CN117004577A

CN117004577A - Klebsiella pneumoniae phage and application and preparation thereof

Info

Publication number: CN117004577A
Application number: CN202310881302.3A
Authority: CN
Inventors: 王承民; 张乐怡; 王雪; 曾君; 郭志良; 左君豪; 季芳; 王学静; 藏莹安; 徐莉莉
Original assignee: Unibond Biotechnology Shanghai Co ltd
Current assignee: Unibond Biotechnology Shanghai Co ltd
Priority date: 2023-07-18
Filing date: 2023-07-18
Publication date: 2023-11-07

Abstract

The application provides klebsiella pneumoniae phage, and application and preparation thereof. Klebsiella pneumoniae phage (Klebsiella pneumoniaphage) pKP-M297-1.1 was deposited at the Guangdong province microbiological bacterial collection center (GDMCC), accession number: the collection number of the building 5 of the Guangzhou city first Zhonglu No. 100 college No. 59 is: GDMCCNo 63520-B1. The application also provides a preparation comprising the klebsiella pneumoniae phage pKP-M297-1.1 and application of the klebsiella pneumoniae phage pKP-M297-1.1 in preparation of a preparation for preventing and treating multi-drug resistant klebsiella pneumoniae infection. The klebsiella pneumoniae phage provided by the application has a wide antibacterial spectrum, and can be applied to the preparation of medicines for preventing and treating multiple drug-resistant klebsiella.

Description

Klebsiella pneumoniae phage and application and preparation thereof

Technical Field

The application relates to the field of microorganisms, and more particularly relates to klebsiella pneumoniae phage and applications and preparations thereof.

Background

Clinically, multi-drug resistant bacteria with resistance to various antibiotics have become an important problem threatening global public health, and klebsiella pneumoniae is one of them. Klebsiella pneumoniae (Klebsiella pneumoniae) is a capsular, gram-negative facultative anaerobic bacterium belonging to the family Enterobacteriaceae (Enterobacteriaceae), which is the second most clinically important pathogenic bacterium of escherichia coli, and is generally capable of surviving in communities and hospitals for a long period of time. When the immunity of the organism is reduced, serious diseases such as pneumonia, hysteritis, septicemia and the like are easy to occur in infection.

In recent years, reports of Klebsiella pneumoniae isolates producing an ultra-broad spectrum of beta-lactamase and carbapenemase have been increasing. In a combined report, the K.varicola SHET-01 strain was found in a urinary catheter from a female patient in a pediatric intensive care unit in a hospital in Shanghai China. The strain showed a high mucilage phenotype (HMV), indicating that it is highly likely to be highly toxic. The drug sensitivity test result shows that the strain shows drug resistance to carbapenem antibiotics meropenem, imipenem and ertapenem, and the Minimum Inhibitory Concentrations (MIC) are respectively 32, 32 and 16 mug/mL; in addition, some hospitals in Guizhou, china also have cases reported that patients die from Klebsiella pneumoniae infection in a short period of time. Because of the continuous appearance of drug-resistant bacteria, great difficulty is caused to the clinical treatment drug selection, and the development of novel antibacterial drugs is urgent.

Phage (bacteriophage or phage) is the most abundant biological population in the biological kingdom, can infect bacteria and replicate, and has the potential advantages of strong sterilization specificity, low development cost, easy separation, self-restriction, few side effects, capability of killing drug-resistant strains and the like. In the situation that drug-resistant strains are continuously appeared and new antibacterial preparations are developed and can not meet the control requirement of drug-resistant bacteria, phage become one of the important choices for the current drug-resistant bacterial infection treatment. In recent years, many successful cases have been reported, including blood flow infection caused by pantopractic acinetobacter baumannii and complicated urinary tract infection caused by pantoprazole klebsiella pneumoniae. There are also clinical phase I/II studies reporting phage therapy at present, showing that administration of phage preparations meeting good operating specifications does not produce observable deleterious effects on the body. The phage is suggested to have better safety as an anti-infective biological agent, and especially has wider application potential in phage treatment at the moment of extremely severe drug-resistant bacteria.

Disclosure of Invention

Aiming at the technical problems, the application provides a Klebsiella pneumoniae bacteriophage and application thereof to the inhibition and killing of multi-drug resistant Klebsiella pneumoniae, and aims to provide a new treatment scheme for the infection of Klebsiella pneumoniae and a potential novel disinfection means for the environmental pollution, feed pollution and the like caused by Klebsiella pneumoniae.

