CN120608027A - A novel lytic bacteriophage and its application - Google Patents

Abstract

The present invention discloses a novel lytic phage and its application, relating to the field of biotechnology. The lytic phage was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 24, 2025, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 66026-B1. The phage can effectively lyse Escherichia coli, especially showing significant lysis ability against Escherichia coli producing extended-spectrum β-lactamase. The present invention provides important technical support for the development of new antibacterial drugs and treatment plans for Escherichia coli, showing broad application prospects and potential value.

Description

Novel cracking phage and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a novel cracking phage and application thereof.

Background

Coli is a symbiotic microorganism important in the human and animal intestinal tract, and its population dynamic balance has a key effect on host health. Multiple pathological processes including gastroenteritis, hemolytic uremic syndrome may be induced when dysbacteriosis leads to hyperproliferation of pathogenic E.coli or abnormal expression of toxigenic strains. Along with the global spread of antibiotic resistance phenomenon, the inhibiting effect of traditional antibacterial drugs on drug-resistant escherichia coli is obviously weakened, and clinical treatment faces serious challenges. The drug-resistant strain not only causes the prolongation of the course of disease and the increase of the treatment cost, but also is easier to form a transmission chain in special environments such as medical institutions and the like, thereby remarkably improving the risk of cross infection.

Phage, as a naturally occurring class of viruses, has the property of precisely recognizing and lysing specific bacteria, and its unique targeted bactericidal mechanism has attracted considerable attention in recent years in the biomedical field. The coliphage is used as a virus specially infecting escherichia coli, has obvious advantages in the aspect of controlling pathogenic escherichia coli infection, and has important research and application values. With the increasing problem of antibiotic resistance, phage therapy is becoming a natural, specific antimicrobial strategy, and is becoming a new therapeutic option against multiple drug resistant bacterial infections. Based on the above, developing a novel high-efficiency coliphage, optimizing the stability of the coliphage, expanding the antibacterial spectrum of the coliphage, and particularly enhancing the cracking capability of the coliphage to multi-drug resistant coliphage strains becomes a key scientific problem to be solved urgently in the current biomedical field.

Disclosure of Invention

The invention aims to provide a novel lytic phage and application thereof, so as to solve the problems in the prior art. The bacteriophage can crack escherichia coli, especially can crack and produce ultra-broad spectrum beta-lactamase escherichia coli, provides technical support for developing novel antibacterial agents and antibacterial schemes for inhibiting escherichia coli, and has good application prospect.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a lytic phage, which is an escherichia coli phage (ESCHERICHIA COLI PHAGE) vB_ EcoS _ GZMU _EI9, wherein the escherichia coli phage vB_ EcoS _ GZMU _EI9 is preserved in the microorganism strain preservation center of Guangdong province at 24 months of 2025, and the preservation address is No. 100 college No. 59 building 5 in Guangzhou city martyr, and the preservation number is GDMCC No:66026-B1.

The invention also provides application of the lytic phage in preparing a medicament for preventing and/or treating escherichia coli infection.

The invention also provides application of the lytic phage in preparing escherichia coli bactericides.

Further, the escherichia coli is beta-lactamase-producing escherichia coli.

The invention also provides a medicine for preventing and/or treating the escherichia coli infection, and the active ingredients comprise the lytic bacteriophage.

Further, the medicament also comprises pharmaceutically acceptable auxiliary materials.

Further, the medicament is in the form of injection, powder, gel, granule or freeze-drying agent.

Further, the medicine also comprises other active ingredients with antibacterial effect on escherichia coli.

The invention also provides an escherichia coli bactericide, and the active ingredients comprise the cracking phage.

Further, the escherichia coli bactericide is in a dosage form of spray, powder, gel, granule or freeze-dried agent.

