WO2019144830A1 - 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用 - Google Patents

一种宽裂解谱绿脓杆菌噬菌体及其消毒应用 Download PDF

Info

Publication number
WO2019144830A1
WO2019144830A1 PCT/CN2019/071883 CN2019071883W WO2019144830A1 WO 2019144830 A1 WO2019144830 A1 WO 2019144830A1 CN 2019071883 W CN2019071883 W CN 2019071883W WO 2019144830 A1 WO2019144830 A1 WO 2019144830A1
Authority
WO
WIPO (PCT)
Prior art keywords
pseudomonas aeruginosa
phage
preparation
environmental
disinfection
Prior art date
Application number
PCT/CN2019/071883
Other languages
English (en)
French (fr)
Inventor
潘强
任慧英
孙虎芝
于晓妍
刘慧�
Original Assignee
青岛诺安百特生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 青岛诺安百特生物技术有限公司 filed Critical 青岛诺安百特生物技术有限公司
Publication of WO2019144830A1 publication Critical patent/WO2019144830A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Definitions

  • the invention belongs to the field of bioengineering, and in particular relates to a phage having environmental disinfection ability and use thereof.
  • Pseudomonas aeruginosa also known as Pseudomonas aeruginosa
  • PA Pseudomonas aeruginosa
  • phage has shown great potential in assisting or replacing antibiotics and disinfectants because of its high efficiency, low cost, easy availability, strong specificity, and difficulty in producing drug resistance. It has been accepted by more and more people. attention.
  • the invention provides a phage having environmental disinfection ability and a use thereof, and aims to control the occurrence of hemorrhagic pneumonia in a leech farm.
  • the main pathogen of leech hemorrhagic pneumonia is Pseudomonas aeruginosa.
  • the occurrence of this disease is mainly concentrated in July-October of each year.
  • the water mink is falling, and the water mink that falls off in the farm flies with the wind and hangs in the cage. Stick to the ground or feces.
  • Pseudomonas aeruginosa is a common bacterium in the environment, especially in animal hair.
  • the otter farm is rarely sprayed and disinfected, and the conventional disinfectant cannot effectively kill Pseudomonas aeruginosa, and it also has side effects such as irritation and corrosivity.
  • domestic researchers have done disinfection tests in a watertight environment, but the environmental disinfection of phage in the mink farm to control the occurrence of hemorrhagic pneumonia has not been reported.
  • the present invention provides a Pseudomonas aeruginosa phage which is a Pseudomonas aeruginosa phage, a Latin name P. aeruginosaphage, which is named Lp14, and has a broad spectrum against Pseudomonas aeruginosa (Pseudomonas aeruginosa).
  • the bactericidal ability of the phage Lp14 is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee. The deposit date is March 30, 2017, and the deposit number is CGMCC No. 13791; the classification is named Pseudomonas aeruginosa phage.
  • the phage Lp14 was isolated from the 17 strains of P. aeruginosa by double-layer plate method. The phage Lp14 was determined to be 88.2% of the 17 host strains, indicating that the phage is a multivalent potent phage. Disinfection has a good application prospect.
  • One aspect of the invention provides a Pseudomonas aeruginosa phage having the accession number CGMCC No. 13791.
  • Another aspect of the present invention provides the use of the Pseudomonas aeruginosa phage of the present invention as a preparation for inhibiting Pseudomonas aeruginosa.
  • Another aspect of the invention provides the use of the Pseudomonas aeruginosa phage of the invention in the preparation of an environmental disinfecting formulation.
  • an environmental disinfectant for killing Pseudomonas aeruginosa more preferably as an environmental disinfectant for animal feeding, food, pharmaceutical, medical device preparation plants, hospitals, and other human activity sites; most preferably as a cockroach Use of environmental disinfectant preparations for poultry or poultry farms.
  • Another aspect of the present invention provides the use of the Pseudomonas aeruginosa phage of the present invention for the preparation of an environmental disinfectant preparation for preventing sputum blood pneumonitis, Pseudomonas aeruginosa umbilical corditis, porcine endometritis, cow mastitis, and the like.
  • an environmental disinfecting preparation comprising the Pseudomonas aeruginosa phage of the present invention, preferably, the Pseudomonas aeruginosa phage concentration is 10 8 PFU/ml or more.
  • the environmental disinfectant preparation of the present invention may be in any of the usual dosage forms including, but not limited to, sprays, aerosols, suspensions, lotions, eluents, dispersions and the like.
