Obligate bacteriophage for targeted lysis of drug-resistant klebsiella pneumoniae and application of obligate bacteriophage

Abstract

The invention discloses a bacteriophage fzNSh6-m5, which is classified and named as Klebsiella phage, is preserved in Guangdong Microbial Culture Collection Center on November 11, 2024, and has a preservation number of GDMCC NO: 65463-B1. The bacteriophage can specifically split klebsiella pneumoniae, and an indoor bacteriostasis test shows that the bacteriostasis rate of the bacteriophage reaches 82%, so that the bacteriophage can be used for splitting drug-resistant klebsiella pneumoniae in the composting maturing period and reducing antibiotic resistance genes. The invention also discloses an application of the phage fzNSh6-m5 in splitting the drug-resistant klebsiella pneumoniae, an application of the phage fzNSh6-m5 in splitting the drug-resistant klebsiella pneumoniae in compost or an application of the phage fzNSh6-m5 in preparing a phage preparation for splitting the drug-resistant klebsiella pneumoniae.

Description

Technical Field

[0001] The invention belongs to the field of harmless composting and relates to a strain of an obligate bacteriophage fzNSh6-m5 capable of targeted lysing drug-resistant Klebsiella pneumoniae and a use thereof. Background Art

[0002] Klebsiella pneumoniae, a genus of Klebsiella, is a Gram-negative pathogenic bacterium that usually colonizes on the mucosal surface of humans or animals, and can cause sepsis, bacteremia, urinary tract infection, pneumonia, etc., endangering the health of humans and animals. At present, the clinical strategy for preventing and treating Klebsiella pneumoniae infection mainly relies on antibiotic therapy. Although antibiotic therapy can play a certain effect in the treatment of Klebsiella pneumoniae infection, there is also a problem of drug resistance. On the one hand, in response to antibiotic stress, Klebsiella pneumoniae can acquire drug resistance through mutation and horizontal gene transfer, making antibiotic therapy ineffective. On the other hand, antibiotics that enter animals and humans (such as sulfonamide antibiotics shared by humans and animals) cannot be completely absorbed, and most of them enter the water and soil environment through excretion, destroying the in situ ecosystem, and inducing the emergence of environmental drug-resistant bacteria, indirectly endangering human health.

[0003] Composting is one of the main ways to recycle livestock waste. The increase in the use of veterinary antibiotics has led to more and more antimicrobial-resistant bacteria (ARB) carrying antibiotic resistance genes being added to the soil through compost, which may enter the food chain directly or indirectly, thus causing potential harm. Although the thermophilic composting process can eliminate more than 80% of ARGs in livestock and poultry manure, recent studies have found that in the cooling stage of the late composting, there are still a few ARGs that remain or even rebound, and these ARGs are often high-risk. Since ARB is the main carrier of ARGs, the reduction in ARB abundance can to a certain extent represent the reduction of ARGs. Therefore, the accurate identification and cleavage of ARB is the key to solving the problem of residual resistance genes in the late composting period.

[0004] Bacteriophages are viruses that can target specific bacteria. Compared with broad-spectrum antibiotics, phages have the characteristics of diversity, specificity and high efficiency, which make them have good application prospects in agriculture, medicine and food industry. Although there have been many research results on phage therapy in human, animal and plant diseases, it has not yet received attention in composting. Therefore, adding phages in the later stage of composting as a means of accurately lysing ARBs and reducing ARGs can provide new ideas for harmless composting. Summary of the invention

[0005] The purpose of the present invention is to provide a lytic bacteriophage fzNSh6-m5 for lysing drug-resistant Klebsiella pneumoniae, which exhibits a significant lysis effect on drug-resistant Klebsiella pneumoniae. The phage can be used to efficiently lyse drug-resistant bacteria in compost, such as drug-resistant Klebsiella pneumoniae, to kill them, and reduce ARGs in the compost, and is non-toxic to the environment.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A strain of drug-resistant Klebsiella pneumoniae obligate phage fzNSh6-m5, classified and named Klebsiella phage, was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 11, 2024, with the deposit number: GDMCCNO:65463-B1.

