A bacteriophage composition and its application and pharmaceutical composition

Abstract

The present invention belongs to the field of microbiology and specifically relates to a phage composition, its application, and pharmaceutical composition. The phage composition comprises the following phages: ФEBU8, ФECL22, and ФEBU30. The present invention highlights the critical importance of early treatment in the treatment of Enterobacter cloacae bacteremia using a phage cocktail, while prophylactic administration may offer the opportunity to delay treatment and salvage bacteremia.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a bacteriophage composition, an application thereof and a pharmaceutical composition. Background Art

[0002] In recent years, the gradual rise of antibiotic resistance among Enterobacteriaceae, particularly the increasing prevalence of carbapenem-resistant strains, has posed a pressing global health challenge. These multidrug-resistant organisms contribute to significant morbidity and mortality in hospital-acquired infections, significantly increasing the complexity of patient management and treatment. Among the Enterobacteriaceae family, the Enterobacter cloacae complex is of particular concern, as it has been identified as one of the leading pathogens of bloodstream, respiratory, and urinary tract infections, particularly in immunocompromised patients. These pathogens, with their propensity to develop resistance and cause severe infections, are classified as "ESKAPEE," a priority group designated by the World Health Organization. Notably, Enterobacter cloacae complex bacteria exhibit alarming rates of carbapenem resistance, further limiting treatment options and driving interest in alternative therapies.

[0003] Phage therapy can specifically target and lyse bacteria without disrupting the host microbiome. Due to its unique mechanism of action, it has become a promising approach to combat multidrug-resistant bacterial infections. Unlike traditional antibiotics, phages are able to replicate themselves at the site of infection, which offers unique advantages in reducing bacterial loads in the body. However, the success of phage therapy depends on factors such as dosage and timing of administration. Several studies have shown that the use of phage cocktails at appropriate doses within the optimal therapeutic window can significantly improve survival and clinical outcomes in infection models. Although a large number of studies have focused on phage therapy against common multidrug-resistant pathogens, phage therapy against Enterobacter spp. remains understudied, partly because relatively few phages of the Enterobacter cloacae complex have been identified.

[0004] Phage combinations, also known as phage cocktails, are often more effective in treating infections than any single phage alone. This is because many phages have a narrow host range, and mixing multiple phages broadens the spectrum of activity. Furthermore, synergistic effects of several phages with different mechanisms of action or receptors can significantly inhibit the emergence of phage-resistant strains. However, in animal models, the bacteriophage killing process can release endotoxins and trigger inflammation, necessitating the development of a safer phage combination. Summary of the Invention

[0005] To solve the above problems, the present invention provides a phage composition, its application and pharmaceutical composition.

[0006] A phage composition comprising the following phages: ФEBU8, ФECL22, and ФEBU30;

[0007] The deposit number of ФECL22 (Enterobacter cloacae bacteriophage) is CGMCC No.46281, and the deposit date is November 18, 2024, deposited in the General Microbiology Center of China Culture Collection of Microorganisms. The deposit number of ФEBU8 (Enterobacter bugandensis bacteriophage) is CGMCC No.46282, and the deposit date is November 18, 2024, deposited in the General Microbiology Center of China Culture Collection of Microorganisms. The deposit number of ФEBU30 (Enterobacter cloacae bacteriophage) is CGMCC No.46283, and the deposit date is November 18, 2024, deposited in the General Microbiology Center of China Culture Collection of Microorganisms (Beijing, China).

[0008] The phage composition is used in the preparation of a medicine for treating Enterobacter cloacae bacteremia.

[0009] Preferably, the volume ratio of ФEBU8, ФECL22 and ФEBU30 in the phage composition is 1-2:1-3:1.

[0010] Preferably, the total content of phages in the phage composition is ≥10 4 PFU / mL.

[0011] The invention relates to an application of the bacteriophage composition in preparing a medicine for combating antibiotic-resistant Enterobacter cloacae bacteremia.

[0012] Preferably, the antibiotic is a carbapenem antibiotic.

[0013] A pharmaceutical composition comprising the phage composition.

[0014] Preferably, it also includes a pharmaceutically acceptable carrier.

[0015] Preferably, the pharmaceutically acceptable carrier is any one of phosphate buffered saline and sterile physiological saline.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention isolated and purified three novel virulent phages with different genome sizes and host ranges, namely ФEBU8, ФECL22 and ФEBU30. Among them, ФEBU8 exhibits broad-spectrum activity and can lyse Enterobacter and Acinetobacter baumannii. ФECL22 also has a relatively broad host spectrum and is stable within a wide temperature range (4-50°C) and pH range (6-10). Its burst capacity is 19 PFU / cell, and its receptor is identified as the outer membrane protein OmpA. ФEBU8 and ФEBU30 can lyse mutants resistant to ФECL22. Therefore, the present invention prepares these three phages into a cocktail, which is safe both in vitro and in vivo.

[0018] The present invention prepares a cocktail based on the broad-spectrum activity of ФEBU8, ФECL22 and ФEBU30 at a volume ratio of 1-2:1-3:1, which can lyse 86.5% (32 / 37) of the tested Enterobacter cloacae complex bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 (A) shows a host spectrum heat map of isolated Enterobacteriaceae phages; dark blue, large and clear plaques; light blue, fuzzy plaques; white, no plaques. Figure 1 (B)-(C) show that Enterobacter cloacae phages ФEBU8 and ФECL23 lyse Acinetobacter baumannii, (B) the two phages are added to Acinetobacter baumannii in gradient droplets; (B) the two phages inhibit the growth curve of Acinetobacter baumannii.

[0020] Figure 2 Showing the morphology of ФEBU8, ФECL22, and ФEBU30; (A) is the results of the lysis test of ФEBU8, ФECL22, and ФEBU30 against Enterobacter cloacae 22 and its derivative ФECL22-resistant mutant R3; (B) is the phage plaques of the three phages on LB agar, where (1) is the respective bacterial host and (2) is the transmission electron microscopy (TEM) image; magnification: ×60,000; scale bar: 100 nm.

