CN113789305B

CN113789305B - Lytic coliphage preparation and its preparation and application

Info

Publication number: CN113789305B
Application number: CN202111074413.0A
Authority: CN
Inventors: 汤芳; 戴建君; 王瑜欣; 薛峰; 李一昊; 曾颃
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-09-10
Filing date: 2021-09-14
Publication date: 2023-05-12
Anticipated expiration: 2041-09-14
Also published as: CN113789305A

Abstract

The invention discloses a lytic escherichia coli phage preparation and application thereof, wherein the preparation comprises phages PNJ1902, PSD2001 and PSD2002; wherein, the deposit number of phage PNJ1902 is GDMCC NO:61890-B1, phage PSD2001 deposited under the accession number GDMCC NO:61891-B1, phage PSD2002 accession number GDMCC NO:61892-B1. The host bacteria of the phage preparation are E.coli O157: h7 (ATCC: 43895) which has an effective removal ability for biofilms of different ages of E.coli ATCC:43895, and examples of application in the intestinal tract of mice have also been confirmed to have a good effect when used as an in vivo bacteriostatic agent.

Description

Lytic coliphage preparation and its preparation and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a lytic escherichia coli phage preparation and application thereof.

Background

Coli O157: h7 (ATCC: 43895) (purchased from Testonio, product number TS 205491) as a zoonotic bacterium can cause human Hemorrhagic Colitis (HC), sometimes complicated with Hemolytic Uremic Syndrome (HUS), serious cases with renal failure and high mortality. The separation rate of E.coli ATCC43895 in pig, cow dung, pork and beef is very high. It is mainly transmitted by food and poses a threat to public health. Meanwhile, it was found that the E.coli ATCC43895 developed a drug resistant strain worldwide since 1997. Thus, there is an urgent need for new antibiotic substitutes to prevent and treat E.coli ATCC:43895 disease.

As a virus which effectively kills bacteria, the application value of the phage in the aspect of treating diseases related to bacterial infection is reevaluated. The phage as a natural antibacterial agent has low inherent toxicity, can replicate itself, is highly specifically combined with a host, can co-evolve with the host, and can effectively treat infection caused by drug-resistant bacteria. Compared with a mouse model for treating escherichia coli infection by antibiotics, phage therapy can reduce the content of escherichia coli in vivo, ensure the normal growth of the mouse, and can not influence intestinal flora. The method provides a new idea for treating gastrointestinal tract infection caused by drug-resistant bacteria in the future. However, at present, the easy generation of resistance of bacteria to single phage makes single phage have great limitation in application, namely the existing single phage has good effect of starting to lyse host bacteria, but resistant bacteria are generated soon, so that the single phage loses the lysis effect of the resistant bacteria, and the production requirement cannot be met.

Disclosure of Invention

The invention aims to provide a lytic escherichia coli phage preparation and application thereof, wherein host bacteria of the phage preparation are escherichia coli O157: h7 (ATCC: 43895) which has an effective removal ability for biofilms of different ages of E.coli ATCC:43895, and examples of application in the intestinal tract of mice have also been confirmed to have a good effect when used as an in vivo bacteriostatic agent.

It is an object of the present invention to provide a lytic E.coli phage preparation comprising phages PNJ1902, PSD2001 and PSD2002; wherein, the deposit number of phage PNJ1902 is GDMCC NO:61890-B1, phage PSD2001 deposited under the accession number GDMCC NO:61891-B1, phage PSD2002 accession number GDMCC NO:61892-B1.

In some embodiments, the phage preparations described herein have a titer ratio of phage PNJ1902, phage PSD2001, and phage PSD2002 of (1-3): (1-3): (1-3), preferably, the titer ratio of phage PNJ1902, phage PSD2001, and phage PSD2002 is 1:1:1.

the applicant isolated phage PNJ1902 from chicken farm in Nanjing, jiangsu province, and screened phage PSD2001 and phage PSD2002 from duck farm in mountain-east, and deposited with the microorganism strain collection in Guangdong province (address: building 5 of No. 59 of Mitsui 100 in Mitsui, guangzhou, md.) on day 26 of 2021, wherein PNJ1902 is named as: coli O157: h7 phage PNJ1902, classified under Escherichia coli phage, deposit No. GDMCC NO:61890-B1; the PSD2001 is named: coli O157: h7 phage PSD2001, class designation Escherichia coli phage, deposit No.: GDMCC NO:61891-B1; the PSD2002 is named: coli O157: h7 phage PSD2002, class designation Escherichia coli phage, deposit No.: GDMCC NO:61892-B1.