The invention discloses the following technical effects:

The invention discovers a novel lytic coliphage (ESCHERICHIA COLI PHAGE) vB_ EcoS _ GZMU _EI9 which shows typical lytic phage characteristics. The phage has stable titer under the conditions of pH value ranging from 4 to 12 and temperature ranging from 4 to 50 ℃, and the highest titer can reach more than 1.35X10 ⁹ PFU/mL. The phage can effectively lyse escherichia coli, and particularly has remarkable lysis capacity for escherichia coli producing ultra-broad-spectrum beta-lactamase. The invention provides important technical support for developing novel antibacterial drugs and treatment schemes aiming at escherichia coli, and has wide application prospect and potential value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a phage morphology map;

FIG. 2 is a phage particle electron microscope image;

FIG. 3 is a phylogenetic analysis of a whole genome sequence based distance method construction;

FIG. 4 is a graph showing the results of thermal stability measurement;

FIG. 5 is a graph showing the results of pH stability measurement;

FIG. 6 is a graph of the results of a determination of optimal multiplicity of infection;

FIG. 7 is a graph showing the detection results of the antibacterial effect at different MOI after 7h and 12 h;

FIG. 8 is a graph showing the results of a cleavage kinetics assay;

FIG. 9 is a graph of phage one-step growth.

FIG. 10 is a graph showing the results of phage-inhibitory biofilm assay.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 isolation, identification and preservation of phages

1. Isolation and purification of phages

The method comprises the steps of collecting a water sample from the Guangdong Hui technology in China, removing most of impurities from the water sample through centrifugation (5000 rpm,10 min), filtering a liquid sample, and performing sterile filtration by using a filter membrane with the diameter of 0.22 mu m to remove bacteria and other macromolecular substances.

The phage were isolated using E.coli strain as host bacteria. And inoculating the treated sample and host bacteria into LB liquid culture medium, and placing the LB liquid culture medium in a shaking table at 37 ℃ for shaking culture until the bacterial liquid OD ₆₀₀ = 0.6, so as to ensure the growth of the host bacteria. Mixing the filtered liquid sample with host bacteria, adding a liquid culture medium, and placing the mixture in a shaking table at a constant temperature of 37 ℃ for co-culture overnight. The culture broth was centrifuged to remove bacterial residues, the supernatant (i.e., phage-containing liquid) was collected, and the bacteria were removed by filtration through a 0.22 μm sterile filter.

And detecting whether target phage exists in the sample through the spot test enrichment liquid, dropping the phage sample to be detected on a host bacterium culture plate, culturing overnight at 37 ℃, and observing whether transparent lysis spots appear at the dropping position, wherein the transparent spots are lysis areas formed after bacteria are infected by the phage. The phage was purified by a double-layer agar plate method until the size of the plaques on the plate became uniform, and a lytic phage (ESCHERICHIA COLI PHAGE) vB_ EcoS _ GZMU _EI9 (hereinafter abbreviated as E19) was obtained by multiple purifications, and the phage morphology was as shown in FIG. 1.

2. Phage titer detection

Phage titers were calculated by double-layer agar plate method. The bottom agar was poured into a plate and after solidification, it was allowed to cool completely. Phage solution was diluted in gradient, diluted solution was mixed with log phase host bacteria solution, incubated at 37 ℃ for 15min, mixed with 0.7% semi-solid LB agar and spread over the bottom plate, and cultured overnight at 37 ℃. If the phage infects bacteria, the bacteria will be lysed to form transparent lysed spots (plaques), and plates with 30-300 plaques in the field of view are selected for counting. Titers (PFU/mL) =number of plaques x 10 x dilution. The result shows that the titer of the phage can reach more than 10 ⁹ PFU/mL.

3. Phage identification

(1) Observation using a Transmission Electron Microscope (TEM)

For the observation of phage morphology, phosphotungstic acid negative staining was used. First, activated phage was dropped onto a 400 mesh carbon film copper mesh, and left to stand for 2 minutes, so that phage was sufficiently adsorbed on the copper mesh. Then, the excess liquid around the copper mesh was gently sucked off with filter paper, 1% phosphotungstic acid solution was added dropwise to carry out negative dyeing on the copper mesh for 1 minute, the excess phosphotungstic acid solution was sucked off again with filter paper, and the copper mesh was left to stand at room temperature and naturally dried. The observation was performed using a Hitachi transmission electron microscope, and the acceleration voltage was set at 80kV. The structural dimensions of phage were measured by Image J software. As shown in FIG. 2, phage E19 is a long-tailed phage with a non-contractile tail, an icosahedral structure with a head length of 61.+ -. 1nm, a tail length of 110.+ -. 1nm, and a tail width of 7.8.+ -. 0.3nm.