  • the environmental disinfectant preparation is in the form of a spray, a lotion or a rinse agent, and comprises a suspending agent such as gellan gum, a protective agent such as 30% glycerin, and a solvent such as a culture solution.
  • a suspending agent such as gellan gum
  • a protective agent such as 30% glycerin
  • a solvent such as a culture solution.
  • the environmental disinfecting formulation is formulated as a liquid composition, it can be mixed with a propellant to aid in the formation of a spray by the spray composition.
  • the applicator can determine the spray dose by setting a valve.
  • the environmental disinfectant preparation may further comprise other active ingredients for virus, bacterial inhibition or elimination in the environment.
  • a method for eliminating Pseudomonas aeruginosa in an environment, or reducing the concentration of Pseudomonas aeruginosa in the environment, comprising administering to the environment the Pseudomonas aeruginosa phage of the present invention, or the environmental disinfecting preparation of the present invention, preferably, The methods are for non-diagnostic and therapeutic purposes.
  • the large-area spraying can be directly performed without sealing the disinfection site.
  • it is sprayed and disinfected as a disinfectant in the dust of the house, it has a very significant disinfection effect on the number of bacteria in the dust and the number of Pseudomonas aeruginosa and is obviously superior to the one hundred poisons.
  • the phage is used as a disinfectant for disinfecting the house dust. And there is obvious disinfection effect.
  • the Pseudomonas aeruginosa phage is used for sputum disinfection, and the mortality caused by hemorrhagic pneumonia can be significantly reduced.
  • the disinfection effect of phage is better than that of chlorpyrifos and control group, and it is not irritating and corrosive, indicating that the phage can provide a safe biological disinfectant for preventing hemorrhagic pneumonia.
  • a carrier is further included.
  • the environmental disinfecting composition according to the present invention may also contain conventional excipients, particularly suspending agents, protective agents, double distilled water, and the like, and may also contain probiotic microorganisms.
  • the vector used in the composition of the present invention is not subject to any limitation as long as the presence of the vector does not interfere with the cleavage of the phage.
  • the Pseudomonas aeruginosa phage in the invention can be formulated into different dosage forms, can kill the environment, and can efficiently lyse Pseudomonas aeruginosa, and can be used for eliminating pathogens and monitoring the contamination of Pseudomonas aeruginosa in animal breeding places.
  • the invention employs a composition comprising an isolated Pseudomonas aeruginosa phage, formulated as a solution, spray, dispersion, suspension, or the like, for use in inhibiting or killing Pseudomonas aeruginosa.
  • composition of the present invention containing the isolated Pseudomonas aeruginosa phage is formulated into a spray or eluent to kill the environment or utensils, to eliminate pathogens, and to monitor the environment of the animal breeding place.
  • the contamination of Pseudomonas aeruginosa is formulated into a spray or eluent to kill the environment or utensils, to eliminate pathogens, and to monitor the environment of the animal breeding place. The contamination of Pseudomonas aeruginosa.
  • the invention provides an isolated Pseudomonas aeruginosa phage of the invention or a composition of the invention comprising one or more isolated Pseudomonas aeruginosa phage of the invention for use in monitoring, inhibiting or killing an animal Application of Pseudomonas aeruginosa in the environment of the breeding ground.
  • the term environment refers to air, appliances, articles, and the like that include a certain space.
  • the article is a feeding device, such as a cage, a food or drinking utensil, and the like.
  • the invention provides an isolated Pseudomonas aeruginosa phage of the invention or a composition of the invention comprising one or more isolated Pseudomonas aeruginosa phage of the invention for inhibiting or killing green Application in the drug of Pseudomonas.
  • the isolated phage of the present invention is applied to eliminate pathogens and monitor animal feeding environment hygiene, including but not limited to mites, especially leeches preventing the contamination of Pseudomonas aeruginosa during feeding.
  • the bacteriophage Lp14 of the present invention is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation date is March 30, 2017, and the deposit number is CGMCC No. 13791.
  • the present invention is the first to apply phage to the prevention of hemorrhagic pneumonia.
