Phage for acinetobacter baumannii and application thereof

Abstract

The invention discloses a bacteriophage for acinetobacter baumannii and application of the bacteriophage. The bacteriophage for the acinetobacter baumannii is vBMZM4AB-P, and the preservation number of the bacteriophage is CCTCC (China Center For Type Culture Collection) CNO: M 20241200. The bacteriophage for the acinetobacter baumannii can specifically split the acinetobacter baumannii 2AB, 4AB and ATCC-17978, is short in incubation period, high in splitting activity, good in stability and high in environmental endurance, provides a new bacteriophage for prevention, control or treatment of the acinetobacter baumannii, and has a broad application prospect. The invention provides a novel scheme and approach for the treatment of the multi-drug-resistant acinetobacter baumannii.

Description

Technical Field

[0001] The present application relates to the technical field of prevention or treatment of Acinetobacter baumannii, and in particular to a bacteriophage targeting Acinetobacter baumannii and its application. Background Art

[0002] Acinetobacter baumannii is one of the most common opportunistic pathogens in hospital infections, causing hospital-acquired infections such as urinary tract infections, bacteremia, and ventilator-associated pneumonia. Acinetobacter baumannii is resistant to multiple drugs, and multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) Acinetobacter baumannii are becoming increasingly prevalent. The latest report from the European Centre for Disease Prevention and Control (ECDC) highlights the serious drug resistance situation of MDR Acinetobacter baumannii, with 74.5% of isolates showing resistance to antibiotics, of which 66.6% are resistant to fluoroquinolones, aminoglycosides, and carbapenems.

[0003] In summary, Acinetobacter baumannii is a pathogen that seriously endangers global health and requires joint efforts to manage and control. Therefore, there is an urgent need to develop new antimicrobial drugs and alternative therapies, as well as strategies to inhibit resistance mechanisms, among which phage therapy combined with antibiotics has great potential.

[0004] Leungtongkam U et al. discovered the relationship between antibiotic resistance, CRISPR / cas system and phage-sensitive bacterial virulence factors, which will help further explore the co-evolution of bacteria and phages. The lysis spectrum of some phages is narrow, which is because bacteria have evolved a variety of mechanisms to hinder phage lysis, such as restriction modification systems, adaptive immunity and spontaneous mutations. Phages can also be used as air purifiers to prevent and control nosocomial infections, especially infections of Acinetobacter baumannii in ICU wards. Chen LK, Chang JC et al. used phage mixtures as aerosols to effectively control the infection rate of carbapenem-resistant Acinetobacter baumannii (CRAB) in hospitals and maintain a safe medical environment. Studies have shown that when used as a combination therapy, phages and antibiotics can work synergistically to achieve greater antibacterial effects. In current medical practice, phage therapy has shown significant therapeutic effects and has been successfully applied to many cases. Grygorcewicz B et al. successfully combined the phage-antibiotic combination therapy (PAC) strategy by conducting research in a human urine model, and further confirmed that the Acinetobacter baumannii phage Aba-1 showed significant effects in reducing biofilm formation. In summary, phage cocktail therapy and PAC therapy can effectively broaden the lysis spectrum and reduce the generation of phage resistance in pathogenic bacteria, providing a new strategy for the treatment of multidrug-resistant Acinetobacter baumannii infections.

[0005] Therefore, the development of phages specific to different Acinetobacter baumannii plays an important role and is of great value in the treatment of Acinetobacter baumannii infections. Summary of the invention

[0006] The purpose of the present application is to provide a new bacteriophage against Acinetobacter baumannii and its application.

[0007] This application adopts the following technical solutions:

[0008] The first aspect of the present application discloses a bacteriophage against Acinetobacter baumannii, the bacteriophage is vB_MZM_4AB-P, and its deposit number is CCTCC NO: M 20241200.

