US20120276612A1 - Phage of acinetobacter baumannii - Google Patents

Phage of acinetobacter baumannii Download PDF

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US20120276612A1
US20120276612A1 US12/854,542 US85454210A US2012276612A1 US 20120276612 A1 US20120276612 A1 US 20120276612A1 US 85454210 A US85454210 A US 85454210A US 2012276612 A1 US2012276612 A1 US 2012276612A1
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phages
acinetobacter baumannii
phage
present
surfactant
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Li-Kuang Chen
Nien-Tsung Lin
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Buddhist Tzu Chi General Hospital
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Buddhist Tzu Chi General Hospital
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Priority claimed from TW098127070A external-priority patent/TWI376418B/zh
Priority claimed from TW098127069A external-priority patent/TWI380777B/zh
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Assigned to TZU CHI BUDDHIST GENERAL HOSPITAL reassignment TZU CHI BUDDHIST GENERAL HOSPITAL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHEN, LI-KUANG, LIN, NIEN-TSUNG
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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • 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
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10131—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • 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
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10231—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Definitions

  • the present invention relates to a novel phage, and more particularly, to a phage of Acinetobacter baumannii.
  • Nosocomial infections are tough issues in Hospitals. Generally, the nosocomial infection rate is about from 3% to 5%. Organisms causing nosocomial infections are usually opportunistic pathogens. In other words, these bacteria are not harmful to hosts with normal immunity, and some of them are even normal flora to human; however, while hosts have weak immunity, the bacteria cause infections, resulting in diseases.
  • Bacteria causing nosocomial infections may exist in stethoscopes, anamnesis papers, tourniquets, grooves, syringe needles, respirators, humidifiers, furniture, floors, vents, monitors, water, soil, food (fruits, vegetables), dirt in drainage, human body such as skin, armpits, mucosal, oral cavity, upper respiratory tract, nasal cavity, gastrointestinal tract, etc.
  • nosocomial infections occur in an intensive care unit since patients in the intensive care unit have weak immunity and have invasive therapies such as being cannulated. According to statistics, the nosocomial infection rate in an intensive care unit is about from 2% to 3%.
  • nosocomial infections include Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, etc.
  • Antibiotics are general therapeutic agents for treating bacterial infections. However, when an antibiotic is overused, bacteria will be selected to have resistance to more antibiotics. In current nosocomial infections, there are more and more bacteria having resistance to antibiotics, and patients infected by these bacteria have to be treated with expensive and novel antibiotics. Further, if the antibiotic resistance keeps developed, there will be no effective antibiotic for therapy.
  • Acinetobacter baumannii (abbreviated as AB, hereafter) belong to Gram negative bacteria. Generally, Acinetobacter baumannii exist in skin, respiratory tract, and gastrointestinal tract in 10% population of human. Acinetobacter baumannii favor warm and humid environment, so as to exist in medical devices, water troughs, beds, bed mats, respiratory devices and even air in a hospital.
  • Acinetobacter baumannii having multiple resistances to gentamicin, amikacin piperacillin/tazobactam, ticarcillin/clavulanate, ceftazidime, cefepime, cefpirome aztreonam, imipenem, meropenem, ciprofloxacin and levofloxacin have been isolated. Since Acinetobacter baumannii easily become having multiple resistances and are capable of living for a while on surfaces of an object, it is a tough issue in prevention and treatment of nosocomial infections.
  • Phages are viruses that infect bacteria, and grow and replicate in bacteria. There are lytic phages and lysogenic phages. Lytic phages infect bacteria, replicate in bacteria, and then are released from bacteria by lysing and killing bacteria. Lysogenic phages are capable of undergoing lytic or lysogenic life cycles, and exist in host cells while in lysogenic life cycles.
  • U.S. Pat. No. 5,688,501, U.S. Pat. No. 5,997,862, U.S. Pat. No. 6,248,324 and U.S. Pat. No. 6,485,902 have disclosed a pharmaceutical composition comprising phages for treating bacterial diseases, group A streptococcal infections, dermatological infections, and control of Escherichia coli O157 infections, respectively.
  • U.S. Pat. No. 6,121,036 has disclosed a pharmaceutical composition having at least one phage.
  • U.S. Pat. No. 6,699,701 has disclosed using Salmonella enteritidis -specific phages for packing food, in which a package material is coated with phages, and food (such as fruit and vegetables) is packed with the package material.
  • the present invention provides an isolated Acinetobacter baumannii phage, comprising one or more genomic sequences selected from the group consisting of sequences of SEQ. ID. NO: 1, 2, 3 and 4 (as shown in sequence listing), and sequences having more than 80% homology thereof.