The application provides a Klebsiella pneumoniae phage (Klebsiella pneumonia phage) pKP-M297-1.1, which is preserved in the Guangdong province microorganism strain collection (GDMCC) with the preservation address: the collection number of the building 5 of the Guangzhou city first Zhonglu No. 100 college No. 59 is: GDMCC No. 63520-B1.

The application also provides a preparation which comprises the klebsiella pneumoniae phage pKP-M297-1.1.

The application also provides application of the klebsiella pneumoniae phage pKP-M297-1.1 in preparation of a preparation for preventing and treating multidrug-resistant klebsiella pneumoniae infection.

Compared with the prior art, the application has the following beneficial effects:

(1) The Klebsiella pneumoniae phage pKP-M297-1.1 is a broad-host-spectrum phage, has a good disinfection effect on multi-drug-resistant Klebsiella pneumoniae M297-1, has a strong disinfection effect on other variable-dwelling Klebsiella pneumoniae, klebsiella pneumoniae and Klebsiella aerogenes, has a wide antibacterial spectrum, and can be applied to preparation of drugs for preventing and treating multi-drug-resistant Klebsiella pneumoniae;

(2) The Klebsiella pneumoniae phage pKP-M297-1.1 can quickly kill host bacteria in a culture medium, has small toxic and side effects, high safety, wide temperature and acid-base tolerance range, good hydrophilicity, easy preparation into spray and injection, good inhibition and killing effects on proliferation of Klebsiella pneumoniae, and good treatment and protection effects on animals infected with multi-drug resistant Klebsiella pneumoniae.

Drawings

FIG. 1 is a plaque photograph of Klebsiella pneumoniae phage pKP-M297-1.1 of the application.

FIG. 2 is a transmission electron microscope image of Klebsiella pneumoniae phage pKP-M297-1.1 of the application.

FIG. 3 is a graph showing the one-step growth of Klebsiella pneumoniae phage pKP-M297-1.1 of the application.

FIG. 4 is a schematic diagram showing the effect of temperature on the activity of Klebsiella pneumoniae phage pKP-M297-1.1 of the application.

FIG. 5 is a schematic representation of the effect of pH on the activity of the Klebsiella pneumoniae phage pKP-M297-1.1 of the application.

FIG. 6 is a schematic diagram showing the sterilization of Klebsiella pneumoniae phage pKP-M297-1.1 of the application in culture.

FIGS. 7 and 8 are schematic diagrams showing the treatment of Klebsiella pneumoniae bacteriophage pKP-M297-1.1 of the present application with respect to Chilo suppressalis infected with Klebsiella pneumoniae.

The Klebsiella pneumoniae phage (Klebsiella pneumonia phage) provided by the application has a pKP-M297-1.1, a taxonomic name of Klebsiella pneumonia phage, and is deposited in the Guangdong province microorganism strain collection (GDMCC) on 2 days of 2023, with a deposit number of: GDMCCNo 63520-B1; the preservation address is: guangzhou city first middle road No. 100 college No. 59 building 5.

Detailed Description

The following examples will enable those skilled in the art to more fully understand the present application and are not intended to limit the same in any way.

MH broth (1L): 21.0g of MH Broth (MH Broth) was weighed and dissolved in 1000mL of ddH ₂ And (3) in O, autoclaving at 121 ℃ for 20min for later use.

0.7% MH (B) semi-solid Medium (1L): weighing 21.0g of MH Broth (MH Broth), 7.0g of agar powder, adding ddH ₂ O to 1000mL,121℃and autoclaved for 20 min.

MH (A) Medium (1L): MH (A) medium (Mueller-Hinton Agar) 36.5g was weighed and 1000mL ddH was added ₂ O,121 ℃, after autoclaving for 20min, cooling to 50 ℃, pouring the flat plate, cooling and solidifying, and inverting for standby.

SM buffer, PBS, DNase I, RNase A, PEG8000, naOH, HCl, naCl are commercially available.

