A bacteriophage cocktail for treating or preventing high-risk clonal strains in companion animals and uses thereof

Abstract

The application discloses a bacteriophage cocktail for treating or preventing high-risk clonal strains of companion animals and application thereof. The application provides a bacteriophage cocktail containing bacteriophages 32M3Y, CRE5643Y, 3BB6Y and S17S. Through simulating the mechanism of resistance occurrence in the bacteriophage treatment process, the second round of bacteriophage screening is carried out on the bacteriophage-resistant strains. The obtained candidate bacteriophages can lyse the resistant strains of high-risk clonal strains of Escherichia coli. The composition composed of the candidate bacteriophages can inhibit the resistance evolution of the bacteria in the bacteriophage treatment process, can avoid the emergence of the bacteriophage-resistant strains with enhanced virulence in the bacteriophage treatment process, has the effect of preventing and treating pet clinical ST410 type strain infection, and can be used for environmental disinfection. Through blocking the transmission chain of drug-resistant bacteria in the domestic environment (pets-human-environment), the risk of human-animal coexistence can be significantly reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to a bacteriophage cocktail for treating or preventing high-risk clonal strains of companion animals and its application. BACKGROUND

[0002] Bacterial drug resistance has become a major threat to the global public health system in the 21st century. The British "Antimicrobial Drug Resistance Assessment" predicts that by 2050, the annual death cases caused by drug-resistant bacteria infections may rise to the level of ten million, and the risk of death will exceed that of malignant tumors. In this public health crisis, the multiple drug resistance (MDR) characteristics of Gram-negative bacteria are particularly prominent, and their complex drug resistance mechanisms have led to a situation where there is no available drug for clinical treatment. It is alarming that high-risk clonal lineages with multiple drug resistance phenotype and strong pathogenicity are rapidly spreading through global transmission networks. Related studies have confirmed that high-risk clonal strains such as ST131-type Escherichia coli and ST258-type Klebsiella pneumoniae have formed regional epidemics worldwide.

[0003] Companion animals serve as an important reservoir of zoonotic pathogens, and their close relationship with humans has created a two-way channel for the cross-species transmission of drug-resistant bacteria. Epidemiological investigations have shown that the use of human antibiotics in pet clinical treatment is widespread, and this irrational drug use model not only accelerates the evolution and selection of multi-drug resistant strains, but also makes companion animals an "amplifier" of horizontal transfer of drug resistance genes. It is particularly worth noting that the cross-transmission of drug-resistant clonal strains between humans and pets has been confirmed to form a sustained cycle of transmission chain. Therefore, it is of urgent public health significance to develop new antibacterial strategies to reduce the use of antibacterial drugs in companion animals and the production and transmission of drug-resistant bacteria.

[0004] Escherichia coli ST410 is an extra-intestinal pathogenic E. coli associated with multi-drug resistance, which has been recognized as a new high-risk clonal strain. In global monitoring of ESBL and CRE drug resistance, ST410 is frequently detected, often carrying important drug resistance genes such as bla OXA-181 , bla CTX-M-15 , bla NDM-5 , etc. In pet clinics, ST410-type E. coli has been detected in multiple countries, and pet ST410 infection cannot be ignored, and efficient alternative antibacterial therapy is urgently needed.

[0005] In the field of research and development of new antibacterial alternative therapies, bacteriophages exhibit unique advantages due to their precise targeting, ecological friendliness, and self-amplification characteristics. Bacteriophages, as a class of viruses that parasitize on bacteria, provide a revolutionary solution to the crisis of multi-drug resistant (MDR) bacteria. Bacteriophage therapy has the characteristics of precise lysis of target bacteria, protection of symbiotic flora, and self-amplification to maintain effective concentration. Compared with economic animals in large-scale breeding, the individualized diagnosis and treatment needs of companion animals are highly consistent with the precise treatment mode and medical philosophy of the 'one bacterium one strategy' of bacteriophage therapy. It not only effectively solves the current clinical 'no drug available' dilemma, but also provides an innovative solution to curb the spread of drug-resistant bacteria between humans and animals.

[0006] However, bacteriophages have limitations such as narrow lysis range and resistance, and a single bacteriophage is difficult to cope with complex drug-resistant bacterial infection scenarios. For example, a bacteriophage Escherichia phage vB_EcoM_32M3Y obtained in previous studies can lyse high-risk clonal strains of Escherichia coli and target multiple serotypes. During the treatment process, the strain will produce resistance to the bacteriophage, leading to treatment failure. Therefore, it is necessary to develop a bacteriophage cocktail therapy, construct a companion animal drug-resistant bacteria monitoring network based on molecular typing, systematically screen potent bacteriophages against ST high-risk clonal strains, establish a standardized bacteriophage resource library, and ultimately form a precise prevention and control system for companion animal drug-resistant bacterial infections based on bacteriophage cocktail therapy. It not only provides an alternative solution for the prevention and control of infectious diseases in companion animals, but also curbs the production and spread of drug-resistant bacteria, ensuring public health safety. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the defects of the existing Escherichia coli bacteriophage 32M3Y, which is prone to produce bacteriophage resistance, and the treatment difficulties of pet multiple drug-resistant clonal strains, as well as the limitation of narrow lysis range of bacteriophage, and to provide a bacteriophage cocktail combination for treating or preventing high-risk clonal strains in companion animals and its application.

[0008] The first object of the present application is to provide a bacteriophage cocktail combination.

[0009] The second object of the present application is to provide the application of the bacteriophage cocktail combination.

[0010] The third object of the present application is to provide a product.

[0011] The fourth object of the present application is to provide a bacteriophage Kagunavirus_CRE5643Y.

[0012] The fifth object of the present application is to provide a bacteriophage Dhakavirus_S17S.

[0013] The sixth object of the present application is to provide a bacteriophage Phapecoctavirus_3BB6Y.

[0014] The sixth object of the present application is to provide the use of the above-mentioned bacteriophage.

[0015] The above-mentioned objects of the present application are achieved by the following technical solutions.

