Phage composition as well as application and pharmaceutical composition thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bacteriophage composition as well as application and a pharmaceutical composition thereof. The bacteriophage composition comprises the following bacteriophages: phi EBU8, phi ECL22 and phi EBU30, the key importance of early treatment in the aspect of treating enterobacter cloacae bacteremia by using the bacteriophage cocktail is proposed, and prophylactic administration possibly provides an opportunity for delayed treatment and rescue of bacteremia.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a bacteriophage composition and an application thereof and a pharmaceutical composition. Background Art

[0002] In recent years, the gradual increase of antibiotic resistance in Enterobacteriaceae, especially the increasing prevalence of carbapenem-resistant strains, has posed an urgent challenge to global health. These multidrug-resistant microorganisms cause high morbidity and mortality in hospital-acquired infections, greatly increasing the complexity of patient management and treatment. Among the Enterobacteriaceae, the Enterobacter cloacae complex is particularly worrying because it has been identified as one of the main pathogens of bloodstream infections, respiratory tract infections, and urinary tract infections, especially in immunocompromised patients. These pathogens have a tendency to develop resistance and cause serious infections, and are classified as "ESKAPEE", which is required by the World Health Organization as a priority. It is worth noting that Enterobacter cloacae complex bacteria show an alarming rate of carbapenem resistance, further limiting treatment options and promoting research interest in alternative therapies.

[0003] Phage therapy can specifically target and lyse bacteria without disrupting the host microbiota. 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 has unique advantages in reducing bacterial loads in the body. However, the success of phage therapy depends on factors such as dose 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, in part because relatively few phages of the Enterobacter cloacae complex have been identified.

[0004] Phage combinations, i.e., phage cocktails, are generally more effective in treating infections than any single phage alone. This is because many phages have a narrow host range, and the mixture of multiple phages broadens the bactericidal spectrum. At the same time, if several phages with different mechanisms of action or different receptors work synergistically, the emergence of tolerant phage strains can be significantly inhibited. However, in animal models, the bacteriophage sterilization process releases endotoxins and induces inflammation, so it is necessary to provide a phage combination with high safety. Summary of the invention

[0005] To solve the above problems, the present invention provides a phage composition and 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 bacteriophage composition is used in preparing 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 bacteriophages in the bacteriophage composition is ≥10 4 PFU / mL.

[0011] The bacteriophage composition is used in preparing a drug for combating antibiotic-resistant Enterobacter cloacae bacteremia.

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

[0013] A pharmaceutical composition comprises the bacteriophage 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 separates and purifies 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 exhibits stability in a wide temperature range (4-50°C) and pH range (6-10). Its burst volume is 19 PFU / cell, and its receptor is identified as the outer membrane protein OmpA. ФEBU8 and ФEBU30 can lyse mutant strains 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 in a volume ratio of 1-2:1-3:1, and 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 the Enterobacter cloacae phages ФEBU8 and ФECL23 lyse Acinetobacter baumannii, (B) the two phages are added to the gradient drop of Acinetobacter baumannii; (B) the two phages inhibit the growth curve of Acinetobacter baumannii.

[0020] Figure 2 Shows the morphology of ФEBU8, ФECL22 and ФEBU30; (A) is the result of 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) are their respective bacterial hosts and (2) are transmission electron microscopy (TEM) images; 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 showing ФECL22; (A) Adsorption test results; differences between groups were analyzed using univariate statistical analysis; ***p<0.001; (B) Introducing OmpA from Enterobacter cloacae into the R3 mutant can restore the phage sensitivity to ФECL22.

[0024] Figure 6 Figure 3 shows the safety evaluation of phage cocktail; (A) CCK-8 assay results of cytotoxicity of the cocktail on liver cells L02; (B) Evaluation of acute toxicity of phage cocktail in mouse model, monitoring clinical scores, where "0" indicates healthy status; (C) Changes in body weight; (D) Determination of endotoxin levels at the 24h checkpoint after injection; (EG) Cytokine levels measured at the same checkpoint; 1×PBS was the negative control; One-way ANOVA was used for statistical analysis of differences among the groups; *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 7 Figure 2 shows the efficacy of different doses of phage cocktail treatment on the mouse bacteremia model induced by Enterobacter cloacae; (A) The mouse bacteremia model was established by different numbers of Enterobacter cloacae22; (B) The bacterial load in the organs was evaluated at 24h POI; (C) The phage titer in the organs was measured at 24h POI; the differences between the groups were analyzed by 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 shows the efficacy of phage cocktail therapy on the mouse bacteremia model induced by Enterobacter cloacae at different time points; (A) Evaluation 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 among groups were statistically analyzed; *p<0.05, **p<0.01, ***p<0.001, ns indicates no statistically significant difference; (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] Fig. 9 To use DNAman software to perform multiple sequence alignment of OmpA protein of Enterobacter cloacae complex strains.

