CN109266621B - New Aerococcus viridis bacteriophage AVP and application thereof - Google Patents

Abstract

The invention provides a new Aerococcus flavescens AVP, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: M2018155; the preservation time is as follows: year 2018, month 3, day 25; the new Aerococcus glaucor bacteriophage AVP can crack the bacteriophage AVP lysate of Aerococcus glaucor, the bacteriophage has wider lysis spectrum, not only can crack the Aerococcus glaucor, but also can crack Staphylococcus aureus and Staphylococcus epidermidis, can crack a standard strain, can crack a clinical strain separated from the environment or the feces of the pathogenic animals, can be used alone or in combination with other substances, and provides a safe and nontoxic bacteriophage disinfectant product for disinfecting and purifying the environment.

Description

New Aerococcus viridis bacteriophage AVP and application thereof

Technical Field

The invention relates to a bacteriophage which is separated from the nature and infects and kills staphylococcus viridis, further discloses a new staphylococcus viridis bacteriophage AVP, also provides the application thereof, and a method for cracking staphylococcus viridis and staphylococcus by using the bacteriophage lysate as an active ingredient composition, belonging to the technical field of microorganism.

Background

Aerococcus viridis (A), (B), (C)Aeroeoccus viridans) (assigned to the family of Streptococcaceae: (Strep) Genus Aerococcus (Aerococcus) It is a model species of the genus Aerococcus, and is formally named by WILLIAMS et al in 1953. Is a bacterium that is intermediate between, and more closely adjacent to, staphylococci and streptococci. Similar to staphylococci and enterococci, they grow in 6.5% sodium chloride broth, but are catalase negative, colony small, alpha-hemolytic, etc. closer to streptococci.

The bacterium is a conditioned pathogen, widely distributed in the nature and generally free from diseases. Can cause serious infection under the conditions of low or incomplete immune function of the body and the like. Such as endocarditis, urinary tract infection, bacteremia, sepsis, meningitis, septic arthritis and the like, aerococcus viridis is an opportunistic pathogen that has been highlighted in recent years.

In recent years, due to the wide application of immunosuppressive agents, the increase of invasive treatment opportunities, clinical antimicrobial drug abuse and other factors, the reports of infections caused by aerococcus viridis in clinical practice are increasing. In addition, the strain can cause infection of fishes and shrimps such as lobsters, tilapia and the like in aquaculture, and huge economic loss is caused to the aquaculture industry. In veterinary clinic, related cases are also endless, which can cause Li in piglets, respiratory diseases of pigs, arthritis, septicemia, meningitis, subclinical mastitis of cattle and the like.

The bacteriophage is a virus for killing bacteria, parasitizes the bacteria, replicates and breeds the bacteria, finally cracks the bacteria to die, is considered as a potential 'effective gram of super bacteria' by scientists due to the natural bactericidal property, has high specificity to host bacteria, strong cracking capability, no toxic or side effect on human and animals (products), no drug residue and the like, and shows wider application prospect and value than antibiotics. As early as 1917, after the first discovery of phages by the falixd Herelle microbiologist of canada, the scientific community has attempted to use them for the treatment of bacterial infections in humans or animals; the Salmonella-directed phage preparation SalmoFresh, developed by Intralitix corporation, U.S. in 2013, was recognized by the U.S. Food and Drug Administration (FDA) as a generally recognized safety grade product (GRAS) and approved for marketing.

However, most of the phage biocontrol studies have focused on the control of escherichia coli, listeria, staphylococcus aureus, and clostridium, and no phage capable of specific control against staphylococcus aureus has been reported so far. Therefore, the development of novel bacteriophage with control effect on the aerococcus viridis is of great significance.

Disclosure of Invention

Aiming at the problems, the invention provides a new Aerococcus glaucor bacteriophage AVP, a bacteriophage AVP lysate capable of cracking Aerococcus glaucor, the bacteriophage has a wider lysis spectrum, not only can crack Aerococcus glaucor, but also can crack Staphylococcus aureus and Staphylococcus epidermidis, can crack a standard strain, can crack a clinical strain separated from the environment or feces of pathogenic animals, can be used alone or in combination with other substances, and provides a safe and nontoxic bacteriophage killing product for disinfecting and purifying the environment.