  • the first successful isolation of the present invention resulted in a Pseudomonas aeruginosa phage having a broad cleavage profile.
  • the phage of the present invention can be used for industrial production, thereby reducing the occurrence of hemorrhagic pneumonia caused by Pseudomonas aeruginosa in leeches culture, and has high safety.
  • the bacterial liquid of Pseudomonas aeruginosa was picked, and the three colonies were streaked on a common agar medium, and single colonies were isolated and cultured in an incubator at 37 ° C for 16-24 hours.
  • a single colony was picked, inoculated into a test tube containing 5 mL of LB broth, and shaken at 200 rpm for 12 hours in an air shaker at 37 ° C to obtain a single bacterial suspension of Pseudomonas aeruginosa.
  • the sample subjected to the above treatment was added to a cultured flask containing a mixed bacterial suspension, and cultured overnight at 200 rpm in an air shaker at 37 °C.
  • the mixture was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken out and filtered through a 0.22 ⁇ m filter to remove bacteria and set aside.
  • the phagocysts of the first isolated phage are usually inconsistent in size and shape. To ensure their purity, further purification is required.
  • the procedure is as follows: single plaques are taken and placed in a sterile EP tube containing 1 mL of physiological saline. 40 ° C water bath for 30 min, the leaching solution was centrifuged at 10,000 rpm for 5 min, the supernatant was taken and the single plaque was obtained by double plate method, so repeated 3-5 times until plaques of uniform size and morphology were obtained.
  • the phage proliferating liquid was further subjected to plaque purification by a double-layer plate method, and repeated 3-5 times to obtain a phage monomer, and the phage was named Lp14.
  • the cleavage spectrum of phage was determined by single spot method. The steps were as follows: one ring of Pseudomonas aeruginosa was picked up by an inoculating loop, and single colonies were streaked on a common agar plate, and cultured in an incubator at 37 ° C for 16-18 h. A single colony was picked up by a sterile tweezers, and the single colonies were picked up, inoculated in a test tube containing 5 mL of nutrient broth, and shaken at 37 ° C for 2 hours in an air bath at 200 rpm to obtain a single bacterial suspension.
  • 200 ⁇ L of the bacterial suspension was uniformly coated on a common agar plate, and 1 ⁇ L of phage proliferation droplets were taken at different positions on the plate, and then naturally dried, and then cultured in an incubator at 37 ° C for 6-8 hours, and the results were observed.
  • the phage culture was clarified for 3 hours, indicating that the phage has a strong cleavage ability to the host bacteria.
  • the phage Lp14 showed a long polyhedral three-dimensional structure, and the head was a long hexagon with a long diameter of about 60 nm, a broad diameter of about 50 nm, and a tail of about 30 nm.
  • 900 ⁇ L of LB broth was mixed with 100 ⁇ L of phage proliferation solution and diluted tenfold to 10 -8 in sequence.
  • Phage titer (pfu/mL) average plaque count ⁇ dilution factor ⁇ 10
  • the titer after phage proliferation is 5.0 ⁇ 10 10 PFU/mL or more.
  • Example 5 disinfection effect of phage on open field dust
  • the sample to be counted for bacteria was placed in a certain proportion of physiological saline, and shaken at room temperature to prepare a bacterial leachate.
  • the bacterial leachate was diluted 10 times with sterile physiological saline, and 1 mL of a certain dilution of the liquid was added to a sterile empty dish, and then added to a nutrient agar medium which was autoclaved and cooled to a non-hot hand (for total bacterial count) Or NAC agar medium (for Pseudomonas aeruginosa count), gently shake to evenly distribute it.
  • the phage Lp14 Due to the large number of Pseudomonas aeruginosa in the open field, the phage Lp14 has significant disinfection effect on the total number of bacteria in the dust in the open field and the number of various Pseudomonas aeruginosa, and the effect is obviously better than the Baidu killing group. Table 1).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Environmental Sciences (AREA)
  • Immunology (AREA)
  • Dentistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Agronomy & Crop Science (AREA)
  • Biomedical Technology (AREA)
  • Pest Control & Pesticides (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一株具有环境消毒能力的噬菌体及其应用，该噬菌体为铜绿假单胞菌噬菌体，拉丁名P.aeruginosaphage，被命名为Lp14，对铜绿假单胞菌(绿脓杆菌)具有广谱的杀菌能力，噬菌体Lp14保藏于中国微生物菌种保藏管理委员会普通微生物学中心，保藏日期为2017年3月30日，保藏编号为CGMCC No.13791；该噬菌体对绿脓杆菌具有强烈的裂解作用，为工业化生产噬菌体及用于消毒杀菌提供了噬菌体来源；该噬菌体还可作为一种安全的生物消毒剂用于貂场环境消毒，从而减少水貂养殖中的绿脓杆菌引起的出血性肺炎的发生，该噬菌体可以用于工业生产，可由宿主菌绿脓杆菌特异性扩增，可作为生物消毒剂应用于貂场环境消毒。