[0008] Another object of the present invention is to provide the use of the bacteriophage fzNSh6-m5 in lysing drug-resistant Klebsiella pneumoniae, in lysing drug-resistant Klebsiella pneumoniae in composting, or in preparing a bacteriophage preparation for lysing drug-resistant Klebsiella pneumoniae.

[0009] Another object of the present invention is to provide a novel and highly effective phage preparation for lysing drug-resistant Klebsiella pneumoniae, wherein the phage preparation comprises the phage fzNSh6-m5.

[0010] The titer of bacteriophage fzNSh6-m5 in the bacteriophage preparation is ≥10 8 PFU / mL.

[0011] Preferably, the titer of phage fzNSh6-m5 in the phage preparation is 10 8 ~10 10 PFU / mL.

[0012] A method for preparing a phage preparation comprises: according to an optimal multiplicity of infection, inoculating a phage suspension into a Klebsiella pneumoniae (K.pneumoniae) bacterial solution in a logarithmic growth phase, co-culturing at 30-40°C and 170-180 r / min for 5-8 hours to proliferate the phage, centrifuging the co-culture solution, taking the supernatant, filtering the supernatant through a 0.22 μm filter membrane to obtain a phage suspension, and adjusting the phage titer with sterile water to obtain a phage preparation.

[0013] The titer of bacteriophage fzNSh6-m5 in the bacteriophage suspension is ≥10 8 PFU / mL, preferably 10 8 ~10 10 PFU / mL.

[0014] The concentration of the Klebsiella pneumoniae (K.pneumoniae) bacterial solution in the logarithmic growth phase is OD 600 =1.2.

[0015] The Klebsiella pneumoniae bacterial liquid is prepared by the following method: streaking and activating the Klebsiella pneumoniae bacterial liquid on a MAC selective medium, and culturing at 30-40° C. until colonies are formed; picking up a single colony formed on the MAC selective medium and transferring it to a TSB liquid medium, culturing at 30-40° C. and 170-180 r / min for 12-16 hours to obtain a Klebsiella pneumoniae bacterial liquid in a logarithmic growth phase.

[0016] The optimal multiplicity of infection is 0.1 to 1, preferably 0.1.

[0017] The centrifugal speed is 8000-12000 r / min, and the centrifugal time is 5 minutes.

[0018] Another object of the present invention is to provide a method for lysing drug-resistant Klebsiella pneumoniae in compost, comprising: inoculating the bacteriophage preparation into the compost in a spraying manner, wherein the amount of bacteriophage added is ≥2×10 8 PFU / mL compost.

[0019] Preferably, the amount of phage added is 2×10 8 PFU / mL compost.

[0020] Preferably, the phage preparation is inoculated into the compost during the compost maturity stage.

[0021] Preferably, the compost is livestock and poultry manure compost.

[0022] Beneficial effects of the present invention:

[0023] The bacteriophage of the present invention can specifically lyse Klebsiella pneumoniae. The activity of the bacteriophage is the highest and the lysis ability is the strongest in the temperature range of 30-40°C and pH=5-10, and both show strong bactericidal ability. The indoor antibacterial test shows that the antibacterial rate reaches 82%, indicating that the bacteriophage of the present invention can be used to lyse drug-resistant Klebsiella pneumoniae in the mature stage of compost and eliminate antibiotic resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The plaques formed by the phage on the double-layer plate; 1, 2, 3, and 4 represent the gradient dilution of the phage suspension to 10 with sterile water. -1 , 10 -2 , 10 -3 , 10 -4 .

[0025] Figure 2 The morphology of bacteriophage fzNSh6-m5 under electron microscope.

[0026] Figure 3 This is the one-step growth curve of bacteriophage fzNSh6-m5.

