CN115029322B - Klebsiella pneumoniae bacteriophage with multi-drug resistance and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and discloses a multi-drug resistant Klebsiella pneumoniae bacteriophage and application thereof. The phage is named Kp_phase_507 and is preserved in China Center for Type Culture Collection (CCTCC) of university of Chinese Wuhan, and the preservation number is CCTCC M20211633. The Kp_phase_507 of the Klebsiella pneumoniae bacteriophage provided by the invention has the advantages of short incubation period, large burst quantity, wide tolerance range to temperature and acid and alkali, wide host spectrum, good hydrophilicity and easiness in preparation of injection, and can rapidly kill host bacteria in a culture medium. Animal experiments prove that the phage has small toxic and side effects and high safety, and has good treatment effect on multi-drug resistant klebsiella pneumoniae infection.

Description

A multidrug-resistant Klebsiella pneumoniae phage and its application

Technical field

The invention relates to the field of biotechnology, and in particular to a multidrug-resistant Klebsiella pneumoniae phage and its application.

Background technique

Klebsiella pneumoniae (KP) is a conditionally pathogenic Gram-negative enterobacteriaceae that is often found in the gastrointestinal tract, skin and nasopharynx of the human body. When the body's immunity is low, it can cause Pneumonia, liver abscess, urinary tract infection, wound infection, sepsis, etc. Klebsiella pneumoniae has become an important pathogen in infections.

Cephalosporins, fluoroquinolones, and trimethoprim-sulfamethoxazole are commonly used drugs against Klebsiella pneumoniae infections, but with the widespread use of a large number of antibiotics, Klebsiella pneumoniae has become resistant to these antibiotics The phenomenon is very common, and a large number of multidrug-resistant strains have emerged. Carbapenem antibiotics are considered the last line of defense in the treatment of multidrug-resistant K. pneumoniae infections, but carbapenem-resistant K. pneumoniae was first reported in 2001. A large number of epidemiological surveys have shown that carbapenem-resistant Klebsiella pneumoniae has become a worldwide epidemic, posing great challenges to clinical treatment. Drug resistance in Klebsiella pneumoniae has posed a major threat to public health.

Bacteriophage is a virus that can specifically infect bacteria. It is a natural antibacterial agent that can produce proteases that degrade polysaccharides on the bacterial surface. The potential therapeutic effect of bacteriophages on bacterial infections was first recognized and applied in 1919. However, due to researchers' lack of understanding of the characteristics of phages, there were many problems with early phage therapy. In addition, the rapid development of antibiotics after the 1940s led to phage therapy being gradually ignored. After 1980, drug-resistant bacteria continued to emerge, posing great challenges to traditional antibiotic treatment, and the development of new antibiotics also slowed down significantly. Therefore, phage therapy has once again entered the attention of researchers. Phage therapy is a method of treating diseases caused by host bacteria by using lytic phages to enter and multiply within the host bacteria and then kill the host bacteria. Phage therapy may become a weapon against "superbugs".

However, because the host specificity of bacteriophages makes their antibacterial spectrum too narrow, individualized treatment must be made for each case rather than a single treatment. Therefore, constantly isolating new phages and analyzing their basic biological characteristics and antibacterial potential are the first prerequisites for the development of phage biological agents. Only in this way can we adapt to the diversity of bacterial biological species and new species produced by continuous mutations.

Contents of the invention

In view of the above problems, the present invention provides a multidrug-resistant Klebsiella pneumoniae phage and its application. The phage Kp_phage_507 has a short incubation period, a large burst volume, can quickly kill host bacteria in the culture medium, has a wide tolerance range to temperature and acid and alkali, has a wide host spectrum, has good hydrophilicity, and can be easily made into injections. It can provide new technical means for the treatment of multidrug-resistant Klebsiella pneumoniae infections.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The invention provides a multidrug-resistant Klebsiella pneumoniae phage, which is named Kp_phage_507 and is deposited in the China Type Culture Collection Center with the deposit number CCTCC M 20211633.

Further, the phage is a long-tailed phage.

Furthermore, the bacteriophage can withstand high temperature of 70°C; the optimal pH of the bacteriophage is 4-11; the incubation period of the growth of the bacteriophage is 0-20min, the outbreak period is 20-100min, and it enters the plateau phase after 100min.

