Phage of a strain of klebsiella pneumoniae with high virulence and multiple drug resistance and application thereof

Abstract

The present application relates to the technical field of biological medicine, and provides a bacteriophage for lysing high-toxicity multi-drug resistant Klebsiella pneumoniae and application thereof.The bacteriophage is Klebsiella pneumoniae phage vB_KpnP_XY3, and the preservation number is CCTCC NO: M 2025022.The bacteriophage can lyse high-toxicity multi-drug resistant Klebsiella pneumoniae 32416 carrying blaTEM, blaSHV, phoQ, pmrB drug resistance genes and rmpA, kfuBC, ybtA, iro-NB, fim-H, ureA, uge, wabG virulence genes, and has great potential in clinical application, and is an ideal choice for replacing antibiotics to treat multi-drug resistant bacteria.The bacteriophage vB_KpnP_XY3 not only widens host spectrum and lysis capacity, but also has broad application potential in clinical treatment of multi-drug resistant Klebsiella pneumoniae infection and environmental purification.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a phage of a highly virulent, multidrug-resistant Klebsiella pneumoniae strain and its application. Background Technology

[0002] Klebsiella pneumoniae (Kp), also known as pneumonia bacillus, is a Gram-negative facultative anaerobic bacterium with a thick capsule. It forms large, grayish-white, mucus-like colonies on ordinary agar plates, which are easily stretched into strings. It commonly causes urinary tract, gastrointestinal, and respiratory tract infections, as well as biofilm-associated pneumonia in immunocompromised patients. It is a major cause of nosocomial infections, ranking second among clinically isolated bacteria, after Escherichia coli.

[0003] In recent years, due to the irrational use of antimicrobial drugs, the number of antibiotic-resistant strains has gradually increased. Currently, Klebsiella pneumoniae has developed resistance to multiple antibiotics, including amoxicillin / clavulanate, tetracycline, polymyxin B, cefotaxime, kanamycin, and cephalosporin. The emergence of highly virulent and multidrug-resistant Klebsiella pneumoniae has brought many difficulties to clinical medication and seriously threatens patients' lives and health. Finding alternative antibiotic treatments has become an urgent necessity.

[0004] Bacteriophages are a class of viruses that parasitize the cells of microorganisms such as bacteria, fungi, and spirochetes, causing infection. Their infection is highly specific and does not disrupt or dysregulate other bacterial communities. In recent years, the overuse of antibiotics has led to the emergence of superbugs, making phage therapy a potential solution for combating antibiotic-resistant infections. Currently, there is no specific phage for the highly virulent, multidrug-resistant Klebsiella pneumoniae Kpn32416; therefore, developing a phage capable of efficiently lysing Klebsiella pneumoniae Kpn32416 is of great significance. Summary of the Invention

[0005] In view of this, the present invention proposes a bacteriophage with a short incubation period, strong lytic activity, no virulence gene, and the ability to efficiently lyse highly virulent multidrug-resistant Klebsiella pneumoniae Kpn32416, and its application in the prevention and control of highly virulent multidrug-resistant Klebsiella pneumoniae infection and environmental disinfection and purification.

[0006] The technical solution of the present invention is implemented as follows: In the first aspect, the present invention provides a phage strain that lyses highly virulent multidrug-resistant Klebsiella pneumoniae, the phage being Klebsiella pneumoniaephage vB_KpnP_XY3, with accession number CCTCC NO: M2025022.

[0007] Secondly, the present invention provides the application of bacteriophages in the lysis of Klebsiella pneumoniae.

[0008] Based on the above technical solutions, the preferred embodiment is Klebsiella pneumoniae 32416.

[0009] Thirdly, the present invention provides the application of bacteriophages in the preparation of drugs against Klebsiella pneumoniae infection.

[0010] The bacteriophage of a highly virulent, multidrug-resistant Klebsiella pneumoniae strain of the present invention and its application have the following advantages over the prior art:

[0011] (1) The present invention uses clinical isolates obtained from the laboratory as indicator bacteria to isolate a Klebsiella pneumoniae phage with strong lytic activity from untreated sewage in the hospital. The phage has a strong inhibitory effect on highly virulent multidrug resistant Klebsiella pneumoniae Kpn32416, which has significant reference value for the treatment and prevention of clinical multidrug resistant Klebsiella pneumoniae.

