Carbapenem-resistant Pseudomonas aeruginosa phage and its application

Abstract

The present invention relates to a carbapenem-resistant Pseudomonas aeruginosa phage and its application. The present invention screens and obtains a carbapenem-resistant Pseudomonas aeruginosa phage, which is named Pseudomonas aeruginosa phage (Pseudo monasaeruginosaphage) vB_PaeP_QSZH. The phage vB_PaeP_QSZH has a strong bactericidal activity against Pseudomonas aeruginosa, can efficiently lyse carbapenem-resistant Pseudomonas aeruginosa from various sources, has a wide lysis spectrum, a wide range of applications, and makes up for the limitation of the narrow phage host spectrum. The phage is specific to carbapenem-resistant Pseudomonas aeruginosa, has a strong lysis ability, a short incubation period, a high burst volume, a strong tolerance to temperature and acid and alkali, and has no virulence genes and drug resistance genes. The present invention can provide a safe and non-toxic phage disinfection product for the treatment of Pseudomonas aeruginosa infection in vivo and in vitro, and for the disinfection and purification of Pseudomonas aeruginosa in aquaculture and other environments.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a carbapenem-resistant Pseudomonas aeruginosa phage and an application thereof. Background Art

[0002] Pseudomonas aeruginosa is a conditionally pathogenic Gram-negative bacillus and a zoonotic pathogen. Due to its metabolic versatility, it lives in various ecological environments such as soil and water, and also exists in poultry, livestock, wild animals, humans and animal-derived foods. Pseudomonas aeruginosa is an opportunistic pathogen for humans and is associated with an increasing number of life-threatening acute and chronic infections, including cystic fibrosis, ventilator-associated pneumonia, urinary tract infections, otitis externa, burns and trauma, bone and joint infections, bacteremia and systemic infections. The rapid spread of carbapenem-resistant Pseudomonas aeruginosa in human clinics poses a huge threat to global human health. Pseudomonas aeruginosa can often be isolated from animals or their breeding environment, causing respiratory symptoms, sepsis, severe diarrhea and death in animals. It is the main pathogen of hemorrhagic pneumonia in minks, causing acute, contagious and fatal lung infections in minks. Infected farms will experience collective, acute and high-mortality outbreaks. After infecting poultry, it can spread rapidly in poultry flocks. Chickens of any age may be infected with this bacterium. Incubation period laying hens infected with Pseudomonas aeruginosa are prone to produce "rotten eggs". Infecting waterfowl such as ducks and geese will cause waterfowl Pseudomonas aeruginosa disease and lead to death. Newborn piglets infected with Pseudomonas aeruginosa will have difficulty breathing, fall to the ground, severe diarrhea and eventually death. In short, single or mixed infections of animals caused by Pseudomonas aeruginosa in the environment often cause group outbreaks of young livestock and poultry, which can cause extremely high lethality to livestock and poultry with low immunity, causing huge economic losses to the livestock and poultry breeding industry. In addition, Pseudomonas aeruginosa in livestock and poultry and the environment, especially carbapenem-resistant strains, can directly endanger human public health through natural transmission and food.

[0003] Currently, carbapenem-resistant Pseudomonas aeruginosa has been listed as a key pathogen that urgently needs new treatment strategies. There is an urgent need to develop new antibacterial drugs for drug-resistant Pseudomonas aeruginosa. Phages, as viruses that can specifically infect bacteria, can replace antibiotics to treat bacterial infections. Compared with antibiotics and chemical antibacterial drugs, phages have the advantages of host specificity, high safety, and no residual effect. They can quickly kill specific pathogens without affecting the normal flora of the body, and they also have a good killing effect on drug-resistant strains. Therefore, it is of great significance to develop new phages that have preventive and therapeutic effects against multidrug-resistant Pseudomonas aeruginosa. Summary of the invention

[0004] The object of the present invention is to provide a carbapenem-resistant Pseudomonas aeruginosa phage and its application, in order to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A carbapenem-resistant Pseudomonas aeruginosa phage, named Pseudomonas aeruginosa phage vB_PaeP_QSZH, was deposited in China Center for Type Culture Collection with a deposit number of CCTCC M20241721.