Phage PNJ1902 in the present invention belongs to the family Long-tail phage (Siphoviridae). There is a long, non-collapsible tail sheath, the shell belonging to a regular polyhedron. Head diameter (L) =60 nm, head transverse diameter (W) =55 nm, L/w=1.09, tail sheath, tail length=196 nm, width=11 nm.

Phage PSD2001 of the present invention is a myophagidae phage (Cercoviridae). PSD2001 has a polyhedral symmetric head and a telescoping tail. Head diameter (L) =118 nm, head width (W) =79 nm, L/w=1.49. Tail shrink (tail length=116 nm, tail width=19 nm).

Phage PSD2002 of the invention belongs to the Myoglyceae phage (Cercoviridae). PSD2002 has a polyhedral symmetric head and a telescoping tail. Head diameter (L) =121 nm, head width (W) =83 nm, L/w=1.46. Tail shrink (tail length=121 nm, tail width=20 nm).

The three phages in the phage preparation have strong cracking performance on escherichia coli ATCC 43895. After mixing the three phages, a synergistic effect between the phages was found to have a more efficient inhibition than that of a single phage.

It is another object of the present invention to provide the use of said phage preparation for the preparation of E.coli inhibitors.

Another object of the present invention is to provide the use of said phage preparation for the preparation of E.coli biofilm remover.

Another object of the present invention is to provide the use of said phage preparation in the manufacture of a medicament for the treatment of diseases caused by E.coli.

The escherichia coli is escherichia coli ATCC 43895.

Another object of the present invention is to provide three lytic E.coli phages, phage PNJ1902, phage PSD2001 and phage PSD2002, respectively; wherein, the deposit number of phage PNJ1902 is GDMCC NO:61890-B1, phage PSD2001 deposited under the accession number GDMCC NO:61891-B1, phage PSD2002 accession number GDMCC NO:61892-B1.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) In the present invention, phage cocktails composed of phages PNJ1902, PSD2001 and PSD2002 have a synergistic effect on the inhibition of planktonic E.coli ATCC43895 in LB medium.

(2) In the invention, the phage cocktail consisting of phages PNJ1902, PSD2001 and PSD2002 has remarkable antibacterial effect on planktonic E.coli ATCC43895 in LB medium for a long time.

(3) In the present invention, phage cocktails consisting of phages PNJ1902, PSD2001 and PSD2002 are effective in removing biofilms of different ages of E.coli ATCC 43895.

(4) Phage cocktails consisting of phages PNJ1902, PSD2001 and PSD2002 can be used for disease treatment 8h after mice were enterically infected with E.coli ATCC 43895.

(5) The phage cocktail consisting of phages PNJ1902, PSD2001 and PSD2002 has higher protection rate for mice infected with E.coli ATCC43895, and can promote weight gain of mice faster and promote recovery of inflammatory factors to normal level compared with antibiotic treatment.

Drawings

FIG. 1 is a transmission electron micrograph (scale bar represents 200 nm) of phages PNJ1902, PSD2001 and PSD2002;

FIG. 2 shows the in vitro lytic capacity of phage cocktail against E.coli ATCC43895 (conditions n=10, P.ltoreq.0.01, P.ltoreq.0.001 per group. The values shown are averages of 3 independent experiments);

FIG. 3 shows the biofilm removal effect of phage cocktails on E.coli ATCC43895 (conditions n=10, P.ltoreq.0.01, P.ltoreq.0.001 per group. The values shown are averages of 3 independent experiments);

FIG. 4 is the protection rate of phage cocktails against mice infected with E.coli ATCC 43895;

FIG. 5 is a graph comparing the effect of phage cocktail and enrofloxacin treatment on mice weight when infected with E.coli ATCC 43895;

FIG. 6 is a graph comparing the inhibition of E.coli ATCC43895 induced inflammatory response by phage cocktail and enrofloxacin.

Description of the preferred embodiments

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

The configuration of the medium used in the following examples:

luria Bertani (LB) liquid medium: to 950mL of deionized water, 10g of tryptone, 5g of yeast extract and 10g of NaCL were added and dissolved. The pH was adjusted to 7.0 with 5 mol/LNaOH. With ddH ₂ O is fixed to volume to 1L. Sterilizing with steam at 121deg.C for 20min.