Example 1

Isolation of Klebsiella pneumoniae phage pKP-M297-1.1

Host bacteria M297-1 (MPEB 0011806, preferably Shanghai) are streaked and inoculated on a MH (A) culture medium, after overnight culture, single clone is selected and inoculated in 5mL of liquid culture medium, and after shaking culture for 12 hours at 37 ℃ is used as a host bacteria culture for standby.

Samples were taken from Zhengzhou zoos, the wastewater samples were centrifuged at 8000rpm for 10min at 4℃and the supernatant was filtered through a 0.22 μm filter. Taking 1000 mu L of filtrate, adding 100 mu L of host bacteria liquid, adding 5mL of MH (B) culture medium, placing in a constant temperature oscillator for culturing for 12h, centrifuging the culture at the temperature of 4 ℃ at the speed of 8000rpm/min for 10min, filtering and sterilizing the supernatant by using a 0.22 mu m filter membrane, and forming stock solution containing phage, namely phage suspension.

Sucking 100 mu L of the standby host bacteria culture into a 10mL sterile centrifuge tube, pouring 8mL of preheated semi-solid culture medium, pouring the mixture into a solid plate after the mixture is inverted and uniformly mixed, dividing the plate into two areas after the mixture is dried, taking 10 mu L of phage suspension, dripping the 10 mu L of phage suspension on one side of the plate, and dripping PBS with the same volume on the other side of the plate to serve as a blank control. After naturally airing, the culture is carried out for 12 hours in an incubator at 37 ℃ in an inverted mode, and whether plaque is formed in the dripping phage area is observed. If plaque formation is present, the presence of phage is demonstrated.

Another 100. Mu.L of the phage suspension was serially diluted 10-fold and 10-fold ^-5 ～10 ^-8 100 mu L of the diluted solution is respectively and uniformly mixed with the host bacterial liquid with the same volume, and is kept stand for 10min, 5-8 mL of 0.7% semisolid MH (B) culture medium with the temperature of about 50 ℃ is added, the diluted solution is uniformly paved on a solid MH (B) plate prepared in advance, and plaque growth is observed after the diluted solution is cultured for 12h at the temperature of 37 ℃. Single clear, uniformly sized plaques were picked into EP tubes containing MH (B) and left at 4℃for 4h. And diluting 100 mu L of the solution by 10 times, and taking 100 mu L of the diluted solution and the host bacteria liquid with the same volume as a double-layer plate. The phage with uniform plaque size can be obtained by repeating the steps for 3 to 5 times, and the phage can be stored at 4 ℃ for standby.

The plaque results are shown in FIG. 1, and the phage can form transparent plaques in agar medium, with clear and regular edges and a diameter of about 1.5mm, which is a typical lytic phage.

Example 2

Amplification and purification of Klebsiella pneumoniae phage pKP-M297-1.1

Taking 0.1mL of phage preservation solution prepared in example 1 and 0.1mL of host bacteria culture prepared in example 1, acting for 15min in a test tube, adding 10mL of MH (B) liquid culture medium, culturing for 12h at 37 ℃, centrifuging at 8000rpm for 20min at 4 ℃, taking the supernatant, filtering with a 0.22 μm filter membrane, and obtaining the filtrate as phage lysate.

PEG purification: adding DNaseI, RNaseA to the phage lysate to a final concentration of 1 μg/mL, standing at room temperature for 30min, adding NaCl to a final concentration of 1mol/L, ice-bathing for 2h, centrifuging at 4 ℃ at 10000rpm/min for 20min, taking the supernatant, adding PEG8000 to a final concentration of 10%, ice-bathing for 12h, centrifuging at 4 ℃ at 10000rpm/min for 20min, carefully discarding the supernatant, inverting for 5min to drain the liquid, adding 2mL of SM buffer to resuspension and precipitate, adding an equal volume of chloroform and gently shaking for 30s, centrifuging at 4 ℃ at 5000rpm/min for 15min to separate an organic phase and a hydrophilic phase, and recovering the hydrophilic phase containing phage particles to obtain a purified phage suspension.

Example 3

Transmission electron microscope observation of Klebsiella pneumoniae phage pKP-M297-1.1

Performing electron microscope observation on the phage suspension purified in the example 2, dripping the phage suspension purified in the example 2 on a copper sheet, naturally precipitating for 2-3 min, sucking redundant liquid by using filter paper, dripping 2% phosphotungstic acid (PTA, 2% w/v) for dyeing, drying at room temperature, and observing by using a transmission electron microscope; the observation results are shown in FIG. 2. Phage pKP-M297-1.1 is a long tail phage with a head of regular icosahedron structure and a head diameter of about 90.91nm.