[0007] Another object of the present invention is to provide a use of a carbapenem-resistant Pseudomonas aeruginosa phage as an active ingredient in the preparation of a drug for preventing and / or treating infectious diseases caused by Pseudomonas aeruginosa. In particular, the use of a carbapenem-resistant Pseudomonas aeruginosa phage in the preparation of a drug for preventing and / or treating infectious diseases caused by Pseudomonas aeruginosa.

[0008] Another object of the present invention is to provide a use of a carbapenem-resistant Pseudomonas aeruginosa phage as an effective ingredient in the preparation of a composition for killing Pseudomonas aeruginosa on the body surface and / or in the body of livestock and poultry.

[0009] Another object of the present invention is to provide a carbapenem-resistant Pseudomonas aeruginosa phage for use as an active ingredient in a drug for killing Pseudomonas aeruginosa in a space environment.

[0010] Preferably, the spatial environment includes at least an animal breeding environment and / or a medical environment.

[0011] Another object of the present invention is to provide a composition for specifically killing Pseudomonas aeruginosa, which comprises the above-mentioned carbapenem-resistant Pseudomonas aeruginosa phage as an active ingredient.

[0012] The present invention also provides an environmental disinfectant, the active ingredient of which is a preparation including the above-mentioned bacteriophage; preferably, the content of bacteriophage is not less than 1×109 PFU / ml, and it can be used for disinfection of cement floors and metal equipment surfaces in farms, hospitals and public places.

[0013] Experiments have shown that spraying 10 9 PFU·mL -1 After the phage vB_PaeP_QSZH cocktail was added, the bacteria were almost completely killed within 1 hour, proving that the phage of the present invention has good application prospects.

[0014] The biological characteristics of the carbapenem-resistant Pseudomonas aeruginosa phage vB_PaeP_QSZH of the present invention were determined by experiments, and the results are as follows:

[0015] 1. The bacteriophage vB_PaeP_QSZH was isolated from the natural environment, and the host bacteria was CR-PA20025438. The bacteriophage vB_PaeP_QSZH is a short-tailed phage, which can form transparent plaques on solid culture medium, with uniform shape and size, clear and regular edges, and a diameter of 7 mm. From the electron microscope photo, it can be seen that the phage head is a regular icosahedron, with a head length of about 62 nm and a tail length of about 15 nm.

[0016] 2. The bacteriophage vB_PaeP_QSZH has a strong bactericidal activity against Pseudomonas aeruginosa, and can efficiently lyse human-derived carbapenem-resistant Pseudomonas aeruginosa, pig-derived carbapenem-resistant Pseudomonas aeruginosa, goose-derived carbapenem-resistant Pseudomonas aeruginosa, duck-derived carbapenem-resistant Pseudomonas aeruginosa, chicken-derived carbapenem-resistant Pseudomonas aeruginosa, etc.

[0017] 3. The optimal MOI of phage vB_PaeP_QSZH was determined by the experiment of determining the optimal MOI. When the phage vB_PaeP_QSZH reacted with the corresponding host bacteria at an MOI of 0.00001, the titer of the progeny phage produced was the highest. That is, the optimal MOI of phage vB_PaeP_QSZH was 0.00001.

[0018] 4. The one-step growth curve showed that the incubation period of the phage vB_PaeP_QSZH was about 10 minutes, the lysis burst period lasted about 140 minutes, and it entered the stable period about 150 minutes after the phage infected the host bacteria. The lysis amount was about 352 PFU·cell -1 .