Furthermore, the phage has a broad lysis spectrum and can lyse 23 of 27 clinically isolated multidrug-resistant Klebsiella pneumoniae strains.

Furthermore, the strain numbers of the 23 multidrug-resistant Klebsiella pneumoniae strains that the phage can lyse are Kpn-ESBL-u1, 507, 346, 676, 491, Kpn-87, Kpn-90, and Kpn-93. , Kpn-96, Kpn-106, Kpn-110, K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, ATCC BAA-2146, ATCC BAA-1705.

Furthermore, the bacteriophage can effectively inhibit the growth of its host bacteria within the range of infection number 1 to 0.001.

The present invention provides a phage preparation comprising the above phage.

Further, the preparation is a medicine, cleaning agent or disinfectant.

The present invention also provides an application of the above-mentioned phage for preparing a preparation for preventing or treating multidrug-resistant Klebsiella pneumoniae infection.

Compared with the prior art, the present invention has the following advantages:

The invention provides a new strain of Klebsiella pneumoniae phage Kp_phage_507, which has a short incubation period, a large burst volume, can quickly kill host bacteria in the culture medium, has a wide tolerance range to temperature and acid and alkali, and has a wide host spectrum. , has good hydrophilicity and is easy to make into injections. Animal experiments have confirmed that the phage has few side effects, is highly safe, and has a good therapeutic effect on multidrug-resistant Klebsiella pneumoniae infections.

Description of the drawings

Figure 1 is a plaque picture of the Klebsiella pneumoniae phage Kp_phage_507 of the present invention.

Figure 2 is a transmission electron microscope image of the Klebsiella pneumoniae phage Kp_phage_507 of the present invention.

Figure 3 is a one-step growth curve of the Klebsiella pneumoniae phage Kp_phage_507 of the present invention.

Figure 4 is a schematic diagram of the effect of temperature on the activity of Klebsiella pneumoniae phage Kp_phage_507 of the present invention.

Figure 5 is a schematic diagram of the effect of pH on the activity of Klebsiella pneumoniae phage Kp_phage_507 of the present invention.

Figure 6 is a schematic diagram of sterilization of Klebsiella pneumoniae phage Kp_phage_507 culture medium of the present invention.

Figure 7 is a schematic diagram of the therapeutic effect of Klebsiella pneumoniae phage Kp_phage_507 of the present invention on Galleria mellonella.

Detailed ways

The technical solution of the present invention will be described in detail below with reference to the embodiments of the present invention and the accompanying drawings. It should be pointed out that for those of ordinary skill in the art, several modifications and improvements can be made without departing from the principle of the present invention, and these should also be regarded as belonging to the protection scope of the present invention. The methods and techniques used are conventional methods and techniques unless otherwise specified.

Bacterial strains, reagents and culture media involved in the examples:

The host bacteria used in the experiment were all multidrug-resistant Klebsiella pneumoniae clinical strain 507.

Brain heart infusion (BHI) liquid culture medium: Weigh 3.6g BHI in 100mL distilled water, autoclave at 121°C for 20 minutes.

Brain heart infusion (BHI) semi-solid culture medium: Weigh 3.6g BHI and 0.75g agar in 100mL distilled water, sterilize by autoclaving at 121°C for 20 minutes.

Brain heart infusion (BHI) solid culture medium: Weigh 3.6g BHI and 1.5g agar in 100mL distilled water, sterilize by high pressure at 121℃ for 20min, then cool to 50℃, pour the plate, cool and solidify, invert and set aside.

Vinegar agar solid culture medium: Weigh 1.5g agar in 100mL distilled water, sterilize by autoclaving at 121°C for 20 minutes, cool to 50°C, pour the plate, cool and solidify, invert and set aside.

SM buffer: Weigh 6.055g Tris and dissolve it in 20mL distilled water. Use concentrated hydrochloric acid to adjust the pH to 7.5 and dilute the volume to 50mL. Then add 5.8g NaCl and 2g MgSO ₄ . Dissolve and dilute to 1L. Autoclave at 121°C for 20 minutes. bacteria.