[0021] Figure 3 Genomic linear analysis of three Enterobacter cloacae phages and their respective homologous phages.

[0022] Figure 4 Figure 3 shows the biological characteristics of ФECL22; (A) heat resistance; (B) pH stability; (C) UV sensitivity; (D) optimal MOI; (E) adsorption rate in LB broth; (F) one-step growth curve; (G) in vitro growth inhibition at different MOIs; (H) in vitro growth inhibition of a phage cocktail consisting of ФEBU8, ФECL22, and ФEBU30.

[0023] Figure 5 Identification of the phage receptor for ФECL22; (A) Adsorption test results; differences between groups were analyzed using univariate statistical analysis; ***p<0.001; (B) Introduction of OmpA from Enterobacter cloacae into the R3 mutant can restore the phage sensitivity to ФECL22.

[0024] Figure 6 Figure 3 Safety evaluation of the phage cocktail; (A) CCK-8 assay results for the cytotoxicity of the cocktail on liver cells L02; (B) Assessment of the acute toxicity of the phage cocktail in the mouse model, monitoring clinical scores, where "0" indicates a healthy state; (C) Changes in body weight; (D) Determination of endotoxin levels at the 24-h checkpoint after injection; (EG) Cytokine levels measured at the same checkpoints; 1× PBS was used as a negative control; Differences between the groups were statistically analyzed using one-way analysis of variance; *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 7 Figure 2: The efficacy of different doses of phage cocktail therapy on the mouse bacteremia model induced by Enterobacter cloacae; (A) The mouse bacteremia model was established using different numbers of Enterobacter cloacae22; (B) The bacterial load in organs was assessed at 24 h POI; (C) The phage titer in organs was measured at 24 h POI; the differences between the groups were analyzed using univariate statistical analysis; *p<0.05, **p<0.01; (D) Clinical scores of treated mice; (E) Changes in body weight; (F) Survival curves of infected mice after receiving different doses of phage cocktail.

[0026] Figure 8 Figure 3: The efficacy of phage cocktail therapy on the mouse bacteremia model induced by Enterobacter cloacae at different time points; (A) Assessment of bacterial load in organs at 24h POI; (B) Determination of phage titers in organs at 24h POI; (C) Routine examination of white blood cell count at 24h POI; (DF) Cytokine levels measured at the same checkpoints; Differences between groups were statistically analyzed; *p<0.05, **p<0.01, ***p<0.001, ns indicates no statistically significant differences; (G) Clinical scores of infected mice treated with cocktail at different time points; (H) Body weight of infected mice treated with cocktail at different time points; (I) Survival curves of infected mice treated with cocktail at different time points.

[0027] Figure 9 To perform multiple sequence alignment of OmpA protein of Enterobacter cloacae complex strains using DNAman software.

[0028] Figure 10(A) shows the phage sensitivity test of bacteria from the spleen, liver and blood of mice treated with different doses of cocktail. Figure 10 (B) HE staining of the liver, spleen, lung, and kidney of mice treated with phage cocktail at different time points. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0030] 1. Strains and culture conditions

[0031] In the present invention, the strains are from the clinical sample library of Nanjing Second Hospital, as shown in Table 1-1. The strains include Acinetobacter baumannii, Enterobacter cloacae complex, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. All strains were cultured on LB agar or LB culture medium at 37°C, and apramycin sulfate (Apramycin sulfate) at a concentration of 100 μg / ml as shown in sequence number 39 in Table 1-1 was added when necessary to ensure the stability of the plasmid. In order to confirm the identity of the clinical isolates, the bacteria were further characterized by 16S rDNA sequencing and MALDI-TOF mass spectrometry using a Clin-TOF-II instrument (China).

[0032] Table 1-1 Bacterial strains used in the present invention

[0033]

[0034]

[0035] Table 1-2 Bacteriophages used in the present invention

[0036] bacteriophage feature ФEBU8 Enterobacter cloacae phage, capable of lysing E. cloacae 22 ФECL13 Enterobacter cloacae phage, unable to lyse E. cloacae 22 ФECL22 Enterobacter cloacae phage, capable of lysing E. cloacae 22 ФECL23 Enterobacter cloacae phage, capable of lysing Acinetobacter baumannii ФEBU30 Enterobacter cloacae phage, capable of lysing E. cloacae 22

[0037] Table 1-3 Plasmids used in the present invention

[0038] plasmids resistance Use or characteristics source pBECAb-Apr Apramycin sulfate resistance Base editing of Bowman chromosome [2] pHERD20T Carbenicillin resistance With arabinose-inducible promoter [3] pWCab24T Apramycin sulfate resistance With arabinose-inducible promoter The present invention <![CDATA[pWCab24T-OmpA ECL ]]> Apramycin sulfate resistance For the expression of Enterobacter cloacae OmpA The present invention <![CDATA[pWCab24T-OmpA ECO ]]> Apramycin sulfate resistance For expression of E. coli OmpA The present invention

[0039] Table 1-4 Primers used in the present invention

[0040]

[0041] Information related to sources in Table 1-1 and Table 1-4:

[0042] 1. Wang, WX, et al., Phage therapy combats pan drug-resistant Acinetobacter baumannii infection safely and efficiently. Int J Antimicrob Agents, 2024.64(2):p.107220.

[0043] 2. Wang, Y., et al., A Highly Efficient CRISPR-Cas9-Based GenomeEngineering Platform in Acinetobacter baumannii to Understand the H(2)O(2)-Sensing Mechanism of OxyR.Cell Chem Biol, 2019.26(12):p.1732-1742.e5.

[0044] 3. Qiu, D., et al., PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp.and other bacteria. Appl Environ Microbiol, 2008.74(23):p.7422-6.

