Method for enhancing treatment effect of salmonella bacteriophage

Abstract

The invention discloses application of a method for enhancing the treatment effect of salmonella bacteriophage, and relates to the technical field of microorganisms. According to the method, bacteriophage is added into salmonella for in-vitro culture, after continuous subculture, the salmonella is dropwise added to an SS plate containing the bacteriophage, and resistant bacteria of the bacteriophage are screened. The split bacteriophage is screened aiming at the resistant bacteria of the bacteriophage, and the split bacteriophage and the initial bacteriophage form a bacteriophage cocktail formula, so that the salmonellosis can be well prevented and treated, and the generation of the resistant bacteria is delayed. The method has the beneficial effects that compared with the existing method for screening the bacteriophage-resistant bacteria through a double-layer plate method, the method is more convenient, the operation is simple, and the screening efficiency of the bacteriophage-resistant bacteria is greatly improved. The bacteriophage cocktail contains the bacteriophage for cracking the resistant bacteria, so that the treatment effect of the bacteriophage is improved, and the practical application scene is quite high.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a method for enhancing the therapeutic effect of Salmonella bacteriophage. Background Technology

[0002] Bacteriophages are viruses capable of lysing bacteria. They are almost ubiquitous in nature and possess significant advantages such as high specificity, rapid self-replication, and low acquisition cost, making them one of the most effective methods for controlling pathogenic bacteria, internationally recognized. In recent years, with the increasing strength of superbugs, bacteriophages have re-emerged as an effective alternative to antibiotics. The development of bacteriophages is progressing rapidly, exhibiting a new, diversified, and international outlook. Since 2014, the United States, the European Union, the Netherlands, France, Belgium, and other countries have successively recognized that bacteriophages can effectively address bacterial resistance and have provided phage-based treatments for some patients. In its "Letter Soliciting Opinions on the Draft National Action Plan for Curbing Antimicrobial Resistance in Animal-Derived Bacteria (2017-2020)," my country for the first time considered bacteriophages as one of the biocontrol agents to replace antibiotics in feed, aiming to protect the health of animal-derived foods.

[0003] As viruses capable of lysing bacteria, bacteriophages have attracted considerable attention. However, host bacteria often develop resistance mutations during phage lysis, significantly limiting their application. Bacteria and bacteriophages have co-evolved for billions of years, during which bacteria have developed various defense strategies to overcome phage attacks. These strategies include: preventing phage adsorption through receptor modification, blocking phage DNA entry, degrading the phage genome through the RM and CRISPR-Cas systems, and preventing further phage proliferation through abortion infection systems.

[0004] Research on phage-resistant bacteria has become an important area of ​​focus. Studies on the generation patterns of phage-resistant bacteria, screening for resistant strains during the infection and lysis process of Salmonella phage, and investigating the differences in the biological characteristics and pathogenicity of resistant strains provide a theoretical basis for solving the problem of resistant bacteria in the application of phage therapy.

[0005] The original screening method for resistant bacteria, the double-layer plate method, is labor-intensive, cumbersome, and costly. The screening method for resistant bacteria provided in this study can greatly improve work efficiency and facilitate the large-scale screening of bacteriophage-resistant bacteria.

[0006] Simultaneously, combining the phage of the lysing resistant bacteria with the original phage in a cocktail form delays the development of resistant bacteria and can improve treatment efficacy. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for enhancing the therapeutic effect of Salmonella phage therapy, offering researchers a rapid and sensitive method for detecting phage-resistant bacteria. Furthermore, by combining the phage that lyses resistant bacteria with the original phage in a cocktail format, the development of resistant bacteria is delayed, thereby improving the therapeutic effect.

