Pseudomonas aeruginosa phage and application thereof

Abstract

The invention relates to a pseudomonas aeruginosa bacteriophage and application thereof, the pseudomonas aeruginosa bacteriophage PA75 has the preservation number as follows: the bacteriophage has strong tolerance to high temperature and acid-base, can effectively split pseudomonas aeruginosa, does not contain antibiotic resistance genes and virulence factors in a genome, and is safe and reliable. The bacteriophage PA75 provided by the invention not only can enrich a germplasm resource library of the pseudomonas aeruginosa bacteriophage, but also can provide a selectable basic material for biological prevention and control of the pseudomonas aeruginosa; the bacteriophage can effectively inhibit the growth of planktonic bacteria in a relatively short time, can be used for daily disinfection of the surfaces of medical apparatuses and instruments and hospital environments especially in medical environments in which drug-resistant bacteria generally exist and common disinfectants have poor effects, and is high in safety coefficient and wide in application range.

Description

Technical Field

[0001] The present invention relates to a Pseudomonas aeruginosa phage and its application, belonging to the technical field of phages. Background Art

[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a conditional pathogen that widely exists in water, air, soil, the intestines and skin of animals. In recent years, with the continuous improvement of the degree of breeding economy, diseases caused by this bacterium, such as hemorrhagic pneumonia in mink, suppurative keratitis in gibbons, and suppurative infections in quails, mostly show acute outbreaks in groups, with an increased mortality rate. Pseudomonas aeruginosa can also cause many pathological reactions in human clinics, such as otitis media, cystic fibrosis pneumonia, ventilator-associated pneumonia, and severe burns, and is one of the main conditional pathogens causing death in hospitals. The reason why Pseudomonas aeruginosa can cause a high mortality rate in livestock, poultry and humans and is difficult to treat is that Pseudomonas aeruginosa has natural and acquired mechanisms to resist various antibiotics, and these mechanisms include efflux pumps, antibiotic degradation, modification enzyme systems, and reduced membrane permeability. In addition, Pseudomonas aeruginosa can also form biofilms, reducing the activity of antibiotics to the threshold of exerting their effects, thereby reducing the efficiency of antibiotic use. For animal production and human health, there is a lack of effective antibacterial agents. At the same time, the implementation of the new regulation "Administrative Measures for the Clinical Application of Antibacterial Agents" in China's "Antibiotic Restriction Order" urgently requires the emergence of new anti-Pseudomonas aeruginosa preparations.

[0003] Phages are a general term for a class of viruses that can infect microorganisms such as bacteria, archaea, and actinomycetes, and are widely present in nature. It is estimated that the number of phages in the Earth's biosphere reaches 1×10 31 individuals, about 1×10 8Phages. As viruses that specifically infect hosts, phages do not infect humans, other animals, or plants, and their lysis effect is not affected by bacterial drug resistance, making their use safe and efficient. Compared with traditional antibiotics, phages have the following significant advantages: 1) Phages can control Pseudomonas aeruginosa in biofilms. Antibiotics are isolated outside the biofilm and only play an antibacterial role against the bacteria on the surface of the biofilm, and cannot kill the persister cells inside the biofilm. Phages can not only infect the bacteria on the surface of the biofilm, but also induce the expression of enzymes that dissolve the biofilm through the host bacteria, enter the biofilm and coexist with the persister cells until the persister cells are activated to control their growth and reproduction. 2) Pseudomonas aeruginosa-specific phages have a relatively broad phage spectrum. Pseudomonas aeruginosa is widely distributed in various ecological environments and has rich polymorphisms. However, although the sensitivity of Pseudomonas aeruginosa isolates in a certain area to individual phage infections shows highly variable characteristics, phages that infect Pseudomonas aeruginosa are widely distributed in all corners of the world, with far less diversity than their host bacteria and limited geographical polymorphisms. 3) Phages are ubiquitous in the natural environment and can be isolated from environments such as sewage, feces, and soil, and the isolation methods are mature. 4) The investment cost is low. Compared with the research and development and clinical trials of new antibiotics, phage isolation is rapid and simple, and the production cost is low during actual use. Summary of the Invention