LB solid medium: to 950mL of deionized water, 10g of tryptone, 5g of yeast extract, 10g of NaCl, and 20g of agar powder were added and dissolved. The pH was adjusted to 7.0 with 5 moL/LNaOH. With ddH ₂ O is fixed to volume to 1L. Sterilizing with steam at 121deg.C for 20min.

LB semisolid culture medium: adding 0.5% agar into LB liquid medium, stirring, sterilizing at 121deg.C for 20min, and cooling to about 50deg.C for immediate use.

Sodium-Magnesium (SM) solution: weighing 0.58g NaCL,0.2g MgSO ₄ ·7H ₂ O, 5mL of 1M Tris-HCl (pH 7.5) and 5mL of 2% gelatin were then added, followed by 80mL of ddH ₂ And (3) in O, stirring uniformly and fully dissolving, then fixing the volume to 100mL, sterilizing at 121 ℃ for 20min, and preserving at 4 ℃ for later use.

PBS buffer: accurately weighing KH ₂ PO ₄ 0.2g、Na ₂ HPO ₄ 2.13g, naCL 8.0g and KCL 0.2g, dissolved in 500mL distilled water, and then subjected to constant volume to 1L, autoclaved at 121 ℃ for 15min, and stored at normal temperature.

The main instrumentation used in the following examples: ultra clean bench (Sujing Antai SW-CJ-2 FD), normal temperature bench centrifuge (Eppendorf 5424), chilled bench centrifuge (Eppendorf 5424R), normal temperature shaker (Wiggens WS-600, germany), microbiological incubator (Thermo Fisher IGS), thermostatic water bath (Thermo Fisher GP 05), high speed bench chilled centrifuge (Thermo Fisher Multifuge X R), vortex mixer (SCILOGEX MX-S), 4 degree refrigerator (Thermo Fisher PLR 386), ultra low temperature refrigerator (Mitsubishi 528L, midday, pure water system (Mitsubix Essential 5+Milli-Q Reference), high capacity thermostatic shaker (shaker) (Taicang Huamei THZ-25), electromagnetic oven, microwave oven, etc.

EXAMPLE 1 morphological observations and sequencing of three phages

1.1 electronic microscope morphological observations

Absorbing 10 mu L of phage suspension on a copper net, precipitating for 10min, absorbing excessive liquid by using filter paper, then dripping 10 mu L of phosphotungstic acid (PTA) for dyeing for 5-10min, naturally drying, and observing phage morphology under a Transmission Electron Microscope (TEM).

1.2 genome sequencing and analysis

Raw sequencing data of phage genomes were mass filtered using trimmonic v0.39 to remove linker sequences and data with a mass less than 15 over a 4bp floating range. The raw data were then aligned with the genome of the isolated host (E.coli ATCC: 43895) to filter any residual contamination of the bacterial genome using BBDuk, a BBmap-pack tool. The filtered raw data were assembled using a Unicycler v0.43 with a minimum contig length of 1000bp. The assembled genome was annotated with RAST server v 2.0. The lytic nature of the phage genome was also checked using VIBRANT, and the presence of antibiotic resistance and virulence genes was checked using various resistance and virulence gene finder databases of phages implemented in ABRicate (https:// gitsub. Com/tseemann/ablicate).

2 results

2.1 morphological observations of phages

PNJ1902 belongs to the family Long-tail phage (Siphoviridae). A long non-contractible tail sheath is arranged, and the shell belongs to a regular polyhedron; head diameter length (L) =60 nm, head transverse diameter (W) =55 nm, L/w=1.09; tail sheath with tail length=196 nm, width=11 nm. PSD2001 and PSD2002 phages belong to the Myoglyceae phage (Cercoviridae). PSD2001 is a regular polyhedral structure with a three-dimensionally symmetric head; head diameter length (L) =118 nm, head width (W) =79 nm, L/w=1.49; with a contracted tail (tail length=116 nm, tail width=19 nm). PSD2002 is also a regular polyhedral structure with three-dimensionally symmetric heads, head diameter length (L) =121 nm, head width (W) =83 nm, L/W=1.46; with a contracted tail (tail length=121 nm, tail width=20 nm). The morphology of phages PNJ1902, PSD2001 and PSD2002 is shown in FIG. 1.