[0019] 5. The results of temperature and pH tolerance experiments showed that the titer of phage vB_PaeP_QSZH remained at a high level after being treated at 40℃ and 50℃ for 80 minutes, and the titer decreased by 44.00% after being treated at 70℃ for 20 minutes. At 80℃, phage vB_PaeP_QSZH was inactivated after 60 minutes. Between pH=3 and 11, the titer of phage vB_PaeP_QSZH remained stable at a high level.

[0020] Beneficial effects: The present invention provides a broad-spectrum lytic phage for carbapenem-resistant Pseudomonas aeruginosa, which has strong bactericidal activity and a wide host spectrum against Pseudomonas aeruginosa. The phage has a good killing effect on carbapenem-resistant Pseudomonas aeruginosa from different types of livestock and poultry as well as human clinical bacteria, has a wide lytic spectrum and a wide range of applications, thus making up for the limitation of the narrow host spectrum of the phage.

[0021] The phage is specific to carbapenem-resistant Pseudomonas aeruginosa, has strong lysis ability, short incubation period, high burst volume, strong tolerance to temperature and acid-base, and no virulence genes or drug resistance genes. It can be used alone or in combination with other substances, for example, it is easy to prepare into a spray and is convenient for large-scale production, and can effectively kill carbapenem-resistant Pseudomonas aeruginosa in the environment.

[0022] The present invention provides a safe and non-toxic phage disinfection product for the treatment of in vivo and in vitro Pseudomonas aeruginosa infection and the disinfection and purification of Pseudomonas aeruginosa in aquaculture and other environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 This is the plaque morphology of bacteriophage vB_PaeP_QSZH.

[0025] Figure 2 The morphology of bacteriophage vB_PaeP_QSZH under transmission electron microscopy.

[0026] Figure 3 This is the one-step growth curve of bacteriophage vB_PaeP_QSZH.

[0027] Figure 4 This is the temperature tolerance of bacteriophage vB_PaeP_QSZH.

[0028] Figure 5 The acid-base tolerance of bacteriophage vB_PaeP_QSZH.

[0029] Figure 6 This is a circle map of the whole genome of bacteriophage vB_PaeP_QSZH.

[0030] Figure 7 The following is a comparison chart of the bactericidal effect of phage preparations on cement surfaces and metal surfaces.

[0031] Biological Deposit Description

[0032] The Pseudomonas aeruginosa phage vB_PaeP_QSZH of the present invention was deposited in the China Center for Type Culture Collection on July 31, 2024, with the deposit address being: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being: CCTCC M20241721. DETAILED DESCRIPTION

[0033] Example 1 Isolation and Identification of Pseudomonas aeruginosa

[0034] 867 fecal samples were collected from laying hens, broilers, ducks, geese, pigs and mink farms in Shandong Province. In the clean bench, the fecal sample and saline mixture in each sampling tube were shaken and mixed, and 100 μL was added to a sterilized centrifuge tube containing 1 mL of NAC broth. The centrifuge tube was placed in a 37°C constant temperature incubator for 16-8 hours to obtain the enrichment solution. Use an inoculation loop to dip the enrichment solution, streak it on a hexadecyltrimethylammonium bromide agar plate, place it in a constant temperature incubator at 37°C for 16-18 hours, observe the number, size, color, and morphology of the colonies, and record the strain colonies of different forms. Pick a single suspected colony that is yellow-green, flat and amorphous, spreads around, and has a moist surface, and inoculate it again on a hexadecyltrimethylammonium bromide agar plate for repeated purification and culture until the colony size, color, and morphology are uniform. A single colony was picked up with a disposable pipette tip and added to a sterilized centrifuge tube containing 1 mL of LB broth and cultured at 37°C for 18 h. The strain was identified as Pseudomonas aeruginosa by 16S rRNA molecular identification. The minimum inhibitory concentration of the strain to colistin, meropenem, cefotaxime, ceftazidime, amikacin, gentamicin, levofloxacin, and ciprofloxacin was determined by agar dilution method.

