NL2026421B1

NL2026421B1 - Strain of clostridium perfringens phage, bacteriostatic agent including the phage, and preparation method and use thereof

Info

Publication number: NL2026421B1
Application number: NL2026421A
Authority: NL
Inventors: Bao Hongduo; Pang Maoda; Wu Liting; Wang Ran; Zhang Hui
Original assignee: Jiangsu Acad Agricultural Sci
Priority date: 2020-05-25
Filing date: 2020-09-07
Publication date: 2023-02-08
Also published as: CN111662880A; NL2026421A; CN111662880B

Abstract

INCLUDING THE PHAGE, 5 AND PREPARATION METHOD AND USE THEREOF The present invention provides a strain of Clostridium perfringens phage, a bacteriostatic agent including the phage, and a preparation method and use thereof, and belongs to the technical field of bioengineering. The Clostridium perfringens phage is deposited in China Center for Type Culture Collection With the accession number of CCTCC M 2020047. The phage of the present invention has a strong lysis effect on Clostridium perfringens, and can be used for inhibiting and/or eliminating Clostridium perfringens. The Clostridium perfringens phage of the present invention can effectively prevent and control the occurrence of an intestinal disease in an animal by inhibiting the overgrowth of Clostridium perfringens. The present invention provides a safe and bacteriostatic phage product source Without toxic and side effects for preventing and controlling an animal intestinal disease caused by Clostridium perfringens in livestock and poultry.

Description

STRAIN OF CLOSTRIDIUM PERFRINGENS PHAGE,

BACTERIOSTATIC AGENT INCLUDING THE PHAGE,

AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD

The present invention relates to the technical field of bioengineering, and in particular to a strain of Clostridium perfringens phage, a bacteriostatic agent including the phage, and a preparation method and use thereof.

BACKGROUD

Clostridium perfringens (Cp), also known as Clostridium welchii, is a gram- positive anaerobic bacterium and is widely distributed in soil, feed, sewage, and the digestive tract and feces of an animal. It is an important food-borne pathogen causing Necrotic Enteritis (NE) and enterotoxemia of an animal, and food poisoning and traumatic gas gangrene of human beings. In the production process of a poultry product, the Cp is a bacterium commonly existed in the intestinal tract of healthy poultry. When the growth environment and daily ration change, it will cause significant proliferation of this bacterium and attachment of this bacterium to the intestinal mucosa to produce a large number of toxins and proteases, which will cause intestinal mucosal damage, thereby causing diarrhea, enteritis and the like diseases, and seriously affecting the production performance of an animal and causing huge economic losses. At present, since the necrotizing enteritis of an animal has been prevented and controlled by adding antibiotics and the like growth-promoting agents into the feed for a long time, drug-resistant strains are constantly produced, which leads to the failure of prevention and control and brings huge economic losses to the livestock and poultry breeding industry. Up to now, there is no record or report on the inhibition of the Cp by the Cp phage.

SUMMARY

An objective of the present invention is to provide a Clostridium perfringens (Cp) phage, a bacteriostatic agent including the phage, and a preparation method and use thereof.

In order to realize the aforementioned objective of the present invention, the present invention provides the following technical solutions.

The present invention provides a Cp phage deposited in China Center for

Type Culture Collection with the accession number of CCTCC M 2020047.

The present invention provides a method for culturing the Cp phage of the aforementioned solution, which includes the following steps: 1) centrifuging culture of Cp and collecting the precipitate; and 2) resuspending the precipitate of step 1) with a TY medium to obtain a bacterial suspension, adding the Cp phage of aforementioned solution into the bacterial suspension, culturing at a condition of 37°C for 6-8 h, then centrifuging, taking the supernatant, and filtering the supernatant through a 0.22 um filter membrane to obtain a Cp phage lysate.

Preferably, the concentration of the Cp culture of step 1) is 1 x 10% cfu/ml to 1 x 10/0 cfu/ml.