[0045] 2. Isolation, purification and host range of bacteriophages

[0046] Phages were isolated and purified from sewage using a double-layer plate method. Specifically, untreated sewage samples were collected from public health and medical centers in Nanjing. Samples were pretreated by filtration through a 0.22 μm membrane to remove environmental bacteria. The 38 bacterial strains listed in Table 1-1, numbered 1 to 38, were used as bacterial hosts. To isolate phages, 100 μL of filtered sewage was mixed with 500 μL of bacterial culture, 0.8% warm agar supplemented with 2 mM CaCl2 was added, and the mixture was plated onto LB plates containing 2.5% agar. The plates were incubated at 37°C for 12 hours, and plaque formation was monitored. Single plaques were selected for enrichment and further purified through six rounds of the double-layer plate method. The purified phages were routinely used at 4°C and stored at -80°C for long-term storage.

[0047] Phage susceptibility was tested using the spot assay using the strains listed in Table 1-1 (numbers 40 to 57). 500 μL of logarithmic-phase bacterial culture was mixed with soft agar and plated onto LB agar plates. After cooling and solidification, 5 μL of each phage solution appeared on the plate surface. The plates were incubated at 37°C for 12 hours, and phage spots were detected. A total of 37 Enterobacter strains were tested, including several randomly selected Bacillus strains, including Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0048] 3. Cesium chloride gradient ultracentrifugation and transmission electron microscopy

[0049] To observe the morphology of phages, the isolated phages were further purified using density gradient centrifugation. Specifically, the phage solution was gradually added to a cesium chloride gradient and centrifuged in a Beckman X-100 ultracentrifuge equipped with a SW 41Ti 109 rotor at 120,000 × g for 2 h at 4°C. The supernatant was collected at the appropriate density layer and transferred to a dialysis membrane (Rui Da Heng Hui, MWCO 5kDa) and dialyzed overnight against 1× SM buffer (Sango Biotechnology). Finally, the purified phages were negatively stained with 2% uranyl acetate and observed using a Hitachi HT 7700112 transmission electron microscope (TEM) at Nanjing Agricultural University.

[0050] 4. Bioinformatics analysis

[0051] Genomic DNA was isolated from phages using the Rapid Nucleotide Isolation Kit (Bioperketus, SDKF60101). The extracted DNA was quality assessed by agarose gel electrophoresis and subsequently sequenced on the Illumina NovaSeq 6000 platform at Beijing NovaSeq Co., Ltd. De novo assembly of the clean data was performed using SPAdes v4.0.0. BLAST analysis was performed using Bowtie2 v2.5.4 to remove host genomic sequences. The circular or linear nature of the phage genome was confirmed using primers targeting the genome ends, as shown in SEQ ID NOs. 12–17 in Tables 1–4, followed by Sanger sequencing. The phage genomes were annotated using Prokka 1.14.6, and GC content was analyzed using GC View. Genome organization and comparison were analyzed using Easyfig. Virulence factor analysis was performed using the Virulence Factor Database (VFDB), and drug resistance was assessed using the Comprehensive Drug Resistance Database (CARD). Phylogenetic trees based on capsid proteins or large subunit terminase proteins were constructed using MEGA 7. The complete genome sequences of ФEBU8, ФECL22, and ФEBU30 have been deposited in the GenBank database with accession numbers PQ227828, PQ227830, and PQ227829, respectively. Single-nucleotide polymorphisms (SNPs) and indel variations between the wild-type strain E. cloacae 22 and its spontaneous phage-resistant mutants were analyzed. Bacteria were cultured to logarithmic phase, and genomic DNA was extracted using a DNA extraction kit. Genomic libraries were constructed and sequenced using PE150 on the Illumina platform. Sequence data were compared with the reference genome using BWA [v0.7.17] and SAMtools [v1.7] for sequence alignment analysis. The genome sequences of E. cloacae 22, R3, R4, and R6 have been deposited in the NCBI database with accession number PRJNA1175430.

[0052] 5. Determination of phage biological characteristics

[0053] To evaluate the pH stability of phage ФECL22, we prepared citrate-phosphate buffer solutions with pH values ​​ranging from 3.0 to 8.0 and Tris-HCl buffer solutions with pH values ​​ranging from 9.0 to 12.0. 100 μL of phage was mixed with 900 μL of the corresponding buffer solution at different pH levels and incubated for 1 hour. After incubation, phage titers were determined using a double-layer agar plate method. The titer in pH 7.0 buffer was used as a control, and the remaining phage counts in the different pH buffer solutions were normalized to this control. The experiment was repeated three times.

[0054] To evaluate the stability of phage ФECL22 at different temperatures, 500 μL of phage was incubated in 1× SM buffer at different temperatures for 1 hour. Phage titers were then determined using a double-layer agar plate assay. Phage counts remaining at different temperatures were normalized to those of the control group using phage incubated at 25°C. The experiment was repeated three times.

[0055] Phage sensitivity to UV: Phages in 1× SM buffer were exposed to UV light. Samples were collected at different time points and phage viability was assessed using a double-layer agar plate assay. The experiment was repeated three times.

[0056] To evaluate the adsorption capacity of ФECL22 to E. cloacae 22 (strain No. 14 in Table 1-1), a logarithmic phase culture of E. cloacae 22 was prepared, washed, and suspended in fresh LB broth. 500 μL of 1×10 7 PFU / mLФECL22 and 5mL Enterobacter cloacae culture (1×10 8 CFU / mL) were mixed and incubated at 37°C with shaking. Samples (100 μL) were collected at different time intervals, and the remaining phage titer in the supernatant was measured and normalized to the phage count initially added. The experiment was repeated three times.

[0057] To determine the optimal infection multiplicity of infection (MOI) of phage ФECL22 against E. cloacae 22, a logarithmic phase culture of E. cloacae 22 was prepared, washed, and suspended in fresh LB broth. 500 μL of ФECL22 solution at different doses was mixed with 5 mL of E. cloacae culture medium (1×10 8 CFU / mL) were mixed and shaken at 37°C overnight. The phage titer in the supernatant was determined, and the MOI that produced the highest phage titer was considered the optimal MOI for the phage. The experiment was repeated three times.