[0035] Among the 867 samples, the detection rate of PA strains sensitive to meropenem was 6.69% (58 / 867), and a total of 70 PA strains sensitive to meropenem were obtained (Table 1). Among them, the detection rate of meropenem-sensitive PA in laying hen samples was significantly higher than that in other sources (p<0.05), which was 11.71% (26 / 222). The detection rates of meropenem-sensitive PA in other samples were: 6.93% (7 / 101) from ducks, 5.8% (12 / 207) from broilers, 5.79% (7 / 121) from geese, and 3.8% (6 / 158) from pigs. Among the 70 PA strains sensitive to meropenem, 50 were carbapenem-resistant Pseudomonas aeruginosa (CRPA). Among the 867 samples, 153 meropenem-sensitive Pseudomonas aeruginosa were also obtained. In addition, 23 human CRPA strains were collected, totaling 93 meropenem-resistant non-sensitive PA strains.

[0036] Table 1 Detection rates and strain numbers of meropenem-sensitive PA and CRPA in samples from different sources

[0037]

[0038] Except for amikacin, the resistance rates of CRPA and meropenem-susceptible PA to various drugs were significantly higher than those of meropenem-susceptible PA (p<0.05). The resistance rates of animal-derived CRPA to cefotaxime, ciprofloxacin, levofloxacin and gentamicin were as high as 86.00%, 86.00%, 74.00% and 62.00%, respectively.

[0039] Example 2 Isolation and Identification of Pseudomonas aeruginosa Phage and Determination of Lysis Rate

[0040] The samples used in the experiment of the present invention are river water collected from rivers in Shandong Province in 2022.

[0041] (1) Isolation of bacteriophage

[0042] Take river water samples and centrifuge them at 4℃, 10000rpm / min for 10min, take the supernatant and filter it through a 0.22μm filter to sterilize it, and place the sample filtrate at 4℃ for use. Take 10mL of each sample filtrate and mix it with an equal volume of 2×LB broth, inoculate all fresh host bacteria at an inoculation rate of 1% of the total volume, mix them evenly and place them in an incubator at 37℃ for overnight culture. Centrifuge the culture solution at 4℃, 10000rpm / min for 10min, take the supernatant and filter it through a 0.22μm filter to sterilize it. Take the filtrate and repeat the above steps, enrich it repeatedly 3 times, and obtain the phage enrichment solution. Take 200μL of host bacteria and inoculate it into 10mL LB broth and culture it to the logarithmic phase. Take 100 μL of logarithmic phase bacterial solution, add 5 mL of LB semi-solid (about 50°C), mix well and quickly pour it onto the marked LB agar. After the agar solidifies, take 3 μL of each sample enrichment solution and spot it on the agar surface with the host bacteria added. After spotting, place the agar plate in a 37°C constant temperature incubator for overnight culture. The next day, observe whether plaques are formed at the spot of the phage enrichment solution. Using the carbapenem-resistant CRPA clinical strain CR-PA20025438 as the host bacteria, a strain of Pseudomonas aeruginosa lytic phage was obtained.

[0043] (2) Phage purification

[0044] Use an inoculation loop to pick up a single clear and transparent plaque and place it in 1mL SM buffer. Let it stand overnight at 4°C and wait for the phage particles to fall off the agar. After filtering through a 0.22μm filter, take 100μL of the filtrate and dilute it tenfold. Take 100μL of each dilution gradient and mix it with the corresponding logarithmic phase host bacterial solution. Incubate at room temperature for 10 minutes to allow the phage to complete adsorption, and then culture the plaques using the double-layer plate method. The method is to add 5mL LB semi-solid to the mixture of phage and host bacteria, mix it and quickly pour it onto the marked LB agar, wait for the agar to solidify, and then culture it at 37°C overnight. Repeat the purification for at least five times until plaques with consistent morphology, size and transparency appear. Obtain phage 25438QSZH. See the plaque morphology for details. Figure 1 .