[0016] The present application provides a bacteriophage cocktail, which contains Escherichia phage vB_EcoM_32M3Y, bacteriophage Kagunavirus_CRE5643Y, bacteriophage Dhakavirus_S17S and bacteriophage Phapecoctavirus_3BB6Y. The Escherichia phage vB_EcoM_32M3Y has been deposited with the China General Microbiological Culture Collection Center on April 24, 2024, and the deposit number is CGMCC No.45969. The bacteriophage Kagunavirus_CRE5643Y has been deposited with the China General Microbiological Culture Collection Center on January 17, 2025, and the deposit number is CGMCC No.46368. The bacteriophage Dhakavirus_S17S has been deposited with the China General Microbiological Culture Collection Center on January 17, 2025, and the deposit number is CGMCC No.46369. The bacteriophage Phapecoctavirus_3BB6Y has been deposited with the China General Microbiological Culture Collection Center on January 17, 2025, and the deposit number is CGMCC No.46370.

[0017] The application is based on the fact that the Escherichia coli phage 32M3Y is easy to produce phage resistance, and the resistant strains of the phage 32M3Y are isolated and studied, the mechanism of resistance occurring in the phage treatment process is simulated, and then several phages are newly isolated and identified in sewage and fecal samples, and the phage resistance strains of the phage are screened for the second time, and the candidate phages can lyse the resistant strains of the high-risk clone of Escherichia coli, inhibit the resistance evolution of bacteria in the phage treatment process, and avoid the emergence of phage-resistant strains with enhanced virulence in the phage treatment process. On this basis, a phage cocktail therapy is established, and a phage cocktail combination composed of the phages 32M3Y, CRE5643Y, S17S and 3BB6Y is provided. The combination has good bactericidal effect on the multiple drug-resistant ST410 type Escherichia coli widely spread in medical and companion animal clinics, can significantly improve the survival state of mice in in vivo treatment, reduce the bacterial load in each organ, and can be better used for preventing and treating companion animals high-risk clone, providing more methods and products for preventing and treating infectious diseases caused by high-risk clone in companion animals, effectively curbing the generation and spread of drug-resistant bacteria, and ensuring public health safety.

[0018] Preferably, the concentration of each phage in the combination is not less than 10 10 PFU / mL.

[0019] The application provides application of the phage cocktail combination in inhibition of the high-risk clone of companion animals for the purpose of non-disease diagnosis and treatment.

[0020] The application provides application of the phage cocktail combination in preparation of a product for inhibiting the high-risk clone of companion animals.

[0021] The application provides application of the phage cocktail combination in preparation of a medicine for preventing and treating the high-risk clone of companion animals.

[0022] Preferably, the high-risk clone is the Escherichia coli ST410 B5 / H24RxC strain, and the pet-derived ST410 type Escherichia coli 39-1PT, 45M, 43M and 11M strains.

[0023] The application provides a product containing the phage cocktail combination.

[0024] In order to solve the problems of rapid development of phage resistance and potential virulence enhancement of drug-resistant strains, the candidate phages isolated and identified from sewage and fecal samples have Escherichia coli resistance, and are further used in the phage cocktail treatment method. Compared with a single phage, the combination has better treatment effect, and the resistance is also improved. Therefore, the application also protects the newly isolated and identified phages having the effect of lysing Escherichia coli and the application of the phages in the phage cocktail treatment method.

[0025] Meanwhile, the research shows that the separated phages CRE5643Y, S17S and 3BB6Y can overcome the resistance of E. coli resistant strains and have good lysis effect on the resistant strains of high-risk clone strains of E. coli obtained in vivo and in vitro. It is shown that the phage CRE5643Y has lysis effect on E. coli 32M, CRE564, 32MS1, 32M12P4, 32M1G22; the phage S17S has lysis effect on E. coli strains 32M, 32M3BB, 32MS1, 32MS2, 32MS3, 32MS4, 32MS5, 32MB4, 32M12P4, 32M102P8, 32M10G13, 32M1P18, 32M10S34, 32M10S41; the phage 3BB6Y has lysis effect on E. coli strains CRE564, 32MS1, 32MS2, 32MS3, 32MS4, 32MS5, 32MB4, 32M12P4, 32M102P8, 32M10G13, 32M1G22, 32M10S34, 32M10S41; except 32M and CRE564, all the above are resistant strains of E. coli phage P32M3Y. Since the newly identified phages CRE5643Y, S17S and 3BB6Y have resistance and lysis effect on these resistant strains, they can make up for the resistance problem in the process of phage therapy, have better therapeutic effect than the use of phage P32M3Y alone, and therefore the application also provides the application of the phages CRE5643Y, S17S or 3BB6Y in lysis of E. coli.

[0026] The application provides a phage Kagunavirus_CRE5643Y, which has been preserved in the China General Microbiological Culture Collection Center on January 17, 2025, and has a preservation number of CGMCC No.46368.

[0027] The application provides a phage Dhakavirus_S17S, which has been preserved in the China General Microbiological Culture Collection Center on January 17, 2025, and has a preservation number of CGMCC No.46369.

[0028] The application provides a phage Phapecoctavirus_3BB6Y, which has been preserved in the China General Microbiological Culture Collection Center on January 17, 2025, and has a preservation number of CGMCC No.46370.

[0029] The application provides the application of the phages CRE5643Y, S17S and 3BB6Y in lysis of E. coli.

[0030] Preferably, the Escherichia coli is a high-risk clone, comprising ST410 type Escherichia coli.

[0031] The application also provides the use of the above-mentioned bacteriophages CRE5643Y, S17S and 3BB6Y in the preparation of a bacteriophage cocktail combination product.

[0032] The application also provides the use of the above-mentioned bacteriophages CRE5643Y, S17S and 3BB6Y in the preparation of a drug for preventing and treating high-risk clones of companion animals.

[0033] The application has the following beneficial effects:

[0034] The application simulates the mechanism of resistance occurring in the process of bacteriophage treatment, carries out a second round of bacteriophage screening on bacteriophage-resistant strains, and develops a bacteriophage cocktail combination (bacteriophages 32M3Y+CRE5643Y+3BB6Y+S17S) with better treatment effect for the multi-drug resistant Escherichia coli ST410 B5 / H24RxC strain based on the candidate bacteriophages isolated and identified from sewage and fecal samples, which has a good bactericidal effect on the multi-drug resistant ST410 type Escherichia coli widely spread in medical and companion animal clinics, can significantly improve the survival state of mice for in vivo treatment, reduce the bacterial load in each organ, can be better used for preventing and treating high-risk clones of companion animals, can inhibit the resistance evolution of bacteria in the process of bacteriophage treatment, and avoid the emergence of bacteriophage-resistant strains with enhanced virulence in the process of bacteriophage treatment; the bacteriophage cocktail combination has the effect of preventing and treating ST410 infection in pet clinics, can be used for environmental disinfection, significantly reduces the risk of zoonosis by blocking the transmission chain of drug-resistant bacteria in the domestic environment (pets-humans-environment), provides more prevention and treatment methods and products for infectious diseases caused by high-risk clones in companion animals, effectively suppresses the generation and spread of drug-resistant bacteria, and ensures public health safety. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The treatment effect of a single bacteriophage P32M3Y.