[0009] It should be noted that the key to this application is that a new phage vB_MZM_4AB-P that can infect multidrug-resistant Acinetobacter baumannii has been isolated. The whole genome similarity between it and the closest Acinetobacter phage vB_AbaP_Acibel007 is only 72.93%, and the similarity between the phage of this application and other phages is less than 6%. In terms of evolutionary classification, it belongs to a new species of the genus Daemvirus of the subfamily Beijerinckvirinae of the family Autographiviridae. The phage vB_MZM_4AB-P obtained by screening in this application can only infect the test strains multidrug-resistant Acinetobacter baumannii 2AB, 4AB and ATCC-17978, and has strong host specificity. When the temperature is as high as 50°C, its survival rate is still 56.8%, showing good temperature tolerance. It can still maintain a survival rate of more than 43% when stored at -20°C, 4°C and 28°C for two months, and has good storage stability. The phage has an incubation period of 10 minutes, can lyse bacteria within 80 minutes, and the lysis amount reaches 746.7pfu / cell, which can effectively inhibit the growth of host bacteria, and its optimal multiplicity of infection is 10. The phage was subjected to genomic analysis, and the results showed that the phage had a newer genetic background. In short, the phage vB_MZM_4AB-P of Acinetobacter baumannii in this application has the remarkable characteristics of novel genetic background, short incubation period, high lytic activity, good stability, strong environmental tolerance, etc., which provides more resources for the combination of "phage cocktail", and has great value in the treatment of multidrug-resistant Acinetobacter baumannii.

[0010] Preferably, the complete genome of the phage vB_MZM_4AB-P of the present application is the sequence shown in SEQ ID NO.1 to SEQ ID NO.5.

[0011] The second aspect of the present application discloses the use of the bacteriophage vB_MZM_4AB-P of the present application in the preparation of an agent for preventing, controlling or killing Acinetobacter baumannii.

[0012] The third aspect of the present application discloses a reagent for preventing, controlling or killing Acinetobacter baumannii, wherein the reagent contains the bacteriophage vB_MZM_4AB-P of the present application.

[0013] It can be understood that the bacteriophage vB_MZM_4AB-P of the present application can specifically lyse Acinetobacter baumannii, and therefore can be used to prepare reagents for preventing, controlling or killing Acinetobacter baumannii. For example, when treating hospital sewage, the reagents of the present application can be used to prevent, control or kill Acinetobacter baumannii.

[0014] The fourth aspect of the present application discloses the use of the bacteriophage vB_MZM_4AB-P of the present application in the preparation of drugs for preventing or treating diseases caused by Acinetobacter baumannii.

[0015] The fifth aspect of the present application discloses a drug for preventing or treating diseases caused by Acinetobacter baumannii, wherein the drug contains the bacteriophage vB_MZM_4AB-P of the present application.

[0016] It can be understood that the bacteriophage vB_MZM_4AB-P of the present application can specifically lyse Acinetobacter baumannii, and thus can be used to prepare drugs for preventing or treating diseases caused by Acinetobacter baumannii.

[0017] Preferably, the medicine of the present application further contains a pharmaceutically acceptable carrier and / or excipient.

[0018] It can be understood that the drug of the present application can be prepared into different dosage forms according to needs. When selecting the corresponding carrier and / or excipient, as long as it does not affect or is beneficial to the phage vB_MZM_4AB-P of the present application to exert its effect, the specific carrier or excipient can refer to the existing phage drugs and will not be repeated here.

[0019] The beneficial effects of this application are:

[0020] The bacteriophage of the present application is directed against Acinetobacter baumannii, which can specifically lyse Acinetobacter baumannii 2AB, 4AB and ATCC-17978, and has a short incubation period, high lysis activity, good stability, and strong environmental tolerance. It provides a new phage for the prevention, control or treatment of Acinetobacter baumannii, and provides a new solution and approach for the treatment of multidrug-resistant Acinetobacter baumannii. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1 is a diagram showing the plaque morphology and microscopic morphology observation results of the bacteriophage vB_MZM_4AB-P in the examples of the present application, wherein Figure A is a plaque photograph and Figure B is a transmission electron microscope observation diagram;

[0022] Figure 2 : The biological property test results of the bacteriophage vB_MZM_4AB-P in the embodiment of the present application, wherein Figure A is the temperature stability test result, Figure B is the acid-base stability test result, Figure C is the storage stability test result, Figure D is the optimal adsorption time test result, and Figure E is the optimal infection multiplicity test result. The bacteriophage and bacteria are mixed in a ratio of 10:1, 1:1, 1:10, 1:1000, and Figure F is a one-step growth curve;