The present invention provides a bacteriostatic agent, which includes the Cp phage of the aforementioned solution or the culture of the Cp phage of the aforementioned solution.

Preferably, the bacteriostatic agent further includes a pharmaceutically acceptable excipient.

Preferably, the pharmaceutically acceptable excipient includes one or more of a buffer solution, a metal ion and a surfactant.

The present invention provides use of the Cp phage or the bacteriostatic agent of the aforementioned solution in preparation of a formulation for inhibiting and/or eliminating Cp.

The present invention provides use of the Cp phage or the bacteriostatic agent of the aforementioned solution in preparation of a medicament for preventing and treating an intestinal disease of an animal.

Preferably, the animal includes a chicken, a duck or a pig.

Beneficial effects of the present invention: the present invention provides a Cp phage deposited in China Center for Type Culture Collection with the accession number of CCTCC M 2020047. The phage of the present invention has a strong lysis effect on Cp, and can be used for inhibiting and/or eliminating Cp.

The Cp phage of the present invention can effectively prevent and control the occurrence of an intestinal disease in an animal by inhibiting the overgrowth of

Cp. The present invention provides a safe phage product source without toxic and side effects for preventing and controlling an animal intestinal disease caused by

Cp in livestock and poultry.

Description of Biological Preservation

The Cp phage vB_CpeS_JS01 was deposited with the accession number of

CCTCC M 2020047 on January 14, 2020 in China Center for Type Culture

Collection at the address of Wuhan University, Wuchang District, Wuhan.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of double-layer plate detection of a purified phage;

FIG. 2 shows the result of observing the phage under an electron microscope; and

FIG. 3 shows the lysis effect of the phage on Cp.

DESCRIPTION OF THE EMBODIMENTS

The present invention provides a strain of Clostridium perfringens (Cp) phage vB_CpeS_JSO01, which is deposited in China Center for Type Culture

Collection with the accession number of CCTCC M 2020047. The Cp phage of the present invention is derived from a fecal sample of a poultry farm.

The phage of the present invention has a strong lysis effect on Cp, and can be used for inhibiting and/or eliminating Cp. The Cp phage of the present invention can effectively prevent and control the occurrence of an intestinal disease in an animal by inhibiting the overgrowth of Cp.

The present invention provides a method for culturing the Cp phage of the above solution, which includes the following steps: 1) centrifuging culture of Cp and collecting the precipitate; and

2) resuspending the precipitate of step 1) with a TY medium to obtain a bacterial suspension, adding the Cp phage of the aforementioned solution into the bacterial suspension, culturing at a condition of 37°C for 6-8 h, then centrifuging, taking the supernatant, and filtering the supernatant through a 0.22 um filter membrane to obtain a Cp phage suspension.

In the present invention, firstly, the culture of Cp is centrifuged to collect the precipitate. The concentration of the culture of Cp is preferably 1 x 105 cfu/ml to 1 x 10'° cfu/ml, and more preferably 1 x 10° cfu/ml. The present invention has no specific limitation on the parameters of the centrifugation, and conventional parameters in the art can be adopted.

In the present invention, after the precipitate is obtained, the precipitate is resuspended with a TY medium to obtain a bacterial suspension, added with the

Cp phage of the aforementioned solution, cultured at a condition of 37°C for 6-8 bh, and then centrifuged, and the supernatant is taken and filtered through a 0.22 um filter membrane to obtain a Cp phage lysate.

The present invention provides a bacteriostatic agent, which includes the Cp phage of the aforementioned solution or the culture of the Cp phage of the aforementioned solution. The bacteriostatic agent preferably also includes a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient preferably includes one or more of a buffer solution, a metal ion and a surfactant.

The present invention provides use of the Cp phage or the bacteriostatic agent in preparation of a formulation for inhibiting and/or eliminating Cp. The Cp preferably includes the Cp in livestock and poultry breeding or the Cp in a production environment.