2.2 phage sequencing

The genomes of phages PNJ1902, PSD2001 and PSD2002 were subjected to nucleic acid sequence alignment at NCBI, which showed that:

the coverage of the PNJ1902 was 91%, 90%,90% and 90% identity of the Salmonella phage vB _sals_abtnlsp9, salmonella phage vB _sals_abtnlsp4, salmonella phage SE8, salmonella phage vB _sen-E22, escherichia phage T5_ev212 and Salmonella phage SE11 alignment was 98.46%, 96.45%, 97.44%, 96.85%, 96.98% and 97.35% respectively. However, the gene of the fiber protein encoded by PNJ1902 has a large difference from the phages with higher similarity, and the homology is <12%. The gene sequence of the fiber protein of PNJ1902 is shown in SEQ ID NO. 1.

Coverage of PSD2001 aligned with phages Escherichia phage P479, escherichia phage UPEC01, escherichia phage MN01, escherichia phage UPEC07 was 92%, 91%, 90%, respectively, and identity was 97.13%, 96.17%, 96.45%, 95.91%, respectively. The gene of the fiber protein encoded by PSD2001 has larger difference with the phages with higher similarity, and the homology is less than 30%. The gene sequence of the fiber protein of PSD2001 is shown as SEQ ID NO. 2.

The coverage of PSD2002 aligned with phages Escherichia phage WG01, escherichia phage vB _EcoM_FB, shigella phage PSD9, enterobacteria phage QL01, enterobacteria phage vB _EcoM_IME540, enterobacteria phage Bp7, enterobacteria phage JS98, escherichia phage MN04, escherichia phage MX01, salmonella phage vB _SalM_ABTNLsp5 is 95%, 92%, 91%, 90%, the identity was 98.36%, 97.14%, 95.36%, 95.38%, 97.92%, 96.42%, 92.77%, 93.44%, 95.36%, 95.35%, respectively.

Genome annotation results and comparative genome analysis results of the three phages show that the phages PNJ1902 and PSD2001 have certain differences with corresponding phages with higher genome similarity in coding and structure assembly, DNA replication, regulation and control functions and the like, and particularly, the genes for coding the fiber proteins are different. Both phages were shown to be new phages. Phage PSD2002 is highly similar to phages Escherichia phage WG01, escherichia phage vB _EcoM_FB and Shigella phage PSD 9.

In addition, the genome of the phage is analyzed for virulence factors and drug resistance genes, genes which code for genes related to virulence or drug resistance are not found, and the result shows that the mixed phage has no potential safety risk in the application of biosafety prevention and control in animal intestinal tracts.

Example 2 determination of the in vitro lytic Capacity of phage cocktail against E.coli ATCC43895

E.coli ATCC43895 cultured in LB liquid to OD _600nm A value of 1, a concentration of about 10 ⁸ CFU/mL. Culturing fresh phage solution by double-layer agar method, and adjusting concentration to 10 ⁷ PFU/mL. mu.L of E.coli ATCC43895 and an equal volume of a single phage or phage mixture 1:1:1 (abbreviated as phage cocktail) were added to a 96-well plate and mixed well, after which the 96-well plate was placed in a thermostatted shaker at 37℃at 180rpm/min. OD of the mixed solution was measured every 1 hour _600nm Values (0 in sterile LB solution) and data were recorded. The experiment was performed in a control group, and an equal amount of LB solution was added.

The test results showed that the phage cocktail significantly inhibited growth of planktonic E.coli ATCC43895 and was less likely to develop resistant strains within 35h compared to the use of a single phage (FIG. 2, E.coli ATCC43895 incubated alone (circles) at 37 ℃ C.; E.coli ATCC43895 incubated with phage PNJ1902 (squares), PSD2001 (right triangles), PSD2002 (reverse triangles) or phage cocktail (diamonds), respectively, per 1h readTake one OD ₆₀₀ _nm Values). And the cracking capacity is stable, the long-time antibacterial effect is achieved, and the good inhibition effect is achieved for at least 35 hours.

EXAMPLE 3 capability of phage cocktail to clear biofilm from E.coli ATCC43895

The three phages were mixed in a ratio of 1:1:1 to prepare a phage cocktail. After culturing E.coli ATCC43895 in 96-well plates for 12 hours, 24 hours, 48 hours and 60 hours, washing with PBS 3 times to remove planktonic bacteria and the medium, respectively, 200. Mu.L of phage cocktail (10 ⁷ PFU/mL) was incubated for 24h. Crystal violet staining was used to confirm the clearance of bacterial biofilms by phage. Biofilm samples treated with phage cocktail were slowly washed 3 times with PBS to remove planktonic bacteria and media. Thereafter, 200. Mu.L of 95% methanol solution was added to each well, and the mixture was fixed at room temperature for 30 minutes, followed by rinsing with PBS 3 times. Each well was added with 0.1% crystal violet solution for 30min, each well was rinsed 3 times with running water, and finally 200. Mu.L of 30% glacial acetic acid was added for 15min. Measuring OD using a spectrophotometer _600nm And records the data.

