Acinetobacter baumannii phage and application thereof

Abstract

The invention relates to an acinetobacter baumannii bacteriophage and application thereof, the acinetobacter baumannii bacteriophage is named PE65, the preservation number of the acinetobacter baumannii bacteriophage is CGMCC NO.46172, the acinetobacter baumannii bacteriophage is preserved in China General Microbiological Culture Collection Center on August 16, 2024, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the postcode is 100101; the invention has the advantages of obvious antibacterial effect, simple fermentation, low cost and good biological safety, and provides a safe and efficient alternative antibody treatment scheme for acinetobacter baumannii infected patients.

Description

Technical Field

[0001] The present invention relates to an Acinetobacter baumannii phage, belonging to the field of biotechnology. Background Art

[0002] Acinetobacter baumannii is a non-fermenting Gram-negative bacillus, widely existing in nature and belonging to conditional pathogens. This bacterium is an important pathogen causing hospital infections, mainly causing respiratory tract infections, and can also cause bacteremia, urinary tract infections, secondary meningitis, surgical site infections, ventilator-associated pneumonia, etc. The resistance rate to commonly used antibiotics shows an increasing trend year by year, which has attracted serious attention from clinicians and microbiologists.

[0003] Domestic data show that Acinetobacter baumannii accounts for more than 70% of the clinically isolated Acinetobacter. The resistance rate of Acinetobacter baumannii to the third and fourth generation cephalosporins has reached 63.0% - 89.9%. The resistance rates to four aminoglycosides (amikacin, gentamycin, netilmicin, tobramycin) and ciprofloxacin all reach 96.3%. At present, the vast majority of strains in China are sensitive to imipenem, meropenem, cefoperazone / sulbactam and polymyxin B, but the effect is poor in the treatment of respiratory tract infections.

[0004] With the increasing drawbacks of bacterial antibiotic resistance, the use of phages to control pathogenic bacteria has received more and more attention. Phages are the general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes. Phages grow and reproduce in bacterial host cells, can cause the lysis of pathogenic bacteria, reduce the density of pathogenic bacteria, thereby reducing or avoiding the chance of pathogenic bacteria infection or disease, and achieving the purpose of treating and preventing diseases. Phage therapy is a new way to treat pathogenic bacterial infections by phage lysis of host bacteria, which lyses the host bacteria while using the host bacteria to synthesize its own genetic material, and has strict host specificity. Compared with traditional antibiotics, phages have the advantages of wide distribution, easy screening, high host specificity, strong proliferation ability, high safety, low R & D cost, etc. There have been cases where phage therapy has been applied to acute and chronic bacterial infections in the oral cavity, otolaryngology and respiratory tract, etc. It can be seen that phage therapy has broad clinical application prospects. Summary of the Invention

[0005] To overcome the defects of the prior art, the present invention provides an Acinetobacter baumannii phage and its application. The technical solution of the present invention is as follows:

[0006] An Acinetobacter baumannii phage named PE65, classified as Acinetobacter baumannii bacteriophage, with a preservation number of CGMCC NO. 46172, was preserved in the China General Microbiological Culture Collection Center on August 16, 2024. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101.

[0007] The titer of this Acinetobacter baumannii phage is 3.0×10 8 -5.0×10 8 PFU / mL. Its head has a polyhedral structure and a slender tail, and it is classified into the Myoviridae family of the Caudovirales order. The major axis of the head is 84 nm, the transverse diameter is 76 nm, the tail length is 110 nm, and the tail diameter is 10 nm.

[0008] The genome of the Acinetobacter baumannii phage belongs to double-stranded linear DNA. The genome size of this phage is 102,988 bp. The contents of bases A, G, T, and C in the whole genome are 31.95%, 21.46%, 30.50%, and 16.09% respectively, and the G+C content is 37.55%.

[0009] The nucleotide sequence of the DNA polymerase of the Acinetobacter baumannii phage is SEQ ID NO.1.

[0010] The nucleotide sequence of the major capsid protein of the Acinetobacter baumannii phage is SEQ ID NO.2.