[0045] (3) Determination of phage lysis rate

[0046] The lysis rate of phage against 93 meropenem-susceptible PA and 153 meropenem-susceptible PA from different sources was determined. Take 100 μL of logarithmic phase bacterial solution and 5 mL of LB semi-solid (about 50 ° C) and mix them well, then quickly pour them onto LB agar, wait for solidification and put them away, then draw 3 μL of phage solution and drop them onto the bacterial agar plate, and be careful to keep the agar plate horizontal. After waiting for the droplet to dry, put it into a 37 ° C constant temperature incubator overnight. The next day, record the lysis of each bacterial agar plate marked point (with or without transparent plaques) and perform statistical analysis. The lysis rate of phage is shown in Table 2. The lysis rate of phage QSZH against 246 strains of Pseudomonas aeruginosa reached 72.95%, the lysis rate against pig and goose strains was as high as 80.0% or more, the lysis rate against duck and laying hen strains was also as high as 70.0% or more, and the lysis rate against broiler strains was 58.0%. It is worth noting that the lysis rate of human CRPA by this phage reached 47.83%.

[0047] Table 2 Lysis rate of Pseudomonas aeruginosa phage against Pseudomonas aeruginosa from different sources

[0048]

[0049] (4) Phage morphology observation

[0050] 20 μL of phage proliferation solution with a titer of 109 pfu·mL-1 was dropped onto the copper mesh and adsorbed for 10 minutes. The excess liquid was removed along the edge of the copper mesh with filter paper, and the copper mesh was covered with 20 μL of 2% phosphotungstic acid negative staining solution for 5 minutes. After the copper mesh was dried, it was observed by electron microscopy on the large instrument sharing platform of Qingdao Agricultural University. From the electron microscope photo ( Figure 2 ) It can be seen that the head of bacteriophage 25438QSZH is a regular icosahedron with a head length of about 62nm and a tail length of about 15nm. It belongs to the family Brachyuraviridae of the order Caudovirales. According to the latest classification standards of the International Committee on Taxonomy of Viruses (ICTV), bacteriophage 25438QSZH is named vB_PaeP_QSZH.

[0051] Example 3 Characteristic Analysis of Pseudomonas aeruginosa Phage

[0052] (1) Determination of the optimal infection multiplicity of phage

[0053] The concentration of each host bacteria was adjusted to 108CFU·mL-1, and 8 infection multiplicity ratios were set to 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, and 0.000001, respectively. The phage titer was adjusted to 102-109PFU·mL-1 according to the infection multiplicity ratio. 100μL of each dilution gradient phage was mixed with 100μL of the corresponding host bacteria, and the LB broth was fixed to 1mL. After culturing for 4 hours in a constant temperature shaker at 37℃ and 180rpm / min, the phage titer was determined. The infection multiplicity ratio with the highest titer was the optimal infection multiplicity (MOI). Three parallel groups were set for each group of infection multiplicity, and the average value was taken. As shown in Table 3, when the phage vB_PaeP_QSZH reacted with the corresponding host bacteria at an infection multiplicity of 0.00001, the titer of the progeny phage produced was the highest, that is, the optimal MOI of the phage vB_PaeP_QSZH was 0.00001.

[0054] Table 3 Optimal multiplicity of infection of bacteriophage vB_PaeP_QSZH

[0055]