[0028] Fig.10(A) shows the bacteriophage sensitivity test of bacteria from the spleen, liver and blood of mice treated with different doses of cocktail. Fig.10 (B) is 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 protection scope 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 creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are 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 on 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 the double-layer plate method. Specifically, untreated sewage samples were collected from the Public Health and Medical Center in Nanjing. The samples were pretreated by 0.22 μm membrane filtration to remove environmental bacteria. The 38 strains of bacteria shown in Serial No. 1 to Serial No. 38 in Table 1-1 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 2mM CaCl2 supplemented with 2mM CaCl2 was added, and poured onto an LB plate containing 2.5% agar. The culture dish was incubated at 37°C for 12h, and the formation of plaques was monitored. Single plaques were selected for enrichment and further purified by 6 rounds of the double-layer plate method. The purified phages were routinely used at 4°C and stored for a long time at -80°C.

[0047] The spot method was used to detect the phage sensitivity of the strains using the strains shown in No. 40 to No. 57 in Table 1-1, that is, 500 μL of the logarithmic phase bacterial culture was mixed with soft agar and poured onto the LB agar plate. After cooling and solidification, 5 μL of each phage solution appeared on the surface of the plate. The plate was incubated at 37°C for 12 hours and the phage spots were detected. A total of 37 strains of Enterobacteriaceae were detected, and several strains of Bacillus were randomly selected, including Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

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

[0049] In order to observe the morphology of phages, the isolated phages were further purified by density gradient centrifugation, specifically: the phage solution was gradually added to the 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 in 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 Biological Nucleic Acid Extraction Kit Rapid Nucleotide Isolation Kit (Bioperketus, SDKF60101). The extracted DNA was quality assessed by agarose gel electrophoresis and then sent to the Illimina NovaSeq 6000 platform of Beijing Novigene Co., Ltd. for sequencing. Clean data were assembled de novo using SPAdes v4.0.0. Bowtie2 v2.5.4 was used for blast analysis to remove host genome sequences. The circular or linear nature of the phage genome was confirmed by primers targeting the ends of the genome, and the primers used were shown in SEQ ID NO.12-17 in Tables 1-4, followed by Sanger sequencing. The phage genome was annotated using Prokka 1.14.6, and the 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 evaluated using the Comprehensive Drug Resistance Database (CARD). Phylogenetic trees based on capsid proteins or large subunit terminalase 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. SNP / Indel variations between the wild-type strain E. cloacae 22 and its spontaneous phage-resistant mutants were analyzed. The bacteria were cultured to the logarithmic phase, and genomic DNA was extracted using a DNA extraction kit. Then, a genomic library was constructed, followed by PE150 sequencing on the Illumina platform. Sequencing 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 numbers PRJNA1175430.

[0052] 5. Determination of biological characteristics of bacteriophage

[0053] To evaluate the pH stability of phage ФECL22, citric acid-phosphate buffers with pH values ​​of 3.0 to 8.0 and Tris-HCl buffers with pH values ​​of 9.0 to 12.0 were prepared. 100 μL of phage was mixed with 900 μL of the corresponding buffer at different pH levels and incubated for 1 h. After incubation, the phage titer was determined by the double-layer agar plate method. The titer in pH 7.0 buffer was used as a control, and the remaining phage counts in different pH buffers were normalized to this control group. 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 h, and then the phage titer was determined by double-layer agar plate method. The phage incubated at 25 °C was used as the control, and the remaining phage counts at different temperatures were normalized to the control group. The experiment was also repeated three times.