Description

一种宽裂解谱绿脓杆菌噬菌体及其消毒应用 技术领域
本发明属于生物工程领域，尤其涉及一株具有环境消毒能力的噬菌体及其用途。
背景技术
铜绿假单胞菌(Pseudomonas aeruginosa,PA)又称作绿脓杆菌，是引起水貂出血性肺炎的主要病原菌之一，在自然界中分布十分广泛，可引起水貂、狐狸、鸡等的急性感染。
近年来随着我国水貂养殖业的快速发展，以及养殖规模的不断扩大，由铜绿假单胞菌引起的水貂出血性肺炎常呈地方性和季节流行性爆发，病死率甚至高达50％，给养殖业带来了极大的经济损失。随着抗生素耐药性的日益严重，由于铜绿假单胞菌引起的感染使用常规抗生素已经无法有效的治疗，尤其是对氨基糖苷类、氟喹诺酮类和β-内酰胺类的敏感性显著降低。
因此，迫切需要研发具有全新作用机制的抗菌产品。而噬菌体以其高效性、低成本、易获得、特异性强、不易产生耐药性等特点，在辅助或者替代抗生素及消毒药方面表现出巨大的发展潜力，已经受到了越来越多人的关注。
本发明提供了一株具有环境消毒能力的噬菌体及其用途，旨在控制水貂养殖场的出血性肺炎的发生。水貂出血性肺炎的主要病原为绿脓杆菌，该病的发生主要集中在每年的7-10月份，此时正值水貂换毛期，养殖场内脱落的水貂毛随风飞扬，挂在貂笼上，粘在地面或粪便上。绿脓杆菌是环境中的常在菌，尤其在动物毛发上有大量的附着。水貂养殖场很少进行喷洒消毒，且常规的消毒药不能有效杀灭绿脓杆菌，还具有刺激性、腐蚀性等副作用。国内的研究者做过水貂密闭环境下的消毒试验，但对水貂养殖场进行噬菌体的环境消毒以控制水貂出血性肺炎的发生尚未见报道。
发明内容
为了解决上述问题，本发明提供一种绿脓杆菌噬菌体，该噬菌体单体为绿脓杆菌噬菌体，拉丁名P.aeruginosaphage，被命名为Lp14，对铜绿假单胞菌(绿脓杆菌)具有广谱的杀菌能力，噬菌体Lp14保藏于中国微生物菌种保藏管理委 员会普通微生物学中心，保藏日期为2017年3月30日，保藏编号为CGMCC No.13791；分类命名为绿脓杆菌噬菌体。噬菌体Lp14经双层平板法以17株水貂源绿脓杆菌为宿主菌，测定出噬菌体Lp14对17株宿主菌的裂解率为88.2％，说明该噬菌体是多价烈性噬菌体，在临床用于貂舍消毒具有很好的应用前景。
本发明一个方面提供了一种绿脓杆菌噬菌体，其保藏编号为CGMCC No.13791。
本发明另一个方面提供了本发明所述的绿脓杆菌噬菌体作为抑制绿脓杆菌的制剂中的用途。
本发明另一个方面提供了本发明所述的绿脓杆菌噬菌体在制备环境消毒制剂中的用途。优选地，在制备杀灭绿脓杆菌的环境消毒制剂中的用途，更优选为作为动物饲养、食品、药品、医疗器械制备工厂、医院、以及其他人类活动场所的环境消毒制剂；最优选作为貂类或禽类饲养场环境消毒制剂的用途。
本发明另一个方面提供了本发明所述绿脓杆菌噬菌体在制备预防貂类血性肺炎、鸡绿脓杆菌性脐炎、猪子宫内膜炎、奶牛乳房炎等的环境消毒制剂中的用途。
本发明再一个方面提供了一种环境消毒制剂，其中包含本发明所述的绿脓杆菌噬菌体，优选地，绿脓杆菌噬菌体浓度为10 8PFU/ml以上。
在本发明的技术方案中，本发明的环境消毒制剂可以为任何常用剂型，所述剂型包括但不仅限于喷雾剂、气雾剂、混悬剂、洗剂、淋洗剂、分散体等。
在本发明的技术方案中，所述环境消毒制剂为喷雾剂、洗剂或淋洗剂的形式，其中含有结冷胶等助悬剂、30％甘油等保护剂、培养液等溶剂。如果所述的环境消毒制剂配制成液体组合物时，其可以与喷射剂混合，以有助于喷雾组合物形成喷雾。如果是喷雾剂，施用装置可通过设置阀门来确定喷雾剂量。
在本发明的技术方案中，所述环境消毒制剂中还可以包含其他用于环境中病毒、细菌抑制或消灭的活性成分。
一种消灭环境中绿脓杆菌，或降低环境中绿脓杆菌浓度的方法，其包括向环境中施用本发明所述的绿脓杆菌噬菌体，或者本发明所述的环境消毒制剂，优选地，所述方法为非诊断和治疗目的的。
在本发明的技术方案中，所述绿脓杆菌噬菌体单独使用时，可以直接进行大面积的喷洒，无须密封消毒场所。作为消毒剂喷洒消毒于貂舍粉尘时，它对粉尘 中的细菌数和绿脓杆菌数均有极显著的消毒效果且极明显优于百毒杀，该噬菌体作为消毒剂用于貂舍粉尘消毒且有明显的消毒效果。
在本发明的技术方案中，所述绿脓杆菌噬菌体用于貂舍消毒，可明显降低出血性肺炎引起的死亡率。噬菌体的消毒效果优于百毒杀和对照组，且没有刺激性和腐蚀性，说明该噬菌体可为预防水貂出血性肺炎提供安全的生物消毒剂。
在本发明的环境消毒组合物中，还进一步包含载体。根据本发明的环境消毒组合物也可以包含常用赋形剂，特别是助悬剂、保护剂、双蒸水等，还可以包含益生微生物。本发明组合物中使用的载体不受任何限制，只要该载体的存在并不干扰噬菌体的裂解作用。