[0036] Figure 2 The strain morphology of a bacteriophage-resistant strain.

[0037] Figure 3 The relative adsorption efficiency comparison of a bacteriophage mutant strain (Note: A: Relative adsorption efficiency of O antigen mutant strain; B: Relative adsorption efficiency of 32M3BB strain).

[0038] Figure 4 The sensitivity change of bacteriophage P32M3Y to yrfF overexpression strain.

[0039] Figure 5 For transcriptome KEGG enrichment analysis (Note: The horizontal axis is the ratio of genes annotated to the function to the total number of differential genes, the vertical axis is the enriched pathway, the scatter plot is represented by points of different colors and sizes, the redder the color, the more significant the enrichment; the larger the point, the more genes enriched; the red box is the significantly enriched pathway).

[0040] Figure 6 For transcriptome up-regulation fold of capsule polysaccharide biosynthesis related genes.

[0041] Figure 7 For mRNA expression level of 32M and 32M3BB.

[0042] Figure 8 For phage resistance trade-off evaluation of 32M3BB (Note: A: Growth curve of 32M3BB; B: Mouse virulence experiment of 32M3BB; C: Anti-macrophage phagocytosis experiment of 32M3BB).

[0043] Figure 9 For plaque morphology and transmission electron microscopy of phage cocktail combination.

[0044] Figure 10 For genome map of phage Escherichia phage vB_EcoM_32M3Y.

[0045] Figure 11 For genome map of phage Escherichia phage vB_EcoS_CRE5643Y.

[0046] Figure 12 For genome map of phage Escherichia phage vB_EcoM_S17S.

[0047] Figure 13 For genome map of phage Escherichia phage vB_EcoM_3BB6Y.

[0048] Figure 14 For phage spectrum of the second round of phage (Note: Red indicates lysis effect, white indicates no lysis effect).

[0049] Figure 15 For in vitro therapeutic effect of phage cocktail combination (Note: A, B: In vitro therapeutic effect of multiple phage combinations of ST410 B5 / H24RxC strains; C: In vitro therapeutic effect of phage cocktail combination 11 on ST410 B5 / H24RxC other pet source strains).

[0050] Figure 16 For in vivo therapeutic survival curve of phage cocktail.

[0051] Figure 17 Comparison of the bacterial load before and after in vivo treatment of the phage cocktail (Note: A: Comparison of the treatment bacterial load of strain 32M; B: Comparison of the treatment bacterial load of strain CRE564). DETAILED DESCRIPTION

[0052] The present application will be further described by the following description of drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0053] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0054] The Escherichia phage vB_EcoM_32M3Y (referred to as P32M3Y for short) used in the examples has been deposited with the China General Microbiological Culture Collection Center on April 24, 2024, and the deposit number is CGMCC No. 45969.

[0055] All experiments of the present application were approved by the Animal Protection and Utilization Committee of South China Agricultural University (APUC number: 2024c028). All experimental methods, animal care and in vivo experiments in the implementation were strictly in accordance with the animal ethics procedures of the People's Republic of China.

[0056] The formula of LB nutrient agar used in the examples is: tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, agar 15.0 g, distilled water 1000 mL.

[0057] The formula of LB broth is: tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, deionized water 1000 mL.

[0058] The formula of SM solution is: sodium chloride 8.5 g, magnesium sulfate 2 g, 1 mol / L Tris-HCl 50 mL, gelatin 0.25 g, deionized water 1000 mL.

[0059] The formula of LB semi-solid agar is: tryptone 7.2 g, yeast extract powder 3.6 g, sodium chloride 7.2 g, agar 7.0 g, deionized water 1000 mL.

[0060] 2mM CaCl2 sterile double-concentration broth: LB broth powder 20 g, CaCl2 88.78 mg, deionized water 400 mL.

[0061] Example 1 Isolation and sequencing of phage P32M3Y-resistant strains

[0062] To solve the problem of rapid development of phage resistance and potential virulence enhancement of drug-resistant strains, we screened the phage-resistant bacteria generated in vitro and in vivo. Due to the generation of bacterial resistance, the effect of treatment with phage P32M3Y was not good, and the results, as shown in Figure 1 , showed that although the phage P32M3Y treatment delayed the death of mice, the mice still died after 2 days due to the generation of phage-resistant bacteria. Therefore, based on previous studies, this embodiment isolated and sequenced different resistant strains of phage P32M3Y to further understand the resistance mechanism of phage P32M3Y.

[0063] Different morphological phage P32M3Y-resistant strains were selected on high MOI phage-bacteria semi-solid plates, which were in vitro resistant strains, numbered 32MB4, 32MS1, 32MS2, 32MS3, 32MS4, 32MS5, and 32M3BB. The phage-resistant strains isolated from the liver, spleen, lung, kidney and other organs of mice that still died after single phage P32M3Y treatment were in vivo resistant strains, numbered 32M1P4, 32M1G22, 32M1P18, 32M12P8, 32M10G13, 32M10S41, and 32M10G34.

[0064] The colony morphology of different phage-resistant strains selected is shown in Figure 2 , and then the bacterial DNA was extracted using the Magen bacterial genome kit, and the microbial small fragment library (350 bp) was constructed by Magen, and Illumina NovaSeq 6000 (PE150) was used for de novo sequencing (second-generation whole genome sequencing). Long-read high-throughput sequencing (third-generation whole genome sequencing) was performed using the Oxford Nanopore MinION sequencer.