[0058] In order to measure the growth curve of bacteriophage ФECL22, the present invention prepared a logarithmic phase culture of Enterobacter cloacae 22, washed it, and suspended it in fresh LB broth. 5 PFU / mLФECL22 and 500μL 1×10 7 CFU / mL of Enterobacter cloacae culture was combined at an MOI of 0.01. After incubation at 37°C for 10 minutes, the cells were centrifuged at 12,000 rpm for 1 minute, washed once, suspended in 1 mL of preheated LB, and then transferred to 100 mL of preheated LB. This time point was designated as 0. Samples (1 mL) were taken at different time points and the phage titer in the supernatant was determined. The burst size was calculated as the total number of phages released at the end of one growth period divided by the number of infected bacteria. The experiment was repeated three times.

[0059] 6. Plasmid construction and transformation

[0060] The OmpA expression plasmid was constructed by reverse PCR on pb-Apr using primers BECAb-qR and BECAb-bF, and the primers are SEQ ID NOs. 3-4 in Tables 1-4. The linearized plasmid was fused with the araC-pBAD promoter fragment and amplified from pHERD20T using primers araC-F and araC-R. pWCab24T was generated by fusion cloning, and the primers are SEQ ID NOs. 5-6 in Tables 1-4. The ompA gene was amplified from Enterobacter cloacae 22 and Escherichia coli Star (TaKaRa Bio) using primers ompA-F and ompA-Ecl-R2 or ompA-Eco-R2, respectively, and the primers are SEQ ID NOs. 7-9 in Tables 1-4. The present invention uses primers ompA-F and ompA-Ecl-R2 to amplify the ompA gene. These fragments were inserted into pWCab24T and digested with EcoRI and HindIII to generate pWCab24T-OmpAECL and pWCab24T-OmpAECO. Sequencing results confirmed the correctness of these plasmids.

[0061] Preparation of electroporation-competent cells of Enterobacter cloacae and Acinetobacter baumannii: Logarithmic-phase cultures of these cells were first prepared in LB broth. For Enterobacter cloacae, 5 μg / mL Polymyxin Bnonapeptide hydrochloride (PMBN) was added to enhance transformation efficiency. The cultures were washed twice with 10% cold glycerol and then resuspended in 10% cold glycerol.

[0062] Competent cells of K. pneumoniae strain 84 were prepared by harvesting the culture directly from agar plates to reduce capsular polysaccharides and then washing twice with cold water. For transformation, 500 ng of plasmid DNA was electroporated using the Gene PulserXcell electroporation system at 2.5 kV, 25 μF, and 200 Ω. To induce OmpA expression, 0.2% arabinose was added to each transformant.

[0063] 7. In vitro bactericidal activity

[0064] In order to study the in vitro bactericidal activity of phage ФECL22, the logarithmic phase culture of Enterobacter cloacae 22 was suspended in fresh LB broth and placed in a 96-well plate, with 100 μL of suspension per well and 1 × 10 6CFU. Different concentrations of purified φECL22 phage solutions obtained by ultracentrifugation were added to wells at different MOIs. Bacteria without phage served as growth controls, and contamination was detected using LB broth alone. Each MOI was tested in eight replicate wells. The OD values ​​at 600 nm (OD) were measured every hour. 600 ) at , and bacterial growth was monitored over 24 h. These experiments were repeated three times.

[0065] To evaluate the bactericidal efficiency of the phage cocktail, ФEBU8, ФECL22, and ФEBU30 were mixed at a ratio of 1:1:1. Equal volumes of bacterial suspension (1×10 6 CFU) and phage cocktail (1×10 6 PFU) were added to a 96-well plate. Eight replicate wells were set up. Each single phage solution served as a control. Bacterial growth was monitored as described above, and the experiment was repeated three times.

[0066] 8. CCK8 detection

[0067] The cytotoxicity of phage suspension to LO2 in normal hepatocytes was detected using Cell Counting Kit 8 (CCK8, Solarbio, CA1210) according to the manufacturer's manual. Briefly, LO2 cells were seeded in DMEM medium containing 10% fetal bovine serum in 96-well plates at 5 × 10 cells per well. 3 Cells were incubated at 37°C in a 5% CO2 atmosphere for 24 hours. The cocktail suspension was then serially diluted with DMEM medium and added to a 96-well plate for 24 hours. CCK8 solution was added to each well and incubated at 37°C for 45 minutes. OD values ​​were measured using an enzyme marker at 450. This experiment was repeated three times.

[0068] 9. Safety evaluation of phage cocktail in vivo

[0069] In this study, all experimental procedures used specific pathogen-free (SPF) Kunming (KM) female mice aged 7–8 weeks and weighing 30–34 g. Mice were obtained from Yuyu Biotechnology (Suzhou) and allowed a one-week acclimatization period, during which they had free access to food and water and were maintained on a standard light / dark cycle at room temperature.

[0070] To evaluate the in vivo safety of the phage cocktail, the mice were randomly divided into five groups of five mice each. The phage cocktail was prepared by mixing purified phages ФEBU8, ФECL22, and ФEBU30 at a 1:1:1 ratio and then diluting them to the desired concentration in 1× PBS. The PBS had a pH of 7.4 and a formulation of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4.

[0071] Phage suspension was administered via two routes: intraperitoneal injection (IP) and intravenous injection (IV). For the IP group, three dose levels were evaluated: 1 × 10 8 PFU / mouse, 1×10 6 PFU / mouse, 1×10 4 PFU / mouse. The intravenous injection group was given a single dose of 1×10 8 PFU / mouse. Each mouse received 200 μL of phage suspension daily for 20 days.

[0072] Clinical observations and body weight measurements were recorded daily by two trained observers starting at the time of injection. Scoring was performed using an adapted mouse sepsis scale (MSS) with a modified scoring framework, with a score of 5 for dead mice and 0 for healthy individuals. At the end of the study, orbital blood samples were collected to assess endotoxin levels and cytokine profiles.