  • sequences of SEQ ID NO. 1 and SEQ ID NO. 2 are DNA sequences encoding RNA polymerase of Acinetobacter baumannii phages. Upon sequence alignment, there is no viral sequence in the gene bank identical or similar to the sequences of SEQ ID NO. 1 and SEQ ID NO. 2 in the present invention.
  • Acinetobacter baumannii phages of the present invention were deposited in DSMZ (German Collection of Microorganisms and Cell Cultures, German), and have deposition numbers as DSM 23599 and DSM23600.
  • Acinetobacter baumannii phages are variants of the above-mentioned deposited phages, and have genomic sequences with homology more than 80% of those in above-mentioned deposited phages.
  • the Acinetobacter baumannii phages of the present invention are lytic phages and specifically infect Acinetobacter baumannii.
  • the phages replicate and propagate in the host cells and lyse cell walls of host cells, and then Acinetobacter baumannii are destructed along with the release of the phages.
  • the phages of the present invention are capable of reducing the amount of Acinetobacter baumannii and disinfecting environments, especially reducing nosocomial infections caused by Acinetobacter baumannii.
  • the Acinetobacter baumannii phages are capable of attaching rapidly to Acinetobacter baumannii, have short latent period, and have large burst size upon lysis of Acinetobacter baumannii.
  • the phages of the present invention have double-stranded DNA having 35 to 40 kb as genetic material.
  • FIG. 1 shows the viral particles of the phage of the present invention, in which the viral particle has a head portion with 20 faces and a tail portion have filament structure for attaching to the surface of host cells.
  • the head portion of the viral particle is about 60 nm, and the tail portion is about 9 to 11 nm
  • the Acinetobacter baumannii phages have acid tolerance and alkali tolerance, and have bioactivity in the environment at pH 4 to 12.
  • bioactivity refers to that the pages are capable of infecting host cells, Acinetobacter baumannii, propagating in the host cells and/or lysing the host cells.
  • the phages of the present invention have bioactivity in a surfactant.
  • the surfactant is one selected from the group consisting of an anionic surfactant, a cationic surfactant, an amphoteric surfactant and a non-ionic surfactant.
  • the anionic surfactant can be, but not limited to, ammonium dodecyl sulfate, disodium laureth sulfosuccinate, disodium octyl sulfosuccinate, linear dodecyl benzene sulfonates, dodecyl phosphates (mono alkyl phosphate, MAP), secondary alkane sulfates (SAS), sodium cocoyl isethionate (SCID), sodium lauryl ether sulfate (SLES), sodium lauroyl sarcosinate, sodium lauryl sulfate (SLS), sodium taurine cocoyl methyltaurate and so on.
  • ammonium dodecyl sulfate disodium laureth sulfosuccinate, disodium octyl sulfosuccinate, linear dodecyl benzene sulfonates
  • the cationic surfactant can be, but not limited to, cetyl trimethyl ammonium chloride, dicocodimonium chloride, didoctyl dimethyl ammonium chloride, diester quaternary ammonium salts, alkyl dimethyl benzyl ammonium chloride, ditallow dimethyl ammonium chloride (DTDMAC), imidazoline quaternary ammonium salts and so on.
  • cetyl trimethyl ammonium chloride dicocodimonium chloride, didoctyl dimethyl ammonium chloride, diester quaternary ammonium salts, alkyl dimethyl benzyl ammonium chloride, ditallow dimethyl ammonium chloride (DTDMAC), imidazoline quaternary ammonium salts and so on.
  • the amphoteric surfactant can be, but not limited to, cocoyl lmidazolinium betaine, cocoamidopropyl hydroxysultaine, cocpamidopropyl dimethyl betaine, disodium cocoamphodipropionate, lauramidopropyl betaine, sodium alkylamphopropionate, tallow dihydroxyethyl betaine and so on.
  • the non-ionic surfactant can be, but not limited to, alkyl polygluoside (APG), cocoamide DEA, lauramine oxide, lauryl ether carboxylic acid, Triton X (such as TX-100, TX-405, etc.), PEG-150 di-stearate, Tween (such as Tween-40, Tween-80, etc.) and Span (such as Span-20, Span-80, etc.) and so on.
  • APG alkyl polygluoside
  • cocoamide DEA cocoamide DEA
  • lauramine oxide such as TX-100, TX-405, etc.
  • PEG-150 di-stearate such as Tween (such as Tween-40, Tween-80, etc.)
  • Span such as Span-20, Span-80, etc.
  • the surfactant is a non-ionic surfactant.
  • the surfactant is a commercial product, especially a detergent.