[0023] Figure 3 is the genome structure diagram of bacteriophage vB_MZM_4AB-P in the examples of the present application;

[0024] Figure 4 It is a whole genome evolutionary tree of bacteriophage vB_MZM_4AB-P in the embodiment of the present application, wherein Figure A is a phylogenetic tree constructed based on the DNA polymerase encoded by gp26 in the vB_MZM_4AB-P genome, Figure B is a phylogenetic tree constructed based on the DNA polymerase encoded by gp28 in the vB_MZM_4AB-P genome, and Figure C is a whole gene evolutionary tree and a similarity heat map of vB_MZM_4AB-P.

[0025] This application is directed to the phage of Acinetobacter baumannii, taxonomic name: Acinetobacter phage vB_MZM_4AB-P, Latin name: Beijerinckvirinae daemvirus vB_MZM_4AB-P, deposited in the China Center for Type Culture Collection on June 13, 2024, address: Wuhan University, Wuhan, China, deposit number CCTCC NO: M 20241200. DETAILED DESCRIPTION

[0026] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources, and the experimental methods used in the following examples are conventional methods unless otherwise specified.

[0028] Example

[0029] 1. Materials and Methods

[0030] 1. Isolation and purification of bacteriophage

[0031] In this study, multidrug-resistant Acinetobacter baumannii 4AB (provided and preserved by Hainan Normal University) was used as the target indicator bacteria to isolate bacteriophages. Sewage from the river under the overpass in Longhua District, Haikou City, Hainan Province was collected, and then centrifuged at 10,000 rpm for 15 minutes at 4°C. 200 mL of the supernatant was taken and added to a three-fold concentrated LB medium containing 2 mL of indicator bacteria. The mixture was cultured in a shaker at 37°C for 18 to 24 hours and then centrifuged again at 10,000 rpm for 15 minutes. The supernatant was sterilized with a bacterial filter with a pore size of 0.22 μm, and the phage was cultured using a double-layer plate technique. The phage was purified multiple times until plaque formation of consistent morphology and size was observed.

[0032] 2. Phage host spectrum determination

[0033] When performing lysis spectrum analysis using the test strains, take 100 μL of the bacterial solution of different strains, mix it thoroughly with LB semi-solid medium, and pour it on the LB agar plate. After the plates solidify, add 3 μL of phage samples on them respectively, and at the same time, use an equal volume of sterile water as a control for spotting, and place these plates in a 37°C constant temperature incubator for incubation. The next day, check whether plaques appear on the surface of the plate, which will be used as a basis for evaluation. The presence or absence of plaques is used to judge the lysis ability of the phage against a specific strain.

[0034] The test strains used for phage host spectrum determination in this example are shown in Table 1.

[0035] Table 1 Test strains

[0036]

[0037]

[0038] 3. Phage morphology observation

[0039] To identify the morphological characteristics of the phage, the fresh eluate of the phage was collected and dripped onto a copper grid covered with a carbon film. The sample was negatively stained with a 2% phosphotungstic acid solution. The negative staining method is referenced in Ding, Y., et al., Virus Res, 2023.326: p.199054.

[0040] The prepared samples were sent to Beijing Zhongke Baice Technology Service Co., Ltd., and the morphology of phage particles was observed using a transmission electron microscope (TEM, JEM-1400, JEOL, Japan) at an accelerating voltage of 100 kV. In order to accurately quantify the size of the head and tail of the phage, the software ImageJ was used for measurement.

[0041] 4. Phage stability assay

[0042] The phage suspension was incubated in a water bath at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 30 minutes to evaluate the thermal stability of the phage, cooled to room temperature, and its titer was determined. The phage suspension incubated at room temperature was used as the control group, and all experiments were repeated three times. The pH value of the phage suspension was adjusted to 1.0, 3.0, 5.0, 7.0, 9.0, and 11.0, and the acid-base stability was determined by incubating at room temperature for 30 minutes. The phage under the initial pH value of the suspension was used as the control, and the experiment was repeated three times.

[0043] In order to study the changes in the titer of phages under different storage conditions, the suspensions were placed in -20°C, 4°C, and 28°C environments, and the titers of phages were measured after storage for 7, 14, 30, 60, 90, 150, and 240 days. All experiments were repeated three times, with the initial concentration as the control.