[0012] (2) The activity of the Klebsiella pneumoniae phage vB_KpnP_XY3 provided by the present invention can remain stable at temperatures of 4-60℃ and pH of 4-11, and is not sensitive to chloroform.

[0013] (3) The optimal multiplicity of infection for the Klebsiella pneumoniae phage vB_KpnP_XY3 provided by the present invention is 0.1.

[0014] (4) The Klebsiella pneumoniae phage vB_KpnP_XY3 provided by this invention has an incubation period of approximately 20 min, a lysis period of approximately 40 min, and enters a plateau phase after 60 min, with a maximum titer reaching 2.7 × 10⁻⁶. 11 The phage concentration was PFU / mL, with an average lysis rate of 340 PFU / cell. This indicates that the phage can amplify rapidly and exhibits highly efficient lytic activity.

[0015] (5) The Klebsiella pneumoniae phage provided by the present invention can specifically kill highly virulent multidrug-resistant Klebsiella pneumoniae (Klebsiella pneumoniae) 32416, which carries four drug resistance genes (blaTEM, blaSHV, phoQ, pmrB) and eight virulence genes (rmpA, kfuBC, ybtA, iro-NB, fim-H, ureA, uge, wabG).

[0016] (6) The Klebsiella pneumoniae phage provided by the present invention has broad application potential in the clinical treatment of Klebsiella pneumoniae infection and environmental pollution disinfection. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a diagram showing the plaque formation results of Klebsiella pneumoniae phage vB_KpnP_XY3 according to the present invention.

[0019] Figure 2 This is a transmission electron microscope (TEM) image of the Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 of this invention.

[0020] Figure 3 The present invention identifies the host bacterium Klebsiella pneumoniae (Klebsiella pneumoniae) 32416 carrying drug resistance and virulence genes using PCR results. Figure A shows the drug resistance genes blaTEM, blaSHV, phoQ, and pmrB, while Figure B shows the virulence genes rmpA, kfuBC, ybtA, iro-NB, fim-H, ureA, uge, and wabG.

[0021] Figure 4 This is a diagram showing the optimal multiplicity of infection for Klebsiella pneumoniae phage vB_KpnP_XY3 according to the present invention.

[0022] Figure 5 This is a one-step growth curve result of Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 of the present invention.

[0023] Figure 6 The thermal stability curve of Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 is shown in the figure.

[0024] Figure 7 This is a pH stability curve of the Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 of the present invention.

[0025] Figure 8 This is a chloroform stability curve of the Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 of the present invention.

[0026] Figure 9 This is a diagram showing the in vitro antibacterial experiment results of Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 of the present invention.

[0027] Figure 10 This is a graph showing the biofilm inhibition rate of Klebsiella pneumoniae bacteriophage vB_KpnP_XY3 according to the present invention. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] The main reagents and instruments used in this invention are as follows: agar powder and tryptone were purchased from Saiguo Biotechnology Co., Ltd.; 0.22μm filter and bacterial culture dish were purchased from Biosharp. The viral genomic DNA / RNA extraction kit was purchased from Tiangen Biotech Co., Ltd.; the 37℃ constant temperature incubator was purchased from Shanghai Boxun Industrial Co., Ltd.; and the constant temperature shaking incubator was purchased from Shanghai Zhichu Instrument Co., Ltd.

[0030] I. Source of the strain

[0031] The 66 different types of test bacteria were provided by the Department of Laboratory Medicine of Xiangyang First People's Hospital.