[0011] The use of the Acinetobacter baumannii phage in lysing Acinetobacter baumannii or preparing a composition for lysing Acinetobacter baumannii.

[0012] The lysed Acinetobacter baumannii includes those resistant to aminoglycosides, penicillins, cephalosporins, carbapenems, tetracyclines, fluoroquinolones, or sulfonamides.

[0013] A disinfectant comprising the Acinetobacter baumannii phage. In the disinfectant, the content of the Acinetobacter baumannii phage is at least 10 7 PFU / mL.

[0014] A pharmaceutical preparation comprising the Acinetobacter baumannii phage for treating infectious diseases caused by Acinetobacter baumannii infection.

[0015] A phage composition comprising the Acinetobacter baumannii phage PE65, characterized in that the phage composition can lyse Acinetobacter baumannii, and its lysis rate is 94.51%.

[0016] The advantages of the present invention are as follows: significant antibacterial effect, simple fermentation, low cost, and good biosafety, providing a safe and efficient alternative antibiotic treatment plan for patients infected with Acinetobacter baumannii. Description of the Drawings

[0017] Figure 1 It is the plaque map of the Acinetobacter baumannii phage;

[0018] Figure 2 It is the electron microscopy image of the Acinetobacter baumannii phage;

[0019] Figure 3 It is the gene map of the Acinetobacter baumannii phage;

[0020] Figure 4 It is the collinearity map of the Acinetobacter baumannii phage;

[0021] Figure 5 It is the phylogenetic tree map of the Acinetobacter baumannii phage;

[0022] Figure 6 It is the heat map of the lysis spectrum of the Acinetobacter baumannii phage;

[0023] Figure 7 It is the graph of the number of viable phages N (logarithmic value) of the Acinetobacter baumannii phage against time.

[0024] Figure 8 Plot log(k) against the reciprocal of temperature (absolute temperature). Detailed Embodiments

[0025] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, and such modifications and substitutions all fall within the protection scope of the present invention.

[0026] In the following embodiments of the present invention, specifically:

[0027] 1.1 Prepare NB solid medium, and the formula is shown in Table 1:

[0028] Component Dosage Beef extract powder 3.0g Peptone 10.0g Sodium chloride 5.0g Agar 20.0g Sodium hydroxide 2 mL Distilled water 1000 mL

[0029] Table 1

[0030] 1.2 Preparation method:

[0031] Weigh each component according to Table 1, heat and dissolve it in distilled water, mix well and make sure that no solid adheres to the bottle wall. Add a silica gel plug and wrap it with kraft paper or newspaper. Sterilize it at 121 °C under high pressure for 15 min, cool it to about 50 °C, and pour it into a sterile petri dish for standby.

[0032] 2.1 Prepare NB liquid medium, and the formula is shown in Table 2:

[0033] Component Dosage Beef extract powder 2.4g Peptone 8.0g Sodium chloride 4.0g Sodium hydroxide 1.6 mL Distilled water 800 mL

[0034] Table 2

[0035] 2.2 Preparation method

[0036] Weigh each component according to Table 2, heat and dissolve it in distilled water. After heating until the agar dissolves, dispense 5 mL into the corresponding test tubes, add a silica gel plug and wrap it with kraft paper or newspaper. Sterilize it at 121 °C under high pressure for 15 min for standby.

[0037] Example 1: Isolation, purification and identification of Acinetobacter baumannii

[0038] Isolation and purification of bacteria: Dip the inoculation loop into the concentrated bacterial solution and streak it on the NB agar plate. Incubate it in a constant temperature incubator at 37 °C for 16 - 18 h to isolate single colonies. The surface of the colonies is smooth, round and convex, and is colorless transparent or milky white on the NB agar plate. Pick out the suspected single colonies with an inoculation loop and streak them in the first zone on the solid NB medium. Replace the inoculation loop and streak them in the second and third zones, and then place them in a constant temperature incubator at 37 °C for 16 - 18 h to continuously purify for three generations. Identification of bacteria: All bacterial identifications need to be based on microscopic morphological identification, and then identified by corresponding PCR. Use specific identification for those with specific primers. For those that cannot be identified, sequence identification is carried out through 16S rRNA (Table 3 is the component table for preparing the PCR system).