[0073] 10. Endotoxin determination

[0074] Using GenScript (China) TM Endotoxin levels in mouse blood were measured using the chromogenic LAL Endotoxin Detection Kit (L00350C). Standards were prepared according to the manufacturer's instructions, and three groups were established: a standard group, a sample group, and a negative control group using pyrogen-free water. To each tube, 100 μL of standard solution, 100 μL of blood sample, and 100 μL of pyrogen-free water were added, followed by the corresponding reagents from the kit. The mixture was then transferred to a 96-well plate, and the absorbance was measured at 545 nm using a microplate reader. A standard curve was constructed based on the OD 545 values ​​of the standards, allowing calculation of the endotoxin content in the samples.

[0075] 11. Measurement of inflammatory factors

[0076] The inflammatory cytokines TNF-α, IL-1β, and IL-6 in mouse serum were quantified using specific ELISA kits: Mouse IL-1β ELISA Kit (EMC001b), Mouse TNF-α ELISA Kit (EMC102a), and Mouse IL-6 ELISA Kit (EMC004), all from Quantitative Analysis (China). Standards were prepared according to the manufacturer's instructions. Each assay included a standard group, a sample group, and a blank diluent as a negative control. To each tube, 100 μL of standard sample, 100 μL of plasma sample, and 100 μL of blank diluent were added, followed by incubation with the corresponding reagents from the kit. The mixture was then transferred to a 96-well plate, and the absorbance was measured at 450 nm using a microplate reader. A standard curve was established based on the OD 450 values ​​of the standards, and TNF-α, IL-1β, and IL-6 levels in the samples were calculated.

[0077] 12. Establishment of a mouse model of Enterobacter cloacae infection and bacteremia

[0078] To investigate the conditions for establishing an E. cloacae infection bacteremia model, 8-week-old female KM mice were divided into four groups of five mice each. A logarithmic phase culture of E. cloacae 22 was collected, washed, and suspended in 1× PBS. Each mouse was intraperitoneally injected with 200 μL of bacterial culture at concentrations of 1×10 8 CFU / mL, 1×10 9 CFU / mL and 1×10 10 CFU / mL. An equal volume of PBS served as a negative control. Two researchers monitored the clinical condition of the mice every 2 hours for the first 12 hours and then daily thereafter. The scoring system used was: a dead mouse was assigned a score of 5, while a healthy mouse was assigned a score of 0. Kaplan-Meier survival curves were analyzed using GraphPad Prism 9.3.0.

[0079] 13. Evaluation of the therapeutic effects of different doses of phage cocktail

[0080] To study the therapeutic effects of different doses of phage cocktail on bacteremia infection, mice were randomly divided into 5 groups, with 8 mice in each group. First, 200 μL of 1×10 10 The bacteremia model was established with 1×10 CFU / mL Enterobacter cloacae 22. Mice receiving an equal volume of 1× PBS served as uninfected controls. At 1 h POI, phage cocktail was intraperitoneally administered at different doses: 1×10 8 PFU / mouse (high dose), 1×10 6 PFU / mouse (medium dose), 1×10 4 PFU / mouse (low dose). An equal volume of 1× PBS was used as a negative control. Two researchers monitored the clinical condition of the mice every 2 hours for the first 12 hours and then daily for 168 hours. Kaplan-Meier survival curves were analyzed using GraphPad Prism 9.3.0. Body weight was also recorded daily.

[0081] To assess the efficacy of treatment, a checkpoint was established at 24 hours post-inoculation (POI). Three mice were randomly selected from each group, euthanized, and autopsied. Blood, liver, and spleen samples were collected and analyzed for phage titer and bacterial load. Six colonies were randomly selected from culture plates to test blood, liver, and spleen samples at three different doses, and their sensitivity to the phage cocktail was assessed using a spot assay.

[0082] 14. Evaluation of the efficacy of phage cocktail therapy at different times

[0083] To investigate the effects of phage cocktail treatment at different time points, mice were randomly divided into 8 groups, each containing 13 mice. The bacteremia mouse model was established as described previously, and mice received an equal volume of 1× PBS as uninfected controls. Infected mice were given 1×10 6 PFU / mouse: 24 h before infection ("-24"), 6 h before infection ("-6"), immediately after infection ("0"), 1 h POI ("1"), 6 h POI ("6"), 24 h before treatment, and 6 h after infection ("-24+6"). An equal amount of 1× PBS served as a negative control. Two researchers monitored the clinical status of mice every 2 h for the first 12 h and then daily for a total of 168 h. Kaplan-Meier survival curves were analyzed using GraphPad Prism 9.3.0.

[0084] To assess treatment efficacy, a checkpoint was established 24 hours post-injection (POI). Three mice were randomly selected from each group, and blood, liver, and spleen samples were collected for analysis of phage titers, bacterial load, and histopathological examination. Blood samples were also collected for assessment of cytokine profiles and routine hematology tests using a Sysmex XN-2800 hematology analyzer.

[0085] 15. Histopathological examination

[0086] Tissue sections of liver, lung, kidney and spleen were fixed with 4% paraformaldehyde, embedded in paraffin and stained with hematoxylin and eosin (H&E). Microscopic examination was performed to evaluate the histological characteristics of these tissues.

[0087] result

[0088] 1. Most isolated Enterobacteriaceae phages exhibit a broad host spectrum

[0089] Isolation of lytic phages from untreated hospital sewage. A total of 14 lytic phages were successfully isolated ( Figure 1(A) in the figure). Using the primers shown in SEQ ID NO.1-2, 16S rDNA sequencing and mass spectrometry analysis revealed that 38 strains of Escherichia coli included 1 strain of Enterobacter bugandensis (EBU), 19 strains of Escherichia coli (Enterobactercloacae, ECL) and 17 strains of Enterobacter hormaechei (EHO), as well as 1 strain of Enterococcus faecalis (EF). Phage dripping experiment analysis showed that the host ranges of isolated Enterobacteriaceae phages were different. The host ranges of ФECL2, ФECL13, ФECL46 and ФEHO37 were narrow, while the host spectra of other phages were broader. Most phages were able to lyse Enterobacter cloacae and Enterobacter hormaechei. Enterobacter cloacae phages ФEBU8, ФECL23 and ФEHO25 showed lytic activity against some Acinetobacter baumannii strains ( Figure 1 (B)-(C)). Meanwhile, E. faecalis phage ФEF24 was able to lyse E. hallii. However, none of the phages tested showed lytic activity against E. coli, K. pneumoniae, P. aeruginosa, or S. aureus.