[0044] 5. Determination of optimal phage adsorption time

[0045] The phage was mixed with the Acinetobacter baumannii 4AB liquid at a ratio of 1:1, and adsorbed at different times to determine the optimal adsorption time of the phage. The supernatant was collected by centrifugation at 10,000 rpm for 1 min, and the titer of the phage was determined. The LB liquid culture medium was used as the control group. The optimal adsorption time of the phage was determined by comparing the titer of the phage at different adsorption time points.

[0046] 6. Optimal Multiplicity of Infection (MOI) Analysis

[0047] In order to determine the optimal infection multiplicity of the phage, the phage was mixed with Acinetobacter baumannii 4AB in the logarithmic growth phase at a ratio of 10:1, 1:1, 1:10, 1:100 and 1:1000, respectively. The mixed sample was allowed to stand at 37°C for 5 minutes to facilitate the adsorption of the phage to the bacteria. The centrifuged sample was centrifuged at 10,000 rpm for 1 minute, and the precipitate was resuspended in 50 mL of fresh LB medium and cultured in a shaker at 37°C and 200 rpm. A control group was also set up, i.e., bacterial culture without the addition of phage. During the experiment, the optical density (OD 595 ) to monitor changes in bacterial growth. By comparing the changes in bacterial growth curves under different phage concentrations, the ability of phage to lyse host bacteria can be further analyzed.

[0048] 7. One-step growth curve of bacteriophage

[0049] Select Acinetobacter baumannii 4AB and phage in the logarithmic growth phase, mix them at a multiplicity of infection of 1.0, and let stand for 5 minutes at 30°C to ensure that the phage can be effectively adsorbed. Centrifuge at 10,000 rpm for 1 minute at room temperature, discard the supernatant, resuspend the resulting precipitate in 50 mL of LB medium, collect samples every 30 minutes, and use spectrophotometry to measure the change in its absorbance at a wavelength of 595 nm to determine the titer of the phage. Repeat the above steps 3 times and record the data. According to the method described in the published literature, a one-step growth curve is drawn to intuitively display the incubation period and lysis characteristics of the phage. The lysis amount calculation formula is as follows: lysis amount = (phage titer at the end of lysis - initial phage titer) / host bacteria concentration at the beginning of infection. Reference, Cao, Y., et al., Arch Virol, 2021.166(2): p.413-426.

[0050] 8. Extraction, sequencing and genome analysis of phage DNA

[0051] The extraction of phage genomic DNA followed the method described by Peng et al., Peng, Q., et al., Sci Rep, 2023.13(1): p.9815. The phage genomic DNA was sent to Sangon (Shanghai Shenggong Biotechnology Co., Ltd.). The phage genomic DNA was sequenced using the HiSeq sequencing platform, and the sequencing quality was preliminarily evaluated using FastQC V0.11.2 software. The raw sequencing data was sheared and spliced ​​using Trimmomatic V0.36 software, and the complete genomic fragments were obtained using SPAdes-3.5.0 software. The coding sequences (CDSs) in the genome were predicted using the FGENESV (Softberry, http: / / linux1.softberry.com) tool and visualized using CGView (https: / / proksee.ca / projects / new), with references to Bardina, C., et al., Front Microbiol, 2016.7: p.545; Stothard, P., JRGrant, and G. Van Domselaar, Brief Bioinform, 2019.20(4): p.1576-1582. The basic local alignment algorithm search tool (BLAST) was used to perform extensive searches for each predicted gene in the NCBI non-redundant protein sequence (NR) database and the CDD database (https: / / www.ncbi.nlm.nih.gov). At the same time, the Pfam database was combined to perform detailed functional annotations on the sequences and functional domains of the predicted proteins. Potential virulence genes were searched using the virulence search program (http: / / www.hpabioinfotools.org.uk / pise / virfactfind_small.html). Potential resistance genes were analyzed using phageleads software (https: / / phageleads.ku.dk / ). Similarity matrix heat maps were prepared using the Microbiological Information Online Platform (www.bioinformatics.com.cn). Functional gene phylogenetic trees were constructed using the neighbor-joining method of MEGA11 software and bootstrap analysis (1000 replicates) using the MUSCLEalignment method. Whole-genome nucleic acid sequence analysis and phylogenetic tree construction were performed using VICTOR online analysis software (ggdc.dsmz.de / victor.php).