[0039] Component Volume (μL) Mix enzyme 12.5 Deionized water 9.5 Forward primer 0.5 Reverse primer 0.5 DNA template 2

[0040] Table 3

[0041] Positive control: System (23 μL) + 2 μL of the determined bacterial solution;

[0042] Negative control: System (23 μL) + 2 μL of deionized water;

[0043] Prepare the colloid (mix 100 mL of buffer and 1 g of agarose, heat and boil until the liquid becomes transparent, cool it with running water to 50 - 60 °C, then add 10 μL of nucleic acid dye, mix well and immediately pour it into the gel tank, load the gel (5 - 6 μL), and run the electrophoresis for 25 min. After obtaining the bands, compare the results and record.

[0044] Design the specific primer sequence of Acinetobacter baumannii as follows (Table 4 is the primer of Acinetobacter baumannii):

[0045]

[0046]

[0047] Table 4

[0048] Example 2. Isolation and Purification of Acinetobacter baumannii Phage

[0049] Resuscitation of strains: Using each host bacterium stocked in a 4°C refrigerator, dip a loop of the bacterium in the cryopreservation solution with an inoculation loop that has been burned and cooled, and perform three-zone streaking (gradually thinning) on a culture dish containing the lower-layer medium. After completion, incubate under specific conditions (incubator at 37°C for 16 - 18 h). Proliferation of bacterial liquid: Use sterilized forceps to pick a single colony from the resuscitated host bacterium medium with a white pipette tip and transfer it to a stoppered test tube containing 5 mL of a specific medium. Bundle 10 test tubes together with a rubber band and kraft paper, and place them in a specific condition for proliferation (incubator at 37°C for 16 - 18 h).

[0050] Bubble sample: Add appropriate amounts of samples such as fecal fluid, sewage, and bedding into a bubble sample bottle. Add 100 μL of bacterial liquid per strain, then add an appropriate amount of specific liquid medium and nutrients, and incubate overnight by soaking under specific conditions (shaker at 37°C, 170 rpm, for 16 - 18 h).

[0051] Verification of phage spot sensitivity:

[0052] (1) Centrifugation: Transfer the samples to 10 mL centrifuge tubes, 8 - 9 mL per tube, centrifuge at 11000 rpm for 10 min, pour the supernatant into a sterilized 10 mL centrifuge tube, and centrifuge again at 11000 rpm for 5 min for standby;

[0053] (2) Take the centrifugate from the last step of the above procedure, dilute it to 0 and -3 gradients, draw grids on the plate and make marks (write down the strain name, dilution gradient, and experiment time respectively); (3) Add 100 μL of the selected strain to a specific upper-layer medium (5 mL, agar concentration of 0.7%) respectively, quickly mix the test tube, and pour it into a culture dish containing a specific lower-layer medium (agar concentration of 1.5%) and wait for it to solidify;

[0054] (4) Dilute the bubble sample filtrate to an appropriate gradient at a 10-fold ratio (generally: 0 and -3 gradients), and pipette 1.5 μL - 2 μL of each sample dilution and drop it onto the corresponding grids on the surface of the medium according to the pre-marked positions;

[0055] (5) After the liquid on the plate with the spots has dried, place it upright in an incubator at 37°C to culture clear and bright plaques, and observe and record the results.

[0056] Leaching phages: Use sterilized forceps to push out a single plaque together with the attached upper medium and place it into a 1.5 mL centrifuge tube containing 1 mL of liquid medium. Use the forceps to crush the attached medium. Place the centrifuge tube on a shaker at 37 °C and 170 rpm for at least 30 min for standby.

[0057] Isolation and purification of phages: Dilute the phage leaching solution by a factor of 10 to an appropriate gradient. Take 120 μL of the phage leaching solution and mix it evenly with 120 μL of the bacterial solution. Incubate at 37 °C for 5 min, then place it on the upper medium at about 50 °C. After mixing evenly, quickly pour it onto a petri dish containing the lower medium. Shake well and place it flat until the medium solidifies. Incubate it inverted in an incubator for an appropriate time, and then obtain a double-layer plate with clear and transparent plaques again. Repeat the purification steps at least 3 times, and the purification time for each time should be the same until the plaque morphology is consistent, the size is uniform, and the transparency degree is consistent.