本发明中的绿脓杆菌噬菌体可配制成不同剂型，对环境进行消杀，能够高效裂解绿脓杆菌，可用于消除病原体、监控动物饲养场所中绿脓杆菌的污染情况。
在一个优选的实施方案中，本发明使用含有分离的绿脓杆菌噬菌体的组合物，配制成溶液剂、喷雾剂、分散体、混悬剂等，以用于抑制或杀灭绿脓杆菌。
在一个优选的实施方案中，将本发明的含有分离的绿脓杆菌噬菌体的组合物配制成喷洒液或淋洗液，对环境或器具进行消杀，用于消除病原体、监控动物饲养场所环境中绿脓杆菌的污染情况。
根据本发明的实施方案，本发明提供本发明的分离的绿脓杆菌噬菌体或本发明的含有一种或多种本发明的分离的绿脓杆菌噬菌体的组合物用于监控、抑制或杀灭动物饲养场所中环境中的绿脓杆菌的应用。
在本发明中术语环境是指包括一定空间之内的空气、器具和制品等。
在一个优选的实施方案中，所述制品为饲养用的器具，例如饲养笼、食物或饮水用用具等。
在本发明的一些实施方案中，本发明提供本发明的分离的绿脓杆菌噬菌体或本发明的含有一种或多种本发明的分离的绿脓杆菌噬菌体的组合物在制备抑制或杀灭绿脓杆菌的药物中的应用。本发明的分离的噬菌体应用于消除病原体、监控动物饲养环境卫生，其包括但不局限于貂类，尤其是水貂在饲养过程中防止绿脓杆菌的污染。
本发明噬菌体Lp14保藏于中国微生物菌种保藏管理委员会普通微生物学中心，保藏日期为2017年3月30日，保藏编号为CGMCC No.13791。
有益效果
1、本发明首次将噬菌体应用于水貂出血性肺炎的预防。
2、本发明首次成功分离获得了绿脓杆菌噬菌体，其具有宽裂解谱。
3、本发明的噬菌体可用于工业生产，作为从而减少水貂养殖中的绿脓杆菌引起的出血性肺炎的发生，安全性高。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。应理解，这些实施例仅用于说明本发明，而不是用来限制本发明的范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
实施例1噬菌体的分离与鉴定
(一)菌种的复苏培养
挑取铜绿假单胞菌的菌液，在普通琼脂培养基上三区划线，分离单菌落，37℃的恒温箱中培养16-24h。
(二)制备细菌增殖液
挑取单菌落，接种盛有5mL的LB肉汤的试管中，37℃的空气振荡器中，200rpm震荡培养12h，得到单一的铜绿假单胞菌的菌悬液。
(三)制备混合菌悬液
分别取200μL单一的菌液，加入到盛有100mL LB肉汤的三角瓶中，37℃的空气振荡器中，200rpm震荡培养12h。
(四)粪样、垫料、皮毛等的初步处理
取粪样、垫料和水貂的皮毛于盛有LB肉汤的三角瓶中，每个三角瓶中加入铜绿假单胞菌的混合菌液，搅拌均匀后，于37℃的恒温箱中浸泡6h,用纱布过滤掉多余的杂质。10000rpm，离心15min，取上清，待用。
(五)制备噬菌体增殖液
取上述处理的样品加入到培养好的盛有混合菌悬液的三角瓶中，37℃的空气振荡器中，200rpm震荡培养过夜。取上述混合液10000rpm，离心5min，取上清，用0.22μm的滤器过滤，除去细菌，待用。
(六)噬菌体的分离
将不同细菌的增殖液100μL分别与200μL相应的菌液混合，37℃恒温孵育5min，再将上述混合液加入到已融化好的上层琼脂中，用手迅速搓动试管，使 之混匀，然后迅速倒入准备好的普通琼脂培养板上，轻轻晃动使其分布均匀，待琼脂凝固之后放入37℃的恒温箱中，6-8h后观察结果。如果有噬菌体的存在，则会在培养基上形成透明、规则的圆形空斑，即为噬菌斑。
(七)噬菌体的纯化
初次分离的噬菌体其噬菌斑，通常其大小、形态不一致，为确保其纯度需要进一步的纯化，步骤如下：抠取单个存在的噬菌斑，放置于盛有1mL生理盐水的无菌EP管中，40℃水浴作用30min，浸出液经10000rpm离心5min，取上清再用双平板法得到单个噬菌斑，如此重复3-5次直至得到大小、形态一致的噬菌斑。
结果将噬菌体增殖液经双层平板法进行进一步噬斑纯化，重复3-5次，得到噬菌体单体，并将该噬菌体命名为Lp14。
实施例2噬菌体裂解谱的测定
采用单斑法测定噬菌体的裂解谱，步骤如下：分别用接种环挑取一环绿脓杆菌菌液，在普通琼脂平板上划线分离单菌落，于37℃的恒温箱中培养16-18h。用无菌镊子夹起灭菌后的白枪头分别挑取单菌落，接种于盛有5mL的营养肉汤的试管中，37℃，200rpm的空气浴震荡培养12h,得到单一的菌悬液，分别取200μL的菌悬液均匀涂布在普通的琼脂平板上，分别取1μL的噬菌体增殖液滴于平板的不同位置，待自然干燥后置37℃的恒温箱中培养6-8h，观察结果。
