CN115029322A - Multi-drug-resistant Klebsiella pneumoniae phage and application thereof - Google Patents
Multi-drug-resistant Klebsiella pneumoniae phage and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and discloses a multi-drug resistant Klebsiella pneumoniae and application thereof. The phage is named Kp _ phase _507 and is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in China, and the preservation number is CCTCC M20211633. The Klebsiella pneumoniae Kp _ phase _507 provided by the invention has short latency, large outbreak amount, wide temperature and acid-base tolerance range, wide host spectrum and good hydrophilicity, can quickly kill host bacteria in a culture medium, and is easy to prepare into injections. Animal experiments prove that the phage has small toxic and side effects, high safety and good treatment effect on multi-drug resistance klebsiella pneumoniae infection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a multi-drug resistant Klebsiella pneumoniae and application thereof.
Background
Klebsiella Pneumoniae (KP) is a conditionally pathogenic gram-negative enterobacter bacterium, which is usually localized in the gastrointestinal tract, skin and nasopharynx of the human body and can cause pneumonia, liver abscess, urinary tract infection, wound infection, septicemia and the like when the immunity of the human body is low. Klebsiella pneumoniae has become an important pathogen of infection.
Cephalosporin, fluoroquinolone and trimethoprim-sulfamethoxazole are common medicines for resisting klebsiella pneumoniae infection, but with the wide use of a large amount of antibiotics, the drug resistance phenomenon of klebsiella pneumoniae to the antibiotics is very common, and a large amount of multi-drug resistant strains appear. Carbapenem antibiotics are considered as the last line of defense in the treatment of multidrug resistant klebsiella pneumoniae infection, but klebsiella pneumoniae resistant to carbapenem antibiotics was first reported in 2001. A large number of epidemiological investigation and research show that the carbapenem antibiotic-resistant Klebsiella pneumoniae has a worldwide epidemic trend, and great challenges are brought to clinical treatment. The problem of drug resistance of klebsiella pneumoniae has posed a significant threat to public health.
The bacteriophage is a virus capable of specifically infecting bacteria, is a natural antibacterial substance, and can produce protease for degrading bacteria surface polysaccharide. The potential therapeutic effect of bacteriophages on bacterial infections was first identified and applied in 1919. But early phage therapy presented many problems due to the unknown nature of the phage by researchers, and antibiotics developed rapidly after the 40's of the 20 th century, resulting in phage therapy being gradually overlooked. After 1980, drug-resistant bacteria have emerged, which has made traditional antibiotic therapy extremely challenging and the development of new antibiotics has slowed down significantly. Thus, phage therapy again entered the field of view of the investigator. The phage therapy is to kill host bacteria by introducing lytic phage into the host bacteria and propagating in vivo, so as to achieve the effect of treating diseases caused by the host bacteria. Phage therapy or becomes a facilitator against "superbacteria".
However, since the host specificity of phages makes their antibiogram too narrow, personalized treatment must be made for each case and no single treatment can be taken. Therefore, continuous isolation of new bacteriophages and their basic analysis of biological properties and antibacterial potential are the first prerequisite for the development of bacteriophage biologics, and only then is it possible to adapt to the diversity of bacterial biological species and the new species generated by continuous mutation.
Disclosure of Invention
Aiming at the problems, the invention provides a multi-drug resistant Klebsiella pneumoniae and application thereof. The phage Kp _ phase _507 has short latency, large outbreak amount, wide temperature and acid-base tolerance range, wide host spectrum and good hydrophilicity, can quickly kill host bacteria in a culture medium, and is easy to prepare into an injection. Can provide a new technical means for the treatment of multidrug resistance Klebsiella pneumoniae infection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a multi-drug resistant Klebsiella pneumoniae phage, which is named as Kp _ phase _507 and is preserved in China center for type culture Collection with the preservation number of CCTCC M20211633.
Further, the bacteriophage is a long-tail bacteriophage.
Further, the phage can tolerate a high temperature of 70 ℃; the optimal pH value of the phage is 4-11; the incubation period of the phage growth is 0-20min, the outbreak period is 20-100min, and the growth enters the plateau period after 100 min.
Furthermore, the bacteriophage has a wide lysis spectrum, and can crack 27 strains to clinically separate 23 strains in the multidrug-resistant klebsiella pneumoniae.
