CN112195160A - Bacteriophage and application thereof - Google Patents
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- CN112195160A CN112195160A CN202011115726.1A CN202011115726A CN112195160A CN 112195160 A CN112195160 A CN 112195160A CN 202011115726 A CN202011115726 A CN 202011115726A CN 112195160 A CN112195160 A CN 112195160A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The application relates to the technical field of biomedicine, and particularly relates to a bacteriophage and application thereof. The bacteriophage can be used for cracking serratia marcescens, and is preserved in Guangdong province microorganism strain preservation center with the preservation number of GDMCC NO. 61140. The bacteriophage provides an effective scheme for clinical treatment of serratia marcescens infection.
Description
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to a bacteriophage and application thereof.
Background
Phages (Phage) are widely distributed in nature and are found in the soil or in the intestinal tract of animals. Bacteriophages are intracellular parasites and have strict host specificity. In recent years, as pathogenic bacteria continuously find out drug resistance phenomena in clinic, even multiple drug resistance, the substitution treatment by using specific phage is the current research direction.
Serratia marcescens (Serratia marcocens) is widely distributed in the nature, exists in water, soil and environment in a large amount, is a common conditional pathogen in clinic, can cause infection of lung, urethra, meninges and wound, and can cause septicemia in severe cases. The use of bacteriophages for the treatment of resistant bacteria has become an alternative because of the abuse and non-regulatory use of antibiotics, which also have developed clinical resistance to Serratia marcescens, and the lengthy and costly development of new antibiotics. However, there is currently no phage against Serratia marcescens.
Disclosure of Invention
The application aims to provide a bacteriophage and application thereof, and aims to solve the technical problem of drug resistance of serratia marcescens.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a bacteriophage which cleaves serratia marcescens, the bacteriophage being deposited at the Guangdong province collection of microorganisms with accession number GDMCC NO. 61140.
The application provides the phage which is separated from the nature and can strongly crack the serratia marcescens, so that the problem of drug resistance of the serratia marcescens in clinic can be effectively solved, and an effective scheme is provided for clinic treatment of serratia marcescens infection.
In a second aspect, the present application provides a use of a bacteriophage as described herein for the preparation of an agent against serratia marcescens.
The phage provided by the application can strongly crack serratia marcescens, so that the phage can be used for preparing an anti-serratia marcescens agent and effectively treating the infection of the serratia marcescens clinically.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph of the in vitro growth kinetics of bacteriophages provided in the examples of the present application;
FIG. 2 is a graph showing the effect of phage on the in vitro lysis of Serratia marcescens provided in the examples of the present application;
FIG. 3 is a graph showing the effect of the phage lytic ability assay provided in the examples of the present application.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
In a first aspect, the embodiments of the present application provide a bacteriophage, wherein the bacteriophage lyses serratia marcescens, and the bacteriophage is deposited in the Guangdong province culture Collection of microorganisms with the deposit number GDMCC NO. 61140.
Specifically, Serratia marcescens is derived from mosquito intestinal tracts, the application provides a bacteriophage which is separated from the nature and can strongly crack Serratia marcescens, and the bacteriophage is classified and named as Serratia marcocens bacteria iophage; has been deposited at the Guangdong province culture Collection (GDMCC) at 12.08.2020, with the following deposition addresses: "building 5", Guangzhou microbial research institute, Zhongluo 100, Mr. 59, Guangdong province; in the example of the application, named as SHENZHEN 01. By utilizing the special performance of the bacteriophage, the problem of drug resistance of the serratia marcescens in clinic can be effectively overcome, and an effective scheme is provided for clinic treatment of serratia marcescens infection.
The bacteriophage also has a lytic effect in insect bodies, and can be used as a substitute treatment mode for drug-resistant bacteria.
Specifically, when the phage is used for the lysis of Serratia marcescens, the addition amount is 1X108PFU/ml。
In a second aspect of the embodiments herein there is provided a use of a bacteriophage of the present application in the preparation of an agent against serratia marcescens.
