K5 capsular serotype klebsiella pneumoniae phage resistant to strong acid, pepsin and trypsin and application of K5 capsular serotype klebsiella pneumoniae phage
By screening for the Klebsiella pneumoniaeφWHPK5 phage, which is resistant to strong acids, pepsin, and trypsin, the problem of insufficient tolerance of existing phages in the gastrointestinal environment has been solved, achieving efficient elimination of intestinal colonizing pathogens and balance of intestinal flora.
Patent Information
- Application Number
- CN202511561034.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-02-10
AI Technical Summary
The existing K5 capsular serotype Klebsiella pneumoniae phage has insufficient tolerance to gastrointestinal digestion conditions, which limits its application in intestinal colonization and elimination, especially due to insufficient tolerance to strong acids, pepsin, and trypsin.
A Klebsiella pneumoniaeφWHPK5 phage was screened and obtained by enriching it from urban sewage and screening it under pepsin and trypsin conditions. The phage was resistant to strong acid, pepsin and trypsin, and is suitable for oral administration and digestive system delivery.
It achieves high activity of bacteriophages in the gastrointestinal environment, effectively inhibits the growth of K5 capsular serotype Klebsiella pneumoniae, maintains intestinal flora balance, is suitable for oral formulations and feed additives, and provides an efficient means of eliminating intestinal colonizing pathogens.
Smart Images
Figure CN121495879A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a K5 capsular serotype Klebsiella pneumoniae phage resistant to strong acids, pepsin, and trypsin, and its applications. Background Technology
[0002] Highly virulent Klebsiella pneumoniae ( hypervirulent Klebsiella pneumoniae HvKp is a relatively recently discovered branch of Klebsiella pneumoniae, with significantly higher pathogenicity than the classic Klebsiella pneumoniae. HvKp highly expresses its capsule and siderophores. The capsule effectively resists phagocytosis by phagocytes and the killing effect of immune substances, while siderophores promote the uptake and storage of iron ions, enabling the bacteria to survive and reproduce strongly within the host. Currently, HvKp has spread globally. Studies have shown that HvKp has a strong ability to adhere to and colonize the intestines, especially in the elderly, immunocompromised individuals, and those on long-term antibiotic use, where intestinal colonization rates are significantly higher. In hospitalized patients, the intestinal colonization rate can be as high as 77%. Typically, HvKp first colonizes the intestines and then acts as a "reservoir" to spread to other parts of the body via the bloodstream, causing serious infections. In recent years, there have been increasing reports of multidrug-resistant (MDR) HvKp infections, posing a significant challenge to clinical treatment. Therefore, eliminating intestinal colonized HvKp is considered one of the important strategies for preventing and controlling such infections.
[0003] Bacteriophages are a class of viruses widely found in nature that can specifically lyse bacteria. They possess unique antibacterial mechanisms and are considered natural "ecological drugs" against drug-resistant bacteria. Compared to antibiotics, bacteriophages have advantages such as high specificity and less likelihood of causing dysbiosis, making them particularly suitable for the colonization and elimination of specific pathogens. However, existing bacteriophage studies targeting Klebsiella pneumoniae mostly focus on broad-spectrum lysins without serotype differentiation, with relatively few reports on bacteriophages targeting the K5 capsular serotype of Klebsiella pneumoniae. K5 serotype strains constitute a certain proportion of hvKp and are associated with higher invasiveness and drug resistance. However, most existing K5 bacteriophages are isolated under in vitro conditions, lacking studies on their tolerance to the complex gastrointestinal environment, particularly insufficient tolerance to strong acids, pepsin, and trypsin digestion conditions. This leads to significantly reduced activity when administered orally, thus limiting their application in intestinal colonization and elimination.
[0004] Therefore, there is an urgent need to develop a K5 capsular serotype Klebsiella pneumoniae phage that can tolerate gastrointestinal digestive conditions and can be used to eliminate intestinal colonizing pathogens. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention proposes a K5 capsular serotype Klebsiella pneumoniae phage resistant to strong acids, pepsin, and trypsin, and its applications.