Further, the 23 multidrug-resistant Klebsiella pneumoniae strains which can be lysed by the bacteriophage are numbered Kpn-ESBL-u1, 507, 346, 676, 491, Kpn-87, Kpn-90, Kpn-93, Kpn-96, Kpn-106, Kpn-110, K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, ATCC BAA-2146 and ATCC BAA-1705, respectively.
Furthermore, the phage can effectively inhibit the growth of host bacteria within the range of infection number of 1-0.001.
The invention provides a phage preparation comprising the phage.
Further, the formulation is a medicament, a cleanser or a disinfectant.
The invention also provides an application of the bacteriophage, which is used for preparing a preparation for preventing or treating multidrug-resistant klebsiella pneumoniae infection.
Compared with the prior art, the invention has the following advantages:
the invention provides a new Klebsiella pneumoniae Kp _ phase _507, which has short latency period, large outbreak amount, wide temperature and acid-base tolerance range, wide host spectrum and good hydrophilicity, can quickly kill host bacteria in a culture medium, and is easy to prepare into injections. Animal experiments prove that the bacteriophage has small toxic and side effect, high safety and good treatment effect on multi-drug resistant klebsiella pneumoniae infection.
Drawings
FIG. 1 is a plaque picture of the Klebsiella pneumoniae Kp _ phase _507 of the present invention.
FIG. 2 is a transmission electron micrograph of Klebsiella pneumoniae Kp _ phase _507 according to the present invention.
FIG. 3 is a graph showing the one-step growth of the Klebsiella pneumoniae Kp _ phase _507 of the present invention.
FIG. 4 is a graph showing the effect of temperature on the activity of the Klebsiella pneumoniae Kp _ phase _507 of the present invention.
FIG. 5 is a schematic diagram showing the effect of pH on the activity of Klebsiella pneumoniae Kp _ phase _507 according to the present invention.
FIG. 6 is a schematic diagram of the sterilization of Klebsiella pneumoniae Kp _ phase _507 medium.
FIG. 7 is a schematic diagram of the therapeutic effect of Klebsiella pneumoniae Kp _ phage _507 galleria mellonella.
Detailed Description
The technical solution of the present invention will be specifically and specifically described below with reference to the embodiments of the present invention and the accompanying drawings. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention. The methods and techniques used are conventional unless otherwise specified.
The examples relate to strains, reagents and media:
the host bacteria used in the experiment are all multidrug-resistant Klebsiella pneumoniae clinical strains 507.
Brain Heart Infusion (BHI) broth: 3.6g of BHI was weighed into 100mL of distilled water and autoclaved at 121 ℃ for 20 min.
Brain Heart Infusion (BHI) semi-solid medium: 3.6g of BHI, 0.75g of agar were weighed into 100mL of distilled water and autoclaved at 121 ℃ for 20 min.
Brain Heart Infusion (BHI) solid medium: 3.6g of BHI and 1.5g of agar are weighed and put into 100mL of distilled water, sterilized at 121 ℃ for 20min under high pressure, cooled to 50 ℃, poured out of a flat plate, cooled and solidified, and inverted for later use.
Solid medium of vegetable agar: weighing 1.5g agar in 100mL distilled water, sterilizing at 121 deg.C for 20min under high pressure, cooling to 50 deg.C, pouring out, cooling to solidify, and standing.
SM buffer solution: 6.055g Tris was weighed into 20mL distilled water for dissolution, pH was adjusted to 7.5 using concentrated hydrochloric acid to a volume of 50mL, then 5.8g NaCl and 2g MgSO 4 Dissolving, and autoclaving at 121 deg.C for 20min to obtain a volume of 1L.
Example 1
Separation and purification of Klebsiella pneumoniae Kp _ phase _507
(1) The separation and purification of phage are carried out by taking the multidrug-resistant Klebsiella pneumoniae clinical isolate 507 as host bacteria. The host bacteria are streaked and inoculated on a BHI solid culture medium, inverted culture is carried out in a constant temperature incubator at 37 ℃ for 12-18h, then, the single clone is selected and inoculated in 5mL of BHI liquid culture medium, and shaking culture is carried out at 37 ℃ until logarithmic phase, and the obtained product is used as a host bacteria culture for standby.