Example 1

Isolation and purification of Klebsiella pneumoniae phage Kp_phage_507

(1) The multidrug-resistant Klebsiella pneumoniae clinical isolate 507 was used as the host bacterium to isolate and purify the phage. Streak the host bacteria onto the BHI solid medium and incubate it upside down in a 37°C constant-temperature incubator for 12-18 hours. Then pick a single clone and inoculate it into 5 mL BHI liquid medium, and culture it with shaking at 37°C until it reaches the logarithmic phase as the host. Bacterial cultures are available for later use.

(2) Collect untreated sewage from a hospital, filter the sewage with double-layer filter paper, centrifuge at 10,000 rpm for 20 minutes, filter the supernatant with a 0.22 μm filter, and collect the filtrate; co-culture the filtrate with the host bacteria in BHI liquid culture medium overnight , centrifuge to take the supernatant, filter it with a 0.22 μm filter, and collect the filtrate to obtain the phage stock solution.

(3) Take 100 μL of host bacteria in the logarithmic growth phase, inoculate it into a semi-solid BHI medium at 55-60°C and mix thoroughly to prepare a double-layer plate. After solidification, drop the above phage stock solution onto the plate and let it dry naturally. Incubate upside down at 37°C for 12-18 hours, and observe whether there are plaques forming on the spot plate. If plaques form, it indicates the presence of specific phages.

(4) The obtained plaques are added to the host bacteria in the logarithmic growth phase, cultured at 37°C at 160 rpm for 10-12 hours, centrifuged at 10,000 rpm for 5 minutes, and filtered with a 0.22 μm filter to obtain the phage stock solution; use SM solution to dissolve the phage stock solution Continuously dilute 10 times, mix 100 μL of dilutions of 10 ^-2 , 10 ^-4 , 10 ^-6 , and 10 ^-8 with 500 μL of logarithmic phase bacterial solution, incubate at room temperature for 10 minutes, and add to semi-solid BHI medium at about 50°C. Mix thoroughly to prepare a double-layer plate. After solidification, incubate at a constant temperature of 37°C for 12-18 hours. Select the plate with plaques and select the larger and isolated plaques to continue culturing. Repeat this step until phage with uniform plaque size are obtained, and store at 4°C for later use.

The above-mentioned spare phage was tested using the double-layer plate method. The results are shown in Figure 1. The phage can form a translucent plaque in the agar medium with no halo around it and a clear and regular edge. It is a typical lytic phage.

Example 2

Enrichment of Klebsiella pneumoniae phage Kp_phage_507

Co-culture the phage prepared in Example 1 and the host bacteria in the logarithmic growth phase at 37°C and 160 rpm until the bacterial liquid becomes clear, centrifuge at 10,000 rpm for 5 minutes to take the supernatant, and filter with a 0.22 μm filter to obtain a phage lysate.

PEG8000 purified phage: Add DnaseI and RNaseA to the phage lysate to a final concentration of 1 μg/mL. After incubation at 37°C for 3 hours, add solid NaCl to a final concentration of 1 mol/L, keep in ice bath for 1 hour, and centrifuge at 4°C and 10,000g for 15 min. , take the supernatant, add solid PEG8000 to a final concentration of 10%, keep in ice bath for 3 hours, centrifuge at 10000g for 15 minutes at 4°C, remove the supernatant, gently resuspend the phage pellet in SM liquid, and extract by adding an equal volume of chloroform. Extract the PEG and cell debris from the phage suspension, shake gently for 30 seconds, and centrifuge at 4°C and 3000g for 15 minutes to separate the organic phase and hydrophilic phase, recover the hydrophilic phase containing phage particles, and obtain a high-concentration enriched phage suspension.

Example 3

Transmission electron microscopy observation of Klebsiella pneumoniae phage Kp_phage_507

Use the high-concentration phage suspension obtained in Example 2 for electron microscopy observation. Take 1 drop of the phage suspension and drop it on the copper mesh. After 5 minutes, use filter paper to absorb the excess phage suspension from the edge of the copper mesh. Use 2% phosphotungstic acid solution to The phage was stained for 10 minutes, dried and then placed under an electron microscope to observe the phage morphology; the observation results are shown in Figure 2. The phage Kp_phage_507 is a long-tailed phage.