[0065] After obtaining the Illumina sequencing data, the data was spliced using SPAde v3.8.7, and the Nanopore sequencing data was assembled and corrected using Unicyclerv0.4.7 to obtain the complete genome sequence. The drug resistance gene, plasmid replicon, and virulence gene analysis were performed using the bioinformatics software ABRicate v0.8. The open reading frame of the genome sequence was annotated using the Bakta v1.9.3 software, and the annotation results were aligned and corrected using NCBI-blastp. Finally, the bacterial core genome SNP (Single Nucleotide Polymorphism) analysis was performed using the Snippy software.

[0066] The 14 selected phage-resistant bacterial strains were subjected to whole genome sequencing analysis, and the results are shown in Table 1. It was found that 10 of the resistant bacteria had frame shift mutations caused by base deletion in the O antigen gene cluster. Four of the bacteria had frame shift mutations in the gmdeaj_09145 gene encoding O antigen polymerase, and six had frame shift mutations in the gmdeaj_09155 gene encoding glycosyltransferase family 8 protein. Both mutations resulted in premature termination of translation of O antigen-related proteins. In addition, two strains had insertions and deletions in the O antigen polymerase gene, and one strain had a point mutation F355I in the gene. In addition to the O antigen mutant strains, one strain, 32M3BB, had a point mutation Y97N in the YrfF gene. It can be seen that the resistance genes and mutations of different phage-resistant strains are completely different, and further analysis of the resistance mechanism is needed.

[0067] Table 1 SNP mutations of phage-resistant bacterial strains

[0068]

[0069] Example 2 Analysis of phage-resistant bacterial resistance mechanism

[0070] 1. Generation of phage resistance

[0071] (1) The bacteria were cultured to the logarithmic growth phase, and the phage solution and bacterial solution (part of the O antigen mutant strains 32M, 32MS1, 32MS2, 32MS3, 32MS4, 32MS5, 32BMB4) were mixed 1:1 at a MOI of 0.01.

[0072] (2) Place at room temperature for 10 min, and centrifuge at 8000g for 1 min in a 2 mL EP tube.

[0073] (3) Take the supernatant and filter through a 0.22 μm filter membrane.

[0074] (4) The filtered liquid was subjected to double-layer plate test to determine the phage titer. Each group was repeated three times. The formula for calculating the relative adsorption rate is:

[0075]

[0076] The adsorption efficiency of phage P32M3Y on part of the O antigen mutant strains at 10 min was compared, and the results are shown in Table A. The results showed that the adsorption efficiency of each group was significantly reduced, indicating that when the O antigen gene cluster is mutated, the synthesis of O antigen is blocked, and the binding force between the phage and the bacterial strain decreases, thereby producing phage resistance. Figure 3

[0077] 2. Analysis of the resistance mechanism of phage-resistant bacterial strain 32M3BB ​

[0078] (1) Analogous to O antigen mutant strain, the adsorption efficiency of phage P32M3Y on 32M and 32M3BB at 10 min was compared, and the method was the same as above.

[0079] The results are shown in Figure 3 B. Compared with 32M, the adsorption efficiency of phage P32M3Y on 32M3BB decreased by 71%, indicating that 32M3BB is also resistant to phage resistance.

[0080] (2) Since 32M cannot be genetically edited by traditional methods (CRISPR / Cas and lambda Red system), overexpression of wild-type yrfF gene in 32M3BB was selected to verify whether it affects colony morphology and phage resistance. The wild-type yrfF gene overexpression vector pBAD24-yrfF was constructed, the recombinant plasmid was transformed into 32M3BB strain, and pBAD24 empty plasmid was used as a control.

[0081] Compared with the empty bacteria 32M3BB-pBAD, the colony morphology of 32M3BB-pBAD-yrfF no longer showed a mucoid phenotype, indicating that the mucoid phenotype of 32M3BB is mediated by yrfF gene, as shown in Figure 2 To verify whether the resistance of phage P32M3Y to 32M3BB is caused by yrfF point mutation, plaque assay was performed with phage P32M3Y on 32M, 32M3BB, 32M3BB-pBAD-yrfF, and 32M3BB-pBAD.

[0082] The results are shown in Figure 4 4 ~10 8 PFU / mL of phage P32M3Y had lytic effect on 32M3BB-pBAD-yrfF, and could produce clear plaques, indicating that after overexpression of yrfF, 32M3BB-pBAD-yrfF could restore sensitivity to phage.

[0083] Based on the above-mentioned decrease in adsorption rate of phage P32M3Y on 32M3BB, relative adsorption experiments were carried out on 32M3BB-pBAD-yrfF and 32M-pBAD, and the method was the same as above.

[0084] The results are shown in Figure 3 B. The relative adsorption efficiency of phage P32M3Y on 32M3BB-pBAD-yrfF could reach 81.75% of 32M, and the relative adsorption efficiency on 32M3BB-pBAD was 32%, close to 32M3BB, indicating that complementation of yrfF restores sensitivity to phage by enhancing adsorption efficiency.

[0085] ​(3) In order to further explore the specific mechanism of 32M3BB resistance, the transcriptome sequencing of 32M and 32M3BB was performed.

[0086] The results are shown in Figure 5 From the correlation coefficient analysis results of each sample, it was found that the expression of 32M3BB in the polysaccharide synthesis and metabolic pathway was significantly up-regulated; among them, the genes involved in the biosynthesis of capsular polysaccharide, such as cpsG, wcaI, wcaF, wcaB, wcaA, wcaK, wcaC, wcaL, wcaE, etc., were up-regulated, as shown in Figure 6 The log2FoldChange value was between 1.74 and 3.72, i.e. the up-regulation multiple was between 3.34 and 13.

[0087] In addition, the differential genes rcsA, rcsB, rcsC were enriched in this pathway, which were related genes in the RcsCDB regulated by yrfF gene, and the log2FoldChange value of rcsA was 1.95, indicating that its expression level was increased by 3.86 times.

[0088] According to the results of transcriptome, part of the differential genes were selected for fluorescence quantitative PCR detection. The results are shown in Figure 7 It was shown that compared with 32M, the expression levels of rcsA and cpsG genes of 32M3BB strain were significantly increased. Among them, the expression level of rcsA was increased by 5 times, the expression level of cpsG was significantly increased by ~ 16 times, and the expression level of wza was also significantly increased (3-8 times).

