CN114164183A - Nocardia south Africa bacteriophage P69 and application thereof - Google Patents
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The invention discloses a nocardia south Africa bacteriophage P69 and application thereof, and belongs to the technical field of biology. The invention discloses a Nocardia south Africa bacteriophage P69 with the preservation number of CGMCC No.23088, which provides a selectable basic material for prevention and control of sludge foaming events caused by Nocardia and enriches a bacteriophage seed bank; through the separation and identification and whole genome sequencing of the phage P69, functional genes and proteins of the phage P69 are further deeply excavated, one or more enzymes having the cracking effect on Nocardia south Africa are provided for phage treatment and biological prevention, and the like, so that the host spectrum is expanded, and the universality and the effectiveness of the phage application are improved; the bacteriophage P69 can rapidly and efficiently crack Nocardia nanensis, and can be used for sludge foaming events caused by excessive propagation of Nocardia in a sewage treatment system.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nocardia south Africa bacteriophage P69 and application thereof.
Background
Nocardia south africana is a rare human pathogen. Like other nocardia, this bacterium causes disease after inhalation or vaccination through the skin. Nocardia south Africa infection is most common in patients with immunodeficiency, such as organ transplant patients or AIDS patients. Mortality in patients infected with nocardia nanafrica is up to 75%, and when the central nervous system is involved, 100%. Studies with experimental animals have shown that this organism is more virulent than nocardia brasiliensis (but less virulent than nocardia asteroides). In addition, nocardia africana has some resistance to aminoglycosides, including amikacin. Currently, trimethoprim sulfamethoxazole (TMP-SMX) is considered to be the most effective drug for treating nocardia infection, but in recent years, reports on drug-resistant strains of the drug are also gradually appeared.
In addition, nocardia south africana is also one of the bacteria that can cause foaming of activated sludge in sewage plants. The surface of the water-repellent gas-bubble stabilizing agent is hydrophobic and can be attached to the surface of the gas bubbles generated by aeration of the aeration tank, and the gas bubbles are stably kept on the water surface under the action of the surfactant. When the nocardia is excessively propagated, a large amount of stable foam floats on the surface of the aeration tank, so that the sludge sedimentation performance is poor, and the effluent is turbid. And when the bubbles are broken, the nocardia will diffuse to the air in the form of aerosol, bringing about a risk of disease. The traditional physical methods (such as salvaging, water spraying defoaming and the like) and chemical methods (such as adding aluminum salt, iron salt or adding disinfectant and the like) can not fundamentally solve the problem of excessive reproduction of the nocardia, and in addition, normal flora in an activated sludge system can be influenced, and the pollutant treatment capacity of the activated sludge is reduced. Therefore, development of new antibacterial agents is imperative.
Generally, bacteriophages have advantages over antibiotics: such as reduced disruption to normal flora (phage versus individual species or bacterial strains), certain self-limiting properties (reduced phage numbers when bacterial numbers are reduced), co-evolution between host and phage (phage can adapt to and infect bacterial resistant mutants that arise during treatment), "self-administration" and large numbers of phage only at the site of infection (they replicate at the site of infection). The phage proved to be safe and well tolerated, with few side effects, and did not appear to have toxic effects on animal cells itself. The type and number of phage is an important factor limiting the further development of phage therapy, and further isolation and identification of phage is required to enrich the phage repertoire.
Therefore, the problem to be solved by the technical personnel in the field is to provide the nocardia south africana bacteriophage P69 and the application thereof.
Disclosure of Invention
In view of the above, the invention provides a nocardia south Africa bacteriophage P69 and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a south African Nocardia bacteriophage P69 with the preservation number of CGMCC No.23088 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, CGMCC for short, China academy of sciences microorganism research institute No. 3, Xilu No. 1, North Chen, south China, at the address of Beijing, the preservation date is 2021 years, 9 months and 7 days, and is classified and named as bacteriophage, in particular to south African Nocardia long-tail bacteriophage.
Further, the application of the Nocardia south Africa bacteriophage P69 in Nocardia lysis.