The invention discloses a new Aerococcus flavescens AVP, which is preserved in China center for type culture Collection with the preservation number: CCTCC NO: M2018155; the preservation time is as follows: year 2018, month 3 and day 25. The phage has a head in a regular icosahedron shape and a flexible tail; the phage can form transparent plaques on 1.5 percent of BHI agar culture medium, the periphery has no halo, the edge is clear and regular, and the diameter is 0.1-1 mm; the restriction map of the genomic nucleic acid shows that the phage nucleic acid is double-stranded dna (dsdna); according to the eighth report of the Committee for Classification of viruses-International Committee for Classification of viruses (ICTV)2005, AVP belongs to the family Myoviridae (ICTV) ((R))Myoviridae）。

The invention further provides application of the novel Aerococcus glaucor bacteriophage AVP and the bacteriophage lysate or the composition containing the bacteriophage as an active ingredient in preparing a medicament for preventing and/or treating infectious diseases caused by Aerococcus glaucor, Staphylococcus aureus or Staphylococcus epidermidis.

The application of the phage lysate of the new aerococcus viridis phage AVP as an effective component in preparing feed additives, drinking water additives, feeds, drinking water, detergents or disinfectants.

The composition can take the bacteriophage lysate of the invention as the only active ingredient; it may also contain other phages than the phages of the invention, which in combination with the phages of the invention can give comparable or better control.

The composition can be prepared into various use forms, such as liquid preparation, freeze-dried preparation or oral solid preparation, and the like, and is used for killing staphylococcus aureus and staphylococcus aureus or staphylococcus epidermidis in oral administration, spraying or injection and other modes.

The pharmaceutically acceptable carrier included in the composition of the present invention is a pharmaceutical carrier generally used for preparing pharmaceutical preparations, and examples thereof are lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but not limited thereto. In addition to the above ingredients, the compositions of the present invention may also include lubricating agents, wetting agents, sweeteners, flavorants, emulsifiers, suspending agents and preservatives.

The invention has the positive effects that: discloses a new Aerococcus viridis bacteriophage AVP and provides the medical application thereof; the bacteriophage of the invention has specific killing activity to the aerococcus viridis, can selectively kill specific pathogenic bacteria, does not kill beneficial bacteria, and does not cause drug resistance. The bacteriophage lysis solution has strong killing activity and wide lysis spectrum, has specific killing activity on the staphylococcus aureus and the staphylococcus epidermidis, and can be used for killing staphylococcus aureus. The AVP bacteriophage has relatively high hydrophilic phase, and may be prepared into spray, medicated bath liquid or injection to kill environment specific pathogenic bacteria effectively.

Drawings

FIG. 1 is a photograph of AVP plaques;

FIG. 2 is a transmission electron micrograph of bacteriophage AVP;

FIG. 3 is a graph of the optimal MOI of phage AVP;

FIG. 4 is a graph of one-step growth of phage AVP.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless otherwise indicated

It is noted that 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.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, there are no specific control bacteriophages reported for infections caused by Aerococcus viridis. Based on the phage lysate, the invention provides a phage lysate with prevention and treatment effects on staphylococcus aureus, staphylococcus aureus and staphylococcus epidermidis and application thereof.

In embodiments of the invention, novel bacteriophages having specific killing activity against Aerococcus viridis are provided.

Bacteriophages are bacteria-specific viruses that are capable of infecting specific bacteria and inhibiting the growth of bacteria, and are viruses that contain single-or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) as genetic material.

Example 1

The phage is a novel phage separated from sewage, and has a head part in a regular icosahedron shape and a telescopic tail part; the phage can form transparent plaques on 1.5 percent of BHI agar culture medium, the periphery has no halo, the edge is clear and regular, and the diameter is 0.1-1 mm; the restriction map of the genomic nucleic acid shows that the phage nucleic acid is double-stranded dna (dsdna); has been preserved in China center for type culture Collection in 2018, 3, 25 and the preservation number is CCTCC NO: M2018155, address: wuhan, Wuhan university, China.