[0055] Phage sensitivity to UV: Phages in 1× SM buffer were exposed to UV light, and samples were collected at different time points to assess phage viability using the double-layer agar plate method. 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) 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 (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) and shaken at 37°C overnight. The phage titer in the supernatant was measured, and the MOI that produced the highest phage titer was considered to be 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, and suspended in fresh LB broth. 500 μL of 1×10 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 min, the cells were centrifuged at 12,000 rpm for 1 min, washed once, suspended in 1 mL of prewarmed LB, and then transferred to 100 mL of prewarmed 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 pb-Apr was subjected to reverse PCR using primers BECAb-qR and primer BECAb-bF to construct an OmpA expression plasmid, and the primers are SEQ ID NO.3-4 in Table 1-4. The linearized plasmid was fused with the araC-pBAD promoter fragment, and amplified from pHERD20T using primers araC-F and araC-R, and pWCab24T was generated by a fusion cloning method, and the primers are SEQ ID NO.5-6 in Table 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 NO.7-9 in Table 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 by internal fusion enzymes to obtain pWCab24T-OmpAECL and pWCab24T-OmpAECO. The sequencing results confirmed the correctness of these plasmids.

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

[0062] Competent cells of Klebsiella 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 used and electroporated using the Gene PulserXcell electroporation system at 2.5 kV, 25 μF, and 200 Ω. To induce the expression of OmpA, 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 bacteriophage ФECL22, the logarithmic phase culture of E. cloacae 22 was suspended in fresh LB broth and placed in a 96-well plate, with 100 μL of the suspension in each well and 1 × 10 6CFU. Different concentrations of purified ФECL22 phage solutions obtained by ultracentrifugation were added to wells at different MOIs. Bacteria without phages served as growth controls, and LB broth alone was used to detect contamination. Each MOI was tested in eight replicate wells. The OD at 600 nm (OD 600 ) to monitor bacterial growth 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 in 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 was used as a control. Bacterial growth monitoring was as described above, and the experiment was repeated 3 times.

[0066] 8. CCK8 detection

[0067] According to the manufacturer's manual, the cell counting kit 8 (CCK8, Solarbio, CA1210) was used to detect the cytotoxicity of phage suspension to LO2 in normal hepatocytes. Briefly, LO2 cells were inoculated in DMEM medium containing 10% fetal bovine serum and placed in 96-well plates with 5×10 cells per well. 3 Cells were incubated at 37°C and 5% carbon dioxide for 24 hours. Then, the cocktail suspension was diluted with DMEM medium and added to a 96-well plate and incubated for 24 hours. CCK8 solution was added to each well and incubated at 37°C for 45 minutes. The OD value was measured with an enzyme marker at 450. This experiment was repeated three times.

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

[0069] In the present invention, all experimental procedures used specific pathogen-free (SPF) Kunming (KM) female mice aged 7 to 8 weeks and weighing 30 to 34 g. The mice were obtained from Yuyu (Suzhou) Biotechnology Company and allowed a one-week adaptation period during which they had free access to food and water and maintained 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 the purified phages ФEBU8, ФECL22, and ФEBU30 in a 1:1:1 ratio and then diluted to the desired concentration in 1× PBS. The pH of PBS was 7.4, and the formula was NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, and KH2PO4: 1.8 mM.

[0071] Phage suspensions were 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 per day for 20 days.

[0072] Clinical observations and body weight measurements were recorded daily by two trained observers starting from the time of injection. The scoring system was adapted from the mouse sepsis scoring scale (MSS) using a modified scoring framework, with 5 points for dead mice and 0 points 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 The chromogenic LAL endotoxin test kit (L00350C) was used to detect the endotoxin level in mouse blood. Standards were prepared according to the manufacturer's instructions, and three groups were established: standard group, sample group, and negative control group, using pyrogen-free water. 100 μL of standard solution, 100 μL of blood sample, and 100 μL of pyrogen-free water were added to each tube, followed by the corresponding reagents in 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 the calculation of the endotoxin content in the samples.

[0075] 11. Measurement of inflammatory factors

[0076] The inflammatory factors 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. In each test tube, 100 μL of standard sample, 100 μL of plasma sample, 100 μL of blank diluent, and then the corresponding reagents were extracted from the kit for incubation. 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 value of the standard, and the levels of TNF-α, IL-1β, and IL-6 in the sample could be calculated.

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

[0078] To explore the conditions for establishing the bacteremia model of E. cloacae infection, 8-week-old female KM mice were divided into 4 groups, with 5 mice in each group. The logarithmic phase culture of E. cloacae 22 was collected, washed, and suspended in 1× PBS. Each mouse was intraperitoneally injected with a total of 200 μL of bacterial culture at a concentration of 1×10 8 CFU / mL, 1×10 9 CFU / mL and 1×10 10 CFU / mL. An equal amount of PBS was used as a negative control. Two researchers monitored the clinical status of the mice every 2 hours during the first 12 hours and then daily. The scoring system used was: a dead mouse was scored as 5 points, while a healthy mouse was scored as 0 points. Kaplan-Meier survival curves were analyzed using GraphPad Prism 9.3.0.