Example 4

Determination of titer of Klebsiella pneumoniae phage Kp_phage_507

Carry out 10-fold gradient dilution of the purified phage stock solution in Example 1, take 100 μl of the 10 ^-6 -10 ^-11 times diluted phage dilution and 500 μl of the host bacteria in the logarithmic phase, mix thoroughly, incubate at room temperature for 10 minutes, and prepare a double-layer plate , incubate at 37°C for 12-18h, and count plaques on each plate. Repeat three times and calculate the phage titer. Phage titer (PFU/mL) = dilution factor × average number/sample volume. The titer of the phage was 3.33×10 ¹¹ PFU/mL.

Example 5

Determination of the optimal multiplicity of infection of Klebsiella pneumoniae phage Kp_phage_507

Count the host bacteria in the logarithmic growth phase. Mix the phage and the host bacteria in the logarithmic phase at a ratio of 10, 1, 0.1, 0.01 and 0.001. After letting it stand at room temperature for 15 minutes, mix it with BHI liquid culture medium and incubate at 37°C at 160rpm. After culturing for 5 hours, centrifuge at 12,000 g for 3 minutes, take the supernatant, filter with a 0.22 μm filter, and collect the filtrate; take 100 μl of the filtrate for each MOI value to determine the phage titer. The MOI value with the highest titer is the optimal multiplicity of infection for the phage. The results are shown in Table 1. The optimal multiplicity of infection for phage Kp_phage_507 is 0.001.

Table 1 Optimum multiplicity of infection for Klebsiella pneumoniae phage Kp_phage_507

Example 5

Determination of one-step growth curve of Klebsiella pneumoniae phage Kp_phage_507

Mix the phage suspension purified in Example 1 with the host bacteria at a ratio of MOI value 0.001, incubate at 37°C for 15 minutes, centrifuge at 5000 rpm for 2 minutes, collect the pellet, resuspend the pellet in 5 mL of BHI liquid culture medium, and culture at 37°C and 160 rpm. Samples were taken at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min time points, the phage titer at each time point was measured, and then the growth curve was drawn. One step growth curve.

The results are shown in Figure 3. 0-20 minutes is the incubation period of K. pneumoniae phage Kp_phage_507, 20-100 minutes is the outbreak period, and it enters the stationary period after 100 minutes. The burst volume is 246PFU/cell.

Example 6

Temperature and pH tolerance experiments of Klebsiella pneumoniae phage Kp_phage_507

Take 5 sterile EP tubes, add 0.5 mL of the purified phage suspension in Example 1 to each, incubate in a water bath at 40°C, 50°C, 60°C, 70°C and 80°C for 1 hour respectively, cool to room temperature, and then measure the elapsed time. Titers of phages after treatment at different temperatures. The results are shown in Figure 4. The phage can withstand high temperatures of 70°C and is rapidly inactivated at 80°C.

Take 12 portions of 0.1 mL of the purified phage suspension in Example 1 and add them to 0.9 mL of BHI liquid culture medium with a pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Incubate at room temperature for 1 hour, and then measure the titer of the phage after being exposed to different pH conditions. The results are shown in Figure 5. The activity of this phage changes little in an environment with a pH value of 4-11. When pH<4 or pH>11, the titer of the phage decreases greatly with the increase of acidity and alkalinity. When pH=2, all phages are inactivated. Therefore, the optimal pH of phage Kp_phage_507 is 4 to 11.

Example 7

Host spectrum analysis of Klebsiella pneumoniae phage Kp_phage_507

Twenty-five multidrug-resistant Klebsiella pneumoniae clinical isolates, one KPC-positive Klebsiella pneumoniae standard strain and one NDM-positive Klebsiella pneumoniae standard strain were selected as the strains to be tested. Use BHI liquid medium to cultivate the strain to be tested to the logarithmic growth phase. Take 100 μL of the bacterial liquid to be tested and mix it with 5 mL of BHI semi-solid medium. Pour the mixed semi-solid onto the plain agar plate and wait until half After the solid solidifies, use a pipette to drop 10 μL Kp_phage_507 onto the solidified double-layer plate, and incubate at 37°C overnight to observe whether plaques appear.

The results are shown in Table 2. Bacteriophage Kp_phage_507 has a broad lysis spectrum and can lyse 23 of the 27 strains tested.