[0032] II. Isolation and Purification of Bacteriophages

[0033] 50 mL of wastewater obtained from the First People's Hospital of Xiangyang City was mixed with 50 mL of LB liquid medium, and 200 μL of logarithmic-phase host bacteria was inoculated. The mixture was incubated overnight at 37°C and 200 rpm. The next day, after centrifugation at 4°C and 10,000 rpm for 10 min, the mixture was filtered through a 0.22 μm filter. The filtered liquid was serially diluted and incubated overnight on double-layer agar plates. Single plaques were picked and immersed in LB medium containing host bacteria, then incubated overnight in a shaking incubator at 37°C and 200 rpm. After centrifugation at 4°C and 10,000 rpm for 10 min, the mixture was filtered through a 0.22 μm filter. The phages were purified 3-5 times using the double-layer agar plate method after serial dilution, until the plaques were uniform in size. The phage titer was determined using the double-layer agar plate method.

[0034] After culturing on a double-layer plate for 12 hours, phage 32416 was able to form phage plaques with a clear center and a semi-transparent halo around it, such as... Figure 1 As shown.

[0035] III. Transmission electron microscopy observation of bacteriophages

[0036] The purified high-potency (>10) 9 20 μL of PFU / mL phage solution was dropped onto a copper grid and allowed to adsorb naturally for 5-10 min. After drying, 20 μL of 2% phosphotungstic acid solution was dropped onto the copper grid and allowed to stand for 3-5 min. Excess droplets were absorbed with filter paper strips, and the grid was dried under an incandescent lamp. The results were then observed and photographed using a transmission electron microscope. Figure 2 .

[0037] Figure 2 As shown, bacteriophage 32416 has an icosahedral head and a long, non-retractable tail. According to the International Committee on Taxonomy of Viruses (ICTV) 2015 report, "Taxonomy of Viruses - Eighth Report of the International Committee on Taxonomy of Viruses", bacteriophage 32416 belongs to the long-tailed virus family.

[0038] According to the naming rules for bacteriophages, it is named Klebsiella pneumoniaephage vB_KpnP_XY3, hereinafter referred to as bacteriophage vB_KpnP_XY3.

[0039] Klebsiella pneumoniaephage vB_KpnP_XY3 was deposited on January 6, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO: M 2025022.

[0040] IV. Detection of host spectrum of bacteriophage vB_KpnP_XY3

[0041] Host spectrum lysis experiments were performed on 66 test bacterial strains (26 Klebsiella pneumoniae strains, 15 Acinetobacter baumannii strains, and 25 Staphylococcus aureus strains) using the droplet method. The test bacteria were cultured to approximately 10⁻⁶ in the logarithmic phase. 8 When the concentration of CFU / mL is 100 μL, spread 100 μL of bacterial culture evenly on an LB solid agar plate using an L-shaped spreader. Add 20 μL of purified phage vB_KpnP_XY3 solution to the plate and incubate overnight at 37℃ for 12 h. Observe the plaque formation afterward.

[0042] Table 1 Results of phage vB_KpnP_XY3 lysis spectrum assay

[0043]

[0044]

[0045] Note: "+" can be split, "++" has a strong splitting ability, and "-" cannot be split.

[0046] Table 1 shows that the specific lysing host bacteriophage of bacteriophage vB_KpnP_XY3 is Kpn 32416, namely Klebsiella pneumoniae 32416.

[0047] V. Host bacterium Klebsiella pneumoniae 32416 (abbreviation of Klebsiella pneumoniae 32416) carries drug resistance and virulence genes.

[0048] The bacterial strain was revived and cultured, and bacterial DNA was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit. Primer sequences for drug resistance and virulence genes were synthesized by Shanghai Sangon Biotech Co., Ltd., referring to relevant literature.

[0049] PCR system 20μL: 1μL DNA template, 0.5μL primer R, 0.5μL primer F, 10μL Premix Taq (TaKaRaPremix TaqVersion 2.0), 8μL ddH2O.

[0050] PCR conditions: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing for 30 s, extension at 72℃ for 1 min, 30 cycles; hold at 72℃ for 10 min.

[0051] PCR products were electrophoresed on a 1% agarose gel (120V, 30 min), photographed and stored using a UV imager, and the PCR identification results are shown below. Figure 3 .

