CN116200345A - A kind of broad-spectrum Klebsiella pneumoniae phage and its training method and application - Google Patents

Abstract

The invention discloses a broad-spectrum Klebsiella pneumoniae bacteriophage (Klebsiella pneumoniae phage), named as P55anc, which is preserved in China Center for Type Culture Collection (CCTCC) NO: M20222087, and the preservation date is 2022, 12 months and 28 days; wherein, phage P55anc can be domesticated to obtain phage P55evo, and the domesticated phage P55evo has stronger lysis effect on the drug-resistant Klebsiella pneumoniae, and the bacteria are not easy to generate phage tolerance and are more stable under ultraviolet sterilization treatment. In addition, phage P55anc and domesticated phage P55evo can be used as antibiotic substitutes for treating the pandrug-resistant klebsiella pneumoniae infection, can also be applied to killing klebsiella pneumoniae in environments such as sewage, animal farms and the like, provides a guarantee for industrialized production of the klebsiella pneumoniae phage, and provides an economic, safe and efficient way for solving the huge threat caused by the pandrug-resistant klebsiella pneumoniae to human and animal health.

Description

A kind of broad-spectrum Klebsiella pneumoniae phage and its training method and application

technical field

The invention relates to the technical field of bioengineering, in particular to a broad-spectrum Klebsiella pneumoniae phage and its training method and application.

Background technique

Klebsiella pneumoniae (Klebsiella pneumoniae) is an opportunistic pathogen that widely exists in the natural environment and is also an important zoonotic pathogen. The antibiotic resistance genes it carries are widely spread around the world. Due to the non-standard use of antibiotics, many Gram-negative bacteria in clinical practice, such as Klebsiella pneumoniae and Escherichia coli, have multi-drug resistance. In the distress situation that antibiotics are difficult to treat multidrug-resistant bacteria, it is imminent to develop new alternatives, and phage therapy is expected to become a candidate to replace or supplement traditional antibiotics.

Phages are the most abundant and diverse microorganisms on the earth, most of which are 10 times the number of microbial cells in the natural environment, and the total number exceeds ¹⁰³⁰ . Phages have the advantages of rapid development, strong specificity, and avoiding damage to probiotics in the body, so they have good development prospects. Although phage therapy has many advantages, phage therapy has limitations. Like most antibacterial agents, bacteria can develop resistance to phages. In the process of phages continuously infecting bacteria, bacteria have evolved different mechanisms to resist phages in order to survive, and phages are also constantly evolving to fight against phage-resistant bacteria. "Antagonistic co-evolution" between bacteria and phages plays an important role in the process of phage therapy and affects the therapeutic outcome.

Contents of the invention

In view of this, the object of the present invention is to propose a broad-spectrum Klebsiella pneumoniae phage that is easy to apply and implement, environmentally friendly and efficient, and has certain economic benefits and market prospects, as well as its training method and application.

In order to realize above-mentioned technical purpose, the technical scheme that the present invention adopts is:

A kind of phage, which is Klebsiella pneumoniae phage (Klebsiella pneumoniae phage), named P55anc, has been preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M20222087, and the preservation date is for December 28, 2022.

In addition, this protocol also provides a method for domesticating phage P55anc, wherein the domesticated phage is named P55evo. The domestication method is as follows: take 20 μL of activated P55anc and add it to a 96-well plate containing 180 μL of liquid LB medium containing host bacteria, cultivate overnight in a 37°C constant temperature incubator, dilute 200 times, and continue to culture in a 37°C constant temperature incubator overnight. A subculture experiment of 200-fold dilution was performed once a day, and a total of 30 repetitions were set up for the above operation. Through the plate spot test, drop the phage filtrates that have been subcultured for 9 days on the LB plate with Klebsiella pneumoniae, culture overnight, pick out the transparent phage plaques and add them to the host bacteria for culture and store them to obtain phages And named it P55evo.

In this scheme, phage P55anc has a lytic effect on 27 strains of Klebsiella pneumoniae, and is a broad-spectrum Klebsiella pneumoniae phage.

In addition, the multiplicity of infection of phage P55anc in this protocol is 0.00001, and the multiplicity of infection of phage P55evo is 0.001.

Further, in this protocol, the incubation period of phage P55anc is 10 minutes, and the incubation period of phage P55evo is 20 minutes.

Further, the preservation method of the phages P55anc and P55evo in this protocol is to mix the phage lysate and 20% glycerol at a ratio of 1:1, and then store them in a temperature environment of -80°C.