[0052] 2. Results and Analysis

[0053] 1. Phage morphology observation

[0054] Using Acinetobacter baumannii 4AB as an indicator bacterium, a bacteriophage was screened and purified from the sewage in the river under the overpass in Longhua District, Haikou City, Hainan Province, named vB_MZM_4AB-P, and preserved in the China Center for Type Culture Collection (CCTCC), with the accession number: CCTCC M 20241200. After 20 hours of constant temperature culture, the bacteriophage vB_MZM_4AB-P formed plaques with a central diameter of 1.25 mm and a halo diameter of 2.1 mm on the Acinetobacter baumannii 4AB plate. The results are as follows Figure 1 As shown in Figure A.

[0055] 2. Transmission electron microscopy observation of phage particles

[0056] Transmission electron microscopy observation of phage particles showed that phage vB_MZM_4AB-P had an icosahedral head with a diameter of 56 nm and a distinct double-layer phage capsid, such as Figure 1 As shown in Figure B.

[0057] 3. Phage host spectrum determination

[0058] The results of host spectrum determination showed that phage vB_MZM_4AB-P had strong host specificity. Among the 25 strains tested, the phage could only infect Acinetobacter baumannii 2AB, 4AB and ATCC-17978, but could not infect Salmonella enterica, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Pseudomonas, Bacillus subtilis, Bacillus pumilus, Vibrio parahaemolyticus and other bacteria, as shown in Table 2. Strong host specificity improves the safety of phages in clinical treatment and avoids interference with other beneficial bacteria.

[0059] Table 2 Phage host spectrum determination results

[0060]

[0061]

[0062] 4. Phage stability assay

[0063] The results of thermal stability showed that the survival rate of phage vB_MZM_4AB-P was still as high as 100% at 40°C and 56.8% at 50°C. At 60°C, the activity of phage decreased sharply, and only 3.2% of phages were active. Figure 2 As shown in Figure A. Phage vB_MZM_4AB-P exhibits a narrow pH tolerance and can only exist stably at pH 7, as shown in Figure 4. Figure 2 As shown in Figure B.

[0064] On the 60th day, the phage vB_MZM_4AB-P could survive stably at -20℃, 4℃, and 28℃, with a survival rate higher than 43%. Among them, at 60 days, the survival rate of the phage vB_MZM_4AB-P at 4℃ was the highest, at 54%, so 4℃ was the ideal condition for preserving the phage vB_MZM_4AB-P. After three months, the phages stored at all temperatures lost their activity. Figure 2 As shown in Figure C. Bacteriophage vB_MZM_4AB-P exhibits good thermal stability and storage stability, which can greatly reduce the cost of transportation and storage in practical applications.

[0065] 5. Determination of optimal phage adsorption time

[0066] When the phage vB_MZM_4AB-P adsorbed with the host bacteria for 3 minutes, the phage content in the mixed solution was the lowest, only 15%, that is, the infection effect of the phage vB_MZM_4AB-P was the best when it adsorbed with the host bacteria for 3 minutes. Figure 2 As shown in Figure D, 3 min is the optimal adsorption time for the phage.

[0067] 6. Optimal Multiplicity of Infection (MOI) Analysis

[0068] The results of the optimal infection multiplicity analysis showed that when the MOI was 10, the titer of phage vB_MZM_4AB-P was the highest, which was 8.0×10 7 CFU / mL, so MOI = 10 is the optimal multiplicity of infection. Figure 2 As shown in Figure E. This phage showed a significant inhibitory effect, and even the lowest MOI = 0.001 was able to inhibit the growth of the host. Therefore, the phage vB_MZM_4AB-P has a strong antibacterial activity.

