ε-polylysine. As a core node in aromatic amino acid metabolism, downregulation of the phenylalanine metabolic pathway may lead to bacterial death by affecting the bacterial antioxidant defense system and protein synthesis efficiency. In summary, ε-polylysine can exert its bactericidal effect by regulating key metabolic pathways in drug-resistant bacteria.

[0027] Example 3 This embodiment investigates the effect of exogenous small molecule metabolites synergistically with ε-polylysine on the bactericidal effect of OTC-A01. The specific experimental protocol is the same as in Example 1, except that ε-polylysine is first added to a final concentration of 12.5 µg / mL, followed by exogenous small molecule metabolites (2-phenylacetamide, uridine, L-threonine, L-lysine, citrulline, uracil, and L-histidine, with 5 experimental groups for each exogenous small molecule metabolite, and final concentrations of 2.5 mM, 5 mM, 10 mM, 20 mM, and 40 mM, respectively). These substances and the bacterial strain are incubated together in M9 medium.

[0028] Seven key downregulated metabolites (2-phenylacetamide, uridine, L-threonine, L-lysine, citrulline, uracil, and L-histidine) identified by S-plot analysis were combined with ε-polylysine for bactericidal effects. The results showed that these metabolites all exhibited varying degrees of synergistic bactericidal effects compared to using ε-polylysine alone. Figure 3Among the various bactericidal agents, 2-phenylacetamide exhibited the most significant synergistic bactericidal effect, with its bactericidal efficacy increasing by 436 times compared to the addition of ε-polylysine alone. Furthermore, uridine, L-threonine, L-lysine, citrulline, uracil, and L-histidine also demonstrated varying degrees of synergistic bactericidal effects, increasing efficacy by 366 times, 127 times, 75 times, 66 times, 65 times, and 4 times, respectively. These findings indicate that metabolic reprogramming can indeed significantly enhance the bactericidal effect of ε-polylysine, particularly with the most prominent synergistic effect of 2-phenylacetamide. This further suggests that the phenylalanine metabolic pathway plays a crucial role in the bactericidal activity of ε-polylysine and provides an important basis for developing bactericidal synergistic strategies based on metabolic reprogramming, opening up new avenues for addressing bacterial resistance issues.

[0029] Example 4 This embodiment investigates the determination of intracellular ε-polylysine content in Aeromonas hydrophila oxytetracycline-resistant bacteria after the synergistic effect of exogenous metabolism with ε-polylysine.

[0030] This study used the TCA-ninhydrin colorimetric method to evaluate the effects of the top three exogenous small molecule metabolites (i.e., 2-phenylacetamide, uridine, and L-threonine) with the best synergistic bactericidal activity with ε-polylysine on the intracellular ε-polylysine content of bacteria. The results showed that... Figure 4 The results showed that the intracellular ε-polylysine content in the untreated control group was only 5.26 μg / mg protein. This result confirms that the method can effectively eliminate the interference of free amino acids in bacterial cells and has good specificity for quantifying intracellular ε-polylysine. Further experimental analysis showed that, compared with the positive control group (ε-polylysine only), the combined use of ε-polylysine with three exogenous small molecule metabolites (2-phenylacetamide, uridine, and L-threonine) significantly increased the intracellular ε-polylysine content in all cases. The intracellular ε-polylysine concentrations in the combined treatment groups were 50.26 μg / mg protein, 45.87 μg / mg protein, and 38.97 μg / mg protein, respectively. Among them, 2-phenylacetamide had the most significant promoting effect on intracellular ε-polylysine accumulation, followed by L-threonine, with uridine showing a decreasing effect in that order. This result is consistent with the findings of Example 3, further confirming the regulatory potential of exogenous small molecule metabolites in reversing bacterial resistance to ε-polylysine. Therefore, the addition of specific exogenous small molecule metabolites can promote the entry of ε-polylysine into bacterial cells, thereby significantly enhancing the bactericidal effect of the drug against drug-resistant strains. This study provides a feasible metabolic-level synergistic strategy for addressing antimicrobial resistance in bacteria.

[0031] Example 5 This embodiment explores the broad-spectrum synergistic bactericidal effect of 2-phenylacetamide and ε-polylysine.

[0032] To evaluate the potential of the broad-spectrum synergistic bactericidal effect of 2-phenylacetamide and ε-polylysine, their synergistic bactericidal effects against Gram-negative bacteria, Gram-positive bacteria, and fungi were further investigated. First, the MIC of ε-polylysine against seven pathogens was determined, and the results are as follows: Figure 5 As shown in Figure a, ε-polylysine exhibited the best antibacterial effect against Salmonella Typhimurium, with a MIC of 12.5 μg / mL; while its inhibitory activity against Escherichia coli and Staphylococcus aureus was weaker, with MICs of 50 μg / mL for both. Further synergistic bactericidal experiments were conducted. Figure 5 (b) indicates that the combined use of 2-phenylacetamide and ε-polylysine significantly enhanced the bactericidal activity against all tested bacterial strains. Specifically, their synergistic bactericidal activity against Aeromonas hydrophila and Staphylococcus aureus was increased by 118-fold and 79.23-fold, respectively, compared to ε-polylysine treatment alone. The synergistic effects against gentamicin-resistant Aeromonas hydrophila, Vibrio parahaemolyticus, Escherichia coli, and Salmonella typhimurium were 49.7-fold, 55-fold, 44.9-fold, and 29.7-fold, respectively. Furthermore, this synergistic combination was highly effective against Gram-negative and Gram-positive bacteria, and also exhibited significant bactericidal activity against fungi. The synergistic bactericidal effect of 2-phenylacetamide and ε-polylysine against Candida albicans reached 42.5-fold. In conclusion, the synergistic bactericidal enhancement effect of 2-phenylacetamide and ε-polylysine on bacterial strains has broad-spectrum performance, indicating that this combination has important application value and research prospects in the development of broad-spectrum and highly effective compound bactericides.

[0033] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A composite bactericidal composition, characterized in that, The composition includes ε-polylysine and small molecule metabolites.

2. The composite bactericidal composition according to claim 1, characterized in that, The small molecule metabolites include one or more of 2-phenylacetamide, uridine, L-threonine, L-lysine, citrulline, uracil, and L-histidine.

3. The composite bactericidal composition according to claim 2, characterized in that, The small molecule metabolite is 2-phenylacetamide.

4. The composite bactericidal composition according to claim 1, characterized in that, The concentration of ε-polylysine was 12.5 µg / mL, and the concentration of small molecule metabolites was 2.5 mM-40 mM.

5. The composite bactericidal composition according to claim 1, characterized in that, The composition promotes bacterial uptake of ε-polylysine.

6. The composite bactericidal composition according to claim 1, characterized in that, The bacteria include Aeromonas hydrophila, gentamicin-resistant bacteria, Vibrio parahaemolyticus, Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Staphylococcus aureus, or Candida albicans.

7. Use of the compound bactericidal composition according to any one of claims 1-6 in the preparation of a reagent for killing oxytetracycline-resistant strains of Aeromonas hydrophila.

Images