[0004] To overcome the defects of the prior art, the present invention provides a Pseudomonas aeruginosa phage and its application. The technical solution of the present invention is as follows: A Pseudomonas aeruginosa phage, the preservation number of the Pseudomonas aeruginosa phage PA75 is CGMCC NO: 46173, and it was preserved in the Institute of Microbiology, Chinese Academy of Sciences on August 16, 2024. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The application of the phage in the preparation of a lysate or the prevention and control of Pseudomonas aeruginosa.

[0006] The pH value during application does not exceed 10.

[0007] The temperature during application is not higher than 70°C.

[0008] The action time of the phage is at least 2 h.

[0009] A drug for preventing and / or treating Pseudomonas aeruginosa infection, comprising the Pseudomonas aeruginosa phage described above.

[0010] A biological agent for preventing and / or treating Pseudomonas aeruginosa infection, comprising the Pseudomonas aeruginosa phage described above.

[0011] A lysate, comprising the Pseudomonas aeruginosa phage described above.

[0012] The advantages of the present invention are as follows: This phage has strong tolerance to high temperature, acid and alkali, can effectively lyse Pseudomonas aeruginosa, and its genome does not contain antibiotic resistance genes and virulence factors, being safe and reliable.

[0013] The phage PA75 provided by the present invention can not only enrich the germplasm resource library of Pseudomonas aeruginosa phages, but also provide an alternative basic material for the biological prevention and control of Pseudomonas aeruginosa; this phage can effectively inhibit the growth of planktonic bacteria in a short time. Especially in the medical environment where drug-resistant bacteria are widespread and the effects of common disinfectants are not good, it can be used for the daily disinfection of the surface of medical devices and the hospital environment, with a high safety factor and a wide range of applications. Brief Description of the Drawings

[0014] Figure 1 It is a morphological diagram of the plaque formed by Pseudomonas aeruginosa phage PA75 on a double-layer plate; Figure 2 It is a morphological diagram of Pseudomonas aeruginosa phage PA75 under a transmission electron microscope; Figure 3 It is a temperature stability diagram of Pseudomonas aeruginosa phage PA75; Figure 4 It is an acid-base stability diagram of Pseudomonas aeruginosa phage PA75; Figure 5 It is a one-step growth curve diagram of Pseudomonas aeruginosa phage PA75; Figure 6 It is a gene phylogenetic tree diagram of Pseudomonas aeruginosa phage PA75; Figure 7 It is an antibacterial effect diagram of Pseudomonas aeruginosa phage PA75; Figure 8 It is an accelerated storage test diagram of Pseudomonas aeruginosa phage PA75; Figure 9 It is a lysis spectrum heat map of Pseudomonas aeruginosa phage PA75. Detailed Embodiments

[0015] 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 the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0016] The present invention provides a phage, and the phage is Pseudomonas aeruginosa phage PA75, CGMCC NO: 46173.

[0017] In the present invention, the phage PA75 was isolated from the water source of a river in Beijing. The preservation number of the Pseudomonas aeruginosa phage PA75 is CGMCC NO: 46173, which was preserved at the Institute of Microbiology, Chinese Academy of Sciences on August 16, 2024. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The genome size of the phage in the present invention is 88,482 bp. The phage in the present invention has strong tolerance to high temperature, acid and alkali, can maintain high infectivity in the range of 60°C to 70°C, has a wide pH tolerance range, and can maintain a high titer in the range of pH 4 to 10. The phage in the present invention can effectively inhibit the growth of Pseudomonas aeruginosa.

[0018] The present invention also provides a lysate of the above phage. As an implementable method, the above phage is cultured in a medium containing the host bacteria, and the host bacteria are removed to obtain the phage lysate. The method for removing the host bacteria can be selected from filtration sterilization or centrifugation sterilization. The host bacteria are Pseudomonas aeruginosa, and the medium can be selected as NB medium.