[0058] Shake the phage proliferation solution: Pick out the plaque and put it into a 1.5 mL centrifuge tube containing 100 μL of broth. Place it on a shaker at 37 °C and 170 rpm for 30 min. Take it out and centrifuge. Take 100 μL of the supernatant and put it into a 5 mL specific liquid medium that has previously been added with 100 μL of the host bacteria (add the bacterial solution first and then the phage leaching solution, 100 μL of phage leaching solution + 100 μL of bacterial solution + 5 mL of specific medium). At the same time, set up a bacterial solution control group (100 μL of bacterial solution + 5 mL of specific medium). Culture it under specific conditions (shaker at 37 °C and 170 rmp for 2.5 - 4 h. Pay attention to controlling the time well. Observe once every 1.5 h first, and then observe once every 0.5 h) until the proliferation solution changes from clear to turbid and then to clear again, and the phage proliferation solution is clearer than the control group. Observe and record the time when it becomes clear again. Centrifuge the phage proliferation solution at 11000 rpm for 10 min, take the supernatant, and filter it using a 0.22 μm sterile microporous filter membrane to obtain the final phage proliferation solution and store it in a 4 °C refrigerator for standby.

[0059] Determination and calculation of phage titer: Dilute the phage proliferation solution by a factor of 10 and spread it on parallel double-layer plates. Aliquot the phage proliferation solution that has become clear into 10 mL sterilized centrifuge tubes and centrifuge (11000 rpm, 5 min). Dilute it by a factor of 10 to 2 appropriate gradients. In newly sterilized 1.5 mL centrifuge tubes, add 240 μL of the host bacterial solution and 240 μL of the phage dilution solution respectively. Mix well and place it in an incubator at 37 °C for 5 min. Take 200 μL and spread it on double-layer plates. Each phage dilution solution is measured in parallel 2 times. Incubate it in an incubator overnight (37 °C, 16 - 18 h), and observe and record (count the plaques and record the plaque size).

[0060] Calculation of phage titer: Phage titer (PFU / mL) = average number of plaques on two parallel double-layer plates (select 30 ≤ number of plaques ≤ 300) × dilution factor × 10 (the result is rounded to three significant figures).

[0061] The titer of this Acinetobacter baumannii phage is 3.0×10 8 -5.0×10 8 PFU / mL. Its head has a polyhedral structure and a slender tail. It is classified into the Myoviridae family of the Caudovirales order. The major axis of the head is 84 nm, the transverse diameter is 76 nm, the tail length is 110 nm, and the tail diameter is 10 nm.

[0062] The genome of the described Acinetobacter baumannii phage belongs to double-stranded linear DNA. The genome size of this Acinetobacter baumannii phage is 102,988 bp. The contents of A, T, G, and C bases in the whole genome are 31.95%, 21.46%, 30.50%, and 16.09% respectively, and the G + C content is 37.55%. After analysis, the phage PE65 genome contains 178 open reading frames (ORFs), and there are repetitive sequences consisting of 127 bases at both ends of the whole genome. 14 tRNAs are found. The proteins are mainly divided into: proteins related to DNA replication, structural proteins, functional proteins, various synthases, and 140 hypothetical proteins. Among them, ORF60 is the major capsid protein, and ORF96 and ORF98 are DNA helicase and DNA polymerase respectively. There are no drug resistance genes and virulence factors.

[0063] Example 3. Biological characteristics of the phage

[0064] The phage obtained in Example 2 was screened to obtain a highly effective and broad-spectrum-lysis Acinetobacter baumannii phage PE65, and its biological characteristics were analyzed.

[0065] Electron microscopy observation of the phage: Take a little of the phage concentrate purified by ultracentrifugation (prepared in Example 2) and drop it on a copper mesh covered with a polyvinyl formaldehyde film, stain it with 2% phosphotungstic acid by mass concentration for 5 - 10 min, place the copper mesh on a dry filter paper, and let it dry naturally. Then observe it with a transmission electron microscope.