Further, the application of the south Africa Nocardia bacteriophage P69 in preventing, treating and removing Nocardia in a sewage treatment system.
According to the technical scheme, compared with the prior art, the invention discloses and provides the nocardia south Africa bacteriophage P69 and the application thereof, provides a selectable basic material for sludge foaming and prevention and control of pathogenic bacteria, and enriches a bacteriophage seed bank; through the separation and identification and whole genome sequencing of the south African Nocardia bacteriophage P69, functional genes and proteins of the south African Nocardia bacteriophage are further deeply excavated, one or more enzymes having the cracking effect on Nocardia are provided for bacteriophage treatment and biological prevention, and the like, so that the host spectrum is expanded, and the application universality and effectiveness of the bacteriophage are improved; the south African Nocardia bacteriophage P69 can rapidly and efficiently crack south African Nocardia, and can be used for pollution control of south African Nocardia and removal of drug-resistant south African Nocardia in a sewage treatment system.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing a two-layer plate plaque morphology of the Nocardia nanafida phage P69 of the present invention;
FIG. 2 is a transmission electron microscope of Nocardia south Africa phage P69 according to the present invention; the scale bar is 100 nm;
FIG. 3 is a circle diagram of the genome of Nocardia south Africa phage P69 according to the invention;
FIG. 4 is a phylogenetic tree of the phage P69 genome of Nocardia south Africa according to the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The phage host bacterium nocardia nanafrica 4.1169 is provided by China general microbiological culture Collection center and is preserved by the laboratory.
Tryptone, yeast extract from OXOID; glucose, anhydrous calcium chloride from mclin; PEG8000 from Amresco; DNase, RNaseA from Sigma; proteinase K was purchased from Amresco; 0.22 μm microporous membrane purchased from New repurification device factories of Shanghai city; chloroform and equilibrium saturated phenol were purchased from biotechnology limited of Beijing ancient China.
EXAMPLE 1 isolation and purification of phage
Preparing a PYCa liquid culture medium:
at 1L ddH2Adding 1g glucose, 1g yeast extract and 15g tryptone into O, mixing uniformly, and autoclaving at 121 ℃ for 20 min. After cooling, 4.5mL of a 1mol/L calcium chloride solution was added.
Preparing a PYCa solid culture medium:
at 1L ddH2Adding 1g of glucose, 1g of yeast extract, 15g of tryptone and 15g of agar powder into the mixture O, uniformly mixing, and then carrying out autoclaving at 121 ℃ for 20 min. 4.5mL of a 1mol/L calcium chloride solution was added in an unsolidified state.
Preparation of PYCa semisolid culture medium:
at 1L ddH2Adding 1g glucose, 1g yeast extract, 15g tryptone and 7.5g agar powder into O, mixing, and autoclaving at 121 deg.C for 20 min. 4.5mL of a 1mol/L calcium chloride solution was added in an unsolidified state.
The sewage sample for the test is collected in a certain fishery in Beijing market in 9 months in 2020 and is used as a water sample for separating the phage.
Taking 10mL of sewage sample, and filtering and sterilizing the sewage sample by using a 0.22 mu m microporous filter membrane; adding 20mL of PYCa liquid culture medium, adding 2.0mL of logarithmic phase host bacterium suspension into a 50mL centrifuge tube, and uniformly mixing; after standing for 15min, it was shaken overnight at 30 ℃ (10 h). Centrifuging at 8000g for 10min at 4 deg.C the next day; and taking the supernatant, wherein the supernatant is the stock solution containing the host bacteriophage. And (3) identifying whether the stock solution contains the phage or not by adopting a spot-test method, dripping a proper amount of the stock solution on a PYCa solid culture medium coated with host bacteria, carrying out inverted culture at 30 ℃ for 48h, and observing whether the phage grows out or not. If clear and bright plaques appear on the plate, the existence of the phage in the stock solution is indicated, otherwise, the phage is not separated, and the sampling and separation are required again.