[0019] The dosage form of the present invention is not particularly limited, and can be a liquid preparation, a solid preparation, a semi-solid preparation, a gas preparation, etc. According to different dosage forms, the preparation of the present invention also includes excipients acceptable for the preparation. The types of excipients of the present invention are not particularly limited, and include one or more of carriers, diluents, excipients, preservatives, surfactants and antioxidants.

[0020] The present invention also provides an application of the above phage, lysate or the above preparation in preventing and controlling Pseudomonas aeruginosa.

[0021] The prevention and control of the present invention includes prevention and treatment; the phage, lysate or preparation of the present invention is used for prevention and control by contacting with Pseudomonas aeruginosa, and can be used in various forms, including but not limited to soaking, smearing, spraying and other methods of applying to the object surface, and the surface can be the contaminated part or the expected contaminated part. As an implementation method, the above phage, lysate or the above preparation of the present invention can be applied to the disinfection of places such as food, medical devices, animal farms, hospitals, etc.

[0022] Since the phage of the present invention has a temperature tolerance of 60°C to 70°C, when the phage, lysate or preparation of the present invention is stored or used, the temperature should not be higher than 65°C. Since the phage of the present invention has a pH tolerance of 4 to 10, when the phage, lysate or preparation of the present invention is stored or used, the pH value should not be higher than 10. When the phage, lysate or preparation of the present invention is used for preventing and controlling Pseudomonas aeruginosa, it can be used under refrigerated or room temperature conditions, and preferably the room temperature is 25 to 37°C. When the phage, lysate or preparation of the present invention is used for preventing and controlling Pseudomonas aeruginosa, it can be used under the condition of pH 4 to 10. Within the above pH range, the phage can maintain high activity against Pseudomonas aeruginosa. The present invention can achieve the effects of effective sterilization and biofilm lysis in as fast as 2 h, and the sterilization effect can last up to 14 h after treatment.

[0023] The present invention also provides an application of the above phage, or the above lysate, or the above preparation in the preparation of a drug or biological agent for preventing and / or treating Pseudomonas aeruginosa infection.

[0024] In the present invention, in the drug or biological agent for preventing and / or treating Pseudomonas aeruginosa infection, the phage, lysate or preparation of the present invention can be used as the only effective ingredient against Pseudomonas aeruginosa, or can also contain other antibacterial active ingredients that inhibit or kill Pseudomonas aeruginosa. The drug or biological agent for preventing and / or treating Pseudomonas aeruginosa infection can be used alone or in combination with other drugs or preparations. As an implementation manner, the drug or biological agent for preventing and / or treating Pseudomonas aeruginosa infection can be used in combination with antibiotics. The drug or biological agent can also include pharmaceutically acceptable excipients.

[0025] The technical solutions provided by the present invention will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present invention.

[0026] In the above examples, unless otherwise specified, they are all conventional methods.

[0027] The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0028] Example 1 Isolation and purification of phage: Weigh 4 g of sodium chloride, 8 g of peptone, 2.4 g of beef extract powder, and 1.6 mL of sodium hydroxide and place them in 800 mL of ultrapure water to prepare NB liquid medium.

[0029] Weigh 5 g of sodium chloride, 10 g of peptone, 3 g of beef extract powder, 20 g of agar, and 2 mL of sodium hydroxide and place them in 1 L of ultrapure water to prepare NB solid medium.

[0030] Weigh 4 g of sodium chloride, 8 g of peptone, 2.4 g of beef extract powder, 6.4 g of agar, and 1.6 mL of sodium hydroxide, and place them in 800 mL of ultrapure water to prepare NB semi-solid medium.

[0031] Mix the above medium components respectively and autoclave at 121 °C for 20 min.

[0032] The host bacterium Pseudomonas aeruginosa A301 used in the present invention is a strain isolated from Beijing Integrated Traditional Chinese and Western Medicine Hospital.