The present invention provides use of the Cp phage or the bacteriostatic agent of the aforementioned solution in preparation of a medicament for preventing and treating an intestinal disease of an animal. The animal preferably includes a chicken, a duck or a pig.

The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are merely a part rather than all of the embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the 5 present invention.

The Cp ATCC 13124 as the host bacterium of the phage used in the examples of the present invention is purchased from the Culture Collection Center of

Guangdong Institute of Microbiology. The Cps CMCC 67422, CMCC 67423 and

CMCC 67424 are purchased from National Center For Medical Culture

Collections.

Example 1 Isolation, preparation and purification of Cp phage 1. Phage separation

The sample of the present invention was collected from a fecal sample of a poultry farm. The sample was added with a peptone solution, and shaken for 2-4 h at room temperature, the supernatant was taken and centrifuged at 5000 xg. min’ ! for 30 min, and then the supernatant was filtered with a 0.22 um filter membrane. 1 ml of the supernatant was taken and added into 50 ml of a TY medium, and then it was added with 1 ml of the overnight culture (BHI overnight culture) of a strain of ATCC 13124 (hereinafter referred to as Cp for short), and then placed at 37°C and cultured with shaking at 150 rpm overnight. On the next day, the aforementioned culture was taken and centrifuged at 10000xg.min™ and 4°C for 30 min to take the supernatant. Then, the supernatant was filtered with a 0.22 un filter membrane to form a phage stock solution. 2 ml of the overnight-cultured bacterial solution was taken and coated on a 20 ml TY agar plate. After the bacterial solution was absorbed by the plate, 0.01 ml of the phage stock solution was pipetted and added dropwise onto the surface of the plate. After air-dried, the plate was placed at 37°C and subjected to anaerobic culture for 16-18 h, and the change of the sample application area was observed. 0.1 ml of the phage stock solution was taken and diluted by 10 times. 0.1 ml of each of the 107-106 diluted solutions was taken and uniformly mixed with 0.1 ml of the overnight-cultured host bacterial solution, added with about 5 ml of a 0.6% TY agar medium, mixed uniformly and then quickly poured into the upper layer of a TY plate, shaken uniformly and placed flat for 5 min until solidified, and then placed at 37°C and subjected to anaerobic culture for 12 h, to observe the formation of plaque. 2. Phage purification 2 ml of the freshly cultured overnight culture of Cp was taken, centrifuged, resuspended in 1 ml of the TY medium, and added with 0.1 ml of the phage [according to the ratios of MOI (multiplicity of infection) of 1:1, 1:10 and 1:100 respectively]; added with 100 ml of the TY medium, and cultured with shaking at 37°C for 6-8 h. The aforementioned culture was taken and centrifuged at 13000xg.min™ and 4°C for 30 min, and the supernatant was taken. Then the supernatant was filtered with a 0.22 um filter membrane to form a phage lysate.

The phage suspension was added with RNase A and DNase I respectively to a final concentration of 1 pg/ml, and incubated at 37°C for 30 min. It was added with 9.3 g of PEG 8000 and 5.8 g of NaCl, shaken well until dissolved, subjected to ice bath for 1 h, and centrifuged at 4°C and 10000xg. min’! for 30 min. The supernatant was discarded. The precipitate was added with 5 ml of a SM solution to fully wash and dissolve the tube wall and the precipitate, and acted at room temperature for 30 min. It was added with an equal volume of chloroform to extract PEG and cell debris from the phage suspension, shaken for 30 s, and centrifuged at 4°C and 3,000xg.min"! for 15 min to recover a hydrophilic phase containing phage particles and thus obtain the purified phage, which is detected by a double-layer plate (as shown in FIG. 1). 3. Detection of phage by electron microscopy

A suspension of the purified phage was taken and observed by an electron microscope. 20 ul of the sample was dropped on a copper net and precipitated for 15 min, and then the excess liquid was absorbed with filter paper. The sample was then stained with 2% phosphotungstic acid for 30 min, and observed by the electron microscope after dried.