[0006] This invention provides a K5 capsular serotype Klebsiella pneumoniae phage, wherein the K5 capsular serotype Klebsiella pneumoniae phage is... Klebsiella pneumoniae φWHPK5 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66323-B1, on May 13, 2025.
[0007] The bacteriophage can effectively inhibit the growth of its host bacteria in the range of infection number from 0.01 to 1.
[0008] The present invention also provides a phage composition comprising the aforementioned K5 capsular serotype Klebsiella pneumoniae phage.
[0009] The present invention also provides a phage preparation, wherein the active ingredient of the phage preparation comprises the K5 capsular serotype Klebsiella pneumoniae phage or the phage composition thereof.
[0010] In some embodiments, the phage formulation is provided in an oral dosage form.
[0011] In some embodiments, the oral dosage form includes tablets, hard capsules, soft capsules, sugar-coated pills, powders, granules, solutions, suspensions, dispersions, syrups, and microgels.
[0012] The present invention also provides a feed, additive or health product for the prevention or treatment of diseases caused by Klebsiella pneumoniae, wherein the active ingredient comprises the K5 capsular serotype Klebsiella pneumoniae phage or the phage composition thereof.
[0013] The present invention also provides a cleaning agent comprising the K5 capsular serotype Klebsiella pneumoniae phage or the phage composition thereof.
[0014] The present invention also provides a disinfectant comprising the K5 capsular serotype Klebsiella pneumoniae phage or the phage composition thereof.
[0015] The present invention also provides the use of the K5 capsular serotype Klebsiella pneumoniae phage in the preparation of medicaments for the prevention or treatment of Klebsiella pneumoniae intestinal colonization.
[0016] The present invention also provides the use of the K5 capsular serotype Klebsiella pneumoniae phage in the preparation of medicaments for the prevention or treatment of Klebsiella pneumoniae infection.
[0017] In summary, compared with the prior art, the present invention achieves the following technical effects: 1. This invention provides a novel Klebsiella pneumoniae bacteriophage. Klebsiella pneumoniaeφWHPK5 exhibits strong acid tolerance by maintaining a high titer at pH=1. Furthermore, the φWHPK5 strain of this invention demonstrates high tolerance to digestive enzymes, resisting the effects of gastric acid and digestive enzymes, making it suitable for applications requiring delivery through the digestive system, such as oral administration and feed additives.
[0018] 2. The φWHPK5 of the present invention has a highly specific lytic effect on K5 capsular serotype Klebsiella pneumoniae, which can effectively eliminate target strains in the intestine, while having little effect on non-target bacteria, thus helping to maintain the balance of intestinal flora. Attached Figure Description
[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a morphological diagram of the plaque of φWHPK5 in Example 1 of the present invention.
[0021] Figure 2 This is an electron microscope image of φWHPK5 in Embodiment 1 of the present invention.
[0022] Figure 3 The results show the tolerance of φWHPK5 at different pH levels in Example 2 of this invention.
[0023] Figure 4 The results of the tolerance of φWHPK5 to pepsin and trypsin in Example 2 of this invention are shown.
[0024] Figure 5 The results of thermal stability tests of φWHPK5 in Example 3 of this invention at 4℃ and 25℃ are shown.
[0025] Figure 6 The results of the thermal stability test of φWHPK5 in Example 3 of this invention at 37°C are shown.
[0026] Figure 7 The results of the thermal stability test of φWHPK5 in Example 3 of this invention at 56℃ are shown.
[0027] Figure 8 This is the experimental result of the host spectrum of φWHPK5 in Example 4 of the present invention (φWHPK5 cannot infect K1 / K2 / K16 / K20 / K54 / K140 capsular serotype Klebsiella pneumoniae).