[0027] Figure 4 This is the temperature tolerance of bacteriophage fzNSh6-m5.

[0028] Figure 5 This is the acid and alkali tolerance of bacteriophage fzNSh6-m5.

[0029] Figure 6 The antibacterial properties of bacteriophage fzNSh6-m5.

[0030] Biodeposit Information:

[0031] Bacteriophage fzNSh6-m5, classified as Klebsiella phage, was deposited on November 11, 2024 at the Guangdong Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong, China, with the deposit number: GDMCC NO: 65463-B1. DETAILED DESCRIPTION

[0032] Example 1

[0033] Screening of obligate bacteriophages against sulfonamide-resistant Klebsiella pneumoniae

[0034] Cow dung compost samples at the mature stage were collected from Fujian Zhiqing Ecological Environmental Protection Co., Ltd. in Fuzhou City, Fujian Province, and a specific bacteriophage fzNSh6-m5 with high efficiency in lysing drug-resistant Klebsiella pneumoniae was screened out by the double-layer plate method.

[0035] 1.1 Prepare the required culture medium:

[0036] TSB liquid medium: tryptone 17g / L, soy peptone 3g / L, glucose 2.5g / L, NaCl 6.5%, K 2 HPO 4 0.25%, deionized water 1000 mL, pH 7.3, autoclave at 115 °C for 30 min.

[0037] TSB semi-solid medium: tryptone 17g / L, soy peptone 3g / L, glucose 2.5g / L, NaCl 6.5%, K 2 HPO 4 0.25%, agar powder 10 g / L, deionized water 1000 mL, pH 7.3, autoclave at 115 °C for 30 min.

[0038] TSB solid medium: tryptone 17g / L, soy peptone 3g / L, glucose 2.5g / L, NaCl 6.5%, K 2 HPO 4 0.25%, agar powder 25 g / L, deionized water 1000 mL, pH 7.3, autoclave at 115 °C for 30 min.

[0039] MacConkey solid medium (MAC selection medium): peptone 20 g / L, porcine bile salt (or bovine or sheep bile salt) 5 g / L, sodium chloride 5 g / L, agar 14 g / L, lactose 10 g / L, 1% crystal violet, 0.3% neutral red, pH 7.1±0.2, autoclave at 115°C for 30 min.

[0040] MHB culture medium: beef extract powder 5 g / L, starch 1.5 g / L, casein hydrolysate 17.5 g / L, pH 7.2±0.2.

[0041] SM buffer: MgSO 4 7H 2 O 2.0g, NaCl 5.8g, 1mol / L Tris-HCl (pH7.5) 50mL, 2% gelatin 5mL, add deionized water to 1000mL, autoclave at 121℃ for 20min after aliquoting, and store at 4℃. SM buffer is used for dilution and storage of bacteriophages.

[0042] 1.2 Isolation and preparation of Klebsiella pneumoniae

[0043] Isolation and identification of Klebsiella pneumoniae fzNSh6:

[0044] Take 1g of the compost sample at maturity and put it into a sterilized 50mL conical flask containing 9mL of deionized water (the ratio of compost to deionized water is 1:9g / mL). Place the conical flask in a shaker and culture at 37°C and 180rpm for 2h to obtain a compost suspension. Take the suspension for 10-fold gradient dilution. The specific steps are as follows: take 100μL of the suspension, add it to a centrifuge tube containing 900μL of sterile water, vortex, and obtain a mixed suspension; take another 100μL of the mixed suspension, add it to a centrifuge tube containing 900μL of sterile water, repeat this step for gradient dilution, and obtain a dilution gradient of 10. -2 , 10 -3 , 10 -4 The dilution gradient is 10 -2 , 10 -3 , 10 -4100 μL of each suspension was applied to the Klebsiella pneumoniae selective medium (MAC selective medium), with 3 replicates for each gradient, and cultured in a constant temperature incubator at 37°C for 24 hours. Pick a single colony and streak it on the MAC selective medium, and culture it in a constant temperature incubator at 37°C for 24 hours. Repeat two to three times to obtain a purified single bacterium. Pick a single colony and inoculate it into TSB liquid medium, and culture it in a shaker at 37°C and 180rpm for 12 to 16 hours to obtain a Klebsiella pneumoniae liquid in the logarithmic growth phase (OD 600 =1.2). Take 600 μL of Klebsiella pneumoniae bacterial solution in the logarithmic growth phase and put it into a 2mL centrifuge tube, mix it with 600 μL of 30% glycerol, and store it in a -80°C refrigerator. Pick the above single colony and culture it in TSB liquid medium, send it to the company for sequencing after bacterial solution PCR (primer sequence 27F: TACGGYTACCTTGTTACGACTT, 1492R: TACGGYTACCTTGTTACGACTT), and compare the egl sequence with the NCBI database. The reference sequence with the highest score belongs to Klebsiella pneumoniae, that is, Klebsiella pneumoniae, and is named Klebsiella pneumoniae fzNSh6.

[0045] Drug resistance test of Klebsiella pneumoniae fzNSh6:

[0046] The KB paper diffusion method was used to conduct a drug sensitivity test on Klebsiella pneumoniae fzNSh6. 200 μL of bacterial solution was dripped onto the MHA plate and evenly coated. After drying, drug sensitivity paper was pasted on the partitions and cultured at 37°C for 16-18 hours. The size of the inhibition zone was measured and the results were determined according to the 2020 version of the CLSI drug sensitivity standard. The experimental results are shown in Table 1. Klebsiella pneumoniae fzNSh6 is resistant to almost all commonly used veterinary drugs and is a multidrug-resistant bacterium.

[0047] Table 1. Antimicrobial resistance of Klebsiella pneumoniae

[0048]

[0049]

[0050] Note: R indicates tolerance to the drug.

[0051] Preparation of Klebsiella pneumoniae fzNSh6 bacterial liquid in the logarithmic growth phase: streak the resistant Klebsiella pneumoniae fzNSh6 bacterial liquid stored in a -80°C ultra-low temperature refrigerator on MAC selective medium for activation, and culture it in a constant temperature incubator at 37°C for 24 hours; pick a single colony and transfer it to 50mL TSB liquid culture medium, shake and culture it in a shaker at 37°C and 180r / min for 12 to 16 hours to obtain the fzNSh6 bacterial liquid in the logarithmic growth phase for use.

[0052] 1.3 Isolation and purification of sulfonamide-resistant Klebsiella pneumoniae obligate phage

[0053] Weigh 10g of the collected compost sample in the mature stage into a sterilized conical flask, add 90mL of sterile water to mix, place in a shaker at 37℃ and 180rpm for 12h, take it out and let it stand for 2h to obtain a compost suspension; transfer the compost suspension to a sterilized 2mL centrifuge tube, centrifuge at 12000r / min for 5min, take the supernatant, and filter it through a 0.22μm microporous filter membrane. The filtrate is the phage stock solution. Take 10mL of fzNSh6 bacterial solution in the logarithmic growth period, mix it with 150mL of TSB semi-solid culture medium cooled to room temperature, and immediately pour it onto a solidified TSB solid culture medium plate to make a double-layer plate (the thickness of the TSB solid culture medium plate is about 1 / 3 of the double-layer plate). After the upper culture medium solidifies, divide the plate into sections and spot-dip 20μL of gradient dilutions (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 ) of the phage stock solution, invert and culture at 37℃ for 16-18h, and observe whether there are plaques. Figure 1 As shown, diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 Plaques can be observed. When plaques appear, pick the largest single plaque and inoculate it into the fzNSh6 bacterial solution in the logarithmic growth phase, shake it and let it stand for 30 minutes, then put it in a shaker, shake and culture it at 37°C and 180rpm for 12 hours to proliferate the phage, centrifuge the co-culture solution at 12000r / min for 5 minutes, take the supernatant, and filter it through a 0.22μm filter membrane, and operate the filtrate through the double-layer plate method. Repeat 3 to 5 times to obtain a relatively pure phage suspension. This phage is named phage fzNSh6-m5 and stored in a 4°C refrigerator for later use.