As shown in FIG. 2, the phage belongs to the family of Myoviridae, and had a symmetrical head, a diameter of about 73 nm, a tail length of about 133 nm, and a tail diameter of 20 nm. The phage was named vB_CpeS_JS01.

Example 2 Detection of host lineage of phage vB_CpeS_JS01

A TY plate was divided into several areas, and different overnight cultures of host bacteria were pipetted onto these areas: 0.1 ml of each of ATCC 13124,

CMCC 67422, CMCC 67423 and CMCC 67424 was dropped onto the TY plate, the bacterial solution was spread evenly and air-dried, then 0.1 ml of the phage stock solution was taken and diluted by 10 times, and 0.01 ml of each of the 10°, 10% and 10° dilutions was added dropwise into the plates coated with different host bacteria. The plate was placed upright and air-dried, and then placed at 37°C and subjected to anaerobic culture for 12 h. Then the lysis of the bacteria by the phage was observed.

The results showed that the phage vB_CpeS_JSO1 had a transparent lysis ring for each of the Cp strains CMCC 67422, CMCC 67423 and CMCC 67424.

That was, the phage was positive for each of the Cp strains.

Example 3 The lysis effect of phage vB_CpeS_JS01 on Cp

The overnight-cultured Cp bacterial solution was adjusted with a TY medium to achieve the ODsmw of about 0.1-0.2, added with the phage vB_CpeS_JS01 (with the MOI of 0.1 and 1, respectively), and placed in an incubator at 37°C for anaerobic culture. Meanwhile, a negative control was set with addition of only the TY medium, and 0.2 ml was taken for detection of the

OD respectively after Oh, 2 h, 4 h, 6 h, 8 h and 10 h, so as to compare the changes of OD after the addition of the phage.

The results were shown in FIG. 3. The ODgoo value in the group added with the phage was changed significantly compared with the control group after action at 37°C for 8 h. The ODsoo value in the group added with the phage was decreased compared with the control group after action at 37°C for 4 h. After 10 h, the control group grew to the ODe of 0.54, while the OD value of the MOI 0.1 group was increased slightly to 0.14, and the OD in the MOI 1 group was only 0.1. Therefore, the phage had a significant lysis effect on the Cp, and all of the different action concentrations can effectively inhibit the growth of the Cp.

Example 4 preventive antimicrobial effect of phage vB_CpeS_JS01 in poultry breeding

Fifty 1-day-old chicks were selected and randomly divided into 5 groups, with 10 chicks in each group. Among the groups, group A was normally fed as a control group, group B was normally fed and meanwhile subjected to gavage of the phage at 1 x 108 pfu/0.1 ml/chick for continuous 7 days, and group C was normally fed and additionally had access to ampicillin-containing drinking water (0.1% W/V) for continuous 7 days; group D was a phage control group that was subjected to gavage of the phage at 1 x 10/0 pfu/0.1 ml/chick for continuous 14 days; and Group E was a blank control group. From day 14 to day 21, the groups were fed with the phage and ampicillin respectively. The groups A, B and C were infected with the Cp bacterium (ATCC 13124) at 105 cfu/0.1 ml/chick on day 19.

On day 21, the chickens were respectively weighed, the intestinal tract was taken to analyze the occurrence of necrotic enteritis for scoring (0 was the small intestine being normal, 1 was the intestinal tract being covered with mucus slightly, and 2 is severe necrotic enteritis), and the total mortality was counted.

The results were shown in Table 1. It could be seen from Table 1 that, the intestinal tract scores and mortalities in the group applied with the phage and the group applied with the antibiotics were both lower than those in the control group, and there was no significant pathological injury in the intestinal tract. There was no death and the intestinal tract was healthy in the group fed only with the phage, which had no significant difference compared with those of the blank group, indicating that the application of the phage at the dose of 1 x 10/9 pfu/0.1 ml/chick had no significant side effects, and it had the characteristics of being safe and having no toxic and side effects.