[0066] Observed by transmission electron microscopy, the morphology of the phage is as Figure 2 shown. Its head has a polyhedral structure and a slender tail. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this phage PE65 is classified into the Myoviridae family of the Caudovirales order. The major axis of the head of this phage is about 84 nm, the transverse diameter is about 76 nm, the tail length is about 110 nm, and the tail diameter is about 10 nm.

[0067] Phage titer determination:

[0068] Determination and calculation of phage titer: The phage proliferation solution was diluted 10-fold serially, and parallel double-layer plates were spread. The well-shaken phage proliferation solution was dispensed into 10 mL sterilized centrifuge tubes and centrifuged (11000 rpm, 5 min). Dilute it to 2 appropriate gradients by 10-fold serial dilution. In newly sterilized 1.5 mL centrifuge tubes, add 240 μL of host bacterial solution and 240 μL of phage dilution solution respectively, mix well and incubate in an incubator at 37 °C for 5 min. Take 200 μL to spread the double-layer plate. Each phage dilution solution was measured in parallel 2 times, and incubated overnight in the incubator (37 °C, 16 - 18 h), and observation records were made (count the number of plaques and record the plaque size).

[0069] Calculation of phage titer: Phage titer (PFU / mL) = average number of plaques on 2 parallel double-layer plates (select 30 ≤ number of plaques ≤ 300) × dilution gradient × 10 (the result is reserved to three significant figures).

[0070] Detected by the double-layer plate method, the titer of Acinetobacter baumannii phage PE65 proliferated in liquid is 5×10 9 PFU / mL.

[0071] Effect of pH on phage:

[0072] Add 4.5 mL of NB liquid medium with pH 4 and pH 10 to sterile test tubes, then place each test tube in a water bath at 37 °C. After the temperature is balanced, add 500 μL of phage concentrate (prepared in Example 2, titer is 3.80×10 8 PFU / mL) and mix well, then react in a water bath at 37 °C for 1 h, 2 h, and 3 h. Determine the phage titer after 10-fold serial dilution. The phage titer remains basically stable after 1 h, 2 h, and 3 h within the range of pH 4 and pH 10, and maintains good stability when acting for 1, 2, and 3 h in the environments of pH 4 and pH 10, showing the characteristic of acid and alkali resistance. It meets the product requirements.

[0073] Example 4. Phage genome analysis

[0074] Use DNAstar software to analyze the base composition and G + C content of the genome. Use the RAST online database (http: / / www.rast.nmpdr.org) to predict the open reading frames (ORFs) of PE65, and comprehensively analyze the results. Use Proksee software (https: / / proksee.ca / ) to draw the phage gene map.

[0075] tRNA prediction uses the tRNAscan-SE Search Server

[0076] (http: / / lowelab.ucsc.edu / tRNAscan-SE / ). The prediction of virulence factor genes and antibiotic resistance genes was performed using VFDB (http: / / www.mgc.ac.cn / VFs / ). The protein sequence of the major capsid protein (ORF60) of phage PE65 was analyzed for homology using BLASTp of NCBI, homologous proteins were selected, and a phylogenetic tree was constructed using MEGA 11.0 software to analyze the evolutionary relationship of phage PE65. Collinearity analysis was performed using Mauve software.

[0077] The genome of phage PE65 belongs to double-stranded linear DNA. The total genome size of phage PE65 is 102,988 bp. The contents of bases A, G, T, and C in the whole genome are 31.95%, 21.46%, 30.50%, and 16.09% respectively, and the G + C content is 37.55%. Through prediction and analysis, the genome of phage PE65 contains 178 open reading frames (ORFs), and there are repetitive sequences consisting of 127 bases at both ends of the whole genome.

[0078] The proteins are mainly divided into: proteins related to DNA replication, structural proteins, functional proteins and various synthases, and 140 hypothetical proteins. Among them, ORF60 is the major capsid protein, and ORF96 and 98 are DNA helicase and DNA polymerase respectively.