[0028] Figure 9This is the result of determining the optimal multiplicity of infection for φWHPK5 in Example 5 of the present invention.
[0029] Figure 10 This is the one-step growth curve of phage φWHPK5 in Example 6 of the present invention. Detailed Implementation
[0030] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
[0031] This invention discloses a highly acid-resistant, pepsin- and trypsin-resistant phage, φWHPK5, targeting Klebsiella pneumoniae K5. This phage was obtained through enrichment in urban sewage and screening under pepsin, trypsin, and strong acid conditions. It exhibits excellent acid tolerance and digestive enzyme stability; the phage maintains high titer stability at both room and low temperatures, making it suitable for long-term storage and application; it has a narrow host spectrum, specifically lysing only Klebsiella pneumoniae K5; it has a low optimal multiple of infection, high lysis efficiency, and short latency period, demonstrating excellent biological properties and antibacterial activity. The φWHPK5 of this invention possesses excellent application potential, providing a highly efficient and stable novel biological agent for the treatment of Klebsiella pneumoniae infection.
[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.
[0033] Example 1: Screening and Identification of Bacteriophages 1. Experimental Materials Host bacteria: Klebsiella pneumoniae strain with capsular serotype K5 was used as the host bacteria.
[0034] Simulated gastric juice: Take 4.5 mL of concentrated hydrochloric acid and add it to 500 mL of distilled water. Take 1 mL of hydrochloric acid solution and add it to 9 mL of distilled water. Then add 0.05 g (30000 U / g) of pepsin powder to obtain 10 mL of simulated gastric juice (pH value is 1.0, pepsin concentration is 150 U / mL).
[0035] Sodium bicarbonate solution: Take 0.75 g of sodium bicarbonate powder and add 100 mL of distilled water to obtain a 0.75% sodium bicarbonate solution, which is used to neutralize simulated gastric juice.
[0036] Trypsin digestion solution: Prepare a 0.5% trypsin-EDTA digestion solution.
[0037] Tris-HCl-MgSO4 solutions with pH=10 and pH=13: Prepare 0.1M Tris and 10 mM MgSO4 solutions, and adjust the pH to 10 and 13 with concentrated hydrochloric acid.
[0038] 2. Phage screening Phage screening process: First, phages are enriched from urban sewage using host bacteria. Then, they are digested with pepsin (pH=1) to eliminate phages that are intolerant to pepsin and strong acid conditions, retaining phages that are resistant to both acid and pepsin. Next, they are digested with trypsin to eliminate phages that are intolerant to trypsin. Finally, phages that are resistant to acid, pepsin, and trypsin are screened to obtain phages.
[0039] The specific screening steps are as follows: (1) Enrichment of bacteriophages Urban domestic sewage is filtered through three layers of qualitative filter paper, and then filtered again using a 0.22μm microporous membrane for later use.
[0040] The host bacteria were cultured in LB liquid medium at 37°C for 12 h. 100 mL of the bacterial suspension was mixed with the filtered wastewater, and then 100 mL of twice-concentration LB liquid medium was added. After thorough mixing, the mixture was incubated at 37°C with shaking at 150 r / min for 16 h. 10 mL of the culture was centrifuged at 12000×g for 10 min, and the supernatant was collected. This supernatant was then centrifuged again at 12000×g for 10 min, and filtered through a 0.22 μm microporous membrane to obtain a culture medium (a) containing bacteriophages.
[0041] (2) Pepsin treatment a. Take 0.5 mL of solution (a) and add it to 10 mL of simulated gastric fluid. After 60 min, take 1 mL of the reactant and add 0.1 mL of sodium bicarbonate solution to neutralize it. Then, centrifuge the mixture at 12000×g for 10 min to obtain solution (b).
[0042] b. Take solution (b) and mix it with an equal volume of host bacterial culture. Incubate overnight at 37°C and 150 rpm for 16 h with shaking to enrich pepsin-resistant bacteriophages. After centrifuging the culture at 12000×g for 10 min, take the supernatant and filter it through a 0.22 μm microporous membrane to obtain a culture medium (c) containing bacteriophages.