The applicant has named phage self-pKP-M297-1.1, collection of microorganisms and cell cultures (GDMCC) from Guangdong province, accession number: building 5, no. 59 of Guangzhou Xian Zhonglu 100 college, with the deposit number of GDMCC No. 63520-B1.

Example 4

Determination of one-step growth curve of Klebsiella pneumoniae phage pKP-M297-1.1

The host bacterial culture prepared in example 1 was mixed with the phage prepared in example 1 (multiplicity of infection: about 0.001), centrifuged at 10000rpm/min for 1min after incubation at 37℃for 10min, the supernatant was discarded, and the MH (B) broth was used to wash the pellet 2 times. Re-suspending the sediment with 5mL of preheated MH (B) liquid culture medium, rapidly placing in a shaking table at 37 ℃ for shaking culture, taking 200 μl of the culture every 10min from 0min, centrifuging at 4 ℃ for 2min at 10000rpm to remove bacteria, taking ten times of gradient dilution of the supernatant to appropriate concentration, and determining phage titer by a double-layer plate method. And drawing a one-step growth curve by taking the sampling time as an abscissa and the logarithm of phage titer as an ordinate. The results of the one-step growth curve are shown in FIG. 3, and the infected host bacteria enter the plateau at 60 min.

Example 5

Temperature and pH tolerance test of Klebsiella pneumoniae phage pKP-M297-1.1

6 sterile EP tubes were taken, 1mL of phage prepared in example 1 was added to each tube, and the phage titer was measured after the reaction was performed at-20℃at 4℃at 37℃at 50℃at 60℃at 70℃for 60 minutes. The detection results are shown in fig. 4: the phage can still keep higher activity after the phage acts for 1h at the temperature of-20 ℃ to 37 ℃, but the phage completely loses activity after the phage is incubated for 1h at 50 ℃.

7 parts of 100. Mu.L of phage prepared in example 1 were placed in MH (B) (900. Mu.L) at pH 2, 4, 6, 7, 8, 10, 12, respectively, and allowed to act at 37℃for 1 hour, and the titer of phage after the reaction was determined by a bilayer method. The detection results are shown in fig. 5: the Klebsiella pneumoniae phage pKP-M297-1.1 has small potency change in the environment with pH value of 7-8, and the activity is basically unchanged. After 1h of action in an environment with pH 4, the potency was significantly lower than in ph=6, and was completely inactivated after 1h in an environment with strong acid at ph=2 and strong base at ph=12.

Example 6

Analysis of the host profile of Klebsiella pneumoniae pKP-M297-1.1

The host profile of Klebsiella pneumoniae pKP-M297-1.1 was analyzed with 15 Klebsiella pneumoniae complex bacteria (Klebsiella pneumoniae, klebsiella pneumoniae-like, klebsiella pneumoniae producing gas), each of which was obtained from Shanghai, inc., and the product numbers are shown in Table 1. The specific operation is as follows: taking 100 mu L of 15 strains of bacteria culture bacteria liquid in a 10mL sterile centrifuge tube, adding a preheated semi-solid culture medium, pouring the mixture into a solid flat plate after the mixture is inverted and uniformly mixed, taking 10 mu L of phage liquid to drop on the surface of the flat plate after the phage liquid is solidified, and using PBS as negative control. After drying, the cells were inverted and incubated at 37℃for 12 hours. The results are shown in Table 1.

TABLE 1

Example 7

Sterilization effect of Klebsiella pneumoniae phage pKP-M297-1.1 in culture medium

EXAMPLE 1 preparation of a culture of host bacterium M297-1 for use to modulate OD ₅₉₅ To 0.1 (viable count is about 5×10) ⁸ cfu), ice bath for standby. Taking 4 branch separation tubes, respectively marking a control group, wherein MOI=100, 1 and 0.01, respectively adding 9mL of culture in an ice bath, adding 9mL of MH (B) into the control group, adding 9mL of phage solution corresponding to each group into an experimental group, rapidly subpackaging to a 1.5mL centrifuge tube, placing into a shaking table at a constant temperature of 37 ℃, and measuring absorbance of the culture at 595nm at intervals of 3 hours. Time as abscissa, OD ₅₉₅ Values are ordinate, and an in vitro bacteriostasis curve is drawn.