[0089] Based on the sequencing analysis of phage-resistant strains, two types of resistance were mainly found. The first type was O antigen mutant strain, which was the main mechanism of phage-resistant strain, and the main reason was that the receptor of phage Escherichia phage vB_EcoM_32M3Y was O antigen. In addition, the mutation type of in vitro resistant strain was highly consistent with that of in vivo screened resistant strain, indicating that the in vitro screened resistant bacteria could reflect the resistance type of reagent in clinic to a certain extent, and had certain representativeness. The second type was the production of capsular polysaccharide. Compared with the original strain JXZ9A32M, the colony morphology of 32M3BB was larger and the surface was wet. Through transcriptome and q-PCR experiment, it was verified that the secretion of capsular polysaccharide of 32M3BB was increased.

[0090] 3. Performance test of phage-resistant strain 32M3BB

[0091] (1) Growth curve determination: single colony was picked and inoculated in 2 mL fresh LB broth, and seed liquid was prepared by overnight culture at 37°C, 180 rpm. The OD 600The inoculum was calculated according to the absorbance, and 50 mL of fresh LB broth was transferred to make the initial culture OD 600 The value was 0.01. Every 1 h, 200 μL was taken in a 96-well plate, and the OD 600 The value was 0.01. Every 1 h, 200 μL was taken in a 96-well plate, and the OD

[0092] As shown in Fig. A, the growth rate of 32M3BB was significantly lower than that of 32M, and the time to enter the plateau phase was about 1 h later than that of 32M, indicating that the adaptability of 32M3BB was reduced compared with the wild-type strain 32M. Figure 8 A, showing that the growth rate of 32M3BB was significantly lower than that of 32M, and the time to enter the plateau phase was about 1 h later than that of 32M, indicating that the adaptability of 32M3BB was reduced compared with the wild-type strain 32M.

[0093] (2) Antibiotic sensitivity test: The antibiotic sensitivity test was performed on the original bacteria and phage-resistant bacteria, and the method was performed according to the guidelines of the American Clinical Laboratory Standardization Committee (Clinical and Laboratory Standards Institute, CLSI). The minimum inhibitory concentration (Minimum Inhibitory Concentration, MIC) of 10 kinds of antibacterial drugs, such as cefotaxime (CTX), imipenem (IMP), ciprofloxacin (CIP), gentamicin (GEN), amikacin (AMK), fosfomycin (FOS), tigecycline (TIG), doxycycline (DOX), florfenicol (FFC) and colistin (CL) on the test strains was determined.

[0094] The results are shown in Table 2, which shows that the MIC value of 32M3BB to antibacterial drugs did not change significantly compared with 32M.

[0095] Table 2 Change of antibiotic sensitivity of 32M3BB (unit: μg / mL)

[0096]

[0097] (3) Mouse survival rate experiment: 5-week-old female C57BL / 6 mice (purchased from Zhuhai Baiti Tong Company) were raised in a SPF animal house (25±2℃, 50% relative humidity, 12:12 h light-dark cycle) for 1 week to adapt to the experimental environment. Except for the blank control group (NC), the mice were randomly divided into 2 groups, i.e. infection model group (32M) and infection model group (32M3BB), each group of 10. The 2 groups of infection groups were injected intraperitoneally with 32M and 32M3BB (1×10 8 CFU), and the survival rate of mice was calculated after 12, 24, 48, 72, 96 and 120 h.

[0098] The results are shown in Fig. B, which shows that the survival rate of mice infected with 32M3BB was significantly lower than that of mice infected with 32M, indicating that the virulence of 32M3BB was reduced compared with the wild-type strain 32M. Figure 8As shown in B, 32M3BB exhibits enhanced pathogenicity potential. In a mouse infection model, 32M3BB caused 70% mortality within 24 hours, which is 40% higher than the mortality rate of 32M.

[0099] (4) Anti-macrophage phagocytosis assay: RAW264.7 cells were cultured to 1-2 × 10⁻⁶ cells per cell line. 5 CFU / mL, vortex to mix, then add 1 mL to each well of a 12-well plate and incubate in a CO2 incubator for no more than 18 hours. Incubate the bacterial culture until OD reaches... 600 =0.5, then dilute the bacteria at an MOI of 10 and resuspend the bacterial culture in DMEM. Discard the complete culture medium in the 12-well plate, gently add 200 μL of PBS or DMEM to the sidewall of the 12-well plate and discard the liquid. Add 200 μL of bacterial culture resuspended in DMEM and incubate in a CO2 incubator for 2 h. Discard the bacterial culture, add 200 μL of a 150 μg / mL apramycin solution dissolved in DMEM, and incubate in a CO2 incubator for 1 h. Discard the drug solution, wash the cells with 200 μL of PBS and discard the liquid. Lyse the cells with 500 μL of pre-chilled 0.1% Triton X-100 for 10 min, thoroughly mix the cells by pipetting, and take 100 μL of serially diluted bacteria for plate counting.

[0100] The results are as follows Figure 8 As shown in C, 32M3BB exhibited a strong immune evasion ability, reducing the phagocytic rate of RAW264.7 macrophages by 20-fold (32M3BB: 0.12% vs 32M: 2.4%).

[0101] In summary, the results indicate that the resistant strain 32M3BB exhibits an adaptive cost of slower growth, but displays an enhanced virulence phenotype, specifically manifested in increased anti-macrophage activity and higher mouse mortality. Growth curve analysis showed that, compared to strain 32M, the growth rate was reduced ( Figure 8 A) indicates a significant adaptive cost. Notably, 32M3BB exhibits enhanced pathogenicity, with a 70% mortality rate within 24 hours in a mouse infection model, a 40% increase compared to the wild-type (p<0.05). Figure 8 B). Simultaneously, this mutant exhibited enhanced immune evasion, reducing the phagocytic rate of RAW264.7 macrophages by 20-fold (0.12% vs 2.4%, p<0.001). Figure 8 C) established a direct link between phage resistance and the evolution of high virulence. The enhanced virulence phenotype of resistant strains is highly detrimental to phage therapy, necessitating the addition of phages that can target these strains to phage cocktails.

[0102] Example 3: Isolation, preparation, purification, and culture of bacteriophages

[0103] 1. Sewage sample pre-treatment

[0104] (1) Collect sewage from Guangzhou Panyu slaughterhouse and Foshan animal clinic, and transport back to the laboratory after low temperature preservation.

[0105] (2) Remove larger particulate impurities by gauze filtration.

[0106] (3) Centrifuge the sample at 4000g for 20 min at low temperature to remove bacteria and other impurities.