[0033] The environmental water sample used for separating phages in the experiment of the present invention was collected from a river water source in August 2022, and this water sample is the raw water of a certain river in Beijing.

[0034] The specific separation method is as follows: Strain resuscitation and proliferation: Use each host bacterium stored in a 4 °C refrigerator. Dip a loop of the bacterium from the cryopreservation solution with an inoculation loop that has been burned and cooled, and perform three-zone streaking (gradually thinning) on the culture dish containing the lower layer of the medium. After completion, place it in a specific condition for cultivation (incubator at 37 °C, cultivate for 16 - 18 h). Use sterilized forceps to pick up 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 NB liquid medium. Bundle 10 test tubes in a group with a rubber band and kraft paper, and place them in a specific condition for proliferation (shaker at 37 °C, cultivate for 16 - 18 h).

[0035] Phage enrichment: Add 20 mL of an appropriate amount of fecal liquid, sewage and other samples into a sample soaking bottle, add 100 μL of bacterial liquid for each strain, and then add 20 mL of NB liquid medium. Incubate overnight under specific conditions (shaker at 37 °C, 170 rpm, 16 - 18 h). The next day, take 6 mL of the sample soaking liquid and place it in a 10 mL EP tube, centrifuge at 11000 rpm for 10 min, take the supernatant and filter it. The obtained filtrate is the phage enrichment solution.

[0036] Verification of phage spot sensitivity: 1) Take the above enrichment solution, dilute it to 0 and -3 gradients, draw grids on the plate and make marks (write the strain name, dilution gradient, and experiment time respectively); 2) Take 100 μL of the selected strain and add it to NB semi-solid medium (5 mL, agar concentration is 0.7%), quickly shake the test tube to mix evenly, pour it into a culture dish containing NB solid medium (agar concentration is 1.5%), and wait for it to solidify; 3) Pipette 1.5 μL - 2 μL of the above 0 and -3 gradient enrichment solutions respectively and drop them onto the corresponding grids on the surface of the medium according to the pre-marked positions; 4) After the liquid on the plate after spotting has dried, place it upright in an incubator at 37 °C and cultivate under specific conditions for an appropriate time to cultivate clear phage plaques, and observe and record the results.

[0037] Phage purification: Use sterilized forceps to push out a single plaque along with the attached upper layer of the medium and place it into a 1.5 mL centrifuge tube containing 1 mL of NB medium. Use 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. Dilute the phage leaching solution at a 10-fold ratio to an appropriate gradient. Take 120 μL of the phage leaching solution and mix it evenly with 120 μL of the bacterial solution. After incubating at 37°C for 5 min, place it in a specific upper layer of the medium at about 50°C. After mixing evenly, quickly pour it onto a petri dish containing a specific lower layer of the medium. Shake well and place it flat until the medium solidifies. Place it in an incubator and incubate it upside down for an appropriate time, and then obtain a double-layer plate with clear 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. The plaque morphology is shown in Figure 1 。

[0038] Example 2 Observation of phage morphology: Take 10 mL of the phage suspension and send it to Qingdao Agricultural University for electron microscopy observation. The results are as Figure 2 shown. Phage PA75 has a polyhedral icosahedral head that encapsulates nucleic acid. The head diameter is about 80 nm, and it has a tail about 70 nm long. The neck connects the head and the tail. According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), this phage is classified as Caudovirales, Myoviridae.

[0039] Example 3 Temperature stability of phage: Temperature stability: Pipette 1 mL of the phage suspension into a 1.5 mL centrifuge tube and keep it in a water bath at 60°C for 20 and 40 min, and at 70°C for 20 min respectively. After gradient dilution, use the double-layer plate method to measure the phage titer at different temperatures, draw a curve, and repeat three times at each temperature.

[0040] The results are as Figure 3 shown. Phage PA75 can maintain a high infectivity in the range of 60°C to 70°C. The phage titer gradually decreases with the increase of temperature. At 60°C for 20 and 40 min, its titer is 3.25×10 8 PFU / mL and 1.68×10 8 PFU / mL, and it will drop to 1.46×10 4 PFU / mL at 70°C.