[0056] (2) Determination of one-step growth curve of phage

[0057] Determine the concentration of host bacteria and adjust the phage titer to 0.01 multiplicity of infection. Add 2 mL of the titered phage and host bacteria to a 5 mL centrifuge tube and mix well. Incubate at 37°C for 10 minutes and centrifuge at 10,000 rpm / min for 1 minute. Discard the supernatant, resuspend the precipitate with 2 mL of LB broth (pre-bathed at 37°C), and centrifuge again at 10,000 rpm / min for 1 minute. Repeat this resuspension and centrifugation step 3 times. Pipette 100 μL of the last resuspended mixture into a 1.5 mL centrifuge tube, and dispense into 18 centrifuge tubes. Make each centrifuge tube up to 1 mL with LB broth. Place 18 1.5 mL centrifuge tubes in a constant temperature shaker, set at 37°C and 180 rpm / min. Set 18 culture times in total, including 0min, 5min, 10min, 15min, 20min, 25min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 110min, 130min, 150min, 180min, and 210min, and take out the corresponding centrifuge tubes and mark them. The centrifuge tubes taken out from the constant temperature shaker were centrifuged at 10000rpm / min for 1min, and the supernatant was aspirated and filtered through a 0.22μm filter membrane. After ten-fold continuous dilution, the phage titer was determined using the double-layer plate method. Three parallel groups were set for each phage and the average value was taken. The one-step growth curve of the phage-infected host bacteria was drawn according to the titer of the phage after different culture times. The calculation formula for the phage lysis amount is phage lysis amount (PFU·cell-1) = phage titer during the lysis burst period ÷ host bacteria concentration at the initial stage of infection. The one-step growth curve results of phage vB_PaeP_QSZH are shown in the figure. Figure 3 As shown in Table 4, the incubation period of bacteriophage vB_PaeP_QSZH after infecting the host bacteria was about 10 minutes, the lytic burst period lasted about 140 minutes, and it entered the stable period about 150 minutes after the phage infected the host bacteria, and the lytic amount was about 352 PFU·cell -1 .

[0058] Table 4 Partial growth curve results of bacteriophage vB_PaeP_QSZH

[0059]

[0060] (3) Determination of phage temperature tolerance

[0061] After mixing 100 μL of phage with the corresponding logarithmic phase host bacterial solution, let it stand for adsorption for 10 minutes, add LB broth to 30 mL, and place it in a constant temperature shaker at 37°C, 180 rpm / min for 4 hours until the liquid is clear. The culture solution was centrifuged at 4°C, 10000 rpm / min for 10 minutes, and the supernatant was filtered and sterilized through a 0.22 μm filter. Take 100 μL of phage filtrate to determine its initial titer, and then take 2 mL of phage filtrate and place it in a constant temperature water bath at 40°C, 50°C, 60°C, 70°C, and 80°C for treatment. Set a total of 4 incubation times of 20min, 40min, 60min, and 80min, and sample 400 μL respectively. After ten-fold continuous dilution, the phage titer was determined using the double-layer plate method. Three parallel groups were set for each phage to take the average value, and the titer change curve of the phage at different temperatures and incubation times was drawn. The temperature tolerance results of this phage are shown in Figure 4 As shown in Table 5, the titer of phage vB_PaeP_QSZH remained at a high level after being treated at 40℃ and 50℃ for 80min, and the titer dropped by 44.00% after being treated at 70℃ for 20min. At 80℃, phage vB_PaeP_QSZH was inactivated after 60min.

[0062] Table 5 Temperature tolerance results of bacteriophage vB_PaeP_QSZH

[0063]

[0064] (4) Determination of phage acid-base tolerance

[0065] After mixing 100 μL of phage with the corresponding logarithmic phase host bacterial solution, let it stand for adsorption for 10 minutes, add LB broth to 30 mL, and place it in a constant temperature shaker at 37°C, 180 rpm / min for 4 hours until the liquid is clear. The culture medium was centrifuged at 4°C, 10000 rpm / min for 10 minutes, and the supernatant was filtered and sterilized through a 0.22 μm filter. Take 100 μL of phage filtrate and mix it with 900 μL of SM buffer with different pH values ​​(pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), and incubate at 37°C for 2 hours. After ten-fold continuous dilution, the phage titer was determined using the double-layer plate method. Three parallel groups were set up for each phage, and the phage titer change curve at different pH values ​​was drawn. The acid-base tolerance results of phages are shown in the figure. Figure 5 As shown in Table 6, between pH=3 and 11, the titer of bacteriophage vB_PaeP_QSZH was stable at a high level, and vB_PaeP_QSZH lost its activity under pH=2 and pH=12.