[0090] Enterobacter cloacae 22 is a carbapenem-resistant clinical strain isolated from cerebrospinal fluid. This study selected it for establishing a mouse bacteremia model. Among the isolated phages, ФEBU8, ФECL22, and ФEBU30 all exhibited relatively broad but distinct host ranges. All three phages were capable of lysing Enterobacter cloacae 22. Furthermore, ФEBU8 and ФEBU30 were also able to lyse the ФECL22-resistant mutant strain R3 (derived from Enterobacter cloacae 22). Figure 2 A); The cocktail of three phage combinations can effectively lyse 86.5% (32 / 37) of the Enterobacter cloacae complex strain and 66.7% (4 / 6) of the Acinetobacter baumannii strain ( Figure 1 ), these three phages formed very small, transparent pinhole plaques without halos ( Figure 2 B). Transmission electron microscopy (TEM) showed that all three phages exhibited the typical structure of E. coli T4 phage, characterized by an elongated icosahedral head, a tail, and long tail fibers ( Figure 2B). Therefore, they were classified as Myoviridae and designated vB_EbuM-8, vB_EclM-22, and vB_EclM-30, respectively. The deposit number of vB_EclM-22 is CGMCC No. 46281, dated November 18, 2024; the deposit number of vB_EbuM-8 is CGMCC No. 46282, dated November 18, 2024; and the deposit number of vB_EclM-30 is CGMCC No. 46283, dated November 18, 2024. For simplicity, they are referred to as ΦEBU8, ΦECL22, and ΦEBU30. In this disclosure, ΦEBU30 is sometimes referred to as ΦECL30. Therefore, in this disclosure, ΦEBU8, ΦECL22, and ΦEBU30 were selected for further identification.

[0091] 2. Genome analysis and annotation

[0092] The full genomes of phages ФEBU8, ФECL22, and ФEBU30 are 172,768 bp, 177,652 bp, and 40,126 bp, respectively, with GC contents of 39.69%, 44.71%, and 52.09%, respectively. PCR using terminal primers (Tables 1-4) confirmed that the genomes of ФEBU8 and ФEBU30 are linear, while that of ФECL22 is circular. ФEBU8 is annotated to contain 298 coding sequences (CDS) and 18 tRNA genes, ФECL22 has 271 CDS and 2 tRNA genes, and ФEBU30 has 48 CDS and 1 tRNA gene.

[0093] The full genome of ФEBU8 has 94% coverage and 96.83% homology with Enterobacteriaceae phage vB_Ent31, and 93% coverage and 88.81% homology with Enterobacteriaceae phage vB_EluP_RZH. ФECL22 has 97% coverage and 99.72% homology with Enterobacteriaceae phage phi5, respectively, and 93% coverage and 91.58% homology with Enterobacteriaceae phage ENC20, respectively. Furthermore, ФEBU30 has 97% coverage and 98.92% homology with Enterobacteriaceae phage IME278, and 85% coverage and 88.82% homology with Enterobacteriaceae phage EP1. Linear comparison of these three phage genomes with their respective homologous phages revealed sequence similarity but different genomic organization, suggesting the possibility of recombination events ( Figure 3 ).

[0094] Furthermore, analysis using the VFDB database revealed that none of the three phages contained virulence genes, and analysis using the CARD database, an antibiotic resistance database, did not detect any antibiotic resistance genes. Therefore, these three phages represent new virulent phages against Enterobacter cloacae and have potential therapeutic applications.

[0095] 3. Biological characteristics of bacteriophage ФECL22

[0096] Given that ФECL22 exhibited a wide host range and was able to lyse 51.4% (19 / 37) of the Enterobacter cloacae complex ( Figure 1 ), the present invention uses it as a representative of Enterobacter cloacae phage to identify its biological characteristics. The thermal stability test shows that ФECL22 can withstand relatively high temperatures. At 50°C, 70% of the phages still maintain activity ( Figure 4 A). After exceeding this temperature, the cleavage activity decreases rapidly. The pH stability test showed that ФECL22 remained stable in the pH range of 6.0 to 10.0, and maintained more than 90% of its cleavage activity within this range ( Figure 4 B). Similar to other phages, ФECL22 is sensitive to UV irradiation. After 20 min of exposure, the phage titer decreased by 2 log units ( Figure 4 C).

[0097] Adsorption experiments showed that within 15 min, about 90% of the ФECL22 particles in LB broth were adsorbed on Enterobacter cloacae 22 ( Figure 4 D). The optimal infection rate (MOI) of ФECL22 is 0.01, which produces approximately 1×10 11 PFU / mL of progeny phage ( Figure 4 E). The incubation period of the phage was 15 minutes, the lysis period was extended to 15 minutes, and the stable period was reached after 30 minutes ( Figure 4 F). The average burst size was approximately 19 phage particles per infected bacterium.

[0098] In the first 6 h, ФECL22 alone inhibited the growth of E. cloacae 22, but then the bacterial growth rebounded ( Figure 4 G). Increasing the number of phages can enhance their inhibitory effect. Although the low MOI did not prevent bacterial growth in the initial stage, the final inhibitory effect after 24 h was comparable to that of the high MOI, which may be due to the proliferation of phages ( Figure 4 G). When combined with the other two phages, ФECL22 was able to completely inhibit bacterial growth within 24 h. ФEBU8 and ФEBU30 showed stronger inhibitory effects than ФECL22, probably because they could inhibit some mutants that were resistant to ФECL22 ( Figure 4H).