[0052] PCR identification revealed that the host bacterium *Klebsiella pneumoniae* Kpn32416 carries the β-lactam resistance genes blaTEM and blaSHV, and the polymyxin resistance genes phoQ and pmrB (see...). Figure 3 A) This bacterium can lead to resistance to carbapenems and colistin antibiotics; it also carries multiple virulence genes, including the capsular polysaccharide synthesis gene rmpA, iron uptake system genes kfuBC, ybtA, and iroNB, fim synthesis genes fimH and ureA, and lipopolysaccharide genes uge and wabG (see [link to relevant documentation]). Figure 3 B).

[0053] VI. Determination of the optimal multiple sensitivity (MOI) of bacteriophage vB_KpnP_XY3

[0054] The host bacterium Kpn 32416 was cultured to the logarithmic growth phase and the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, bacteriophage vB_KpnP_XY3 was added at MOI ratios of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100. After incubation at 37°C for 15 min, the mixture was added to 1 mL of LB liquid medium and cultured at 37°C and 200 rpm for 6 h with shaking. The mixture was then centrifuged at 4°C and 1000 g for 10 min. The supernatant was filtered through a 0.22 μm filter, and the bacteriophage titer was determined using the double-layer agar plate method. The highest titer represented the optimal MOI. The experiment was repeated three times in parallel. The results are shown in […]. Figure 4 .

[0055] like Figure 4As shown, the optimal multiplicity of infection for bacteriophage vB_KpnP_XY3 is 0.1, at which point the titer can reach 3.1 × 10⁻⁶. 10 PFU / mL.

[0056] VII. Determination of one-step growth curve of bacteriophage vB_KpnP_XY3

[0057] The corresponding bacteriophage vB_KpnP_XY3 was mixed with the host bacterium Kpn 32416 at the optimal MOI (0.1), incubated at 37℃ for 15 min, centrifuged at 4℃ and 1000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in 10 mL of LB liquid medium. The mixture was then incubated at 37℃ and 200 rpm with shaking. Samples were taken at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min of incubation, and the phage titer was determined using the double-layer agar plate method. The experiment was repeated three times in parallel. The results are shown in the figure. Figure 5 .

[0058] like Figure 5 As shown, the lysis latency of bacteriophage vB_KpnP_XY3 in lysing the host bacterium Kpn 32416 was 20 min, the lysis period was 40 min, and there was a plateau phase of about 60 min. This indicates that bacteriophage vB_KpnP_XY3 can be amplified in large quantities in a short time and has highly efficient lysis activity.

[0059] 8. Temperature stability test of bacteriophage vB_KpnP_XY3

[0060] The phage fluid was incubated in water baths at different temperatures (4℃, 25℃, 37℃, 50℃, 60℃, and 70℃) for 60 min. After incubation, the fluid was immediately cooled in an ice bath and then serially diluted. The titer was determined using the bilayer plate method. The results are shown in [Figure number missing]. Figure 6 .

[0061] like Figure 6 As shown, bacteriophage vB_KpnP_XY3 is relatively stable in environments with temperatures ranging from 4 to 60°C, with a titer of 10. 9 -10 10 PFU / mL, bacteriophage vB_KpnP_XY3 is inactivated when the temperature reaches 70℃.

[0062] IX. Determination of pH stability of bacteriophage vB_KpnP_XY3

[0063] The pH of LB liquid medium was adjusted to different ranges from 2 to 12. 1 mL of purified phage solution was added to LB medium at different pH ranges and incubated at 37°C for 60 min. The solutions were then serially diluted, and the phage titers were determined. The results are shown in the table below. Figure 7 .

[0064] like Figure 7 As shown, bacteriophage vB_KpnP_XY3 is relatively stable in an environment with pH 4-11, with the highest titer at pH 3, approaching 3 × 10⁻⁶. 9 PFU / mL; bacteriophage vB_KpnP_XY3 was inactivated at pH 3 and 12.