Based on the above phage characteristics, the present invention also provides a drug for killing or inhibiting Klebsiella pneumoniae, which includes the phage P55anc and/or phage P55evo.

Adopting the above-mentioned technical scheme, compared with the prior art, the present invention has the beneficial effects that: this scheme provides a broad-spectrum phage P55anc, which has a certain bactericidal ability against pan-drug-resistant Klebsiella pneumoniae and Also has UV bactericidal stability. In addition, after domestication of phage P55anc by a specific domestication method, the evolved strain of phage P55evo can be obtained, which has stronger bactericidal ability, better UV stability, larger optimal MOI value, stable PFU, and one-step growth curve stability and other advantages.

The phage P55anc and P55evo of the scheme of the present invention have a strong bactericidal effect on pan-drug-resistant Klebsiella pneumoniae, which can reduce the abundance of pan-drug-resistant Klebsiella pneumoniae in clinical practice and the huge threat it poses to the health of the body; Factory production, and as a good antibiotic substitute for killing pan-drug-resistant Klebsiella pneumoniae, its application method is not only easy to operate, easy to implement, but also environmentally friendly and efficient, and has certain economic benefits.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art. The experimental method is highly sensitive and easy to operate, and can obtain more potent evolved phages in a short period of time, which can be widely used in the evolution of phages and improve their antibacterial and bactericidal effects.

Fig. 1 is the phage plaque figure of phage P55anc (a) and P55evo (b);

Fig. 2 is the electron microscope morphology diagram of phage P55anc;

Figure 3 is a characterization diagram of the bactericidal ability (OD ₆₀₀ ) of phages P55anc and P55evo;

Fig. 4 is the ultraviolet stability characterization figure of bacteriophage P55anc and P55evo;

Figure 5 is a graph showing the multiplicity of infection of phages P55anc and P55evo;

Fig. 6 is a one-step growth curve characterization graph of phages P55anc and P55evo.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. In particular, the following examples are only used to illustrate the present invention, but not to limit the scope of the present invention. Likewise, the following embodiments are only some but not all embodiments of the present invention, and all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

In the present invention, pan-drug-resistant Klebsiella pneumoniae was donated by Fujian Medical University, and the strain was isolated and preserved from a patient's sputum sample; wherein, the technical means for isolating Klebsiella pneumoniae from sputum is an existing Known means, it will not be repeated here.

Example 1

Isolation and identification of Klebsiella pneumoniae phage P55anc

(1) Sewage sample treatment: take 2mL of sewage filtrate, centrifuge (1,2000rpm, 3min), take the supernatant and filter (0.22μm sterile water filter membrane), to obtain a sterile phage suspension.

(2) Preparation of host bacterial solution: 50 μL of purified Klebsiella pneumoniae bacterial solution was inoculated into 5 mL of LB broth, and incubated at 37° C. and 200 rpm for 2-3 hours to reach the logarithmic phase of growth.

(3) Phage isolation (double-layer agar plate method): Take 1 mL of the phage filtrate in step (1) and add 1 mL of Klebsiella pneumoniae in the logarithmic phase, then add 2 mL of LB broth, and culture overnight at 37° C. with 200 rpm shaking. The co-cultivation solution was centrifuged and filtered, and the supernatant was collected as the phage stock solution. The above phage stock solution was serially diluted from 10 ^-1 to 10 ^-8 , and 100 μL of each serial dilution and 100 μL of the corresponding host bacteria were placed in a 5mL EP tube. After standing for 15 minutes, 3.5mL 0.6% LB semi-solid agar was added, and poured into In the prepared lower agar (1.5% LB agar medium). Place it upside down in a constant temperature incubator at 37°C and culture overnight until a single transparent phage plaque grows. Pick a single phage plaque with a pipette tip and add 500 μL of host bacteria, then add 2 mL of LB broth, incubate with shaking at 37°C for 8-12 hours, centrifuge and filter, take the filtrate for gradient dilution, and then culture and purify the phage on a double-layer agar plate, repeat 3 times Second, the enriched and purified phage P55anc was obtained, and its plaques are shown in Figure 1(a).

The phage obtained in this implementation is Klebsiella pneumoniaephage (Klebsiella pneumoniaephage), named as P55anc, which has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO:M20222087, and the preservation date is December 28, 2022. day.