[0066] Table 6 Acid-base tolerance results of bacteriophage vB_PaeP_QSZH

[0067]

[0068] (5) Phage whole genome sequencing

[0069] The phage was sent to Guangdong Meige Gene Technology Co., Ltd. for DNA single virus sample second-generation sequencing library construction, and the whole base group sequencing and assembly of the phage were completed. The software Soapnuke (v2.0.5) was used to evaluate the data quality of the sequencing and remove low-quality data; the BWA (v0.7.17) software was used to align the cleanreads to the Pseudomonas aeruginosa genome and remove the host bacteria sequence; the assembly software Megahit (v1.1.2) was used to assemble the high-quality reads of each phage to obtain the contigs sequence. MetaGeneMark was used to predict the gene sequence of the phage. The whole phage genome was uploaded to the online websites Resfinder 3.1 (https: / / cge.cbs.dtu.dk / services / ResFinder / ), VirulenceFinder 2.0 (https: / / cge.food.dtu.dk / services / VirulenceFinder / ) and VFDB (http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi) to analyze the presence of resistance genes and virulence genes; the whole phage genome was annotated using the RAST website (https: / / rast.nmpdr.org / ); the genome features are shown in Figure 6 As shown in Table 7, the phage vB_PaeP_QSZH does not carry drug resistance genes and virulence factors, indicating that it has the potential for safe application and clinical or production. The total length of vB_PaeP_QSZH is 43346 bp, the G+C content is 62.24%, and the predicted genome encodes 49 proteins, including 30 hypothetical proteins. 19 genes are annotated in the database, including the large subunit of the phage terminase, the phage endonuclease, the phage exonuclease, and proteins with functions such as DNA replication, transcription, packaging, repair, and phage morphology, and contains a perforin-lysin system.

[0070] Table 7 Genome-wide characteristics of bacteriophage vB_PaeP_QSZH

[0071]

[0072] Example 4 Bacteriophage vB_PaeP_QSZH Environmental Disinfection Experiment

[0073] (1) Phage spray disinfection cement surface test

[0074] According to the phage lysis, a human clinical CRPA strain (CR-PA20026836) and a laying hen CRPA strain (JMA210PA) were selected as test bacteria. The concentration of the two strains was adjusted to 10 6 CFU·mL-1, adjust the concentration of CRPA (1:1) mixed bacterial solution to 1mL, add the bacterial solution to the sterile spray bottle for later use. Adjust the titer of the phage to 10 9 PFU·mL -1 , and the phages were respectively drawn into 1mL to 10mL sterilized spray bottles to prepare phage preparations. A transparent storage box disinfected with alcohol was used to simulate a closed space infected with Pseudomonas aeruginosa, and two sterilized cement blocks were placed in the storage box to simulate the external environment. The bacterial solution with adjusted concentration was evenly sprayed on the surface of the cement block, and the phage preparation with corresponding titer was used for spray disinfection. 2mL of phage was evenly sprayed on the cement block, and the lid of the storage box was closed to allow the phage in the box to settle naturally for 30 minutes. Take a sterilized cement block that has not been sprayed and disinfected as a negative control. After 30 minutes, it was rinsed with 5mL PBS buffer. After the rinsing, it was concentrated or diluted, and a suitable gradient was selected to be coated on the hexadecyltrimethylammonium bromide agar solid culture medium. After coating, it was placed in an incubator for culture, and the surface disinfection effect of the phage spray was evaluated according to the number of colonies on the plate. The data used for the disinfection effect was the bacterial concentration in PBS after rinsing with the PBS buffer used for washing. The disinfection effect is shown in Figure 7 As shown, the negative control group without phage disinfection had a large number of colonies growing, and the CRPA concentration in the PBS buffer was 3×10 3 CFU·mL -1 The number of colonies in the group treated with phage disinfection was significantly reduced. The concentrations of the three replicates were 1×10 1 CFU·mL -1 , 1×10 1 CFU·mL -1 , 3×10 1 CFU·mL -1 The CRPA concentration in the phage-treated group was significantly lower than that in the negative control group (p<0.05), which proved that phage spray had a significant disinfection effect on CRPA contamination from both human and livestock sources on the cement surface.