[0043] c. Take 0.5 mL of solution (c) and add it to 0.5 mL of 0.5% trypsin digestion solution. After digestion for 60 min, perform a 10-fold serial dilution to 10. -9 Take 10 -6 10 -7 10 -8 and 10-9 Mix 100 μL of each dilution gradient liquid with 50 μL of the host bacterial culture in the culture dish, let stand for 5 min, add 15 mL of 0.8% LB agar medium (55-60℃), mix thoroughly, allow to solidify, and incubate at 37℃ for 12 h to observe plaque formation. Select culture dishes with 30-100 plaques, cut off the agar at the plaque location with a blade, transfer to a clean, sterile test tube, chop it with a blade, and add it to 2 mL of pre-cultured highly virulent Klebsiella pneumoniae. Incubate overnight at 37℃ and 150 rpm with shaking for 16 h. Centrifuge the culture at 12000×g for 10 min, collect the supernatant, and filter through a 0.22 μm pore size filter membrane to obtain purified bacteriophages. Repeat this purification process 3-5 times until morphologically consistent plaques are obtained, thus obtaining purified bacteriophages.
[0044] (3) Determination of phage titer Phage titer refers to the number of phages contained per milliliter of liquid, also known as plaque forming unit (PFU). Add 50 μL of the phage stock solution to be tested to a disposable sterile culture dish. Dilute the phage 10-fold to a final volume of 10. -9 Take 10 -6 10 -7 10 -8 and 10 -9 Dilute the phage solution with gradients and prepare phage plaques according to step (2)c above. Select plates with 30 to 100 plaques. The plaques on the plates should be evenly distributed, not confluent, and have clear edges. Repeat the experiment 3 times and take the average value, which is the phage titer (PFU / mL) to be tested.
[0045] (4) Morphological identification of bacteriophage plaques Following the above screening steps, this invention obtained a pepsin-resistant bacteriophage against K5 capsular serotype Klebsiella pneumoniae, named φWHPK5. The highest titer achievable with φWHPK5 is 3 × 10⁻⁶. 10 PFU / mL. After 24 hours of incubation, φWHPK5 plaques (such as...) Figure 1 (As shown) It consists of two transparent layers, an inner and an outer layer. The inner transparent layer is more transparent than the outer transparent layer and has a diameter of about 2 mm. The outer transparent layer has a diameter of about 10 mm.
[0046] (5) Transmission electron microscopy morphological observation of bacteriophages Take 20 μL of purified bacteriophage and place it on a 200-mesh copper grid, then negatively stain it with 2% phosphotungstic acid. Observe the morphology of the bacteriophage using a transmission electron microscope at an accelerating voltage of 80 kV.
[0047] Electron micrographs as shown Figure 2 As shown, φWHPK5 is a tailed bacteriophage with an icosahedral head and a short tail. The head diameter of φWHPK5 is 60 nm, and the tail is about 10 nm long.
[0048] Example 2: Test of phage tolerance to strong acid conditions, pepsin, and trypsin. φWHPK5 was placed at 37°C and digested with simulated gastric juice or trypsin as described in Example 1 for 30 min, 60 min, 90 min, 120 min and 150 min respectively. The phage titer was determined by the plaque formation test to evaluate the phage's resistance to strong acid, pepsin and trypsin.
[0049] Depend on Figure 3 The results showed that the bacteriophage maintained a high titer after 3 hours at pH 1; however, the titer decreased rapidly at pH 10 and pH 13. Therefore, this bacteriophage exhibits good acid stability. Figure 4 The results showed that even after 3 hours of trypsin digestion, the titer of the bacteriophage did not change significantly, while after 3 hours of pepsin digestion, the titer decreased by 0.8 orders of magnitude. These results demonstrate that the bacteriophage of the present invention has strong resistance to acidic conditions and the ability to be digested by trypsin and pepsin.