[0107] (4) Perform bacterial sterilization filtration using 0.45 μm and 0.22 μm microporous filters in sequence.

[0108] (5) Use small tangential flow to ultrafiltrate and concentrate the filtered sewage sample to about 50 mL of liquid, obtaining a bacteriophage stock solution for use, which can add a small amount of chloroform.

[0109] 2. Feces sample pre-treatment

[0110] (1) Collect pig feces from a breeding farm in Yunfu, Guangdong, and transport back to the laboratory after low temperature preservation.

[0111] (2) Add fresh feces to SM buffer and mix well by shaking for 4 h.

[0112] (3) Centrifuge the sample at 4000g for 20 min at low temperature to remove bacteria and other impurities.

[0113] (4) Perform bacterial sterilization filtration using 0.45 μm and 0.22 μm microporous filters in sequence.

[0114] 3. Bacteriophage culture

[0115] (1) Take 10 mL of sterile double-concentration broth containing 2 mmol / L CaCl2, and add 10 mL of the above-mentioned clear (or filtered) water sample.

[0116] (2) Inoculate 0.1 mL of overnight culture of host bacteria with 10 mL of the above-mentioned clear (or filtered) water sample, and incubate at the appropriate growth temperature (usually 37°C) with gentle shaking (50 rpm / min).

[0117] (3) After 24-48 h of culture, centrifuge at 10000g for 10 min. Pour the supernatant into a small bottle with a screw cap or a test tube with a stopper. Add 0.5 mL of chloroform to the clear crude lysate, shake gently and store at 4°C.

[0118] 4. Bacteriophage spotting test

[0119] (1) Take 100 μL of the bacterial body of the overnight culture in the above step and 5 mL of the semi-solid medium melted and kept at 50°C, mix them, and pour them onto the bottom agar plate to prepare the bacterial lawn of each strain.

[0120] (2) Add several drops of 5 μL of the sample enrichment liquid to be tested on the bacterial lawn until the drops are completely dried.

[0121] (3) Incubate the plate in a 37°C constant temperature incubator overnight. Check whether there is a clear and turbid lysis zone.

[0122] 5. Isolation of bacteriophage

[0123] (1) Add 0.9 mL of SM Buffer to a sterile test tube or centrifuge tube, and number the centrifuge tubes according to the dilution.

[0124] (2) Add 0.1 mL of the bacteriophage enrichment liquid to the first tube, mix well, and transfer 0.1 mL to the second tube. Perform 10-fold dilution in this way.

[0125] (3) Transfer 0.1 mL of the bacteriophage dilution to a warm semi-solid medium test tube, immediately add 0.1 mL of the host bacteria cultured to the logarithmic growth phase, mix well, and introduce it into the surface of the solid medium; after the semi-solid layer is solidified, culture it at the optimum culture temperature with inversion. Culture for 8-10 h, and select the plate with a single plaque for the purification of the bacteriophage.

[0126] 6. Purification of bacteriophage

[0127] (1) Use a high-pressure sterilized gun tip to pick up a single plaque, suspend it in 1 mL of SM Buffer, and mix it thoroughly by vortexing to fully dissolve the bacteriophage in the SM Buffer.

[0128] (2) Filter it using a filter membrane with a pore size of 0.22 μm to remove impurities.

[0129] (3) Dilute the SM Buffer mixed with the plaque appropriately, and use the double-layer plate culture method to perform continuous passage of the bacteriophage.

[0130] 7. Preservation of bacteriophage

[0131] In a 2 mL preservation tube, mix 0.5 mL of the bacteriophage stock solution with 0.5 mL of sterile glycerol to a final concentration of 50%. Store it in a -80°C refrigerator.

[0132] The purified bacteriophage individuals were obtained when the observed plaques were substantially consistent in size and morphology. The bacteriophages were isolated from the sewage sample and the fecal sample according to the above separation and purification steps, respectively, and the obtained candidate bacteriophages were named as PCRE5643Y, PS15B, PS15S, PS17S, P3BB12S, P3BB5Y, P3BB6Y, and P3BB7Y, respectively.

[0133] Example 4 Observation of bacteriophages

[0134] (1) Take the bacteriophage culture prepared in Example 3 and bacteriophage P32M3Y, and use a 100 kDa Amicon Ultra centrifugal ultrafiltration tube to concentrate 50 mL of the bacteriophage liquid to 2 mL.

[0135] (2) Take 15 μL of the concentrated bacteriophage liquid and drop it on a copper mesh, and precipitate for 15 min.

[0136] (3) Use filter paper to gently absorb the excess liquid on the copper mesh, then add 2% phosphotungstic acid (PTA, pH = 7.0) on the copper mesh, and stain for 10 min. After the copper mesh is dried, electron microscope observation and photography are performed.

[0137] The results of the electron microscope observation of some bacteriophages are shown in Table 1. Figure 9 As shown in Table 1, under transmission electron microscope observation, the head structure of the bacteriophage P32M3Y was found to have a diameter of about 85 nm, and a tail structure having a length of about 126 nm.

[0138] Under transmission electron microscope observation, the head structure of the bacteriophage 3BB6Y was found to have a diameter of about 85.38 nm, and a tail structure having a length of about 85.13 nm.

[0139] Under transmission electron microscope observation, the head structure of the bacteriophage CRE5643Y was found to have a diameter of about 55 nm, and a tail structure having a length of about 109.5 nm.

[0140] Under transmission electron microscope observation, the head structure of the bacteriophage S17S was found to have a diameter of about 83 nm, and a tail structure having a length of about 121.1 nm.

[0141] Example 5 Extraction and sequencing of bacteriophage genomes

[0142] (1) Take the bacteriophage culture prepared in Example 3, and use a 100 kDa Amicon Ultra centrifugal ultrafiltration tube to concentrate 50 mL of the bacteriophage liquid to 2 mL.

[0143] (2) Take 2 mL of the phage sample which has passed through a 0.22 μm microporous filter into a 15 mL centrifuge tube which has been subjected to high pressure sterilization and has a smooth surface.

[0144] (3) Add 2 μL of DNase I and RNase (1 mg / mL final concentration 1 μg / mL), mix gently, and incubate at 37°C for 1 h.