Example 2

Domestication of phage P55 anc

Take 20 μL of activated P55anc and add it to a 96-well plate containing 180 μL of liquid LB medium containing host bacteria. After overnight incubation in a 37°C constant temperature incubator, dilute 200 times and continue to culture in a 37°C constant temperature incubator overnight. After that, dilute once a day 200-fold passage experiment. The phage filtrates subcultured for 9 days were dropped on the LB plate with Klebsiella pneumoniae, and the results were observed overnight. The transparent phage plaques were picked out and added to the host bacteria for culture and preserved to obtain phages, which were named P55evo, and the phage plaques were shown in Figure 1(b).

Since the phage P55evo can be directly domesticated from the phage P55anc and has reproducibility in acquisition channels, the application does not deposit the phage P55evo.

Example 3

Electron microscope morphology of phage P55anc

Pick a single phage plaque of phage P55anc and put it in sterile water, and mix it by gently pipetting. Take 20 μL of the phage filtrate and drop it on a 200-mesh copper grid, let it stand for 5 minutes, absorb the excess liquid with filter paper, and dry it for 1 minute. Add 1 drop of 1% phosphotungstic acid to the copper grid, let it stand at room temperature for 10 minutes, and observe the morphology of the phage under a Hitachi HT7700 transmission electron microscope. The characterization diagram obtained is shown in Figure 2.

Example 4

Determination of Bactericidal Ability of Phage P55anc and Phage P55evo (OD ₆₀₀ )

Pick Klebsiella pneumoniae for overnight culture, take 3 replicates of each cultured bacterial solution, dilute 200 times and take 180 μL of bacterial solution, add 20 μL of P55anc and P55evo phage filtrate, mix well and measure the 24-h growth curve. The growth curve of Klebsiella pneumoniae was used as a control.

The bactericidal ability (OD ₆₀₀ ) of phage P55anc and phage P55evo is shown in FIG. 3 . It can be seen from Figure 3 that the OD ₆₀₀ of P55anc began to rise at 6 hours, and it was similar to Klebsiella pneumoniae at 18 hours. At this time, phage P55anc had no bactericidal ability and the host bacteria showed phage resistance; phage P55evo had no obvious OD ₆₀₀ during the 24-hour sterilization process The change was always kept at 0.1, at this time the host bacteria did not develop phage resistance to the phage. The results show that the phage evolution strain phage P55evo has obvious bactericidal effect on bacteria Kp55 (Klebsiella pneumoniae), which is necessary for the evolution of phage P55anc.

Example 5

UV Stability of Phage P55anc and Phage P55evo

UV sensitivity: Take 1 mL of phage P55anc and P55evo respectively in a sterile blank petri dish, and irradiate them for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min at a distance of 30 cm from the UV lamp. The biological stability was reflected by measuring the phage titers of phage P55anc and phage P55evo, and the results are shown in FIG. 4 .

It can be seen from Figure 4 that the PFU titer of phage P55anc was lower than that of phage P55evo after 15 minutes of ultraviolet light, and the PFU of phage P55evo was always higher than that of phage P55anc after 15 minutes. Overall, phage P55anc was less UV stable than phage P55evo.

Example 6

Determination of optimal multiplicity of infection (MOI) of phage P55anc and phage P55evo

Pick a single colony of Klebsiella pneumoniae and add it to LB broth for 2-3 hours, and then use LB broth to serially dilute to 10 ^-8 , take 10 μL of each gradient to spot the plate, put it into a 37°C constant temperature incubator for overnight culture, and calculate the CFU. The cultured phages P55anc and P55evo were centrifugally filtered, diluted to 10 ^-8 in sequence, and then 10 μL of each gradient phage was dropped into a plate containing the host bacteria, cultivated overnight to calculate PFU. The phage stock solution and host bacteria were stored in a refrigerator at 4°C. After calculating CFU and PFU, mix according to the ratio of phage concentration (PFU/mL)/bacteria concentration (CFU/mL) of 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 and 0.000001, 100 μL of phage + 100 μL of host bacteria + 2 mL of LB meat The soup was mixed, shaken and cultivated at 37°C and 200rpm for 5 hours, and the co-cultivation solution was centrifugally filtered and then diluted to 10 ^-8 in turn. Take 100 μL of phage at each gradient concentration and add 100 μL of host bacteria to stand for 10 minutes. Use the double-layer plate method to measure the phage titer and count the results. Repeat 3 times, and the MOI with the highest titer is the optimal MOI. It can be seen from Figure 5 that the optimal MOI of phage P55anc is 0.00001, and the optimal MOI of phage P55evo is 0.001.