[0065] 10. Chloroform stability test of bacteriophage vB_KpnP_XY3

[0066] Five phage aliquots (500 μL each) were prepared according to chloroform:phage volume ratios of 0.01, 0.05, 0.1, 0.5, and 1. Each aliquot was vigorously mixed with 5 μL, 25 μL, 50 μL, 250 μL, and 500 μL of chloroform, respectively, for 1 min. The mixtures were then incubated at room temperature for 30 min, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. The phage titers were determined using the bilayer agar plate method after serial dilution. Results are shown in the figure. Figure 8 .

[0067] like Figure 8 As shown, vB_KpnP_XY3 is not sensitive to chloroform, and its titer is higher than 10 when the chloroform:phage volume ratio is 0.01, 0.05, 0.1, 0.5, and 1, respectively. 10 PFU / mL.

[0068] XI. In vitro antibacterial detection of bacteriophage vB_KpnP_XY3

[0069] After the host bacterium Kpn 32416 was cultured to the exponential phase, the concentration was adjusted to 10. 8 CFU / mL was used to infect bacteriophages with MOIs of 10, 1, 0.1, 0.01, and 0.001. LB broth was used to infect host bacteriophage Kpn 32416 as a positive control. The cultures were incubated at 37℃ and 200 rpm. The wavelength at 580 nm was measured at 0, 2, 4, 6, 8, 10, and 12 h. Results are shown in the table below. Figure 9 .

[0070] like Figure 9 As shown, bacteriophage vB_KpnP_XY3 has a significant inhibitory effect on the host bacterium Kpn 32416, and the activity of the host bacterium gradually weakens over time.

[0071] XII. Detection of biofilm inhibition rate of bacteriophage vB_KpnP_XY3

[0072] The purified phage titer was adjusted to 10. 9 PFU / mL, host bacterial Kpn 32416 concentration adjusted to 10 8CFU / mL, 180 μL of bacterial suspension and 20 μL of phage suspension were added to 96-well plates at MOI (phage concentration / bacterial concentration) ratios of 1, 0.1, 0.01, and 0.001, respectively, with three replicates for each titer. A mixture of 180 μL of bacterial suspension and 20 μL of SM buffer was used as a negative control, and 200 μL of LB liquid was used as a blank control. 200 μL of PBS was added to the outermost ring of the 96-well plate to prevent evaporation of the inner liquid. The plates were incubated at 37°C for 6, 12, 24, and 48 h, at which point the mixture was discarded. After gently rinsing three times with PBS, 200 μL of 99% formaldehyde was added for fixation for 15 min. Each well was then stained with 200 μL of 1% crystal violet solution for 5 min. After rinsing with running water, 160 μL of 33% glacial acetic acid was added to each well to dissolve the phage. The absorbance at 570 nm was measured using a microplate reader. Biofilm inhibition rate = (OD570 of negative control - OD570 at each MOI) ÷ OD570 of negative control × 100%, results are shown in [link to results]. Figure 10 .

[0073] like Figure 10 As shown, bacteriophage vB_KpnP_XY3 effectively inhibited the growth of host bacterial biofilms, with an inhibition rate approaching 90% after 48 hours. This indicates that bacteriophage vB_KpnP_XY3 possesses persistent antibacterial activity and will play an important role in the future prevention and control of Klebsiella pneumoniae.

[0074] 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 bacteriophage of a highly virulent, multidrug-resistant Klebsiella pneumoniae strain, characterized in that: The bacteriophage is Klebsiella pneumoniae bacteriophage (Klebsiella pneumoniae bacteriophage). Klebsiella pneumoniae phage vB_KpnP_XY3, with accession number CCTCC NO: M2025022, the described Klebsiella pneumoniae is Klebsiella pneumoniae ( Klebsiella pneumoniae )32416.

2. The application of the bacteriophage as described in claim 1 in the lysis of Klebsiella pneumoniae, characterized in that: The Klebsiella pneumoniae mentioned is Klebsiella pneumoniae ( Klebsiella pneumoniae )32416, the application described is not a method for diagnosing or treating diseases.

3. The use of the bacteriophage as described in claim 1 in the preparation of drugs against Klebsiella pneumoniae infection, characterized in that: The Klebsiella pneumoniae mentioned is Klebsiella pneumoniae ( Klebsiella pneumoniae )32416.

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