结果显示用17株绿脓杆菌为宿主菌进行裂解谱的测定，噬菌体Lp14的裂解率达88.2％(15/17)。
实施例3透射电镜观察噬菌体的形态
(一)样品的制备
分别取200μL的宿主菌液和噬菌体的增殖液，加入到盛有5mL营养肉汤的试管中，37℃振荡培养2-3h，直至发现混合液由浑浊变为澄清，并且有碎片存在，加入200μL的氯仿，继续震荡培养30min，经过10000rpm离心5min，得到噬菌体的增殖液，将制备好的噬菌体的增殖液用0.22μm的滤器过滤，除去可能残留的细菌。
结果加有噬菌体的培养物3h即可澄清，说明噬菌体对宿主菌具有很强的裂解能力。
(二)样品的处理
取样品悬液20μL滴于微孔铜片上，沉淀15min左右，用滤纸吸去多余的液体。在铜片上滴加15μL的2％的磷钨酸(PTA)。染色5min，用滤纸吸去多余的染液，干燥后用投射电子显微镜观察并拍照。
结果可见噬菌体Lp14呈长多面体立体结构，平面观察头部呈长六角形，长径约为60nm，宽径约为50nm，尾部约为30nm。
实施例4噬菌体效价的测定
(一)制备宿主菌菌液
挑取一环绿脓杆菌，在普通的琼脂板上划线分离单菌落，37℃恒温箱中培养12-16h。分别挑取单菌落，接种到盛有5mL营养肉汤的试管中，37℃空气震荡器中，200rpm震荡培养12h，得到宿主菌的菌悬液。
(二)噬菌体的稀释
900μL的LB肉汤和100μL的噬菌体的增殖液混合，依次十倍比稀释至10 -8。
(三)双平板法测效价
取两个合适稀释度的噬菌体各100μL与200μL的宿主菌混合，37℃恒温箱中温育5min，将混合液加入到已融化好的上层琼脂培养基中，混匀后倒入底层琼脂板中，轻轻转动平板，使上层琼脂培养基均匀的铺满平板，待其凝固后，倒置放于37℃的恒温箱中，培养6-8h。每个稀释度做3个平行，取噬菌斑数在30-300之间的稀释度来计数，取3个平行的平均值，并计算噬菌体的效价。
噬菌体的效价(pfu/mL)＝平均噬菌斑数×稀释倍数×10
噬菌体增殖后的效价达5.0×10 10PFU/mL以上。
实施例5噬菌体对貂场粉尘的消毒效果
(一)噬菌体计数方法(效价)
按实施例4中的方法进行噬菌体计数。
(二)细菌计数方法
将需进行细菌计数的样品放入一定比例的生理盐水中，室温充分振荡制成细菌浸出液。将此细菌浸出液用无菌生理盐水按10倍比例稀释，取一定稀释比例的液体1mL加入无菌空平皿，然后加入已高压灭菌并冷却至不烫手的营养琼脂培养基(用于细菌总数计数)或NAC琼脂培养基(用于绿脓杆菌计数)，轻轻摇晃使之均匀分布。室温放置30min使之凝固，37℃倒置培养10-12h，取合适稀释比例的平皿计算相应细菌数。每个稀释度设三个重复，进行平板计数。
(三)噬菌体Lp14对貂场粉尘的消毒试验
采集貂场粉尘，包括貂笼及其附近物品上粘附的动物毛发、粉尘。称取10g粉尘放入含有90mL无菌生理盐水的锥形瓶中，室温振荡混匀，将粉尘浸出液用无菌双层纱布过滤，取8mL滤液滴加于已高压灭菌并烘干的直径为15cm的滤纸上，将其干燥制成菌片，对其进行喷雾消毒，分为生理盐水对照组、Lp14噬菌体组和百毒杀消毒剂组。消毒之前调整噬菌体Lp14效价为1×10 8pfu/mL。消毒结束后37℃烘干滤纸，以滤纸为样品进行细菌计数。
由于貂场中存在的绿脓杆菌的种类较多，结果可见噬菌体Lp14对貂场粉尘中的细菌总数和各种绿脓杆菌数均有显著的消毒效果，且效果明显优于百毒杀组(表1)。
表1噬菌体对貂场粉尘消毒效果
Figure PCTCN2019071883-appb-000001
同行肩标字母相同表示差异不显著(P>0.05)，不同表示差异显著(P<0.05)。
实施例6噬菌体消毒对水貂出血性肺炎的预防效果试验
在出血性肺炎发病高峰期间(7～10月份)采用喷雾方式在貂场施用绿脓杆菌噬菌体。选择相邻两栋貂舍作为噬菌体消毒舍，间隔两栋舍，再选择相邻两栋貂舍作为百毒杀消毒舍，再间隔两栋舍，选择相邻两栋貂舍作为对照舍，每栋貂舍500只水貂。噬菌体消毒舍所用噬菌体浓度为10 8PFU/ml，百毒杀消毒舍所用浓度为常规使用浓度。每2周消毒一次，记录水貂死亡情况，对疑似出血性肺炎的死亡水貂进行剖检，核实由出血性肺炎引起的死亡，统计死亡率(表2)。
表2噬菌体对水貂出血性肺炎的预防效果
Figure PCTCN2019071883-appb-000002
结果表明，噬菌体消毒舍水貂出血性肺炎引起的死亡率为11.2％，百毒杀消毒舍的死亡率为18.8％，对照舍的死亡率为25.6％，说明噬菌体的消毒效果优于百毒杀和对照舍，且没有刺激性和腐蚀性。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (9)