(2) Collecting untreated sewage in a certain hospital, filtering the sewage by using double-layer filter paper, centrifuging at 10000rpm for 20min, taking supernate, filtering by using a 0.22 mu m filter, and collecting filtrate; co-culturing the filtrate and host bacteria in BHI liquid culture medium overnight, centrifuging to obtain supernatant, filtering with 0.22 μm filter, and collecting filtrate to obtain bacteriophage stock solution.
(3) Taking 100 mu L of host bacteria in logarithmic growth phase, inoculating the host bacteria in semisolid BHI culture medium at 55-60 ℃, fully and uniformly mixing to prepare a double-layer plate, after solidification, dripping the phage stock on the plate, naturally airing, carrying out inverted culture at 37 ℃ for 12-18h, and observing whether plaque is formed at the point plate. If plaque is formed, it is indicated that specific phage is present.
(4) Adding the obtained plaque into host bacteria in logarithmic growth phase, culturing at 37 deg.C and 160rpm for 10-12 hr, centrifuging at 10000rpm for 5min, and filtering with 0.22 μm filter to obtain phage stock solution; continuously diluting phage stock solution by 10 times with SM solution, and respectively taking 10 times -2 、10 -4 、10 -6 、10 -8 Mixing 100 mu L of the diluted solution with 500 mu L of logarithmic phase bacterial solution, incubating at room temperature for 10min, adding into semi-solid BHI culture medium at about 50 ℃ and mixing well to prepare double-layer plate, culturing at 37 ℃ for 12-18h after solidification, selecting plate with bacteriophage, selecting larger and isolated bacteriophage, and continuing culturingAnd (5) nourishing. This procedure was repeated until phage with uniform plaque size were obtained and stored at 4 ℃ for future use.
The phage prepared for use is detected by a double-layer plate method, and the result is shown in figure 1, and the phage can form a bright plaque in an agar culture medium, has no halo around and has clear and regular edges, and is a typical lytic phage.
Example 2
Enrichment of Klebsiella pneumoniae Kp _ phase _507
The phage prepared in example 1 and the host bacteria in logarithmic growth phase were co-cultured at 37 ℃ and 160rpm until the bacterial fluid became clear, centrifuged at 10000rpm for 5min to obtain the supernatant, and filtered through a 0.22 μm filter to obtain phage lysate.
PEG8000 purification of phage: adding DnaseI and RnaseA into a phage lysate until the final concentrations are 1 mu g/mL, incubating for 3h at 37 ℃, adding solid NaCl until the final concentration is 1mol/L, carrying out ice bath for 1h, centrifuging for 15min at 4 ℃ and 10000g, taking a supernatant, adding solid PEG8000 until the final concentration is 10%, carrying out ice bath for 3h, centrifuging for 15min at 4 ℃ and 10000g, removing the supernatant, gently suspending a phage precipitate in SM solution, extracting PEG and cell debris in a phage suspension by adding chloroform with the same volume, carrying out mild oscillation for 30s, centrifuging for 15min at 4 ℃ and 3000g to separate an organic phase and a hydrophilic phase, recovering the hydrophilic phase containing phage particles, and obtaining a high-concentration enriched phage suspension.
Example 3
Transmission electron microscope observation of Klebsiella pneumoniae Kp _ phase _507
Observing the high-concentration phage suspension obtained in the embodiment 2 by using an electron microscope, dripping 1 drop of the phage suspension on a copper mesh, sucking redundant phage suspension from the edge of the copper mesh by using filter paper after 5min, dyeing the phage by using 2% phosphotungstic acid solution for 10min, drying and then placing under the electron microscope to observe the form of the phage; as shown in FIG. 2, the phage Kp _ phase _507 was a long-tail phage.
Example 4
Determination of Klebsiella pneumoniae Kp _ phase _507 titer
The phage stock purified in example 1 was diluted in a 10-fold gradient,respectively take 10 -6 -10 -11 Fully and uniformly mixing 100 mul of the diluted phage diluent and 500 mul of the host bacteria in the logarithmic phase, incubating for 10min at room temperature, preparing double-layer plates, culturing for 12-18h at 37 ℃, and counting plaques on each plate. Three replicates were performed and phage titers were calculated. Titer of phage (PFU/mL) is dilution times mean/sample volume. The titer of the phage was 3.33X 10 11 PFU/mL。
Example 5
Determination of optimal infection complex number of Klebsiella pneumoniae Kp _ phase _507
Counting host bacteria in logarithmic phase, mixing phage and host bacteria in logarithmic phase at the ratio of 10, 1, 0.1, 0.01 and 0.001, standing at room temperature for 15min, mixing with BHI liquid culture medium, culturing at 37 deg.C and 160rpm for 5h, centrifuging at 12000g for 3min, collecting supernatant, filtering with 0.22 μm filter, and collecting filtrate; mu.l of filtrate was taken for each MOI value and the phage titer was determined. The highest MOI value of the titer is the optimal multiplicity of infection of the phage. As a result, as shown in Table 1, the optimum multiplicity of infection of the phage Kp _ phase _507 was 0.001.