[0054] Pick the above-purified plaques and add them to 100mL of Klebsiella pneumoniae fzNSh6 bacterial solution in the logarithmic growth phase. Add DNase1 and RNaseA to the mixed solution to a final concentration of 1 μg / mL. Shake and culture in a shaker at 37°C and 180 r / min for 30 min. Take it out, add NaCl (5.84 g / 100 mL), shake until completely dissolved, ice bath for 1 h, and then centrifuge at 12000 r / min for 5 min to remove bacteria and impurities. Collect the supernatant, add solid PEG8000 to a final concentration of 10% (w / v), gently knead and shake until completely dissolved, continue ice bath for 1 h, so that the phage particles form a precipitate, centrifuge at 4°C and 12000 r / min for 10 min, discard the supernatant, add 2mL SM buffer per 100mL fzNSh6 bacterial solution to suspend the precipitate, and obtain crude phage particles for standby use. Take 20 μL of crude phage particles, dissolve them with sterile water and drop them on the copper mesh. Let it stand for natural precipitation for 15 minutes. Use filter paper to absorb the excess liquid on the side, then drop a drop of 2% phosphotungstic acid on the copper mesh to stain the phage particles. Let it stand for 10 minutes, use filter paper to absorb the excess staining liquid on the side, and let it stand for about 5 minutes to dry the sample. Observe the morphology of the phage under an electron microscope, such as Figure 2 As shown, the head of bacteriophage fzNSh6-m5 is an icosahedral structure with a short tail, belonging to the Brachyura phage family.

[0055] The phage genomic DNA was extracted using a λ phage DNA extraction kit (Abigen, China) according to the operating procedures. The purity and concentration of the genomic DNA were detected using a NanoDrop 2000 ultra-micro spectrophotometer (NanoDrop, USA) and sent to Shanghai Lingen Biotechnology Co., Ltd. for sequencing.

[0056] Bacteriophage fzNSh6-m5, classified as Klebsiella phage, was deposited on November 11, 2024 at the Guangdong Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong, China, with the deposit number: GDMCC NO: 65463-B1.

[0057] Example 2

[0058] 2.1 Determination of optimal infection multiplicity of phage

[0059] The multiplicity of infection (MOI) refers to the ratio of the number of phages to the number of host bacteria before the phages adsorb and infect the host bacteria. The optimal multiplicity of infection is the MOI that can produce the highest yield of progeny phages in the end.

[0060] Take the Klebsiella pneumoniae fzNSh6 culture medium that has been cultured to the logarithmic growth phase and measure the OD value of the culture medium at this time. 600 The value was recorded, the bacterial solution was added to a 48-well plate in sequence, and then the phage suspension purified in Example 1 was added according to MOI of 0.001, 0.01, 0.1, 1, 10, and 100, the mixed solution was shaken and mixed, and placed in a shaking incubator at 37°C and 180 r / min for 12 h, the co-culture suspension in the 48-well plate was extracted into a sterilized 2 mL centrifuge tube, centrifuged at 13000 r / min for 5 min, the supernatant was taken, and then filtered with a 0.22 μm filter membrane to obtain a phage suspension, and the phage suspension was titrated and recorded by a double-layer plate method to obtain the optimal infection multiplicity. As can be seen from Table 2, the optimal infection multiplicity of phage fzNSh6-m5 is 0.1.