Table 1 Effect of phage on growth performance of chicken

Groups Intestinal tract Mortality

A 0.8 2/8

B 0.4 0/10

C 0.3 0/10

D 0.1 0/10

E 0.1 0/10

Results were shown in Table 2, and respectively on day 14 and day 21, the weight gains of chicks in the group applied with the phage and the group applied with the antibiotics were both higher than that in the control group. During 21 days of culture, 2 chicks died in the control group, while there was no death in both the phage group and the antibiotic group, and the intestinal tract scores in both the phage group and the antibiotic group were lower than that in the control group. It could be seen that the application of the phage could inhibit the growth of the bacteria, and the phage had the same action ability as the antibiotics and can effectively prevent the occurrence of enteritis.

Table 2 Effect of phage on weight gain of chick

Groups Bars AEC gain rs a

A 30.38 281 684

B 30.78 305 795

C 30.59 301 712

D 30.56 291 721

E 30.74 279 702

Example 5 safety experiment of phage vB_CpeS_JS01

A total of 20 female SPF-grade BALB/c mice aged 6-8 weeks and weighed 27 g + 2 9, were purchased from the Comparative Medicine Center of Yangzhou

University. The mice were randomly divided into 2 groups, with 10 mice in each group. One group took the phage at 10/0 pfu/0.1 ml/mouse orally, while the control group took an equal volume of PBS orally. After continuous oral administration for 14 days, the mice were killed by cervical dislocation, to observe the changes of internal organs, the digestive tract and mucosa.

The results showed that this amount of phage had no effect on the daily behavior of the mice, and there was no abnormality in anatomical examination, and no phage could be detected in the feces of the mice two days after completion of feeding.

The above description is only preferred embodiments of the present invention. It should be pointed out that, for those of ordinary skills in the art,

several improvements and modifications can be made without departing from the principle of the present invention.

These improvements and modifications should also be considered as falling into the claimed scope of the present invention.

Claims

Conclusions

1. Strain of a Clostridium perfringens phage preserved at the China Center for the Collection of Type Cultures with accession number CCTCC M 2020047.

A method of culturing the Clostridium perfringens phage according to claim 1, comprising the steps of: 1) centrifuging the Clostridium perfringens phage, and collecting the precipitate; and 2) resuspending the precipitate of step 1) with a TY medium to obtain a bacterial suspension, adding the Clostridium perfringens phage according to claim 1 into the bacterial suspension, culturing at 37°C for 6 -8 hours, then centrifuge, skim off the supernatant, and filter the supernatant through a 0.22 µm filter membrane to obtain a lysate of Clostridium perfringens phage.

The culture method of claim 2, wherein the concentration of the Clostridium perfringens culture of step 1) is 1 x 10 3 colony-forming units/ml to 1 x 10 colony-forming units/ml.

A bacteriostatic agent comprising the Clostridium perfringens phage of claim 1 or the culture of the Clostridium perfringens phage of claim 1.

The bacteriostatic agent of claim 4, further comprising a pharmaceutically acceptable excipient.

The bacteriostatic agent of claim 5, wherein the pharmaceutically acceptable excipient comprises one or more of a buffer solution, a metal ion and a surfactant.

Use of the Clostridium perfringens phage of claim 1 or the bacteriostatic agent of any one of claims 4-7 in the preparation of a formulation for inhibiting and/or eliminating Clostridium perfringens.

Use of the Clostridium perfringens phage of claim 1 or the bacteriostatic agent of any one of claims 4-7 in the manufacture of a medicament for the prevention and treatment of an intestinal disease in an animal.

The use according to claim 8, wherein the animal comprises a chicken, a duck or a pig.