[0050] Example 3: Thermal stability test of bacteriophages First, the plaque formation assay was used to determine the titer of the phage stock solution. The dilution gradient was repeated on 3 plates for counting, and the average number of plates was used for counting.
[0051] Stability test at 4℃: The phage was placed at 4℃ for 7 consecutive days, and samples were taken at fixed times each day to test the phage titer.
[0052] Stability test at 25℃: The samples were taken at a fixed time each day for 7 consecutive days at 25℃ to test the phage titer.
[0053] Stability test at 37℃: Samples were taken every 12 hours to detect phage titers, and sampling was conducted continuously for 7 days.
[0054] Stability test at 56℃: 56℃ is the complement inactivation temperature. Sampling times were 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, and 24 h, and phage titers were measured.
[0055] Test results are as follows Figures 5-7As shown, the phage maintained good activity and high titers for one week at temperatures of 4℃, 25℃, and 37℃. At 56℃, the phage titer decreased slightly after 3 hours, began to decrease after 6 hours, and decreased significantly after 12 hours. This demonstrates that φWHPK5 exhibits good thermal stability and is suitable for storage at room temperature and under refrigeration.
[0056] Example 4: Determination of the host range of φWHPK5 Since the host range of bacteriophages is an important characteristic, it needs to be determined. φWHPK5 was mixed with host bacteria of strains K1, K2, K20, K54, K140, and K16, respectively, and cultured overnight at 37°C with shaking at 150 rpm for 16 hours. The mixture was then centrifuged at 12000×g for 10 minutes, and the supernatant was collected. A plaque formation test was performed according to step 1 of Example, and the mixture was incubated at 37°C for 12 hours. The presence or absence of plaques was observed to determine whether the obtained bacteriophage could infect other capsular serotypes of Klebsiella pneumoniae.
[0057] Figure 8 The results showed that φWHPK5 could only form plaques on strains with the K5 capsular serotype, but not on strains with the K1, K2, K20, K54, K140, and K16 capsular serotypes. Therefore, this indicates that bacteriophage φWHPK5 has a narrow host spectrum and strong specificity.
[0058] Example 5: Determination of the optimal multiple of infection (MOI) of φWHPK5 MOI is the ratio of infecting phage to the number of susceptible host bacteria. At the optimal MOI, phage lysis of the same number of bacteria produces the highest number of progeny phages.
[0059] The optimal multiplicity of infection (MLI) is determined as follows: The host bacteria are cultured at 37°C to the logarithmic phase. The bacterial suspension is then diluted, and an OD600 value of 0.2 (1 × 10⁻⁶ host bacteria) is measured. 8 Phages were added at concentrations of CFU / mL in the following proportions: 10, 1, 0.1, 0.01, and 0.001. After incubation at 150 rpm for 3 h, the phages were centrifuged at 15000×g for 10 min. 0.1 mL of the supernatant was collected, filtered through a 0.22 μm pore size filter, and continuously diluted 10-fold with LB liquid medium. 0.1 mL of each solution was incubated with 0.1 mL of the host bacterial culture at 37℃ for 15 min. Liquid LB agar medium (55~60℃) was added and evenly spread in a disposable sterile culture dish. The mixture was incubated at 37℃ for 12 h, and the phage titer was measured. The above experiment was repeated three times, and the average value was taken to determine the highest number of phages as the optimal MOI.
[0060] Determining the optimal multiplicity of infection yields the largest number of progeny phages. Results are as follows: Figure 9 As shown, when the MOI of φWHPK5 is 0.1, the number of progeny phages released after the host is infected by the phage is the largest, that is, the optimal multiplicity of infection of φWHPK5 is 0.1.
[0061] Example 6: One-step growth curve of φWHPK5 This embodiment systematically evaluates the replication dynamics and replication efficiency of φWHPK5 through a one-step growth curve experiment.