(2) Phage whole genome analysis

Phylogenetic tree was constructed using the MEGA 11.0.13 software by 1000 boottrap replicates using the distance method as shown in fig. 3. According to the international committee on viral classification (ICTV) standards, two phages should be classified as different species when their genomic sequence similarity is below 95%. Comparative analysis of the similarity of phage E19 to other phages using VIRDIC, E19 showed the highest sequence similarity to ESCHERICHIA PHAGE vB-EcoM-SHAK 7858 (Genbank number: OR 594183.1), 78.9% genomic similarity. Referring to the classification guidelines for BAVS, nucleotide sequences in a virus population that are more than 50% similar can be categorized as the same genus, the new species differs from the existing species by more than 5% at the nucleotide level, i.e., the similarity is no more than 95%. Since the similarity of the genome of E19 to other phages in the genus Kagunavirus falls within a range of more than 50% and less than 95%, E19 was determined to be a new species of the genus Kagunavirus.

4. Preservation of phages

Phage (ESCHERICHIA COLI PHAGE) vB_ EcoS _ GZMU _EI9 was deposited at the microorganism strain collection of Guangdong province at 24 th month of 2025, at accession number GDMCC No:66026-B1, building 5, university 100, guangzhou, martyr.

Example 2 phage host profiling

The host range of phage E19 was determined by testing different strains by spot experiments. 100. Mu.L of log phase host bacteria and 0.7% semisolid LB agar were mixed and spread on a culture dish to prepare a bacteria-carrying plate. mu.L of phage was dropped onto the surface of the plate and incubated overnight in a 37℃incubator, and if plaque appeared, it was shown that the phage infects the corresponding host bacteria. As a result, the cleavage efficiency of phage E19 was 42.37% (25/59), which includes 13 E.coli producing an ultra-broad spectrum of beta-lactamase (labeled ESBL in Table 1).

Table 1 phage vs. 59 E.coli host profile assay ("+" for lytic, "-" for non-lytic)

EXAMPLE 3 biological Properties of phage E19

1. Thermal stability

100. Mu.L of phage solution was added to 900. Mu.L of sterile LB liquid medium, and different temperature conditions (4 ℃, 37 ℃, 50 ℃,60 ℃, 70 ℃) were set, and the corresponding buffers were pre-cooled or pre-heated, respectively. After the mixture was allowed to stand at a specified temperature for 1 hour, phage titers were detected by a double-layer agar plate method. As a result, as shown in FIG. 4, phage E19 maintained a higher titer in the range of 4℃to 50℃and the titer was decreased at 60℃while phages were inactivated at 70℃or higher.

2. PH stability

Hydrochloric acid or sodium hydroxide solution was added to the sterile LB liquid medium, the pH was adjusted to a range of 1 to 13, and the bacteria were filtered using a microporous filter membrane of 0.22. Mu.m. 100. Mu.L of phage were added to 900. Mu.L of LB liquid medium at different pH values, incubated at 37℃for 1 hour, and phage titers were determined using the double-layer agar plate method. As a result, as shown in FIG. 5, phage E19 remained active at pH values ranging from 2 to 12.

3. Optimal multiplicity of infection

To determine the optimal multiplicity of infection of phages, the host bacterial liquid in logarithmic growth phase was diluted to 1X 10 ⁷ CFU/mL and infected with phages of different titers. Phage titers at different multiplicity of infection (MOI) were detected using a double-layer agar plate method. As shown in FIG. 6, phage E19 had an optimal multiplicity of infection of 0.01 and a titer of 1.35X10 ⁹ PFU/mL.