[0075] (2) Phage spray disinfection metal surface test

[0076] Similarly, human clinical CRPA strain CR-PA20026836 and laying hen CRPA strain JMA210PA were selected as test bacteria. The concentration of the two strains was adjusted to 10 6CFU·mL-1, mix the two strains of bacteria in a ratio of 1:1, then draw out 1 mL of bacterial solution and add the bacterial solution to a sterile spray bottle for later use. Draw out 1 mL of bacteriophage into a 10 mL sterile spray bottle to prepare a bacteriophage spray preparation.

[0077] Select a metal door handle, spray alcohol on it for disinfection, and wipe it with sterile gauze. Divide the door handle into two areas, spray a certain amount of bacterial solution evenly on each area, and spray a certain amount of bacteriophage on the other area. Let the bacteriophage and bacterial solution settle naturally for 30 minutes. After 30 minutes, wipe the two areas with sterile gauze soaked in PBS, and wash the gauze with 5mL PBS. Take an appropriate amount of PBS washing solution and apply it on the solid culture medium. Culture at 37℃ for 20 hours, and evaluate the disinfection effect of phage spray on the metal surface according to the number of colonies on the plate. The data used for the disinfection effect is the CRPA bacterial concentration in the PBS washing solution. The spray disinfection test results are as follows: Figure 7 As shown in the figure, a large number of Pseudomonas aeruginosa colonies grew on the culture medium of the control group that was not disinfected with phage spray, and the bacterial concentration in the PBS washing solution was 3×10 3 CFU·mL -1 However, only a few Pseudomonas aeruginosa colonies grew on the culture dishes of the phage spray disinfection group. The CRPA concentrations in the PBS washing solution for the three repeated treatments in this group were 4×10 1 CFU·mL -1 , 3×10 1 CFU·mL -1 , 1×10 1 CFU·mL -1 The CRPA concentration in the phage-treated group was significantly lower than that in the negative control group (p<0.05), which proved that phage spray had a significant disinfection effect on human- and livestock-derived CRPA contamination on metal surfaces in farms and living areas.

[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A carbapenem-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Bacteriophage (Pseudomonas aeruginosa phage) , named as Pseudomonas aeruginosa phage vB_PaeP_QSZH, deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20241721; the lysis rate of the phage against carbapenem-resistant Pseudomonas aeruginosa from pigs and geese is ≥80%; the incubation period of the phage is 10 minutes, the lysis burst period is 140 minutes, and the lysis amount is 352 PFU•cell⁻¹; the phage does not carry virulence genes and drug-resistant genes.

2. Use of the carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1 as an active ingredient in the preparation of a drug for treating infectious diseases caused by Pseudomonas aeruginosa.

3. Use of the carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1 as an effective ingredient in the preparation of a drug for treating infectious diseases caused by carbapenem-resistant Pseudomonas aeruginosa.

4. Use of the carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1 as an effective ingredient in the preparation of a composition for killing Pseudomonas aeruginosa on the surface of livestock and poultry and / or in livestock and poultry.

5. Use of the carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1 as an active ingredient in a drug for killing Pseudomonas aeruginosa in a space environment.

6. The spatial environment in the application described in claim 5 at least includes an animal breeding environment and / or a medical environment.

7. A composition for specifically killing Pseudomonas aeruginosa, characterized in that: The active ingredient of the composition comprises the carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1.

8. An environmental disinfectant, characterized in that The active ingredient comprises the carbapenem-resistant Pseudomonas aeruginosa phage according to claim 1; the content of the phage is not less than 1×109 PFU / ml.

9. The environmental disinfectant according to claim 8, characterized in that: The environmental disinfectant is used in disinfecting farms, hospital cement floors and metal equipment surfaces.

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