[0145] (4) In a 15 mL centrifuge tube, add 2 mL of formamide, 20 μL of EDTA, 200 μL of 2M Tris HCl / 0.2M EDTA (TE), mix gently, and stand at room temperature for 30 min.

[0146] (5) Use the phage genome extraction kit (M13 Isolation Kit D6900) to complete the extraction of genomic DNA according to the instructions.

[0147] (6) Send the phage product to Meiji Bio for sequencing to obtain the nucleotide sequence of the phage.

[0148] The genome annotation results of part of the phage are shown in Table 1, which shows that the genome of Escherichia phage P32M3Y is linear double-stranded DNA, with a size of 152897 bp and a G+C content of 49%, and 187 open reading frames (ORF) are successfully annotated. The prediction results of VirulenceFinder and ResFinder show that P32M3Y genome does not contain virulence factors and drug resistance genes, and belongs to Ackermannviridae virus family, Aglimvirinae subfamily, and Agtrevirus genus. Figures 10-13 The genome of phage Escherichia phage vB_EcoS_CRE5643Y is linear double-stranded DNA, belongs to Guernseyvirinae virus subfamily, Kagunavirus genus, has a size of 44881 bp, a G+C content of 51%, and 80 open reading frames (ORF) are successfully annotated. The prediction results of VirulenceFinder and ResFinder show that the genome of Escherichia phage vB_EcoS_CRE5643Y does not contain virulence factors and drug resistance genes.

[0149]

[0150] ​The bacteriophage Escherichia phage vB_EcoM_3BB6Y genome is a linear double-stranded DNA, belonging to the Stephanstirmvirinae subfamily, Phapecoctavirus genus, with a size of 146712 bp and a G+C content of 39%, and 278 Open Reading Frames (ORFs) are successfully annotated. The prediction results of VirulenceFinder and ResFinder show that there are no virulence factors and drug resistance genes in the Escherichia phage vB_EcoS_3BB6Y genome.

[0151] The bacteriophage Escherichia phage vB_EcoM_S17S genome is a linear double-stranded DNA, belonging to the Straboviridae family, Tevenvirinae subfamily, Dhakavirus genus, with a size of 169396 bp and a G+C content of 40%, and 278 Open Reading Frames (ORFs) are successfully annotated. The prediction results of VirulenceFinder and ResFinder show that there are no virulence factors and drug resistance genes in the Escherichia phage vB_EcoS_S17S genome.

[0152] Example 6 Determination of the lysis spectrum of bacteriophages and preservation

[0153] 1. Determination of the lysis spectrum of bacteriophages

[0154] ST410 type Escherichia coli isolates and bacteriophage-resistant strains in Example 1 were selected and cultured to the logarithmic growth phase, 200 μL of bacterial solution was added to LB agar plates, and 4.8 mL of semi-solid was added, shaken well, and cooled; 5 μL of bacteriophage liquid with a titer of 1 x 10 8 PFU / mL of bacteriophage liquid was dropped in the center of the semi-solid, and after the liquid was completely absorbed, it was placed in a 37°C incubator overnight. The bacteria that could form plaques were recorded as “+”, which was a positive result, and vice versa, which was a negative result.

[0155] Through bacteriophage screening by bacteriophage-resistant strains, Escherichia coli bacteriophages that overcome resistance were obtained, and receptors were speculated by tail protein analysis. The specific information is shown in Table 3, which shows that 87.5% (7 / 8) of the bacteriophages isolated in the second round have a wide coverage of bacteriophage-resistant strains obtained in vivo and in vitro, and the bacteriophage spectrum results are shown in Figure 14 Table 4, which have good lytic effects on different Escherichia coli resistant strains, indicating that these bacteriophages have good therapeutic effects on bacteriophage-resistant strains in vivo and in vitro.

[0156] Table 3 Information of phages separated in the second round

[0157]

[0158] 2. Preservation of phages

[0159] The above resistant phages were then biologically preserved, and the preservation information is as follows:

[0160] (1) Escherichia phage vB_EcoS_CRE5643Y, deposited on January 17, 2025 at the China General Microbiological Culture Collection Center, taxonomically named Kagunavirus, and the preservation number is CGMCC No. 46368.

[0161] (2) Escherichia phage vB_EcoM_S17S, deposited on January 17, 2025 at the China General Microbiological Culture Collection Center, taxonomically named Dhakavirus, and the preservation number is CGMCC No. 46369.

[0162] (3) Escherichia phage vB_EcoS_3BB6Y, deposited on January 17, 2025 at the China General Microbiological Culture Collection Center, taxonomically named Phapecoctavirus, and the preservation number is CGMCC No. 46370.

[0163] Example 7 In vitro therapeutic effect of Escherichia phage

[0164] Using the 8 candidate phages separated in Example 3 and phage P32M3Y, phage cocktail therapy was used to treat Escherichia coli ST410 B5 / H24RxC strain 32M and CRE564, as well as other pet-derived ST410 type Escherichia coli 39-1PT, 45M, 43M and 11M. According to Table 3, the design of phage cocktail combination was carried out, and in vitro bactericidal experiments were carried out. The design principle of the phage cocktail is: it includes 2 strains of phage P32M3Y and PCRE5643Y effective against ST410 B5 / H24RxC strain, and other phages in the combination are phages of Dhillonvirus, Dhakavirus, Phapecoctavirus genus in Table 3 against phage-resistant bacteria, and only one strain of each genus phage because the receptors of phages of the same genus are similar, different combinations are set.

[0165] The combinations of the phage cocktails are set as combination 1, combination 2, combination 3, combination 4, combination 5, combination 6, respectively, and the ratio of the phages used in the phage cocktails is 1:1:1:1, wherein the phage combination of combination 4 is P32M3Y+PCRE5643Y+P3BB6Y+PS17S. Meanwhile, single phage 32M3Y or PCRE5643Y is used for treatment. The specific operation method is as follows:

[0166] (1) The host bacteria are cultured to the logarithmic growth phase, and the phage cocktail and the bacterial solution are mixed at a ratio of 1:1 at MOI=10. The blank group is mixed with LB broth and the first round of phage P32M3Y, and cultured at 37°C in a growth curve instrument.

[0167] (2) The OD 600 value is measured every 1h. Each experimental group is repeated 3 times.