The experimental results showed a significant decrease in biofilm at ages 24h,36h,48h and 60h compared to the control (figure 3). The results of the significance analysis showed that: 24h biofilm (P.ltoreq.0.01), 36h biofilm (P.ltoreq.0.01), 48h biofilm (P.ltoreq.0.001) and 60h biofilm (P.ltoreq.0.001). In addition, phage PSD2002 has little biofilm removal capacity. However, phages PNJ1902, PSD2001 and phage cocktails of the present application have a strong capacity to remove biofilms of E.coli ATCC43895 of different ages.

EXAMPLE 4 determination of the therapeutic Effect of phage cocktail on mice infected with E.coli ATCC43895

The three phages were mixed in a ratio of 1:1:1 to prepare a phage cocktail. 40 4 week old SPF grade ICR mice (females) were divided into 4 groups (10 mice/group): group 1, mice were orally gavaged with 200. Mu.L of E.coli ATCC43895 (1.29X 10) ⁹ CFU/mL/patient), 200. Mu.L phage cocktail (3.6X10) was lavaged after 8h ⁸ PFU/mL/treatment (E.coli+phage cocktail group); group 2, mice were orally gavaged with 200 μl PBSThe same volume of phage cocktail treatment (PBS + phage cocktail group) was given after 8 hours; group 3, mice were orally gavaged with equal volumes of E.coli ATCC43895, treated with equal volumes of PBS after 8h (positive control group); group 4, mice were orally perfused with equal volumes of PBS. It is adapted for 2-3d before lavage.

At test day 0, mice were orally subjected to 100. Mu.L of 1M sodium bicarbonate to neutralize gastric acid for 5min, and after neutralization with 200. Mu.L of bacterial culture (1.29X 10) ⁹ CFU/mL) were inoculated with post-challenge phage cocktail treatment group and post-challenge PBS control group mice. After 8h phage cocktail (3.6X10) ⁸ PFU/mL), phage cocktail treatment group after gastric lavage and bacterial cocktail group alone, and after 24 hours, gastric lavage phage cocktail was repeated 2 times. At the same time 200. Mu.L PBS after gastric lavage and sterilization and PBS control group and simple PBS control group, 24 hours later, the test was repeated 4 times. After 2 weeks of continuous observation, survival of each group of mice was determined and survival curves were drawn.

Test results: after the phage cocktail is orally taken, the mice in the PBS+phage cocktail group and the negative control group are not dead, and the survival rate of 4 tests is 100%; the survival rate of the coliform+phage cocktail group was 100% for 2 times and 90% for 2 times. The positive control group of 10 mice all died, with a survival rate of 0% for four trials (fig. 4, number of mice per group of 10. For each group of mice, the results shown were from at least 2 independent experiments), indicating that the phage cocktail had a higher protection rate for mice infected with e.coli ATCC 43895.

Example 5 Effect of phage cocktail treatment on weight gain in mice

The three phages were mixed in a ratio of 1:1:1 to prepare a phage cocktail. Experiment 30 female SPF ICR mice (4 weeks old) were randomly divided into 3 groups (10 mice/group): (1) Coli + phage cocktail (phage treatment group); (2) E.coli+enrofloxacin (antibiotic treatment group); (3) PBS+PBS (negative control group). Prior to the experiment, all mice were gavaged with 100. Mu.L of 1M NaHCO ₃ To neutralize stomach acid. After 5min, mice were perfused with 200. Mu.L of E.coli (1.29X 10) ⁹ CFU/mL) or PBS. After 8h, the mice of the different groups were respectively gavaged with 200. Mu.L phage cocktail (3.6X)10 ⁸ PFU/mL), enrofloxacin (10 mg/kg), or PBS. The same dose of phage cocktail and enrofloxacin was repeated 2 times at 24h intervals for the 2 nd lavage. The body weight of the mice was monitored daily for the following 2 weeks.