[0041] Example 4 Acid-base stability of phage: pH tolerance: Add 4.5 mL of NB broth with different pH values (4, 10) to sterile test tubes, 6 tubes for each pH value. Then place the test tubes in a 37°C incubator for cultivation. After the temperature stabilizes, add 500 μL of phage proliferation solution to each tube, mix well, and incubate in the 37°C incubator for 1 h, 2 h, and 3 h. After the reaction, perform gradient dilution and use the double-layer plate method to measure the phage titer at different pH values, draw a curve, and repeat three times for each pH value.

[0042] The results are as Figure 4 shown. The pH tolerance range of phage PA75 is 4 - 10, and a relatively high titer is maintained within this range. The titer remains above 2.45×10 9 PFU / mL after acting for 1 - 3 h in an acidic environment. When the pH is 10, the phage titer remains above 3.53×10 9 PFU / mL after acting for 1 - 3 h.

[0043] Example 5 One-step growth curve of phage: One-step growth curve: Mix 1 mL of phage proliferation solution with an infection multiplicity of 10 and 1 mL of fresh proliferation solution of the host bacterium thoroughly (start timing at this moment), incubate at 37°C for 5 min, centrifuge at 12000 rpm for 30 s, use a micropipette to aspirate the supernatant as much as possible, then wash once with 5 mL of NB broth (centrifuge at 12000 rpm for 30 s), and discard the supernatant. Suspend the precipitate with pre-warmed BHI broth (total volume is 5 mL) and mix thoroughly, quickly place it in a 37°C shaker and shake at 170 rpm for cultivation. Take out 150 μL at 0 min and every 10 min, centrifuge at 10000 rpm for 1 min, perform gradient dilution and use the double-layer plate method to measure the phage titer at different time periods, draw a curve, and repeat three times for each time period.

[0044] The results are as Figure 5 shown. The first 10 min of the growth curve of phage PA75 is the latent period, 10 - 110 min is the logarithmic growth phase, 110 - 150 min is the stationary phase, and its burst size is 6.24.

[0045] Example 6 Phage genome analysis: Send 10 mL of phage solution to Beijing University of Chemical Technology for sequencing. Use Megahit software for sequence splicing to obtain the complete phage genome sequence; use the NCBI database for online gene alignment, and use MEGA software to draw the phage gene phylogenetic tree.

[0046] The results after phage genome splicing show that the genome size of phage PA75 is 88482 bp. The phylogenetic tree based on the TerL gene of phage PA75 is as Figure 6As shown, the results indicate that phage PA75 is closely related to Pseudomonas aeruginosa phage PAK-P5 and distantly related to other phages.

[0047] Example 7 Bacteriostatic Effect of Phage: Adjusting Bacterial Concentration: The bacterial solution was serially diluted 10-fold to an appropriate gradient. 20 µL of the diluted solution was pipetted at multiple points onto a specific solid medium culture dish. The plate was inverted and cultured under specific conditions to obtain colonies. The total number of colonies was counted to obtain the average number of colonies. Then, the bacterial solution concentration was adjusted to 10 6 CFU / mL.

[0048] Bacterial Concentration (CFU / mL) = Average Number of Colonies × Dilution Factor × 10 × 5.

[0049] Suspension Quantitative Bactericidal Test: The phage (3 mL) was mixed with the adjusted bacterial solution (3 mL); they were allowed to act at room temperature for 2 h. Every 20 minutes, a portion of the mixed solution was taken for centrifugation, the supernatant was removed, and it was resuspended with physiological saline. The remaining bacterial concentration in the obtained resuspended solution was measured using the method described above. A control group was prepared by mixing the bacterial solution with the culture medium.

[0050] The results are as Figure 7 shown. The bactericidal rate of the phage was 98.67% at 20 min, 99.25% at 40 min, and 99.9% at 60 - 120 min. This indicates that phage PA75 can effectively inhibit the growth of the host bacterium.