[0079] 13. Evaluation of 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 that received an equal amount of 1× PBS served as uninfected controls. At 1 h POI, phage cocktails were 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 amount of 1× PBS was used as negative control. Two researchers monitored the clinical status of the mice every 2 hours during the first 12 hours and then once a day for the entire 168 hours. Kaplan-Meier survival curves were analyzed using GraphPad Prism 9.3.0. Body weight was also recorded daily.

[0081] To evaluate the efficacy of treatment, a checkpoint was established at 24h POI. Three mice were randomly selected from each group, euthanized, and autopsied. Blood, liver, and spleen samples were collected and analyzed for phage titers and bacterial loads. Six colonies were randomly selected from the 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 including 13 mice. The bacteremia mouse model was established as described previously, and mice received an equal volume of 1× PBS as an uninfected control. Infected mice were given 1×10 6 PFU / mouse: 24 h before infection ("-24"), 6 h before infection ("-6"), immediately after infection ("0"), 1 hPOI ("1"), 6 hPOI ("6"), 24 h before treatment, and 6 h after infection ("-24+6"). An equal amount of 1× PBS was used as a negative control. Two researchers monitored the clinical status of mice every 2 h during the first 12 h of the mice and then once a day for the entire 168 h. Kaplan-Meier survival curves were analyzed using GraphPad Prism 9.3.0.

[0084] To evaluate the efficacy of treatment, a checkpoint was established at 24 h 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 used to evaluate cytokine profiles and perform routine hematological examinations, which were performed 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 the 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 (Enterobacter hormaechei, EHO), as well as 1 strain of Enterococcus faecalis (EF). Analysis of phage drip experiments showed that the host ranges of isolated Enterobacter 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. hominis. However, none of the phages showed lytic ability against the tested E. coli, K. pneumoniae, P. aeruginosa, or S. aureus.

[0090] Enterobacter cloacae 22 is a carbapenem-resistant clinical strain isolated from cerebrospinal fluid. The present invention selects it for establishing a mouse bacteremia model. Among the isolated phages, ФEBU8, ФECL22 and ФEBU30 all show relatively wide but different host ranges, and all three phages can lyse Enterobacter cloacae 22. At the same time, ФEBU8 and ФEBU30 can also lyse the ФECL22-resistant mutant strain R3 (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 strains and 66.7% (4 / 6) of the Acinetobacter baumannii strains ( 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 are classified into the Myoviridae family and are named vB_EbuM-8, vB_EclM-22 and vB_EclM-30, respectively. The deposit number of vB_EclM-22 is CGMCC No.46281, and the deposit date is November 18, 2024. The deposit number of vB_EbuM-8 is CGMCC No.46282, and the deposit date is November 18, 2024. The deposit number of vB_EclM-30 is CGMCC No.46283, and the deposit date is November 18, 2024. For simplicity, they are referred to as ФEBU8, ФECL22 and ФEBU30. In the present invention, ФEBU30 is sometimes recorded as ФECL30. Therefore, in the present invention, ФEBU8, ФECL22 and ФEBU30 are selected for further identification.

[0091] 2. Genome analysis and annotation

[0092] The full genome lengths of phages ФEBU8, ФECL22, and ФEBU30 were 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 were linear, while ФECL22 was circular. ФEBU8 was annotated to contain 298 coding sequences (CDS) and 18 tRNA genes, ФECL22 had 271 CDS and 2 tRNA genes, and ФEBU30 had 48 CDS and 1 tRNA gene.

[0093] The whole genome of ФEBU8 has a coverage of 94% and a homology of 96.83% with Enterobacteriaceae phage vB_Ent31; it has a coverage of 93% and a homology of 88.81% with Enterobacteriaceae phage vB_EluP_RZH. The coverage and homology of ФECL22 with Enterobacteriaceae phage phi5 are 97% and 99.72%, respectively, and the coverage and homology with Enterobacteriaceae phage ENC20 are 93% and 91.58%, respectively. In addition, the coverage of ФEBU30 with Enterobacteriaceae phage IME278 is 97% and the homology is 98.92%, and the coverage with Enterobacteriaceae phage EP1 is 85% and the homology is 88.82%. Linear comparison of the three phage genomes with their respective homologous phages revealed that their sequences are similar, but the genomic organization is different, which indicates that recombination events may have occurred ( Figure 3 ).