Example 7

One-step growth curve determination of phages P55anc and P55evo

Klebsiella pneumoniae and its bacteriophage were mixed in 200 μL each under the optimal MOI conditions, added to 20 mL of liquid LB medium, cultivated in a 37°C constant temperature incubator for 5 minutes, centrifuged at 12,000 rpm for 1 minute, discarded the supernatant, and resuspended in 5 mL of LB broth. Discard the supernatant by centrifugation, add 20mL LB broth, and culture with shaking at 37°C. Take the sample taken before the shaking culture as 0 min, and then take a sample every 10 min, and measure the phage titer by spot plate test, repeating 3 times. Taking time (min) as the abscissa and phage titer as the ordinate to draw a one-step growth curve, the incubation period, lysis period and plateau period of P55anc and P55evo infecting the host bacteria are obtained, and the results are shown in Figure 6.

It can be seen from Figure 6 that the titer of phage P55anc has no obvious change within 0-10 min, so the incubation period of the phage is about 10 min; within 10-50 min, the titer of the phage increases sharply, indicating that the lysis period of the phage is about 40 min; The titer tends to be stable and enters the plateau period. The titer of phage P55evo does not change significantly within 0-20 minutes, so the incubation period of the phage is about 20 minutes; within 20-70 minutes, the titer of the phage increases sharply, indicating that the lysis period of the phage is about 50 minutes; the titer tends to be stable after 70 minutes , entering the plateau period.

Based on the above, the phages P55anc and P55evo of this protocol can be used to kill pan-drug-resistant Klebsiella pneumoniae. The application technical idea roughly includes the following steps:

1) Sewage from Fuzhou Fucun Sewage Treatment Plant was taken as a sample to isolate Klebsiella pneumoniae phage.

2) Study on the physiological characteristics of phages P55anc and P55evo.

The inventors of the present application have shown through experiments that bacteriophage P55anc and P55evo can be used as a good antibiotic substitute for killing clinical pan-drug-resistant Klebsiella pneumoniae, and this method can effectively reduce clinical Klebsiella pneumoniae infection, reduce threat to body health. Compared with the untreated Klebsiella pneumoniae Kp55, phage P55anc showed a strong bactericidal ability in the first 6 hours, and then the bacteria gradually tolerated phage P55anc and recovered their growth rate after 18 hours. The evolved phage P55evo has stronger bactericidal ability, can completely kill bacteria within 24 hours and inhibit the production of bacterial resistant phage. The method is simple to operate, easy to implement, environmentally friendly and efficient, economical and safe, and is conducive to large-scale use.

The above descriptions are only part of the embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process conversion made by using the description of the present invention and the contents of the accompanying drawings, or directly or indirectly used in other related All technical fields are equally included in the scope of patent protection of the present invention.

Claims (10)

1. A phage is characterized in that the phage is Klebsiella pneumoniae phage (Klebsiella pneumoniae phage), named P55anc, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20222087 and the preservation date of 2022, 12 months and 28 days.

2. The phage of claim 1, wherein the phage multiplicity of infection is 0.00001.

3. The phage of claim 1, wherein the phage has a latency period of 10 minutes.

4. The phage of claim 1, wherein the phage is stored by mixing phage lysate with 20% glycerol in a ratio of 1:1 and then storing at-80 ℃.

5. An antibiotic substitute for killing or inhibiting klebsiella pneumoniae comprising phage P55anc according to any one of claims 1 to 4.

6. Phage P55evo, characterized in that it is domesticated from phage P55anc according to one of claims 1 to 4, and the domesticated phage is designated P55evo.

7. The phage P55evo of claim 6, wherein the domestication method is: 20. Mu.L of activated phage P55anc was added to a 96-well plate containing 180. Mu.L of liquid LB medium of host bacteria, and cultured overnight in a 37℃incubator in 30 replicates; the continuous culture is carried out for 9 days after once-a-day 200-fold dilution, the purified repeated phage filter liquid is dripped on an LB plate with klebsiella pneumoniae for overnight culture, and then transparent plaques are picked out and added into a host bacterium for culture and then are preserved, so that phage P55evo is obtained.

8. The phage P55evo of claim 6, wherein the phage multiplicity of infection is 0.001.

9. The phage P55evo of claim 6, wherein the phage has a latency period of 20min.

10. A medicament for killing or inhibiting klebsiella pneumoniae, characterized in that: comprising the phage P55anc of one of claims 1 to 4 and/or the phage P55evo of one of claims 6 to 9.

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