[0062] The one-step growth curve method is as follows: The host strain was cultured to the logarithmic growth phase, and 0.1 mL of bacterial suspension (concentration 1×10⁻⁶) was taken. 8 The phage (CFU / mL) was mixed with the phage at the optimal multiplicity of infection and added to 5 mL of liquid LB medium. The mixture was incubated at 37°C for 15 min, then centrifuged (4°C, 13000×g, 1 min). The supernatant was discarded, and the mixture was precipitated twice with LB liquid medium. Centrifugation was repeated to remove free, unadsorbed phage. The mixture was then added to 5 mL of preheated 37°C liquid LB medium and incubated at 37°C with shaking at 150 rpm for 120 min. 100 μL samples were taken every 10 min over 1 h to determine the phage titer. This experiment was repeated three times, and the average value was used. An adsorption-growth curve was plotted with infection time on the x-axis and the logarithm of the phage titer (base 10) on the y-axis. The burst size of phage φWHPK5 was defined as the phage titer during the plateau phase divided by the titer during the latent phase, approximately 1950 (n¼3) PFU per infected cell.
[0063] When bacteriophage φWHPK5 was mixed with host bacteria at different MOIs, the maximum titer produced when φWHPK5 infected the host bacteria at an MOI of 0.1 was significantly higher than that of other groups. Figure 9 This indicates that 0.1 is the optimal MOI for phage φWHPK5. A one-step growth curve for phage φWHPK5 was plotted based on the experimental data. The results show that the latency period of phage φWHPK5 is 30 minutes, after which the phage titer gradually increases, reaching a plateau phase at 90 minutes. Figure 10 The burst size of bacteriophage φWHPK5, defined as the plateau phase phage titer divided by the latent phase titer, is approximately 1950 (n¼3) PFU per infected cell.
[0064] All quantitative experiments were repeated three times. Phage titers were presented as logarithms, and data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA combined with Dunnett's multiple comparison test. *P<0.05, **P<0.01, ****P<0.0001.
[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A K5 capsular serotype Klebsiella pneumoniae phage, characterized in that, The K5 capsular serogen Klebsiella pneumoniae phage is Klebsiella pneumoniae φWHPK5 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66323-B1, on May 13, 2025.
2. A bacteriophage composition, characterized in that, Includes the K5 capsular serotype Klebsiella pneumoniae phage as described in claim 1.
3. A phage preparation, characterized in that, The active ingredient of the phage preparation includes the K5 capsular serotype Klebsiella pneumoniae phage as described in claim 1 or the phage composition as described in claim 2.
4. The phage preparation according to claim 3, characterized in that, The phage formulation is provided in an oral dosage form.
5. The phage drug formulation according to claim 4, characterized in that, The oral dosage forms include tablets, hard capsules, soft capsules, sugar-coated pills, powders, granules, solutions, suspensions, dispersions, syrups, and microgels.
6. A feed, additive, or health product for the prevention or treatment of diseases caused by Klebsiella pneumoniae, characterized in that, The active ingredient includes the K5 capsular serotype Klebsiella pneumoniae phage as described in claim 1 or the phage composition as described in claim 2.
7. A cleaning agent, characterized in that, Includes the K5 capsular serotype Klebsiella pneumoniae phage as described in claim 1 or the phage composition as described in claim 2.
8. A disinfectant, characterized in that, Includes the K5 capsular serotype Klebsiella pneumoniae phage as described in claim 1 or the phage composition as described in claim 2.
9. The use of the K5 capsular serotype Klebsiella pneumoniae phage according to claim 1 in the preparation of a medicament for the prevention or treatment of Klebsiella pneumoniae intestinal colonization.
10. The use of the K5 capsular serotype Klebsiella pneumoniae phage according to claim 1 in the preparation of a medicament for the prevention or treatment of Klebsiella pneumoniae infection.