[0168] The results are shown in Figure 15 A-B, which show that all combinations have effective bactericidal activity within the first 10 hours. Although phage resistance appears in some groups around 12 hours, it is obviously later than single phage treatment. Among them, combination 4 (phage P32M3Y+PCRE5643Y+P3BB6Y+PS17S) has good bactericidal effect on ST410 Escherichia coli, including good bactericidal effect on other pet-derived ST410 type Escherichia coli 39-1PT, 45M, 43M and 11M, as shown in Figure 15 C.

[0169] Example 8 In vivo treatment effect of Escherichia coli phage

[0170] Take 70 C57BL / 6 mice of 29-35 days old, and divide them into 5 groups, i.e. 6 blank groups, 16 resistant strain 32M control groups, 16 32M phage treatment groups, 16 CRE564 control groups, and 16 CRE564 phage cocktail treatment groups. The phage:bacteria ratio (MOI) is 10:1, and except for the blank groups, the rest of the mice are injected intraperitoneally with 10 8 CFU of Escherichia coli solution. 2h later, the mice start to show symptoms of listlessness, the phage treatment groups are injected intraperitoneally with 10 9 PFU of phage cocktail at a ratio of MOI=10:1, and the blank groups and the control groups are injected intraperitoneally with the same volume of PBS.

[0171] The results are shown in Figure 16As shown, it is shown that after 12h, the mortality rate of 32M control group mice is more than 50%, and 70% at 24h; while after phage treatment, the survival of mice is obviously improved, showing only 10% death at 24h and 30% death at 36h. The control group of CRE564 reaches half death at 24h, but the mice in the phage cocktail treatment group are all cured.

[0172] Further, the phage cocktail treatment group is further measured for the bacterial load before and after treatment in vivo, and the sample is ground in 1mL sterile PBS using a Shanghai Jingxin cryogenic grinder, 4℃, 60Hz, 2min, and repeated once after a 1min pause. The homogenate solution is continuously diluted in PBS. Then 50μL of the diluent is evenly dropped on the LB agar plate, and incubated at 37℃ for 18h.

[0173] The determination results are as shown in Table 2. Figure 17 As shown, it is shown that after phage cocktail treatment, the bacterial load of each organ of the treatment group is significantly reduced.

[0174] In summary, based on the poor treatment effect and the defects and deficiencies of drug resistance of the phage Escherichia phage vB_EcoM_32M3Y, through the isolation and research of the resistant strains of the phage 32M3Y, it is shown that the resistance types of different resistant strains are completely different, and it is found that the phage resistant mutant strain 32M3BB grows slowly, the virulence is enhanced, and the mortality rate to mice is increased. In order to solve the rapid development of phage drug resistance and the potential virulence enhancement of drug-resistant strains, 8 candidate phages with Escherichia coli resistance are isolated and identified from sewage and fecal samples; combined with the phage 32M3Y, we developed a therapeutic phage cocktail combination (phages P32M3Y+PCRE5643Y+P3BB6Y+PS17S) for Escherichia coli ST410 B5 / H24RxC strains, a phage cocktail therapy for pet-derived multi-drug resistant Escherichia coli ST410, by simulating the mechanism of resistance appearing in the phage treatment process, a second round of phage screening is carried out for phage-resistant strains, the resistance evolution of bacteria in the phage treatment process is inhibited, and the emergence of phage-resistant strains with enhanced virulence in the phage treatment process is avoided. The phage cocktail combination has the treatment ability for anti-pet clinical ST410 infection and can be used for disinfection of pet hospitals and hospitals, significantly reduces the transmission chain of drug-resistant bacteria in the domestic environment (pets-humans-environment), and significantly reduces the risk of zoonosis, providing more methods and products for treating or preventing diseases caused by high-risk clonal strains of companion animals.

[0175] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A cocktail of bacteriophages, characterized in that, Escherichia coli phage Escherichia phage vB_EcoM_32M3Y, phage Kagunavirus _CRE5643Y, phage Dhakavirus _S17S, phage Phapecoctavirus _3BB6Y; the Escherichia coli phage Escherichia phage vB_EcoM_32M3Y has been deposited with the China General Microbiological Culture Collection Center on April 24, 2024, and has the accession number CGMCC No. 45969; the phage Kagunavirus _CRE5643Y has been deposited with the China General Microbiological Culture Collection Center on January 17, 2025, and has the accession number CGMCC No. 46368; the phage Dhakavirus _S17S has been deposited with the China General Microbiological Culture Collection Center on January 17, 2025, and has the accession number CGMCC No. 46369; the phage Phapecoctavirus _3BB6Y has been deposited with the China General Microbiological Culture Collection Center on January 17, 2025, and has the accession number CGMCC No. 46370.

2. The bacteriophage cocktail of claim 1, wherein, The concentration of each bacteriophage in the combination is not less than 10 10 PFU / mL.

3. Use of the phage cocktail combination according to claim 1 or 2 for the inhibition of high-risk clonal strains of companion animals for non-diagnostic therapeutic purposes or for the manufacture of a product for the inhibition of infection with high-risk clonal strains of companion animals, characterized in that, The high-risk clonal strain of the companion animal is Escherichia coli of ST410 type.

4. Use of the phage cocktail combination according to claim 1 or 2 for the manufacture of a medicament for the treatment of a high risk clonal strain infection in a companion animal, characterized in that, The high-risk clonal strain of the companion animal is Escherichia coli of ST410 type.

5. A product characterized by, The phage cocktail according to claim 1 or 2.

6. A bacteriophage Kagunavirus _CRE5643Y, characterized in that, The phage has been preserved in the China General Microbiological Culture Collection Center on January 17, 2025, and the preservation number is CGMCC No. 46368.

7. A bacteriophage Dhakavirus S17S, characterized in that, The phage has been preserved in the China General Microbiological Culture Collection Center on January 17, 2025, and the preservation number is CGMCC No. 46369.

8. A bacteriophage Phapecoctavirus 3BB6Y characterized in that, The phage has been preserved in the China General Microbiological Culture Collection Center on January 17, 2025, and the preservation number is CGMCC No. 46370.

9. Use of the phage according to any one of claims 6-8 in the preparation of a phage cocktail.

10. Use of the bacteriophage according to any one of claims 6 to 8 for the preparation of a medicament for the treatment of an infection with a high-risk clonal strain in a companion animal, characterized in that, The high-risk clonal strain of the companion animal is Escherichia coli of ST410 type.

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