[0051] Example 8 Accelerated Storage Test of Phage: Establishment of the Arrhenius Equation during Accelerated Storage: 1) Determination of the detection temperature. Based on the temperature stability of the phage, groups of detection temperatures and action times were set; at least 3 effective temperatures were selected, and at least 3 time points were detected at each temperature; it is advisable to conduct a preliminary experiment before the experiment to explore and ensure that the titer can be detected at each time point; 2) Determination of the degradation rate constant. The logarithm of the number of surviving phages N was plotted against time, and the slope of the linear equation was obtained from the trend line of the data points in the graph. The slope at the above temperatures was plotted as log(k) against the reciprocal of the temperature (absolute temperature). The degradation rate constants at other temperatures were calculated from the linear equation of the trend line of the data points in the graph to obtain a quadratic regression equation . 3) Calculation of the survival rate at any temperature. Under the same storage temperature condition, the number of surviving samples conforms to the first-order reaction kinetic equation, LnN0 - LnN = Kt, where N0 is the initial number of live phages in 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).

[0052] The results are as Figure 8 shown. The degradation rate constants at other temperatures are calculated according to the linear equation of the trend line of the data points in the figure, and a quadratic regression equation logK = -8621.4 / T + 25.494 is obtained. The degradation rate constant at 37°C calculated from the above equation is: 4.97×10 -3 . Under the same storage temperature condition, the survival number in the sample conforms to the first-order reaction kinetic equation: LnN0 - LnN = Kt. The time at 37°C calculated from the above equation is 3102.71 h, which is 129 days.

[0053] Example 9 Bacteriophage lysis rate: Determination of bacteriophage lysis rate: The lysis rate of the bacteriophage was determined by the spot assay method. Take 1 mL of fresh bacteriophage proliferation solution and centrifuge it at 10,000 rpm for 10 min to sediment the bacterial debris. Initially, the original bacteriophage solution and 10 -1 fold dilutions were used for the experiment. Single colonies of 98 Pseudomonas aeruginosa strains clinically isolated 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 solutions of each strain. Take 100 μL of the bacterial solution and add it to the upper layer of NB medium. After thorough mixing, spread it evenly on the lower agar plate. After solidification, take 1.5 μL of the bacteriophage proliferation solution and drop it on the plate. When adding the samples, there should be no contact between the various bacteriophage proliferation solutions to avoid affecting the test results. After natural drying, culture at 37°C for 16 - 18 h and observe the results.

[0054] Through the determination of the lysis rate, it was found that the bacteriophage in the present invention has a lysis effect on 48 strains among the 98 clinically isolated Pseudomonas aeruginosa strains, and the lysis rate is 48.98%, indicating that the bacteriophage has strong lysis ability and broad-spectrum property. The lysis rate of the present invention against drug-resistant strains from a certain hospital is as high as 60%, which is much higher than that of general Pseudomonas phages, solving the problem of bacterial tolerance.

[0055] The above is only the preferred specific implementation manner 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, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A Pseudomonas aeruginosa phage, characterized in that, The preservation number of the Pseudomonas aeruginosa phage PA75 is CGMCC NO: 46173, and it was preserved in the Institute of Microbiology, Chinese Academy of Sciences on August 16, 2024. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the phage according to claim 1 in the preparation of a lysate or for preventing and controlling Pseudomonas aeruginosa.

3. The use according to claim 2, wherein The pH value during use does not exceed 10.

4. The use according to claim 2, characterized in that, The temperature during use is not higher than 70°C.

5. The use according to claim 2, characterized in that, The action time of the phage is at least 2 h.

6. A drug for preventing and / or treating Pseudomonas aeruginosa infection, characterized in that, It includes the Pseudomonas aeruginosa phage according to claim 1.

7. A biological agent for preventing and / or treating Pseudomonas aeruginosa infection, characterized in that, It includes the Pseudomonas aeruginosa phage according to claim 1.

8. A lysis solution, characterized in that, It includes the Pseudomonas aeruginosa phage according to claim 1.

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