[0008] A method for enhancing the therapeutic effect of Salmonella phage includes the following steps: Step (1) Screening of resistant strains: Salmonella phage SP8 was cultured in LB broth medium to produce resistant strains; then, a 1×10⁸ phage-containing medium was prepared. 8 -1×10 10 Prepare PFU / mL SS medium plates. Cool the boiled SS medium to approximately 45°C and add the phage liquid according to the specified ratio. Gently mix, pour into plates, and allow to solidify. Spot 20µL of the appropriate dilution of the bacterial and phage co-culture solution onto SS medium containing the phage, and incubate at 37°C for 18 hours. The bacterial strain that grows on the plate is the phage-resistant strain. Step (2) Screening of new phages capable of lysing resistant strains: New phages capable of lysing resistant strains are screened and used as one of the components in the phage cocktail. The original phage is combined with the newly screened phage to make a cocktail. It can delay the development of resistant bacteria during phage therapy, greatly improving the therapeutic effect.

[0009] This study involves a Salmonella phage SP8 strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified as Salmonella phage, with a deposit date of August 12, 2022, and accession number CGMCC No. 45256; the strain is in a viable state.

[0010] This study also involves a Salmonella phage, Salmonella phage PC111, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified as Salmonella phage, with a deposit date of March 24, 2025, and accession number CGMCC No: 46431; the strain is in a viable state.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention overcomes the shortcomings of traditional detection methods, such as long detection cycles, high time and labor costs, and large quantities of experimental materials. It significantly reduces operational steps and labor intensity, achieving time-saving, labor-saving, rapid, and sensitive results. It can perform large-scale screening of bacterial suspensions, the proportion of resistant bacteria produced in bacterial and phage co-cultures, feces, and animal tissues, providing a basis for subsequent phage therapy. Simultaneously, by combining the phages that lyse resistant bacteria with the original phages in a cocktail format, the production of resistant bacteria is delayed, improving treatment efficacy. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a schematic diagram of SS culture medium plates with added bacteriophages used in an embodiment of the present invention for screening resistant bacteria (the upper part shows a normal SS culture medium plate, and the lower part shows an SS culture medium plate with added bacteriophages). Figure 2 This is a schematic diagram of the synergistic effect of the phage cocktail in an embodiment of the present invention. Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should also be noted that, to make the embodiments more comprehensive, the following embodiments are the best and preferred embodiments, and those skilled in the art can use other alternative methods to implement some well-known technologies; moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0015] It should be noted that the use of terms such as "an embodiment," "an embodiment," "an exemplary embodiment," and "some embodiments" in the specification indicates that the described embodiment may include a specific feature, structure, or characteristic, but not every embodiment necessarily includes that specific feature, structure, or characteristic. Furthermore, when a specific feature, structure, or characteristic is described in connection with an embodiment, implementing such a feature, structure, or characteristic in conjunction with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the art.

[0016] Generally, terms can be understood at least partly from their use in context. For example, depending at least partly on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in a singular sense, or a combination of features, structures, or characteristics in a plural sense. Additionally, the term "based on" can be understood not necessarily to convey an exclusive set of factors, but rather, alternatively, depending at least partly on the context, to allow for the presence of other factors that are not necessarily explicitly described.

[0017] A method for enhancing the therapeutic effect of Salmonella phage includes the following steps: Step (1) Screening of resistant strains: Salmonella phage SP8 was cultured in LB broth medium to produce resistant strains; then, a 1×10⁸ phage-containing medium was prepared. 8 -1×10 10 Prepare PFU / mL SS medium plates. Cool the boiled SS medium to approximately 45°C and add the phage liquid according to the specified ratio. Gently mix, pour into plates, and allow to solidify. Spot 20µL of the appropriate dilution of the bacterial and phage co-culture solution onto SS medium containing the phage, and incubate at 37°C for 18 hours. The bacterial strain that grows on the plate is the phage-resistant strain. Step (2) Screening of new phages capable of lysing resistant strains: New phages capable of lysing resistant strains are screened and used as one of the components in the phage cocktail. The original phage is combined with the newly screened phage to make a cocktail. It delays the development of resistant bacteria during phage therapy, which can greatly improve the therapeutic effect.