[0094] In addition, analysis using the VFDB database revealed that none of the three phages had virulence genes in their genomes, and analysis using the antibiotic resistance database CARD did not detect any antibiotic resistance genes. Therefore, these three phages represent new virulent phages of Enterobacter cloacae and have potential therapeutic applications.

[0095] 3. Identification of biological characteristics of bacteriophage ФECL22

[0096] Given that ФECL22 exhibited a wide host range, it 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 remain active ( Figure 4 A). After exceeding this temperature, the cleavage activity decreases rapidly. The pH stability test shows that ФECL22 remains stable in the pH range of 6.0 to 10.0, and maintains more than 90% of its cleavage activity within this range ( Figure 4 B). Similar to other phages, ФECL22 was sensitive to UV irradiation, and 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, at which 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 increased their inhibitory effect. Although low MOI did not prevent bacterial growth in the initial stage, the final inhibitory effect after 24 h was comparable to that of high MOI, probably 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] In order to identify the receptor of Enterobacter cloacae phage ФECL22, the present invention isolated three spontaneous phage-resistant mutants from Enterobacter cloacae 22, as shown in sequence numbers 58 to 60 in Table 1-1, and named R3, R4 and R6 respectively. Adsorption experiments 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 their SNP / Indel variations were analyzed. Compared with the wild-type strain, mutants R3, R4, and R6 had 125, 127, and 130 SNP sites, respectively. Both R3 and R4 had two stop mutations in the ompA gene, resulting in truncation of the OmpA protein. Although SNP and indel analysis did not reveal 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, considering that some T-even phages use OmpA as a receptor in Escherichia coli, it is speculated that ФECL22 may also use 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 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 Escherichia coli cannot replace its homolog, despite the 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 ( Fig. 9 ). In addition, 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 secondary receptor.

[0101] 5. Enterobacter cloacae phage cocktail is safe both in vivo and in vivo

[0102] Since E. cloacae rapidly develops resistance to individual phages, the present invention investigated the therapeutic effect 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 evaluated before starting phage therapy. CCK8 assay 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 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 within 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 injection 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 34h POI, while the remaining four mice survived. 7 There was no death in mice with CFU within 6 days ( Figure 7 A). Therefore, 2×10 9 CFU dose was used to establish the acute infection model.

[0107] The present invention first explored the protective effects of different doses of the cocktail given 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). 24h 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, low dose group was 4.1×10 5 Consistent with this, the medium dose also significantly reduced bacterial loads 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 ( Fig.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 high doses 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 phage and bacterial loads, as well as cytokine levels and leukocyte counts in the liver, spleen, and blood were evaluated. Bacterial loads were significantly reduced in the “-6”, “0”, and “1” treatment regimens compared to untreated infected mice. The “-24+6” group showed stronger 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 vascular congestion. This level of recovery is better than that of the "6" regimen ( Fig.10 At the 168h POI endpoint, 1h POI rescued 80% (8 / 10) of the 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 6h POI significantly reduced the efficacy, resulting in only a 50% (5 / 10) survival rate. Prophylactic administration of the phage cocktail ("-24") 24h 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 dose and treatment time. The present invention found that when 1×10 10 When the number of CFU / mouse Enterobacter cloacae was 100%, 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 mouse organ slices and organ bacteria collection that after 24h 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 time points of treatment, the present invention found that prophylactic administration of phages 24 hours before infection did not protect any mice; the survival rate of the group that prophylactically administered phages 6 hours before infection was 80%; the survival rate of mice was 100% when phage treatment was performed immediately after infection; the survival rate of mice was 80% when phage treatment was performed again 1 hour after infection; and the survival rate of mice was 50% when phage treatment was performed again 6 hours after infection. According to literature reports, in the absence of a host, phages stimulate the body's immune response when entering the body, thereby gradually removing the phages. However, the present invention's combined treatment of prophylactic administration of phages 24 hours before infection and delayed treatment for 6 hours saved 100% of the mice. The prophylactic use of phages can provide a valuable time window 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 a preferred embodiment.

[0115] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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 can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A bacteriophage composition, characterized in that: The phage composition includes the following phages: ФEBU8, ФECL22 and ФEBU30; 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 National Microorganism Culture Collection Administration. 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 National Microorganism Culture Collection Administration. 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 National Microorganism Culture Collection Administration.

2. Use of the bacteriophage 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 volume ratio of ФEBU8, ФECL22 and ФEBU30 in the phage composition is 1-2:1-3:

1.

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

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

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

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

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

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

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