  1. 一种用于环境消毒的绿脓杆菌噬菌体，其保藏编号为CGMCC No.13791。
  2. 如权利要求1所述的绿脓杆菌噬菌体作为抑制绿脓杆菌的制剂中的用途。
  3. 如权利要求1所述的绿脓杆菌噬菌体在制备环境消毒制剂中的用途；优选地，在制备杀灭绿脓杆菌的环境消毒制剂中的用途，更优选为作为动物饲养、食品、药品、医疗器械制备工厂、医院、以及其他人类活动场所的环境消毒制剂；最优选作为貂类或禽类饲养场环境消毒制剂的用途。
  4. 如权利要求1所述的绿脓杆菌噬菌体在制备预防貂类出血性肺炎、鸡绿脓杆菌性脐炎、猪子宫内膜炎、奶牛乳房炎的环境消毒制剂中的用途。
  5. 一种环境消毒制剂，其中包含权利要求1所述的绿脓杆菌噬菌体，优选地，绿脓杆菌噬菌体浓度为10 8PFU/ml以上。
  6. 如权利要求5所述的环境消毒制剂，环境消毒制剂的剂型选自喷雾剂、气雾剂、混悬剂、洗剂、淋洗剂、分散体、溶液剂。
  7. 如权利要求5所述的环境消毒制剂，其中还包含其他用于环境中病毒、细菌抑制或消灭的活性成分。
  8. 如权利要求5所述的环境消毒制剂，其中包含载体，所述载体为赋形剂、助悬剂、保护剂、溶剂。
  9. 一种消灭环境中绿脓杆菌，或降低环境中绿脓杆菌浓度的方法，其包括向环境中施用权利要求1的绿脓杆菌噬菌体，或者权利要求5-8的环境消毒制剂，优选地，所述方法为非诊断和治疗目的的。
PCT/CN2019/071883 2018-01-25 2019-01-16 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用 WO2019144830A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810071265.9A CN108070572A (zh) 2018-01-25 2018-01-25 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用
CN201810071265.9 2018-01-25