The phage sterilization experiment results are shown in FIG. 6, in which the control group (M297-1+MHB) had an OD within the first 21h ₅₉₅ The value is in an ascending trend, the bacterial load is gradually increased at the moment, and the bacterial load enters a platform stage after 21 h; when phage pKP-M297-1.1 was added at different transfection complex, the trend of the first 12h curve was approximately the same: within the first 6h, OD ₅₉₅ Values less than 0.05 and significantly lower than the control group indicate that klebsiella pneumoniae M297-1 was largely lysed. The test group moi=100 showed an increasing trend after 12h, but the values were always lower than the control group. Experiment group moi=0.01, moi=1 OD at 24h ₅₉₅ The value is smaller than 0.06 and is obviously lower than that of a control group, which indicates that the Klebsiella pneumoniae pKP-M297-1.1 has better antibacterial effect on the Klebsiella pneumoniae M297-1 under the infection complex.

Example 8

Klebsiella pneumoniae phage pKP-M297-1.1 protection experiment based on Chilo suppressalis larva model

Phage protection experiments were performed using the larvae of Chilo suppressalis as a model to evaluate the potential of phages for anti-infection. Healthy larvae weighing about 300mg were selected, starved for treatment one day prior to the test, and their immunity was reduced. The larvae were surface sterilized with a cotton swab containing 75% ethanol at the time of the test. Groups were made according to table 2, 10 per group, corresponding liquids were injected using microinjectors: 10 μl of the first injection was injected through the first left foot of the tail, and after 0.5h, the second injection was injected on the opposite side. Pigmentation and mortality of the larvae were recorded every 3 hours, for 72 hours. (if the larvae move, they respond to physical stimulus at the pipette tip and there is no sign of melanin formation, i.e., the larvae are considered to survive, if they do not respond to stimulus or the larvae are considered to die).

TABLE 2

Numbering device	First injection	Post injection for 0.5h	Remarks
				1	Physiological saline	Physiological saline	-
2	Physiological saline	Phage	-
				3	Bacterial liquid (M297-1), 5X 10 ⁴ CFU	Physiological saline	-
4	Bacterial liquid (M297-1), 5X 10 ⁴ CFU	Phage	MOI＝100
				5	Bacterial liquid (M297-1), 5X 10 ⁴ CFU	Phage	MOI＝1
6	Bacterial liquid (M297-1), 5X 10 ⁴ CFU	Phage	MOI＝0.01

The white control and phage safety test groups were free of blackening and death of the Chilo suppressalis larvae (A, B in FIG. 7). The larvae of the affected group had died within 72 hours after injection of M297-1 with a survival rate of 20% (C in fig. 7). While the larvae of Chilo suppressalis from the phage-treated group appeared slightly blackened (D, E and F in FIG. 7) compared to the placebo group, the mortality rate was significantly reduced within 72 hours. Moi=100, moi=1, and moi=0.01 group survival rates of 90%, 70%, and 60%, respectively; kaplan-Meier analysis showed significant differences between the three phage-treated groups and the infected group (moi=100, moi=1, moi=0.01) (P <0.05, fig. 8).

In conclusion, the Klebsiella pneumoniae phage pKP-M297-1.1 has a good application prospect in preventing, controlling and treating Klebsiella pneumoniae infection.

It should be understood by those skilled in the art that the above embodiments are exemplary embodiments only and that various changes, substitutions and alterations can be made hereto without departing from the spirit and scope of the application.

Claims

1. Klebsiella pneumoniae phage (Klebsiella pneumoniaphage) pKP-M297-1.1 deposited at the Guangdong province microbiological bacterial collection center (GDMCC), accession number: the collection number of the building 5 of the Guangzhou city first Zhonglu No. 100 college No. 59 is: GDMCCNo 63520-B1.

2. A formulation comprising klebsiella pneumoniae phage pKP-M297-1.1 of claim 1.

3. Use of klebsiella pneumoniae phage pKP-M297-1.1 according to claim 1 for the preparation of a formulation for preventing and treating multiple drug resistant klebsiella pneumoniae infection.