[0061] Table 2. Results of the optimal infection multiplicity of phage

[0062]

[0063] 2.2 Phage one-step growth curve determination

[0064] Preparation of phage preparation: According to the optimal infection multiplicity, the phage suspension purified in Example 1 was inoculated into the bacterial suspension of Klebsiella pneumoniae fzNSh6 in the logarithmic growth phase (OD 600 =1.2), mix well, place in a shaker at 37°C, 180r / min for 5h to allow phage proliferation, centrifuge the co-culture suspension at 12000r / min for 5min, take the supernatant, and filter it through a 0.22μm filter membrane to obtain a phage suspension. Use sterile water to adjust the phage titer to 10 8 PFU / mL.

[0065] The Klebsiella pneumoniae fzNSh6 bacterial culture (OD 600 =1.2) were added to a 2 mL sterilized centrifuge tube in sequence, and the phage preparation was added according to the optimal infection multiplicity, and the mixture was incubated in a 37°C water bath for 30 min. The mixture was taken out and centrifuged at 13000 r / min for 1 min. The supernatant was discarded and washed 3 to 5 times with TSB liquid culture medium. The precipitate was resuspended with TSB liquid culture medium preheated at 37°C and placed in a shaking incubator at 37°C and 180 r / min for shaking culture. Starting from 0 min, 100 μL was taken out every 15 min, and the titer was determined by the double-layer plate method after continuous 10-fold dilution with sterile water. The one-step growth curve of the phage was plotted with the logarithm of the phage titer as the ordinate and the culture time as the abscissa, as shown in FIG. Figure 3 As shown, the incubation period of bacteriophage fzNSh6-m5 was 30 min, the burst period lasted about 15 min, and the lysis amount was 1.46×10 7(PFU / mL) / min.

[0066] 2.3 Phage thermal stability assay

[0067] Take 150mL of Klebsiella pneumoniae fzNSh6 cultured to the logarithmic growth phase and add it to a sterile 48-well plate, 700μL per well, and measure the OD at this time. 600 value, and change the OD 600 Value named as pre-OD 600 value (0H), set aside. Take 8 phage preparations (prepared according to "Phage preparation preparation" in "2.2 Phage one-step growth curve determination"), 2 mL each, and place them in a water bath at 0℃, 20℃, 30℃, 37℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 2 hours; according to the optimal multiplicity of infection, add the treated phage preparations to a 48-well plate and mark them. There are three parallel replicates under each temperature treatment, with no phage preparation as a control. Place in a shaker at 37℃, 180r / min for 8 hours, and measure OD with an enzyme marker. 600 Value, recorded as the late OD 600 Value. Figure 4 It can be seen that the phage fzNSh6-m5 has strong activity at 0-40℃. At 30-40℃, the activity of the phage fzNSh6-m5 is the highest and the lysis ability is the strongest. When the treatment temperature reaches 50℃, the OD 600 The value was basically the same as that of the control, indicating that the phage activity was lost when the temperature was above 50℃.

[0068] 2.5 Phage acid-base sensitivity assay

[0069] Take 150mL of Klebsiella pneumoniae fzNSh6 cultured to the logarithmic growth phase and add it to a sterile 48-well plate in sequence, 700μL per well, mark them respectively and measure OD 600 value, and change the OD 600 Value named as pre-OD 600 Value (0H), set aside. Add the phage preparation (prepared according to "Phage preparation preparation" in "2.2 Phage one-step growth curve determination") to sterilized TSB liquid culture medium (pH adjusted with hydrochloric acid and sodium hydroxide) with pH 3, 4, 5, 6, 7, 8, 9, 10, respectively, and place in a 37°C water bath for 2h; according to the optimal multiplicity of infection, add the treated phage preparation to a 48-well plate and mark it. Use the control treatment without adding phage preparation. There are 3 parallel replicates for each pH treatment. Place in a shaker at 37°C and 180r / min for 8h, and use an enzyme marker to measure the OD at this time. 600 Value, recorded as the late OD 600 Value (8H). Figure 5 It can be seen that in the pH range of 5 to 10, bacteriophage fzNSh6-m5 has a strong inhibitory ability on the growth of pathogen fzNSh6, and its antibacterial activity is highest and most suitable for growth in an environment of pH 9.