[0099] 4. OmpA is the receptor of bacteriophage ФECL22

[0100] To identify the receptor for the Enterobacter cloacae phage ФECL22, the present invention isolated three spontaneous phage-resistant mutants from Enterobacter cloacae 22, as shown in SEQ ID NOs. 58 to 60 in Table 1-1, and named R3, R4, and R6, respectively. Adsorption tests showed that compared with Enterobacter cloacae 22, ФECL22 could not effectively attach to these mutants ( Figure 5 A), suggesting that their phage resistance is due to impaired adsorption. To compare the genomic differences between the wild-type strain and these mutants, their genomes were sequenced and analyzed for SNP / Indel variations. Compared to the wild-type strain, mutants R3, R4, and R6 had 125, 127, and 130 SNPs, respectively. R3 and R4 both harbored two stop mutations in the ompA gene, resulting in truncation of the OmpA protein. Although SNP and indel analysis did not reveal any mutations in the ompA gene of R6, PCR and Sanger sequencing revealed a stop mutation in R6, in which the codon for the 82nd tryptophan (TGG) was replaced by a stop codon (TAG). Therefore, given that some T-even phages use OmpA as a receptor in Escherichia coli, it is speculated that ФECL22 may also utilize OmpA as its receptor. To verify this, the present invention expressed OmpA from Enterobacter cloacae in the R3 mutant, which restored the sensitivity of the bacterium to ФECL22, making it comparable to that of the wild-type strain ( Figure 5 B). However, when OmpA from Escherichia coli was introduced into the R3 mutant, the bacteria still had phage resistance. These results indicate that OmpA in Enterobacter cloacae is a specific receptor for ФECL22, and that OmpA from E. coli cannot replace its homolog, despite its 86% sequence homology. In order to determine whether variations in the OmpA protein sequence affect the sensitivity of Enterobacter cloacae complex strains to ФECL22, the present invention randomly selected 6 ФECL22-tolerant and 7 ФECL22-sensitive strains, amplified them, and the amplification primers were the primers shown in SEQ ID NO.10-11 in Table 1-4, and their OmpA genes were sequenced. Sequence analysis showed that the sequences between these strains were almost identical ( Figure 9 Furthermore, introduction of OmpA from E. cloacae 22 into E. coli, K. pneumoniae, and A. baumannii did not result in susceptibility to ФECL22. These findings suggest that the presence of an intact OmpA alone does not guarantee successful ФECL22 infection, suggesting that E. cloacae may have a co-receptor.

[0101] 5. Enterobacter cloacae phage cocktail is safe both inside and outside the body

[0102] Because E. cloacae rapidly develops resistance to individual phages, the present invention investigated the therapeutic efficacy of a 1:1:1 combination of a 3-phage cocktail consisting of purified ФEBU8, ФECL22, and ФEBU30. The cytotoxicity of this phage cocktail was assessed before initiating phage therapy. CCK8 assays showed that even at a high concentration of 1×10 9 PFU / mL, the phage cocktail did not show any adverse effects on the human liver cell line LO2 ( Figure 6 A).

[0103] Subsequently, the in vivo safety of the phage cocktail was evaluated by administering it to mice once daily for 20 consecutive days via intraperitoneal (IP) or intravenous (IV) injection. The mice were monitored for clinical signs, and health scores showed that they remained healthy with no obvious symptoms ( Figure 6 B). In addition, the body weight of mice injected with phage solution showed no significant change over 20 days ( Figure 6 C) On the last day after injection, all mice were sacrificed and blood was collected to measure endotoxin levels and cytokines. Compared with the control group, each mouse was injected intravenously and IP with 1×10 8 PFU can lead to a significant increase in endotoxin levels ( Figure 6 D). Intravenous administration of high-dose phages resulted in increased IL-1β and IL-6 levels, but not TNF-α levels. However, 1×10 6 PFU did not cause any adverse reactions ( Figure 6 FG).

[0104] Overall, the phage cocktail of the present invention demonstrated safety both in vitro and in vivo.

[0105] 6. Phage cocktail rescues E. cloacae bacteremia in mice in a dose-dependent manner

[0106] KM mice were infected intraperitoneally with different doses of Enterobacter cloacae 22 to establish a bacteremia model. 9 CFU, all 5 mice died within 44 h post infection (POI). 8 CFU, only one mouse died at 34 h POI, while the remaining four mice survived. 7 No mice with CFU died within 6 days ( Figure 7 A). Therefore, 2×10 9 The acute infection model was established by using the dose of CFU.

[0107] The present invention first explored the protective effects of different doses of the cocktail administered 1 h after infection, including high doses (1×10 8 PFU / mouse), medium dose (1×10 6 PFU / mouse) and low dose (1×10 4 PFU / mouse). 24 h after POI, three mice in each group were sacrificed and the phage and bacterial loads in the liver, spleen, and blood were measured. The medium dose resulted in the highest phage titers in the liver, spleen, and blood ( Figure 7 B); Specifically, 9.4×10 5 PFU / g, compared with 1.1×10 5 PFU / g, the low-dose group was 4.1×10 5 PFU / g. Consistent with this, the middle dose also significantly reduced the bacterial load in the liver, spleen, and blood compared with the other two groups ( Figure 7 C). A total of 54 strains were isolated from mice treated with phage cocktail, all of which were sensitive to it ( Figure 10 (A) in the figure.

[0108] Compared with untreated mice, treated mice showed milder initial symptoms and gradually recovered after 48 hours, eventually returning to an active and healthy state comparable to that of uninfected controls ( Figure 7 D). Body weight data confirmed these observations, showing that treated mice began to regain body weight after 48 h ( Figure 7 E). However, using a high dose of phage cocktail (1×10 8 PFU) caused the most severe symptoms and weight loss within the first 48 h, while the moderate dose (1 × 10 6 PFU) resulted in milder symptoms and faster recovery. At the 168h POI endpoint, 80% (4 / 5) of infected mice treated with phage cocktail survived, regardless of the phage dose ( Figure 7 F). These findings suggest that, although the final outcomes were similar across treatment groups, the recovery trajectory was dose-dependent, with the intermediate-dose regimen showing particular efficacy.