[0069] 7. One-step growth curve of bacteriophage

[0070] The one-step growth curve results of phage vB_MZM_4AB-P in this study showed that the incubation period of phage vB_MZM_4AB-P was 10 min, the lysis period lasted about 80 min, and the highest phage concentration could reach 1.27×10 8 PFU / mL, the lysis amount is 746.7pfu / cell, such as Figure 2As shown in Figure F, the incubation period of most known Acinetobacter baumannii phages is usually between 10 min and 30 min, and the lysis amount is between 150 and 788 pfu / cell. Compared with these Acinetobacter baumannii phages, vB_MZM_4AB-P has the characteristics of short incubation period and large lysis amount, indicating that the phage has efficient infection and lysis capabilities. References Wang, R., et al., Virol, 2024.21(1).283; Ndiaye, l., et al., BMC Microbiol., 2024.24(1).449; Rastegar, S., et al., Pathog. Dis., 2024.82; Li, S., et al. al.,Appl.Microbiol.Biotechnol.,2024.108(1).428; Zheng,X.,et al.,Virol Sin,2024.39(5).767-781;Wang,Z.,et al.,Front.Cell.Infect.Microbiol.,2024.14;Luo,J.,etal.,Antimicrob.Aqents.Chemother.,2024.e0044824;Tian,X.,etal. al.,Front.Microbiol.,2024.15.1379400; Evseev,P.,et al.,Int.J.Mol.Sci.2024.25(4);Zhang,L.,et al.,Virus Res.,2022.320.198889;Wintachai,P.,et al., Viruses, 2022.14(11); Mardiana, M., et al., Viruses, 2022.14(6).

[0071] 8. Whole genome analysis of bacteriophage vB_MZM_4AB-P

[0072] The genome of bacteriophage vB_MZM_4AB-P is a linear double-stranded DNA with a genome size of 42975 bp ( Figure 3), with a G+C content of 40.57%, Genbank accession: PP091972. The phage encodes a total of 53 open reading frames (ORFs), of which 1 is forward transcribed and 52 are reverse transcribed; 30 of them are known putative functional proteins and 23 are hypothetical proteins. Among the predicted functional proteins, 12 ORFs are annotated as phage structural proteins, of which gp4 encodes internal virion proteins, gp5 and gp6 encode tail proteins, gp8 encodes coat proteins, gp9 encodes head scaffold proteins, gp10 encodes head-tail adapters, gp11, gp25, gp42, gp43, gp44 encode structural proteins, and gp53 encodes tail fiber proteins, with the same transcription direction.

[0073] Genes gp2, gp3, gp51, and gp52 are annotated as cell lytic protein encoding genes. gp2 encodes a protein inside the virion with a lytic enzyme domain, and gp3 encodes a lysozyme motif inside the virion. gp51 encodes a lytic enzyme that catalyzes the hydrolysis of the peptidoglycan network structure and destroys the physical integrity of the bacterial cell wall. gp52 encodes perforin, which is highly similar to the gene encoding perforin of Acinetobacter phage vB_AbaP_Acibel007, with a similarity of 90.83%. Most phages use perforin and lytic enzymes to lyse and kill host cells, and together they form the perforin-lytic enzyme system.

[0074] There are 13 genes in the phage genome that encode DNA replication-related proteins. By searching the NR database of NCBI, it is predicted that the gp26 and gp28 genes encode DNA polymerase. DNA polymerase is a conserved protein of bacteriophages and can reflect the evolutionary relationship of bacteriophages. DNA polymerase sequences with a similarity of more than 56% to Gp26 were selected in the NCBI database, and a phylogenetic tree was constructed based on them ( Figure 4 A), which shows that Gp26 has the highest similarity with the DNA polymerase of Acinetobacter phage vB_AbaP_Acibel007 (96.82%) and is on the same evolutionary branch. Similarly, DNA polymerase sequences with a similarity of more than 71% to Gp28 were selected and a phylogenetic tree was constructed ( Figure 4B), which shows that Gp28 has the highest similarity (95.36%) with the DNA polymerase of Acinetobacter phage vB_AbaP_Acibel007, and is located in the same evolutionary branch. From the perspective of conserved protein DNA polymerase, phage vB_MZM_4AB-P and Acinetobacter phage vB_AbaP_Acibel007 are most closely related, belonging to the genus Daemvirus of the subfamily Beijerinckvirinae of the family Autographiviridae. gp14 and gp16 genes encode N4-like RNA helicase, gp18 gene encodes deoxynucleoside monophosphate kinase, gp22 gene encodes recombinant endonuclease VII, gp23 gene encodes nucleotidyl transferase, gp24 gene encodes exonuclease, gp27 gene encodes HNN homing endonuclease, gp30 gene encodes ATP-dependent DNA ligase, gp31 gene encodes DNA helicase, gp33 gene encodes DNA primase, and gp34 gene encodes endonuclease. In addition, the phage genome does not contain resistance genes or virulence factors, so the phage has good safety in actual human application.