TABLE 1 optimal multiplicity of infection for Klebsiella pneumoniae Kp _ phase _507
Example 5
Determination of Klebsiella pneumoniae Kp _ phase _507 one-step growth curve
The phage suspension purified in example 1 was mixed with host bacteria at a ratio of MOI value of 0.001, incubated at 37 ℃ for 15min, centrifuged at 5000rpm for 2min, the pellet was collected, resuspended with 5mL of BHI broth, cultured at 37 ℃ at 160rmp, sampled at time points of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120min, and the phage titer at each time point was determined, and then a growth curve was plotted. One step growth curve.
As shown in FIG. 3, the incubation period of Klebsiella pneumoniae Kp _ phase _507 was 0-20 minutes, the outbreak period was 20-100 minutes, and the stationary period was reached after 100 minutes. The burst size was 246 PFU/cell.
Example 6
Temperature and pH tolerance experiment of Klebsiella pneumoniae Kp _ phase _507
0.5mL of the phage suspension purified in example 1 was added to each of 5 sterile EP tubes, and the mixture was allowed to react in water baths of 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 1 hour, cooled to room temperature, and then the titer of phage treated at different temperatures was determined. As shown in FIG. 4, the phage was able to tolerate a high temperature of 70 ℃ and the phage was rapidly inactivated at 80 ℃.
12 portions of 0.1mL of the phage suspension purified in example 1 were added to 0.9mL of BHI broth at pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, respectively, and allowed to act at room temperature for 1 hour, and then the titer of phage after the different pH conditions were measured. As a result, as shown in FIG. 5, the activity of the phage was less changed in the environment of pH 4 to 11; when pH <4 or pH >11, the titer of the phage decreases very much with the increase of the acidity and basicity; when pH 2, phages were all inactivated. Therefore, the optimum pH value of the phage Kp _ phase _507 is 4-11.
Example 7
Host spectrum analysis of Klebsiella pneumoniae Kp _ phase _507
25 multidrug-resistant Klebsiella pneumoniae clinical isolates, 1 KPC-positive Klebsiella pneumoniae standard strain and an NDM-positive Klebsiella pneumoniae standard strain are selected as strains to be detected. Culturing the strain to be detected to logarithmic growth phase by using a BHI liquid culture medium, uniformly mixing 100 mu L of bacterial liquid of the strain to be detected with 5mL of BHI semisolid culture medium, pouring the mixed semisolid on a plain agar plate, after the semisolid is solidified, sucking 10 mu L of Kp _ phase _507 by using a liquid transfer gun and dripping the Kp _ phase _507 on the solidified double-layer plate, culturing at constant temperature of 37 ℃ overnight, and observing whether the bacterial liquid is formed by the occurrence of the plaque.
As shown in Table 2, the bacteriophage Kp _ phase _507 has a broad lysis spectrum and can lyse 23 of the 27 strains to be tested.
TABLE 2 host spectra results for Klebsiella pneumoniae Kp _ phase _507
Example 8
Sterilization effect of Klebsiella pneumoniae Kp _ phase _507 in culture medium
Adding the phage suspension into 5mL of host bacteria in logarithmic phase according to MOI value 1 and MOI value 0.001, culturing at 37 ℃ and 160rpm, measuring OD600 of co-culture bacteria liquid every 1h, and measuring for 5 h; and setting host bacteria added with SM liquid to 5mL of logarithmic phase as experimental control group.
As shown in FIG. 6, the phage Kp _ phase _507 has strong in vitro killing capability to Klebsiella pneumoniae 507, good lysis effect is achieved within 2h, and OD600 of the culture solution is always maintained at an extremely low level within 2-5 h. The result shows that the phage Kp _ phase _507 can effectively inhibit the growth of host bacteria within the range of infection number of 1-0.001.