Table 2 Host spectrum results of Klebsiella pneumoniae phage Kp_phage_507

Example 8

Bactericidal effect of Klebsiella pneumoniae phage Kp_phage_507 in culture medium

Take the phage suspension and add it to 5 mL of host bacteria in the logarithmic growth phase according to MOI values 1 and 0.001 respectively. Cultivate at 37°C and 160 rpm. Measure the OD600 of the co-cultured bacterial solution every 1 hour for a total of 5 hours. Alternatively, add only SM solution to 5mL of host bacteria in logarithmic growth phase was the experimental control group.

The results are shown in Figure 6. Bacteriophage Kp_phage_507 has a strong ability to kill Klebsiella pneumoniae 507 in vitro. It has achieved good lysis effect in 2h, and the OD600 of the culture medium has been maintained at an extremely low level in 2-5h. s level. This result shows that phage Kp_phage_507 can effectively inhibit the growth of its host bacteria in the range of infection number 1 to 0.001.

Example 9

Safety experiment of Klebsiella pneumoniae phage Kp_phage_507

Take 20 Galleria mellonella larvae weighing 250-350mg and randomly divide them into 2 groups, 10 in each group. In the experimental group, each Galleria mellonella larvae was injected with 10 μL of 1×10 ⁷ PFU/mL phage into the right hind foot (replace Example 1 The obtained phage stock solution was adjusted to a concentration of 1×10 ⁷ PFU/mL), the control group was injected with an equal volume of PBS, and the death of Galleria mellonella larvae was observed for 3 consecutive days. repeat three times.

The results showed that the mortality rates of the phage injection group and the PBS injection group were similar, indicating that phage Kp_phage_507 has a certain degree of safety.

Example 10

Experiment on controlling multidrug-resistant Klebsiella pneumoniae infection with Kp_phage_507

Forty Galleria mellonella larvae weighing 250–350 mg were taken and randomly divided into 4 groups of 10 larvae in each group. In the first group (Kp), each Galleria mellonella larvae was injected with 10 μL of host bacteria at 1×10 ⁵ CFU/mL in its right hind foot; in the second group (phage+Kp), each larvae was injected with 1×10 ⁵ CFU/mL of host bacteria. 10 μL after 1 hour, then inject 10 μL of the purified phage in Example 1 at 1×10 ⁷ CFU/mL; each of the third group (phage) was only injected with 10 μL of the purified phage in Example 1 at 1×10 ⁷ CFU/mL; Each of the four groups (PBS) was injected with an equal amount of PBS. The death of Galleria mellonella larvae was observed for 3 consecutive days. repeat three times.

The results are shown in Figure 7. The survival rate of the second group was significantly higher than that of the first group, and the mortality rates of the third and fourth groups were similar, indicating that K. pneumoniae phage Kp_phage_507 can better control multidrug resistance. Klebsiella pneumoniae infection, reduced mortality.

Claims (7)

1. A multi-drug resistant klebsiella pneumoniae bacteriophage, which is characterized in that: the phage is named Kp_phase_507 and is preserved in China Center for Type Culture Collection (CCTCC) M20211633.

2. The multi-drug resistant klebsiella pneumoniae bacteriophage of claim 1, wherein: the phage is a long tail phage.

3. The multi-drug resistant klebsiella pneumoniae bacteriophage of claim 1, wherein: the phage can withstand high temperatures of 70 ℃; the optimum pH of the phage is 4-11; the incubation period of phage growth is 0-20min, the burst period is 20-100min, and the phage enters the platform period after 100 min.

4. The multi-drug resistant klebsiella pneumoniae bacteriophage of claim 1, wherein: the phage can effectively inhibit the growth of host bacteria within the range of 1-0.001 of infection number.

5. A phage preparation comprising the phage of claim 1.

6. The phage preparation of claim 5, wherein: the preparation is a medicine, a cleaning agent or a disinfectant.

7. Use of a bacteriophage of claim 1, wherein: the preparation is used for preparing a preparation for preventing or treating multidrug-resistant klebsiella pneumoniae infection; the strain numbers of the multi-drug resistant klebsiella pneumoniae which can be lysed by the phage are ATCC BAA-2146 and ATCC BAA-1705 respectively.