[0075] Figure 3 The genome structure of bacteriophage vB_MZM_4AB-P. Different gene modules are marked with different colors, including packaging gene module (blue), structural gene module (orange), cell lysis-related gene module (pink) and replication-related gene module (green). Genes with unknown functions are indicated in gray.

[0076] The whole genome of the bacteriophage vB_MZM_4AB-P in this example is the sequence shown in SEQ ID NO.1 to SEQ ID NO.5. Since the whole genome sequence is long, it is divided into five parts, namely the sequences shown in SEQ ID NO.1 to SEQ ID NO.5; therefore, the five sequences are directly combined in order to form the whole genome sequence. SEQ ID NO.1 to SEQ ID NO.5 simply divide the whole genome sequence into five parts, without distinguishing them by function or gene, and the five sequences should be treated as a whole.

[0077] SEQ ID NO.1:

[0078]

[0079] SEQ ID NO.2:

[0080]

[0081] SEQ ID NO.3:

[0082]

[0083] SEQ ID NO.4:

[0084]

[0085] SEQ ID NO.5:

[0086]

[0087] 9. Phage comparative genomics analysis

[0088] By comparing the whole genome sequence of phage vB_MZM_4AB-P, the whole genome evolutionary tree and similarity heat map of the phage were constructed. The results showed that vB_MZM_4AB-P and Acinetobacter phage vB_AbaP_Acibel007 were in the same evolutionary branch ( Figure 4 C), with the closest genetic relationship, with a similarity of 72.93% (Query Cover = 82.00%, Per.Ident = 88.94%). This finding is consistent with the results of the evolutionary tree constructed based on the conserved protein DNA polymerase ( Figure 4 A and 4B), further confirming their genetic relationship. According to the phage classification method of Dann Turner et al., two phages with genome similarity higher than 95% are classified as the same species, and phages with similarity higher than 70% are classified as the same genus. The whole genome BLASTn similarity of vB_MZM_4AB-P and Acinetobacter phage vB_AbaP_Acibel007 is lower than 95%, and they belong to a new species in evolutionary classification. The similarities between vB_MZM_4AB-P and Acinetobacter phage APK37.1, vB_Ab_1137_KEN_07, and vB_Ab_1137_KEN_06 are 6.39%, 4.65%, and 4.64%, respectively. They are on different evolutionary branches and have distant relationships. The similarities with other phages are all less than 4%. Therefore, vB_MZM_4AB-P is a new member of the genus Daemvirus in the domain Duplodnaviria, kingdom Heunggongvirae, phylum Uroviricota, class Caudoviricetes, family Autographiviridae, subfamily Beijerinckvirinae.

[0089] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A bacteriophage against Acinetobacter baumannii, characterized in that: The bacteriophage is vB_MZM_4AB-P, and its deposit number is CCTCC NO: M 20241200.

2. The bacteriophage according to claim 1, characterized in that: The complete genome of the bacteriophage is the sequence shown in SEQ ID NO.1 to SEQ ID NO.

5.

3. Use of the bacteriophage according to claim 1 or 2 in the preparation of an agent for preventing, controlling or killing Acinetobacter baumannii.

4. A reagent for preventing, controlling or killing Acinetobacter baumannii, characterized in that: The reagent contains the bacteriophage according to claim 1 or 2.

5. Use of the bacteriophage according to claim 1 or 2 in the preparation of a medicament for preventing or treating a disease caused by Acinetobacter baumannii.

6. A drug for preventing or treating a disease caused by Acinetobacter baumannii, characterized in that: The medicine contains the bacteriophage according to claim 1 or 2.

7. The drug according to claim 6, characterized in that: The medicine also contains pharmaceutically acceptable carriers and / or excipients.

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