Example 9
Safety experiment of Klebsiella pneumoniae Kp _ phase _507
20 large galleria mellonella larvae with the weight of 250-350 mg are taken and randomly divided into 2 groups of 10 in each group, and the right hind paw of each galleria mellonella larva of the experimental group is injected with 1 multiplied by 10 7 10. mu.L of phage (PFU/mL) (phage stock solution obtained in example 1 was adjusted to a concentration of 1X 10) 7 PFU/mL), the control group was injected with an equal volume of PBS and the death of the larvae of galleria mellonella was observed for 3 consecutive days. This was repeated three times.
The results show that the mortality rates of the phage injection group and the PBS injection group are equivalent, which indicates that the phage Kp _ phase _507 has certain safety.
Example 10
Multiple drug-resistant klebsiella pneumoniae infection experiment controlled by klebsiella pneumoniae phage Kp _ phase _507
Taking 40 large galleria mellonella larvae with the weight of 250-350 mg, randomly dividing the larvae into 4 groups, wherein each group comprisesGroup 10 only. The first group (Kp) is injected with 1X 10 of the right hind paw of each galleria mellonella larva 5 10 mu L of CFU/mL host bacteria; second group (phase + Kp) of each injection was 1X 10 5 After 10. mu.L of CFU/mL host bacteria for 1 hour, 1X 10 was injected 7 CFU/mL of phage purified in example 1 at 10. mu.L; the third group (phase) was injected with 1X 10 injections each 7 CFU/mL of phage purified in example 1 at 10. mu.L; the fourth group (PBS) was each injected with an equal amount of PBS. The death of the larvae of the greater wax moth was observed for 3 consecutive days. This was repeated three times.
The results are shown in fig. 7, the survival rate of the second group is significantly improved compared to that of the first group, and the mortality rates of the third group and the fourth group are equivalent, which indicates that the klebsiella pneumoniae phage Kp _ phase _507 can better control the infection of multidrug-resistant klebsiella pneumoniae and reduce the mortality rate.
Claims (9)
1. A multi-drug resistant Klebsiella pneumoniae phage is characterized in that: the phage is named Kp _ phase _507 and is preserved in China center for type culture Collection with the preservation number of CCTCC M20211633.
2. The multidrug resistant klebsiella pneumoniae phage of claim 1, wherein: the phage is a long-tail phage.
3. The multidrug resistant klebsiella pneumoniae phage of claim 1, wherein: the phage can tolerate a high temperature of 70 ℃; the optimal pH value of the phage is 4-11; the incubation period of the phage growth is 0-20min, the outbreak period is 20-100min, and the growth enters the plateau period after 100 min.
4. The multidrug resistant klebsiella pneumoniae phage of claim 1, wherein: the bacteriophage has a wide cracking spectrum, and can crack 27 strains to clinically separate 23 strains in the multidrug-resistant klebsiella pneumoniae.
5. The multidrug-resistant klebsiella pneumoniae phage according to claim 4, wherein: the bacterial strain numbers of the 23 strains of multidrug-resistant Klebsiella pneumoniae which can be cracked by the bacteriophage are Kpn-ESBL-u1, 507, 346, 676, 491, Kpn-87, Kpn-90, Kpn-93, Kpn-96, Kpn-106, Kpn-110, K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, ATCC BAA-2146 and ATCC BAA-1705 respectively.
6. The multidrug resistant klebsiella pneumoniae phage of claim 1, wherein: the phage can effectively inhibit the growth of host bacteria within the range of infection number of 1-0.001.
7. A phage preparation comprising the phage of claim 1.
8. The phage preparation of claim 7, wherein: the preparation is a medicament, a cleaning agent or a disinfectant.
9. Use of a bacteriophage according to claim 1, wherein: is used for preparing a preparation for preventing or treating multidrug resistant Klebsiella pneumoniae infection.
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| CN112662634A (en) * | 2020-12-30 | 2021-04-16 | 瑞科盟(青岛)生物工程有限公司 | Drug-resistant Klebsiella pneumoniae RDP-KP-20004 and application thereof |
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| AU2017204331A1 (en) * | 2010-09-17 | 2017-07-13 | Technophage, Investigação E Desenvolvimento Em Biotecnologia, Sa | Antibacterial phage, phage peptides and methods of use thereof |
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