[0018] Example 1: A rapid detection method for phage-resistant bacteria includes the following steps: (1) culturing Salmonella phage SP8 in LB broth medium to induce the production of resistant strains; (2) preparing a phage-resistant strain containing 1×10⁻⁶ phages. 8 -1×10 10 (2) Heat the boiled SS medium to about 45°C and add the phage liquid according to the ratio. After gently mixing, pour the mixture onto the plate and let it solidify for later use. (3) Spot 20µL of the bacterial solution at an appropriate dilution onto the SS medium containing the phage and incubate it in a 37°C incubator for 18h. (4) The strain that grows on the plate is the resistant strain of the phage.

[0019] The results showed that this method can quickly screen out a large number of resistant strains. In this study, 60 resistant strains were screened out, numbered A1-A20, B1-B20, and C1-C20, respectively, and none of them had their own SP8 phages lysed.

[0020] Example 2: Screening of new phages for resistant bacteria: In Example 1, 60 resistant strains were screened as host bacteria. The lysis of other bacteriophages was detected by spot method. Among them, bacteriophage PC111 had a lysis rate of up to 98.33% (59 / 60) against resistant strains, while the lysis rates of other bacteriophages against resistant strains were all below 80%.

[0021] The specific pyrolysis details are shown in Table 1.

[0022] ; ; ; ; ; ; ;

[0023] Example 3: Synergistic effect of phage cocktail: A 200 μL system was prepared in a 96-well plate to verify whether there was a synergistic effect between two phage cocktails. For a single phage, 190 μL of broth, 5 μL of bacterial culture, and 5 μL of phage propagation stock solution were added to the 96-well plate. For a combination of two phages, 185 μL of broth, 5 μL of bacterial culture, and 10 μL of phage propagation stock solution were added to the 96-well plate in a 1:1 mixture. Negative broth control and positive bacterial culture control were set up, and each sample was replicated in triplicate.

[0024] Seal the 96-well plate with the added liquid around its edges with sealing film, being careful not to touch the top and bottom surfaces. Place the plate in a SCIENTZ microbial growth curve analyzer, incubate for 23 hours, then export the results and use Prism8 software to plot and analyze them.

[0025] The results of 96-well plate studies showed that SP8 and PC111 had a synergistic effect in a 200 μL system, prolonging the growth time of resistant bacteria by at least 4 hours. This suggests that the SP8+PC111 cocktail can achieve better therapeutic effects in animals by lysing SP8-resistant bacteria and delaying the emergence of resistant bacteria.

[0026] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0027] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for enhancing the therapeutic effect of Salmonella phage, characterized in that, Includes the following steps: Step (1) Salmonella phage SP8 was cultured in LB broth medium to obtain a co-culture solution; the solution was spotted on SS medium plates containing phage at a concentration of 1×10^8-1×10^10 PFU / mL and cultured for 18 hours to screen for phage-resistant strains. Step (2) By screening for new phages that can lyse resistant strains, the new phages are mixed with the original Salmonella phage SP8 to obtain a phage cocktail.

2. The method according to claim 1, characterized in that, The bacteriophage SP8, belonging to the long-tailed bacteriophage family, with accession number CGMCC No. 45256, has a lytic effect on Salmonella. The applicant has already reported on this effect in the patent "A Salmonella bacteriophage SP8, a bacteriophage composition and its application". It exhibits strong lytic activity and a wide lytic spectrum against Salmonella, and can effectively control Salmonella infection in the intestines of poultry.

3. The method according to claim 1, wherein the novel phage is Salmonella phage PC111.

4. The method according to claim 1 or 2, wherein the phage cocktail comprises Salmonella phage SP8 and Salmonella phage PC111.

5. The method according to claim 1, wherein in the resistant strain screening step, SS medium plates containing bacteriophages are used, and the resistant strains are screened by spotting in co-culture medium and culturing for 18 hours.

6. The method according to claim 1, wherein the method can improve the lysis effect of bacteriophage on Salmonella and reduce the production of resistant bacteria.

7. The method according to claim 1, wherein in step (2), a new phage for lysing resistant strains is screened using the spot method, and the new phage is mixed with Salmonella phage SP8 to obtain a phage cocktail.

8. The method according to claim 1, wherein the method is applicable to Salmonella phage treatment and can effectively improve the treatment effect.

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