[0109] 7. Prophylactic administration provides an opportunity to delay treatment to save bacteremia

[0110] To determine the optimal timing of phage cocktail administration, its effect was evaluated at different time intervals: 24 h before infection ("-24"), 6 h before infection ("-6"), immediately after infection ("0"), 1 h POI ("1"), 6 h POI ("6"), and a combination of 24 h before infection and 6 h after infection ("-24 + 6"). After 24 h POI, three mice per group were sacrificed and the phage and bacterial loads, as well as cytokine levels and white blood cell counts in the liver, spleen, and blood were evaluated. Compared with untreated infected mice, bacterial loads were significantly reduced in the "-6", "0", and "1" treatment regimens. The "-24 + 6" group showed greater efficacy than the "6" group, with significantly reduced bacterial levels in organs ( Figure 8 A). However, no phages were detected in the organs of the "-24" group, indicating that there were no residual phages at all 24 h after administration ( Figure 8 B). Routine blood tests revealed that E. cloacae infection caused a decrease in white blood cell counts, while administration of the phage cocktail restored these levels ( Figure 8 C). In addition, the white blood cell count in the "-24+6" regimen was significantly increased compared with the "-6" group. Similarly, phage cocktail administration reduced the levels of inflammatory factors IL-1β, IL-6, and TNF-α, and the cytokine levels in the "-24+6" regimen were significantly lower than those in the "-6" group ( Figure 8 DF). Hematoxylin and eosin (HE) staining of untreated infected mice showed obvious pathological changes, including glomerular atrophy and interstitial congestion. There were patchy hemorrhages in the alveolar cavity, and the spleen showed hemorrhage and congestion in the medullary area, with a clear distinction between the medulla and the cortex. Hepatocytes showed mild swelling and vascular congestion. Compared with untreated infected mice, the "-24" treatment regimen did not significantly alleviate these symptoms. However, after treatment with the "-24+6" regimen, significant improvements were observed: the boundary between the splenic cortex and medulla became less obvious, medullary congestion decreased, cortical hyperplasia was obvious, and necrosis in the cortex almost completely disappeared. In addition, the alveolar hemorrhage area showed obvious absorption, the glomeruli showed slight swelling, the hepatocytes showed more obvious swelling, and the interstitial vessels were congested. This level of recovery is better than that of the "6" regimen ( Figure 10 At the 168h POI endpoint, 1h POI rescued 80% (8 / 10) of infected mice ( Figure 8 G); When treatment was started immediately after bacterial infection, a 100% (10 / 10) survival rate was observed. However, delaying treatment until 6 h POI significantly reduced the efficacy, resulting in only a 50% (5 / 10) survival rate. Prophylactic administration of the phage cocktail ("-24") 24 h before infection had no protective effect, resulting in the death of all infected mice. The combined use of "-24 + 6" achieved a 100% survival rate (10 / 10) ( Figure 8 G).

[0111] The present findings emphasize the critical importance of early and timely treatment in treating E. cloacae bacteremia using phage cocktails, while prophylactic administration may provide an opportunity to delay treatment to salvage bacteremia.

[0112] Sepsis caused by Enterobacter cloacae has become a serious clinical threat to humans. The present invention established a mouse bacteremia model caused by Enterobacter cloacae infection and optimized the phage cocktail dosage and treatment time. The present invention found that when 1×10 10 CFU / mouse Enterobacter cloacae, the mortality rate of KM mice was 100%, and death occurred between 20 and 48 hours. One hour after bacterial infection, mice were intraperitoneally injected with 1×10 phage cocktail. 4 PFU / mouse, 1×10 6 PFU / mouse, 1×10 8 PFU / mouse, the survival rate of all mice was 80%. It was observed from the mouse organ slices and organ bacteria collection that after 24 hours of treatment, 1×10 6 The organ recovery of the PFU / mouse group was better and the bacterial load was relatively low. The weight fluctuation of this group was not so significant compared with the other two groups. 6 PFU / mouse was the optimal dose for subsequent experiments.

[0113] At different treatment time points, the present invention found that prophylactic administration of phages 24 hours before infection did not protect any mice; the group that received prophylactic phages 6 hours before infection had an 80% survival rate; mice treated with phages immediately after infection had a 100% survival rate; mice treated with phages 1 hour after infection had an 80% survival rate; and mice treated with phages 6 hours after infection had a 50% survival rate. Literature reports suggest that, in the absence of a host, phages stimulate an immune response upon entry, gradually eliminating the phages. However, the present invention's combined treatment of prophylactic phage administration 24 hours before infection and delayed treatment for 6 hours saved 100% of the mice. Prophylactic phage administration may provide a valuable window of opportunity for delayed treatment.

[0114] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0115] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0116] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A bacteriophage composition, characterized in that: The phage composition includes the following phages: ФEBU8, ФECL22 and ФEBU30; The deposit number of ФECL22 is CGMCC No.46281, and the deposit date is November 18, 2024, deposited in the Center for General Microorganisms of China Culture Collection Administration; the deposit number of ФEBU8 is CGMCC No.46282, and the deposit date is November 18, 2024, deposited in the Center for General Microorganisms of China Culture Collection Administration; the deposit number of ФEBU30 is CGMCC No.46283, and the deposit date is November 18, 2024, deposited in the Center for General Microorganisms of China Culture Collection Administration; The volume ratio of ФEBU8, ФECL22 and ФEBU30 in the phage composition is 1:1:

1.

2. Use of the phage composition according to claim 1 in the preparation of a medicament for treating Enterobacter cloacae bacteremia.

3. The use according to claim 2, characterized in that The total content of bacteriophages in the bacteriophage composition is ≥10 4 PFU / mL.

4. Use of the phage composition according to claim 1 in the preparation of a medicament for combating antibiotic-resistant Enterobacter cloacae bacteremia.

5. The use according to claim 4, characterized in that The antibiotic is a carbapenem antibiotic.

6. A pharmaceutical composition, characterized in that The invention comprises the phage composition according to claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that Also included are pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutically acceptable carrier is any one of phosphate buffered saline and sterile physiological saline.

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