Example 2

Phage isolation and preparation

The process of phage isolation is described in detail below. The fecal sewage sample in the invention is collected from a burnt plum sewage ditch in Changchun city; the host bacterium is Aerococcus viridis AV-XHY. Collecting sewage, filtering with gauze, or centrifuging at 6000r/min for 10min, and collecting supernatant; BHI medium (100 mL) was prepared with treated wastewater instead of ddH 2O; adding 1mL of overnight cultured bacteria into the culture medium, and culturing at 37 ℃ for 10-12 h; the culture was centrifuged at 12000r/min for 5min in 1mL, the supernatant was filtered through a 0.22 μm filter and stored, and the resulting filtrate was used for a spot test to check whether phages capable of killing Aerococcus viridis were included.

The plaque test was performed as follows: 2% of the culture broth was inoculated with Aerococcus viridis AV-XHY in BHI broth, and cultured overnight at 37 ℃ with shaking. 100. mu.L (OD 600 ═ 1.5) of the bacterial culture broth prepared above was applied to BHI plates. The plates were placed in a room to dry for about 30 minutes. After drying, 10. mu.L of the resulting filtrate was dropped directly on the surface of pale green balloonflower lawn and dried for about 30 minutes. After drying, the plates were incubated at 37 ℃ for one day and then examined for the formation of transparent areas on the surface of the bacterial lawn. If a transparent region is formed where the filtrate drips, it can be judged that the filtrate contains phages capable of killing Aerococcus flavescens AV-XHY. By the above procedure, a filtrate containing phages having the ability to kill A. viridis AV-XHY can be obtained.

Thereafter, phages were isolated from the above-mentioned filtrate confirmed to have phages capable of killing Aerococcus viridis. Diluting the obtained filtrate by a multiple ratio with sterile PBS, adding 100 mu L of the diluted filtrate and 200 mu L of overnight cultured host bacterium AV-XHY into 7mL of 45 ℃ BHI semisolid culture medium, pouring the mixture on a solid agar culture medium to prepare a double-layer plate after fully and uniformly mixing, placing the double-layer plate in a 37 ℃ incubator for incubation for 4-6h, and observing the growth of plaques.

Example 3

Phage amplification and purification

On the double-layer flat plate with the formed plaques, picking single plaques which are relatively round and bright and have larger diameters by using a tip of a sterile pipette, inoculating the single plaques into 5ml of BHI liquid culture medium, adding 200 mu L of phage host bacterial liquid, uniformly mixing, acting for 15min at room temperature, culturing for 10-14 h at 37 ℃, centrifuging for 10min at 12000rpm and 4 ℃, and taking supernatant; the double-layer plate experiment is repeated, so that 4-5 times of single plaques are picked repeatedly, and the phage is purified into plaques with the same size.

1mL of freshly cultured host bacteria was taken and 300. mu.L of phage lysate was added (in a ratio of 1:1, 1:10 and 1:100 for individual phage cultures to host bacteria, respectively). Incubating at 37 deg.C for 20min to make phage particles adsorbed to host bacteria; adding 800mL of BHI liquid culture medium, and adding CaCl₂And (3) shaking and culturing the mother liquor to a final concentration of 1.25mM at 37 ℃ for 6-8 h, centrifuging at 12000rpm at 4 ℃ for 10min, and taking the supernatant, namely the phage lysate.

PEG purification: adding RNase A and DNase I into the phage lysate until the final concentration is 1 mug/mL, and standing at room temperature for 30 min; adding NaCl to the final concentration of 1mol/L, uniformly mixing, and carrying out ice bath for 1-2 h; centrifuging at 4 deg.C for 15-20min at 8000r/min, and collecting supernatant; adding 10g PEG-8000 per 100ml, stirring gently to dissolve, ice-cooling for more than 2 hr (preferably overnight) to allow bacteriophage to form precipitate under the action of PEG-8000; centrifuging at 12000r/min for 10-20min at 4 deg.C, recovering precipitated phage particles, adding 2mL SM solution, washing precipitate thoroughly, and acting at room temperature for 1 h; adding chloroform with the same volume for extraction, and carrying out mild oscillation for 30 s; the organic phase and the hydrophilic phase were separated by centrifugation at 5000rpm for 10min at 4 ℃ and the hydrophilic phase containing the phage particles was recovered to obtain purified phage.