[0079] Through the whole-genome bioinformatics analysis of Acinetobacter baumannii phage PE65, a phage polymerase gene of Acinetobacter baumannii was obtained. The nucleotide sequence of the phage polymerase gene is shown in SEQ ID NO.1:

[0080]

[0081]

[0082] The online prediction software tRNAscan-SE found that Acinetobacter baumannii phage PE65 has 14 tRNAs. The VFDB prediction software did not find any known virulence factor genes or antibiotic resistance genes.

[0083] Comparative genomic analysis: The whole genome of Acinetobacter baumannii phage PE65 was aligned using the BLASTn program on the NCBI website. The results showed that Acinetobacter baumannii phage PE65 has a high genomic similarity with

[0084] phages of the genus Saclayvirus in the family Viruses; Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Saclayvirus; unclassified Saclayvirus. The phage Acinetobacter phage TAC1 (MK170160.1, coverage 94%, identity 98.91%, E = 0) with the highest Score value was selected, and the Mauve software was used to perform collinearity analysis on this phage. The results are as Figure 4 shown. Acinetobacter baumannii phage PE65 has a similar conserved framework with MK170160.1 and strong collinearity.

[0085] Phylogenetic tree of Acinetobacter baumannii phage PE65:

[0086] The protein sequence of the major capsid protein (ORF60) with evolutionary significance in Acinetobacter baumannii phage PE65 was selected, and protein alignment was performed using blastp in the NCBI database. Related protein sequences with similar protein sequences were selected to construct a phylogenetic tree using MEGA11.0 software. The results are as Figure 5 shown.

[0087] Determination of the phage lysis spectrum: The spot assay was used to determine the phage lysis spectrum. Take 1 mL of fresh phage proliferation solution (prepared in Example 2), and centrifuge at 10,000 rpm for 10 min to sediment bacterial debris. The phage stock solution was initially selected for the experiment. Single colonies of 91 strains of Acinetobacter baumannii preserved in the laboratory were separately inoculated into 5 mL of NB liquid medium and cultured at 37°C for 16 - 18 h to obtain the bacterial suspensions of each strain. Take 200 μL of the bacterial suspension and add it to the NB upper medium, mix well, and evenly spread it on the lower agar plate. After solidification, 1.5 μL of the phage proliferation solution (prepared in Example 2) was separately dropped onto the plate. When adding the samples, there should be no contact between the phage proliferation solutions to avoid affecting the test results. After natural drying, culture at 37°C for 16 - 18 h and observe the results.

[0088] The lysis spectrum assay showed that the phage of the present invention had a lysis effect on 51 of the 91 clinically isolated Acinetobacter baumannii strains, with a lysis rate of 56.04%. The phage composition had a lysis effect on 86 of the 91 Acinetobacter baumannii strains, with a lysis rate of 94.51%. Figure 6 ). This indicates that the phage combination has stronger lytic and broad-spectrum properties and is not easily tolerated.

[0089] Example 5: Accelerated storage test

[0090] Establishment of the Arrhenius equation during accelerated storage: 1) Determination of the detection temperature, grouping the detection temperature and action time according to the temperature stability of the phage; at least 3 effective temperatures are selected, and at least 3 time points are tested at each temperature; it is best to do a preliminary experiment before the experiment to ensure that the effective titer at each time point can be detected; 2) Determine the degradation rate constant and plot the number of surviving phages N (logarithmic value) against time ( Figure 7 ), and the slope of the linear equation is obtained according to the trend line of the data points in the figure. LogN=Kt+bThe slope at the above temperature is plotted as log(k) versus the inverse of temperature (absolute temperature) ( Figure 8 ), the degradation rate constants at other temperatures were deduced from the linear equation of the trend line of the data points in the figure, and the quadratic regression equation LogK = a / T + b was obtained. 3) The survival rate at any temperature was deduced. The number of survivors in the sample under the same storage temperature conditions conformed to the first-order reaction kinetic equation, LnN0-LnN = Kt, where N0 is the number of live phages at the beginning of the test (mL -1 ), N is the number of live phages at time t (mL -1 ), k is the rate constant (h-1), and t is the sampling time (h).