The phage provided by the application can strongly crack serratia marcescens, so that the phage can be used for preparing an anti-serratia marcescens agent and effectively treating the infection of the serratia marcescens clinically.
Specifically, the anti-serratia marcescens agent is a serratia marcescens cracking agent, and the bacteriophage can strongly crack serratia marcescens, so the anti-serratia marcescens agent can be a medicament for treating serratia marcescens infection.
In one embodiment, the anti-serratia marcescens agent is a medicament for treating a serratia marcescens infection.
In one embodiment, the anti-serratia marcescens agent is a medicament for treating pulmonary infection caused by serratia marcescens.
In one embodiment, the anti-serratia marcescens agent is a medicament for treating urinary tract infection caused by serratia marcescens.
In one embodiment, the anti-serratia marcescens agent is a medicament for treating wound infection caused by serratia marcescens.
In one embodiment, the anti-serratia marcescens agent is a medicament for treating meningitis caused by serratia marcescens.
In one embodiment, the anti-serratia marcescens agent is a medicament for treating septicemia caused by serratia marcescens.
The following description will be given with reference to specific examples.
EXAMPLE 1 screening and purification of phages
1. Treatment of sewage
Taking the sewage of the Hebian of Shenzhen, centrifuging at 4000rpm for 15min, filtering and sterilizing by using a filter membrane of 0.22 mu m, and filling the filtrate into a sterile culture bottle.
2. Phage culture
4mL of the filtered supernatant is added with 200 mu L of logarithmic phase host bacterium culture solution, placed at 37 ℃ and cultured in a 220rpm incubator for 3 hours, and filtered by a microporous filter membrane to obtain phage stock solution.
3. Screening and identification of unique phages
Selecting single independent plaques with consistent shape and size on a double-layer agar plate, puncturing the selected plaques by using a sterile inoculating needle, inoculating 3mL LB (Luria-Bertani) broth culture medium, slightly stirring for several times, adding 0.2mL of a Serratia marcescens-containing bacterial liquid, shaking uniformly, standing for 1h at room temperature, culturing for 12h at 37 ℃, centrifuging for 30min at 12000 Xg at 4 ℃, then sterilizing by using a 0.22 mu m filter membrane, and storing the supernatant at 4 ℃.
Specifically, the phage Serratia marcescens bacteriophage Shenzhen01 was deposited at the Guangdong province culture Collection (GDMCC) at 12/08 in 2020 at the deposition address: "building 5", Guangzhou microbial research institute, Zhongluo 100, Mr. 59, Guangdong province; the microbial preservation number is as follows: GDMCC No. 61140.
4. Kinetics of phage growth
(1) Mixing the above selected phage with 1x108After incubating Serratia marcescens (CFU/ml) at a multiplicity of infection (MOI) of 0.1 at 37 ℃ for 20min, centrifuging at 12000g for 1min, and removing the supernatant; washing with sterile LB medium for three times, and blotting to remove liquid.
(2) Adding the bacteria adsorbed with bacteriophage into 20ml LB liquid culture medium, mixing well, culturing at 37 deg.C, sampling 200ul at 0min and every 10min, centrifuging at 12000g for 1min, and collecting supernatant for determination.
(3) Phage titers were determined using double-layer agar plates, in triplicate for each time point.
As shown in FIG. 1, it is understood from FIG. 1 that the latency of the Shenzhen01 phage is about 20min, the burst time is about 50min (latency to stationary phase), and the burst amount is 350 PFU/cell. The burst size is the final phage titer at the end of the burst ÷ concentration of host bacteria at the initial stage of infection, i.e., 3.5X 1010(PFU/mL)/1.0×108(CFU/mL)＝350PFU/cell。
Example 2 verification of bacteriophage lysis of Serratia marcescens
1. In 5ml LB liquid medium according to 1% (i.e. 100ul of Serratia marcescens bacterial liquid inoculated to 10ml of LB liquid medium without antibiotic) inoculated Serratia marcescens, cultured for 4 h. The bacterial liquid is divided into two parts, one part is continuously cultured, and the other part is inoculated with the phage according to 0.1 percent.