CsCl isopycnic gradient centrifugal purification, namely preparing CsCl gradient liquid according to the table, and sequentially adding 1mL of each gradient liquid into a 5mL translucent polyacrylamide high-speed centrifugal tube according to the sequence from high density to low density; slowly adding 700 mu L of phage concentrate on the CsCl gradient solution, placing the solution in a high-speed centrifuge at 4 ℃, and horizontally centrifuging at 35000r/min for 3 h; after the centrifugation is finished, opening a bin door when the vacuum is reduced to 0, taking out a sample, and shutting down; the lower end of the sample is provided with a layer of blue-color tape, and a thin needle head is inserted from the side surface of the tape and carefully sucked; the sample was placed in a dialysis bag and dialyzed against 10mM Tris-HCl, pH 7.4, 100mM MgCl 2 buffer, 2L once (10-14 kd); finally the sample was aspirated and the phage titer was determined (FIG. 1).

The phage titer is detected by adopting a double-layer plate method: diluting the purified phage solution by 10 times gradient, and collecting

And fully and uniformly mixing 100 mu L of each of the phage diluents with 200 mu L of the host bacterium liquid, paving double-layer agar plates, culturing at the constant temperature of 37 ℃ for about 10 h, and counting plaques of each agar plate. Selecting a plate with the plaque of about 100-200, and calculating the initial concentration of the phage according to the dilution multiple to obtain the titer of the phage.

The purified phage is preserved in China center for type culture Collection with the preservation number of CCTC NO: m2018155, date of deposit 3/25/2018, address of depository: wuhan university, Wuhan, China, zip code 430072.

Example 4

Phage AVP Transmission Electron microscopy

The phage purified by PEG in the embodiment 2 is taken for electron microscope observation, and the specific operation steps are as follows: dropping 10 μ L sample on copper mesh, precipitating for 15min, removing excessive liquid with filter paper, dyeing with 2% phosphotungstic acid (PTA) for 1-2min, drying, and observing with transmission electron microscope; the observation result is shown in FIG. 2, in which the head is a regular icosahedron and the diameter of the head is about

94nm and a tail length of about 189 nm. According to the eighth report of the Committee for Classification of viruses-International Committee for Classification of viruses (ICTV)2005, AVP belongs to the family Myoviridae (ICTV) ((R))Myoviridae ）。

Example 5

Optimal MOI and one-step growth curves for bacteriophage AVP

Optimal MOI determination: adjusting the concentration of the bacterial liquid cultured to logarithmic phase to 10⁷cfu/mL, then mixing phage and bacteria according to the ratio of phage/bacteria of 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10, transferring into BHI liquid medium, and culturing at 37 deg.C for 8h with shaking. Centrifuging the culture solution at 4 deg.C for 15min at 10000g, filtering the supernatant with disposable filter with pore diameter of 0.22 μm to obtain bacteriophage value-added solution, and measuring titer of the solution by double-layer plate method to obtain bacteriophage with highest concentrationThe best MOI was obtained with the bacteria ratio, and the results are shown in FIG. 3.

One-step growth curve determination: mixing the host bacteria cultured to the logarithmic phase with the phage according to the optimal MOI proportion, standing at 4 ℃ for 15min, suspending the precipitate with a fresh BHI liquid culture medium, carrying out shake culture on the suspension at 37 ℃, and taking a sample every 10min to determine the titer of the phage, thereby drawing a one-step growth curve of phage-infected bacteria, wherein the result is shown in FIG. 4.

Example 6

Analysis of phage AVP host spectra

The phage AVP titer obtained in example 2 was adjusted to 10 ⁸ pfu/mL for use. Selecting a plurality of staphylococcus aureus and staphylococcus epidermidis as objects in the test, and analyzing the host spectrum of the phage AVP, wherein the specific operations are as follows: 100 mu L of overnight culture of the strains to be detected are respectively taken and dripped in the center of a 1.5 percent BHI medium flat plate, and the strains are respectively coated into uniform lawn by a coating rod. And (3) dropwise adding 10 mu L of phage AVP on the surface of the lawn, drying the liquid drops, and then inversely culturing at 37 ℃ for 12-16 h, wherein the observation result shows that if the plaque is generated, the result is marked as "+", otherwise, the result is "-", and the more the "+" number shows that the plaque transparency is stronger. The results are shown in table 1:

TABLE 1

1	Bacterial strain	Bacterial strains	Sxhy-1p
				2	Gold grape	R6186	+++
3	Gold grape	19685	+++
				4	Gold grape	R6491	+++
5	Gold grape	n10	+
				6	Gold grape	W11	+++
7	Gold grape	ATCC25923	+++
				8	Gold grape	ATCC29213	+++
9	Gold grape	4B	+++
				10	Gold grape	R6046	+++
11	Gold grape	n25	+++
				12	Gold grape	R23	+++
13	Gold grape	R238	+++
				14	Gold grape	n38	++
15	Gold grape	3659	+++
				16	Gold grape	n4	+
17	Gold grape	R89	++
				18	Gold grape	R15	++
19	Gold grape	R196	+++
				20	Gold grape	W903	++
21	Gold grape	R3784	+++
				22	Gold grape	Wood46	+++
23	Gold grape	ATCC49525	+++
				24	Gold grape	208	+++
25	Gold grape	B6994	+
				26	Gold grape	R3790	+++
27	Gold grape	J2	++
				28	Gold grape	R5886	+++
29	Gold grape	CVCC2261	+++
				30	Gold grape	W4727	+++
31	Gold grape	R6040	+++
				32	Gold grape	R6199	+++
33	Gold grape	R6012	+++
				34	Gold grape	R81	+++
35	Gold grape	W1	+++
				36	Gold grape	USA300	+++
37	Gold grape	R6166	+++
				38	Gold grape	n 26	+
39	Gold grape	YB57	+++
				40	Gold grape	n14	+++
41	Gold grape	n 13	++
				42	Gold grape	JP1	++
43	Gold grape	JP2	+++
				44	Gold grape	JP3	+++
45	Gold grape	JP4	+++
				46	Gold grape	JP5	+++
47	Gold grape	JP6	+++
				48	Gold grape	JP7	+++
49	Gold grape	JP8	+++
				50	Gold grape	JP9	+++
51	Gold grape	JP10	+++
				52	Gold grape	JP11	+++
53	Gold grape	JP13	+++
				54	Gold grape	JP14	+++
55	Gold grape	JP15	+++
				56	Gold grape	JP16	+++
57	Gold grape	SL1	++
				58	Gold grape	SW1	++
59	Gold grape	SH1	++
				60	Epigon grape	BP9	++
61	Epigon grape	BP10	++
				62	Epigon grape	BP11	++
63	Epigon grape	BP12	++
				64	Gold grape	n3	+++
65	Gold grape	W4661	++
				66	Gold grape	ATCC26003	+++
67	Gold grape	W3275	+++
				69	Gold grape	ATCC44515	+++
70	Gold grape	206	+++
				71	Gold grape	J4	+++
72	Gold grape	SAZ4	+++
				73	Gold grape	SAAT	+++
74	Gold grape	SA1545	+++
				75	Gold grape	n26	+
76	Aerococcus sp	AV-XHY	+++

Of the 76 strains used for the determination of the lysis buffer, 1 strain was a host strain of AVP, 4 strains were Staphylococcus epidermidis, and the remaining 71 strains were all Staphylococcus aureus. As can be seen from the table, in the plaque test, the phage AVP lysate can cause all 76 strains to be tested to generate plaques, and most of the plaques are very transparent, which indicates that the phage has the characteristic of wider lysis spectrum, and can lyse both aerococci and staphylococci.

Claims (4)

1. A strain of aerococcus viridis (A)Aerococcus viridans) The phage AVP is preserved in the China center for type culture Collection in 2018, 3 months and 25 days, and the preservation name isAerococcus viridansphase AVP with the preservation number: CCTCC NO: M2018155.

2. The use of the Aerococcus glaucor bacteriophage AVP and bacteriophage lysate of claim 1 in the preparation of a medicament for the prevention and treatment of infectious diseases caused by Aerococcus glaucor and Staphylococcus aureus.

3. Use of the Aerococcus glaucor bacteriophage AVP and bacteriophage lysate of claim 1 in the preparation of a medicament for the prevention and treatment of infectious diseases caused by Staphylococcus epidermidis.

4. Use of a phage lysate of Aerococcus glaucor phage AVP according to claim 1 as an active ingredient in the preparation of a feed additive, a drinking water additive, a feed, drinking water, a detergent or a disinfectant.

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