[0091] The results are as follows Figure 7 , 8 As shown, the degradation rate constants at other temperatures are deduced based on the linear equation of the trend line of the data points in the figure, and a one-dimensional quadratic regression equation logK=-10443 / T+31.297 is obtained.

[0092] The survival number in the sample under the same storage temperature condition conforms to the first-order reaction kinetic equation: LnN0-LnN=Kt

[0093] It can be deduced from the above equation (as shown in Table 5):

[0094] Degradation rate constant Inactivation time <![CDATA[10 8 > For each one-titer decrease 37℃ <![CDATA[5.93×10 -3 > 3456.11 h, 144 days 14.49 days 16.2 days 25℃ <![CDATA[3.94×10 -4 > 2166.74 days, 5.94 years 243.59 days 243.59 days 4℃ <![CDATA[1.95×10 -6 > 438178.23 days, 1200.49 years 120.78 years 134.96 years

[0095] Table 5

[0096] The present invention also relates to a phage composition, comprising Acinetobacter baumannii phage PE65 as described above and other Acinetobacter baumannii phages. The lysis rate of the Acinetobacter baumannii phage composition is much higher than that of a single Acinetobacter baumannii phage, broadening its bactericidal spectrum, increasing bactericidal activity, and also solving the problem of bacterial variation and tolerance.

[0097] The Acinetobacter baumannii phage PE65 of the present invention has good acid-base stability, and the titer remains basically stable after 1 - 3 h at pH 4 and 10.

[0098]

[0099]

[0100] Table 6

[0101] The Acinetobacter baumannii phage PE65 of the present invention establishes an accelerated storage Arrhenius equation and exhibits good storage stability at 4°C and 37°C.

[0102] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

Claims

1. A bacteriophage of Acinetobacter baumannii, characterized in that The Acinetobacter baumannii phage is named PE65, classified as Acinetobacter baumannii bacteriophage, and its preservation number is CGMCC NO.46172. It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on August 16, 2024, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101.

2. The Acinetobacter baumannii phage according to claim 1, characterized in that The titer of the Acinetobacter baumannii phage was 3.0×10 8 -5.0×10 8 PFU / mL, the head is a polyhedral structure with a slender tail, classified as the Myocaudaceae family of the order Caudata, the head has a long diameter of 84nm, a transverse diameter of 76nm, a tail length of 110nm, and a tail diameter of 10nm.

3. The Acinetobacter baumannii phage according to claim 1 or 2, characterized in that The genome of the Acinetobacter baumannii phage belongs to double-stranded linear DNA. The genome size of the Acinetobacter baumannii phage is 102988bp, and the A, G, T, and C base contents in the whole genome are 31.95%, 21.46%, 30.50%, and 16.09%, respectively, and the G+C content is 37.55%.

4. The Acinetobacter baumannii phage according to claim 3, characterized in that The nucleotide sequence of the DNA polymerase of the Acinetobacter baumannii phage is SEQ ID NO.

1.

5. The Acinetobacter baumannii phage according to claim 4, characterized in that The nucleotide sequence of the major capsid protein of the Acinetobacter baumannii phage is SEQ ID NO.

2.

6. Use of the Acinetobacter baumannii phage according to any one of claims 1 to 5 in lysing Acinetobacter baumannii or preparing a composition for lysing Acinetobacter baumannii.

7. The use according to claim 6, characterized in that Lysed A. baumannii include A. baumannii that are resistant to aminoglycosides, penicillins, cephalosporins, carbapenems, tetracyclines, fluoroquinolones, or sulfonamides.

8. A disinfectant comprising the Acinetobacter baumannii phage according to any one of claims 1 to 5, wherein the content of the Acinetobacter baumannii phage in the disinfectant is at least 10 7 PFU / mL.

9. A pharmaceutical preparation, characterized in that The Acinetobacter baumannii phage according to any one of claims 1 to 5 is used for treating infectious diseases caused by Acinetobacter baumannii infection.

10. A phage composition comprising the Acinetobacter baumannii phage PE65 according to claim 1, characterized in that: The phage composition can lyse Acinetobacter baumannii, and the lysis rate is 94.51%.

Images