2. Standing and culturing for 12h in an incubator at 37 ℃, and carrying out shaking culture for 4h at 120 r/min.
3. Dividing a common nutrient agar plate into 2 areas by using a marker pen, sucking 100ul of the bacterial liquid respectively, dripping the bacterial liquid on the center of the common nutrient agar plate, uniformly coating the bacterial liquid by using a sterile coating rod, naturally airing, and then culturing in a 37 ℃ incubator for 12 hours.
As a result, as shown in FIG. 2, the region coated with the phage-free bacterial suspension showed uniform growth of Serratia marcescens, while the region coated with the phage-added bacterial suspension showed no colonies (region marked with D1 in FIG. 2). From this, it was determined that the phage of this example was able to lyse Serratia marcescens.
Example 3 lytic Capacity characterization of phages
1. Female aedes mosquitoes which emerged for 3-5 days were picked and fed with medical absorbent cotton stained with antibiotics (20 units penicillin and 20 mg streptomycin per ml) for 3 consecutive days (daily replacement of medical absorbent cotton containing antibiotics).
2. After 3 days, the cotton balls were removed and allowed to starve for 24 hours, maintaining the temperature and humidity (temperature 28 ℃, humidity 80%).
3. Then, the medical absorbent cotton stained with Serratia marcescens was fed for 3 consecutive days (the medical absorbent cotton containing the host bacteria was changed every day). The adult female mosquitoes were divided into 10 control groups fed with medical absorbent cotton soaked with 10% sucrose water and 10 experimental groups fed with phages (1X 10) containing the above example 18PFU/ml) of medical absorbent cotton.
4. After feeding for 24 hours, 4 aedes mosquitoes in each group are picked for experiment. Firstly, cleaning the surface of the aedes by using 75% ethanol for 3 times, and cleaning by using sterilized PBS (phosphate buffer solution) once; the midgut of aedes was dissected under a stereomicroscope, placed in a sterile EP tube containing 400 μ L PBS, ground with a sterile grinding rod, and diluted 103Plating the plate and culturing in an incubator at 37 ℃ for 12 h.
The results are shown in FIG. 3: control group (i.e. mock1 group, 10 in FIG. 3)-3Shows dilution 103Double plating) mosquito midgut plated with serratia marcescens, and experimental groups (C and D2 groups in fig. 3, 10-3Shows dilution 103Double coated plate) plates fed phage did not grow with serratia marcescens, indicating that the phage of the present application are in mosquitoesThe insects have activity in vivo.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A bacteriophage which cleaves Serratia marcescens and is deposited at the Guangdong province culture Collection with the deposit number GDMCC NO. 61140.
2. The bacteriophage of claim 1, wherein said serratia marcescens is derived from the mosquito gut.
3. The bacteriophage of claim 1, wherein said bacteriophage is added in an amount of 1x10 when used to lyse serratia marcescens8PFU/ml。
4. Use of a bacteriophage according to any one of claims 1 to 3 for the preparation of an agent against serratia marcescens.
5. The use according to claim 4, wherein the anti-serratia marcescens agent is a medicament for the treatment of a serratia marcescens infection.
6. The use according to claim 5, wherein the anti-serratia marcescens agent is a medicament for treating pulmonary infections caused by serratia marcescens.
7. The use according to claim 5, wherein the anti-serratia marcescens agent is a medicament for treating urinary tract infections caused by serratia marcescens.
8. The use according to claim 5, wherein the anti-serratia marcescens agent is a medicament for treating wound infection caused by serratia marcescens.
9. The use according to claim 5, wherein the anti-Serratia marcescens agent is a medicament for treating meningitis caused by Serratia marcescens.
10. The use according to claim 5, wherein the anti-serratia marcescens agent is a medicament for the treatment of sepsis caused by serratia marcescens.
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