Test results: the monitoring result of the weight change of the mice shows that the weight of the mice is obviously reduced (P is less than or equal to 0.01) compared with the negative control group on the 3 rd day after the mice are infected with escherichia coli (figure 5). After treatment, the weight of the mice in the phage-treated group on day 17 was not significantly different from that in the negative control group (P.gtoreq.0.05), while the weight of the antibiotics-treated group was significantly lower than that in the negative control group (P.gtoreq.0.001). Test results indicate that the phage cocktail treatment is more beneficial to recovery of mouse body weight than antibiotic treatment.

Example 6 Effect of phage cocktail treatment on expression levels of inflammatory factors in mice

Test 30 female SPF ICR mice (4 weeks old) were randomly divided into 3 groups (10 mice/group): the test groups and treatments were the same as in example 5, and then IL-6, IL-1. Beta. And TNF-. Alpha.inflammatory factor levels in the duodenum and colon of the mice were monitored every 2 days, and the detection of inflammatory factor transcription levels was performed using ELISA kit (NJCBIO, china) 3 times per test.

Test results: the results showed that the levels of inflammatory factors IL-1. Beta., IL-6 and TNF-. Alpha.in mice were significantly increased (P.ltoreq.0.05) on day 1 compared to the negative control group after infection with E.coli (FIG. 6). While after treatment, all inflammatory factor levels in phage-treated groups decreased faster than in antibiotic-treated groups and more closely approached the level of negative control groups (fig. 6). The results indicate that phage treatment can return expression of inflammatory factors to healthy levels faster in mice than antibiotics.

Sequence listing

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ggatattcaa tggccaacaa ctgggctggt aagtatgtgg ccggtggtgg tggtggtcgt 540

ccatatggtt taggcggtac taatggtgcc gcttggcccg gtggtaatgc ttcttatgaa 600

agtccaggag ctggtggtaa tacagggcaa tattgggccg gtgcaggtgg cgatgctggt 660

gcccctggtc aggctgcaaa tcctggtcat ggttattcta cccctcctat ggccgccgga 720

cgcgcggtgt tcggaagttc tcctgcttgg ggggcaacgg gcactatcta tggctcatgg 780

ttatag 786

Claims

1. A lytic escherichia coli phage preparation, characterized in that said preparation comprises phages PNJ1902, PSD2001 and PSD2002; wherein the class of phage PNJ1902 is designatedEscherichia coli phageDeposit No. GDMCC NO:61890-B1, class of phage PSD2001 is designated asEscherichia coli phageDeposit No. GDMCC NO:61891-B1, class of phage PSD2002 is namedEscherichia coli phageDeposit No. GDMCC NO:61892-B1.

2. The lytic escherichia coli phage preparation of claim 1, wherein the titer ratio of phages PNJ1902, PSD2001 and PSD2002 in the phage preparation is (1-3): (1-3): (1-3).

3. The lytic escherichia coli phage preparation of claim 2, wherein the titer ratio of phages PNJ1902, PSD2001 and PSD2002 in the phage preparation is 1:1:1.

4. use of a lytic coliphage preparation according to any one of claims 1-3 for the preparation of an coliform inhibitor.

5. The use according to claim 4, wherein the E.coli is E.coli ATCC 43895.

6. Use of a lytic coliphage preparation according to any one of claims 1-3 in the preparation of a biofilm remover for escherichia coli.

7. The use according to claim 6, wherein the E.coli is E.coli ATCC 43895.

8. Use of a lytic coliphage preparation according to any one of claims 1-3 for the preparation of a medicament for the treatment of a disease caused by escherichia coli.

9. The use according to claim 8, wherein the E.coli is E.coli ATCC 43895.

10. Coli phage PNJ1902, classified and namedEscherichia coli phageDeposit No. GDMCC NO:61890-B1.

11. Coli phage PSD2001, classified and namedEscherichia coli phageDeposit No. GDMCC NO:61891-B1.

12. Coliphage PSD2002, classified and namedEscherichia coli phageDeposit No. GDMCC NO:61892-B1.

13. Use of phage PNJ1902 of claim 10, phage PSD2001 of claim 11 or phage PSD2002 of claim 12 in the preparation of an escherichia coli inhibitor, in the preparation of an escherichia coli biofilm remover or in the preparation of a medicament for the treatment of a disease caused by escherichia coli.

14. The use according to claim 13, wherein the E.coli is E.coli ATCC 43895.