The separated phage is not necessarily single phage, and the invention adopts a double-layer plate method for further purification. Picking up the plaque on the plate, immersing the plaque in a sterile tube containing 1mL of SM solution, standing the plaque at room temperature for 1h, and diluting the plaque to 10 degrees in a ten-fold gradient manner-6. Take 0.5mL of the diluted solution and 1mL of the host suspension were added to a 5mL tube, followed by 3.5mL of PYCa semisolid medium, which was then quickly poured into PYCa solid medium and spread over the bilayer plate. Culturing at 30 deg.C for 48h, and observing the growth of plaque. A single plaque was then picked and this step repeated 5-6 times until the size and morphology of each plaque in the double-layered plate was essentially identical, as shown in FIG. 1.
Example 2 morphological Observation of phages
And (3) dripping 20 mu L of phage suspension on a copper net, after the phage suspension is naturally precipitated for 10min, sucking the phage suspension from the side by using dry filter paper, airing for about 1min, adding 1 drop of 1% uranyl acetate on the copper net, dyeing for 2min, carefully sucking the excess dye from the side by using the dry filter paper, naturally airing for 30min in a dark place, and observing by using a transmission electron microscope (JEM-1400).
The results of transmission electron microscopy, as shown in FIG. 2, show that phage P69 has a spherical head with a diameter of about 68.47nm and a longer tail with a length of about 239.25nm and a tail diameter of about 13.12 nm. According to the eighth report of the international organization for taxonomy of viruses (ICTV) classification of viruses, this strain of bacteriophage P69 was initially classified as the long tail phage family.
Example 3 phage genome analysis identification
Phage whole genome sequencing and analysis: DNA of the samples was sequenced using illuminainnextseq 500 for PE 2 x 150. And splicing the optimized sequences by using spades v.3.11.1 splicing software to obtain an optimal assembly result. The open reading frame of the genome is subjected to predictive analysis by using biological software PHASTER, NCBI Blastp is used for completing the primary annotation of functional genes, tRNAscan-SE (http:// lowelab. ucsc. edu// tRNAscan-SE /) is used for predicting tRNA on line, CGView Server software (http:// CGView. ca /) is used for completing the drawing of a whole genome atlas (see figure 3), and a system evolutionary tree is constructed by using MEGA X software based on whole genome data.
The result showed that the genome size of phage P69 was 68913bp, and the G + C content was 65.56 mol%. From the Blast alignment results, it appears to be a highly novel phage, the most homologous of which is the phage Pleakley (78.48%), the host of which is Gordonia. The CDS region was predicted by prokka, and a total of 90 CDS and one tRNA-Ser were found. From the matching results, the genome has a typical phage modular structure. Comprises a DNA replication module, a structural protein gene, a lysis module and a DNA packaging gene. Homology alignment and phylogenetic tree analysis (see FIG. 4) showed that Phage P69 has a closest relationship to Gordonia Phage Faxboro. Through experimental determination of host spectra (see table 1), the phage P69 can only crack part of Nocardia, and the specificity is strong.
TABLE 1 host Spectroscopy assay of bacteriophage P69
Note: after infection with phage, "+" indicates the presence of plaques and "-" indicates the absence of plaques;
CGMCC＝China General Microbiological Culture Collection Center。
the previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. A Nocardia south Africa bacteriophage P69 is characterized in that the preservation number is CGMCC No. 23088.
2. The use of a nocardia south africana bacteriophage P69 according to claim 1 for lysing nocardia.
3. The use of a nocardia south africana bacteriophage P69 according to claim 1 for the control and removal of nocardia in a wastewater treatment system.
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| CN114774370A (en) * | 2022-04-01 | 2022-07-22 | 中国科学院大学 | Catfish Edwardsiella phage EIP-1 and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994025572A1 (en) * | 1993-04-29 | 1994-11-10 | Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University | Mycobacterial species-specific reporter mycobacteriophages |
| US20060094076A1 (en) * | 2004-11-01 | 2006-05-04 | Stave James W | Bacteriophages as selective agents |
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2021
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| CN114774370A (en) * | 2022-04-01 | 2022-07-22 | 中国科学院大学 | Catfish Edwardsiella phage EIP-1 and application thereof |
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