Publications (1)

Publication Number Publication Date
WO2019144830A1 true WO2019144830A1 (zh) 2019-08-01

Family

ID=62156988

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/071883 WO2019144830A1 (zh) 2018-01-25 2019-01-16 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用

Country Status (2)

Country Link
CN (1) CN108070572A (zh)
WO (1) WO2019144830A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4344399A4 (en) * 2022-08-11 2024-04-03 Phagelab Chile Spa BACTERIOPHAGES AND ASSOCIATED COMPOSITIONS FOR REGULATING THE GROWTH OF PSEUDOMONAS LUNDENSIS IN MEAT PRODUCTS

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108070572A (zh) * 2018-01-25 2018-05-25 青岛诺安百特生物技术有限公司 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用
CN109280650B (zh) 2018-08-22 2019-07-12 中国科学院南京土壤研究所 一株噬菌体及其在土壤修复中的应用
CN110144333B (zh) * 2019-05-27 2020-04-17 青岛诺安百特生物技术有限公司 一株铜绿假单胞菌噬菌体及其应用
CN111296493A (zh) * 2020-03-09 2020-06-19 苏州十一方生物科技有限公司 一种噬菌体消毒剂及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199576A (zh) * 2010-11-23 2011-09-28 天津科技大学 降解铜绿假单胞菌生物被膜噬菌体的分离与筛选方法
CN103981154A (zh) * 2014-05-28 2014-08-13 大连理工大学 一种铜绿假单胞菌噬菌体及其在水貂出血性肺炎预防中的应用
WO2015059298A1 (en) * 2013-10-25 2015-04-30 Pherecydes Pharma Phage therapy of pseudomonas infections
CN106929481A (zh) * 2017-02-20 2017-07-07 大连理工大学 一种铜绿假单胞菌噬菌体及其应用
CN108070572A (zh) * 2018-01-25 2018-05-25 青岛诺安百特生物技术有限公司 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100363489C (zh) * 2005-06-16 2008-01-23 华中科技大学同济医学院附属同济医院 一株耐亚胺培南绿脓杆菌噬菌体及其用于治疗耐亚胺培南绿脓杆菌感染的用途
CN101962631A (zh) * 2010-09-03 2011-02-02 黑龙江省科学院微生物研究所 耐药绿脓杆菌宽噬噬菌体的制备方法
CN104140957B (zh) * 2013-05-07 2017-04-26 兰州大学 一株可裂解多重耐药铜绿假单胞菌的噬菌体及其治疗感染的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199576A (zh) * 2010-11-23 2011-09-28 天津科技大学 降解铜绿假单胞菌生物被膜噬菌体的分离与筛选方法
WO2015059298A1 (en) * 2013-10-25 2015-04-30 Pherecydes Pharma Phage therapy of pseudomonas infections
CN103981154A (zh) * 2014-05-28 2014-08-13 大连理工大学 一种铜绿假单胞菌噬菌体及其在水貂出血性肺炎预防中的应用
CN106929481A (zh) * 2017-02-20 2017-07-07 大连理工大学 一种铜绿假单胞菌噬菌体及其应用
CN108070572A (zh) * 2018-01-25 2018-05-25 青岛诺安百特生物技术有限公司 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AL SAFFAR, M. F. ET AL.: "The Molecular and Biological Study of the Isolated Bacteriophages Infecting Pseudomonas aeruginosa From Sewage water", MESOPOTEMIA ENVIRONMENTAL JOURNAL., vol. 2017, no. C, January 2017 (2017-01-01) - 31 December 2017 (2017-12-31), pages 64 - 82, XP055627655 *
YANG, MEI ET AL.: "The Isolation and Identification of Pseudomonas Aeruginosa Lytic Phage YH6and Its Biological Characteristics", AGRICULTURE, CHINA MASTER'S THESES FULL- TEXT DATABASE, 15 September 2015 (2015-09-15) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4344399A4 (en) * 2022-08-11 2024-04-03 Phagelab Chile Spa BACTERIOPHAGES AND ASSOCIATED COMPOSITIONS FOR REGULATING THE GROWTH OF PSEUDOMONAS LUNDENSIS IN MEAT PRODUCTS

Also Published As

Publication number Publication date
CN108070572A (zh) 2018-05-25

Similar Documents

Publication Publication Date Title
WO2022048061A1 (zh) 一种耐高温宽裂解谱的沙门氏菌噬菌体及其组合物
WO2019144830A1 (zh) 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用
WO2019154032A1 (zh) 一种宽谱沙门氏菌噬菌体及其应用
CN110438091B (zh) 一株新的肺炎克雷伯氏菌噬菌体及其应用
CN106754745B (zh) 一种肺炎克雷伯氏菌噬菌体及其应用
CN111662880B (zh) 一株产气荚膜梭菌噬菌体、包括噬菌体的抑菌剂及制备方法和应用
CN112280749B (zh) 大肠杆菌噬菌体vB_EcoM_swi3及其应用
CN110129283B (zh) 一种短尾大肠杆菌噬菌体及其应用
CN110607284A (zh) 大肠杆菌噬菌体vB_EcoM_swi3及其应用
CN113755450B (zh) 一株大肠杆菌噬菌体gn4-1及其应用
CN113583971B (zh) 一株可同时裂解大肠杆菌的沙门氏菌噬菌体及其应用
CN113583972A (zh) 一株可降低抗生素耐药性的大肠杆菌噬菌体及其应用
WO2014019485A1 (zh) 广藿香酮的新用途
CN111690620A (zh) 一株魏氏梭菌噬菌体、其噬菌体组合物及其应用
CN115786279A (zh) 一种耐高温鸽源鼠伤寒沙门氏菌噬菌体、其噬菌体组合物及其应用
CN104845943B (zh) 一种链球菌噬菌体及其应用
KR20110117172A (ko) 공기-청정 소독제 및 이의 제조방법 및 용도
CN106939302B (zh) Etec噬菌体、基于该噬菌体的生物消毒剂及其应用方法
CN107794232B (zh) 拮抗痤疮丙酸杆菌和革兰氏阳性菌生长的链球菌及其代谢产物
CN109750003A (zh) 一种宽裂解谱绿脓杆菌噬菌体及其消毒应用
CN108103029A (zh) 一株可裂解牛源无乳链球菌的噬菌体及其应用
CN109266621A (zh) 一株新浅绿色气球菌噬菌体avp及其用途
CN106754751B (zh) 一种肠出血性大肠杆菌噬菌体及其应用
RU2435864C1 (ru) Способ пробиотической обработки клеток с содержащимися в них мелкими непродуктивными животными в условиях ветеринарного госпиталя
KR101842668B1 (ko) 신규한 살모넬라균 특이 박테리오파지 sg2 및 이를 포함하는 항균 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19743385

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19743385

Country of ref document: EP

Kind code of ref document: A1