[0070] Example 3

[0071] Indoor antibacterial test

[0072] Take 50 mL of Klebsiella pneumoniae fzNSh6 culture medium that has been cultured to the logarithmic growth phase and adjust the OD with physiological saline. 600 to 0.5, and added to a sterile 96-well plate in sequence, 190 μL per well, and added phage preparations (prepared in "Phage preparation preparation" in "2.2 Phage one-step growth curve determination" in Example 2) in sequence according to the optimal infection multiplicity of phage, with no phage preparation added as the control (CK), and 3 parallel replicates were set under each treatment. Place in a shaking incubator at 37°C and 180 r / min for 24 h, and measure OD with an ELISA reader every 0.5 h 600 The value is recorded. The antibacterial effect of phage is characterized according to the growth curve of Klebsiella pneumoniae. Figure 6 It can be seen that after adding bacteriophage fzNSh6-m5, the OD of Klebsiella pneumoniae 600 The value continued to decrease in the first 5 hours, then increased and stabilized after 12 hours, while the OD of Klebsiella pneumoniae without the addition of phage 600 It has been showing an upward trend until it stabilized. Compared with CK, bacteriophage fzNSh6-m5 has a better inhibitory effect on Klebsiella pneumoniae, and the 5h inhibition rate reaches 82%.

[0073] The results of the indoor antibacterial test show that the bacteriophage fzNSh6-m5 of the present invention can be used to lyse drug-resistant Klebsiella pneumoniae in the mature stage of compost and eliminate antibiotic resistance genes.

Claims

1. A bacteriophage fzNSh6-m5, classified and named Klebsiella phage, was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 11, 2024, with the deposit number: GDMCC NO: 65463-B1.

2. Use of the bacteriophage fzNSh6-m5 according to claim 1 in lysing drug-resistant Klebsiella pneumoniae, in lysing drug-resistant Klebsiella pneumoniae in composting, or in preparing a bacteriophage preparation for lysing drug-resistant Klebsiella pneumoniae.

3. A bacteriophage preparation for lysing drug-resistant Klebsiella pneumoniae, characterized in that: The phage preparation comprises the phage fzNSh6-m5 described in claim 1.

4. The bacteriophage preparation according to claim 3, characterized in that: The titer of bacteriophage fzNSh6-m5 in the phage preparation is ≥10 8 PFU / mL.

5. The bacteriophage preparation according to claim 3 or 4, characterized in that: The titer of bacteriophage fzNSh6-m5 in the phage preparation was 10 8 ~10 10 PFU / mL.

6. A method for preparing the bacteriophage preparation according to claim 3, characterized in that: include: According to the optimal multiplicity of infection, the phage suspension was inoculated into the Klebsiella pneumoniae liquid in the logarithmic growth phase, and co-cultured at 30-40°C and 170-180 r / min with shaking for 5-8 h. The co-culture liquid was centrifuged, and the supernatant was taken and filtered through a 0.22 μm filter membrane to obtain a phage suspension. The phage titer was adjusted with sterile water to obtain a phage preparation.

7. The method for preparing a bacteriophage preparation according to claim 6, characterized in that: The optimal multiplicity of infection is 0.1-1.

8. The method for preparing a bacteriophage preparation according to claim 6 or 7, characterized in that: The optimal multiplicity of infection is 0.

1.

9. A method for reducing drug-resistant Klebsiella pneumoniae in compost, characterized in that: include: The phage preparation according to claim 3 is inoculated into the compost by spraying, and the amount of phage added is ≥ 2×10 8 PFU / mL compost.

10. The method for eliminating drug-resistant Klebsiella pneumoniae in compost according to claim 9, characterized in that: The amount of phage added was 2×10 8 PFU / mL compost.

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