4. Kinetics of cleavage

The log phase host bacteria and phage solution were mixed in a 1:1 ratio to prepare 7 groups of different infectious complex numbers (1000, 100, 10,1, 0.1, 0.01, 0.001) and added to 96-well plates. The log phase host bacterial liquid is used as a control group, and is subjected to shaking culture at 37 ℃ and 220rpm, sampling is carried out every 1 hour, OD ₆₀₀ values are measured, and the experiment is repeated 3 times. As shown in FIGS. 7 to 8, the absorbance of the pure bacterial liquid in the control group rapidly increased, the host bacteria reached the logarithmic phase at 4 hours, and the host bacteria entered the stationary phase after 14 hours, with a statistical difference from the OD ₆₀₀ value of each phage group. The phage can inhibit the growth of the escherichia coli for 7 hours, and has stronger inhibition capability, but after 7 hours, the escherichia coli generates phage resistance, a phage group strain grows rapidly, the OD ₆₀₀ value is leveled with that of a control group about 16 hours, and the resistance appears earlier, so that the escherichia coli has stronger capability of generating the resistance to the phage.

5. One-step growth curve

Phage solution and log phase bacterial solution were mixed at the ratio of moi=0.01, and shake-cultured at 37 ℃ for 15 minutes. The mixture was then centrifuged for 10 minutes, the supernatant was discarded, and the pellet was washed by resuspension with LB liquid medium and incubated on a shaker at 37℃at 220 r/min. Samples were taken every 10 minutes to determine phage titer. As can be seen from FIG. 9, the incubation period of phage E19 was about 30 minutes, 30-40 minutes entered the burst phase, and about 110 minutes entered the plateau phase, and the final phage concentration was maintained at about 10 ⁹ PFU/mL.

6. Phage inhibition of biofilm effects

And judging the inhibition condition of the biological film by adopting a crystal violet staining method. Mixing log-phase host bacteria and phage solution 1:1, adding into 96-well plate, MOI is 100, 10, 1, taking equal amount of log-phase bacterial solution as positive control group (PC), taking equal amount of LB culture solution as blank control (NC), culturing in 37 deg.C incubator for 24 hr to form biofilm, and repeating three times per well. After incubation, the in-well broth was discarded and washed once with 200 μl PBS to remove planktonic bacteria. Washing, air drying, adding 200 mu L of 1% crystal violet solution for dyeing for 30 minutes, discarding and washing for three times, adding absolute ethyl alcohol for decoloration for 5 minutes after air drying, transferring the eluent to a new sterile 96-well plate, and using a multifunctional enzyme-labeled instrument to determine the OD value at 595nm wavelength. The results are shown in FIG. 10, where the OD values of all phage groups were statistically significantly different from that of the PC group, indicating that phage E19 effectively inhibited host biofilm formation.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (10)

1. A lytic phage, characterized in that the lytic phage is Escherichia coli phage vB_EcoS_GZMU_EI9; the Escherichia coli phage vB_EcoS_GZMU_EI9 was deposited in the Guangdong Provincial Microbiological Culture Collection on April 24, 2025, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 66026-B1. 2. Use of the lytic bacteriophage according to claim 1 in the preparation of a medicament for preventing and/or treating Escherichia coli infection. 3. Use of the lytic bacteriophage according to claim 1 in the preparation of an Escherichia coli bactericide. 4. The use according to claim 2 or 3, characterized in that the Escherichia coli is a β-lactamase-producing Escherichia coli. 5. A drug for preventing and/or treating Escherichia coli infection, characterized in that the active ingredient comprises the lytic bacteriophage according to claim 1. The drug according to claim 5 , further comprising a pharmaceutically acceptable excipient. 7. The drug according to claim 6, characterized in that the dosage form of the drug is an injection, powder, gel, granule or lyophilized agent. 8. The drug according to claim 5, characterized in that it further comprises other active ingredients having an antibacterial effect on Escherichia coli. 9. An Escherichia coli bactericide, characterized in that the active ingredient comprises the lytic bacteriophage according to claim 1. 10 . The E. coli fungicide according to claim 9 , wherein the E. coli fungicide is in the form of a spray, a powder, a gel, a granule or a lyophilized agent.