  • the present invention provides Acinetobacter baumannii phages for sterilizing Acinetobacter baumannii, and for preparing a pharmaceutical composition for treating diseases caused by Acinetobacter baumannii.
  • Acinetobacter baumannii phages are used as a sterilizing agent in health care centers (such as home care nursing), medical centers (such as hospitals, sanitaria, etc.) and medical research institutes, so as to reduce the amount of Acinetobacter baumannii in the environment.
  • Acinetobacter baumannii phages of the present invention can be used in health care centers, medical centers and medical research institutes, for example, but not limited to, intensive care units, surgeries, recovery rooms, consulting rooms and conference rooms. Also, Acinetobacter baumannii phages of the present invention can be applied to equipments in hospitals and sanitaria, for example, but not limited to, stethoscopes, anamnesis papers, tourniquets, grooves, syringe needles, respirators, humidifiers, furniture, floors, vents and monitors.
  • Acinetobacter baumannii phages of the present invention can be directly or indirectly sprayed or applied on the objects (such as lotion for human skin).
  • the objects can be immersed in the composition having Acinetobacter baumannii phages of the present invention.
  • FIG. 1 shows SEM images of Acinetobacter baumanni phages according to the present invention
  • FIG. 2A shows DNA pulsed-field gel electrophoresis patterns of restriction digests of Acinetobacter baumannii phage according to one embodiment of the present invention, using short (0.2-12 s for 6.5 h, left panel) and long (0.2-0.5 s for 16.5 h, middle and right panel) running conditions, in which M is molecular standard, 1 to 9 respectively indicate DNA samples treated with HincII, HindIII, SnaBI, SspI, EcoRV, BglII, MluI, XbaI, and EcoRI;
  • FIG. 2B shows the restriction enzyme map of DNA of Acinetobacter baumannii phage according to one embodiment of the present invention
  • FIG. 3 shows SDS-polyacrylamide gel electrophoresis of viron protein of Acinetobacter baumannii phage according to one embodiment of the present invention, in which M is molecular standard;
  • FIG. 4 shows the absorption of Acinetobacter baumannii phage according to the present invention to Acinetobacter baumannii ATCC 17978;
  • FIG. 5 shows the one-step growth curve of Acinetobacter baumannii phages according to the present invention on Acinetobacter baumannii ATCC 17978;
  • FIG. 6 shows the viability of Acinetobacter baumannii phages according to the present invention in surfactants
  • FIG. 7A shows the viability of Acinetobacter baumannii phages according to the present invention at different temperatures
  • FIG. 7B shows the viability of Acinetobacter baumannii phages according to the present invention at different temperatures and thaw conditions
  • FIG. 8 shows the viability of Acinetobacter baumannii phages according to the present invention at different pH
  • FIG. 9 shows the viability of Acinetobacter baumannii phages according to the present invention in chemicals.
  • strains of Acinetobacter baumannii phages obtained in the present invention which were named as ⁇ AB1 (deposition number: DSM 23599), ⁇ AB2 (deposition number: DSM 23600), ⁇ AB3 (a variant of ⁇ AB2) and ⁇ AB4 (a variant of ⁇ AB2), wherein ⁇ AB3 and ⁇ AB4 respectively have more than 80% of homology to ⁇ AB2.
  • the four strains of phages were all capable of infecting Acinetobacter baumannii with different infectivity.
  • Acinetobacter baumannii strains listed in Table 1 were used, in which 35 Acinetobacter baumannii strains were collected from Buddhist Tzu Chi General Hospital, Hualien, and 2 strains were obtained from ATCC (American Type Culture Collection).
  • Bacterial lawns were prepared by covering 1.8% of LB agar plate with a layer of 0.7% of LB agar having host cells (strains as listed in Table 1).
  • phage solution 10 10 PFU/ml obtained from example 1 was dropped into the bacterial lawns.
  • the agar plate was dried for 10 minutes in the laminar flow, and then incubated at 37° C. for 18-20 hours. Subsequently, the production of plaques was observed.
  • MDRAB Acinetobacter baumannii have multiple resistances to gentamicin, amikacin, piperacillin/tazobactam, ticarcillin/clavulanate, ceftazidime, cefepime, cefpirome, aztreonam, imipenem, meropenem, ciprofloxacin, and levofloxacin.
  • Amp ampicillin; Imi: imipenem; Mer: meropenem; r resistant; s sensitive
  • the phages obtained from example 1 formed no plaque on the bacterial lawns of A. calcoaceticus, 10 strains of E. coli, 6 strains of K. pneumoniae and 3 strains of P. aeruginosa, and plaques were only formed on the bacterial lawns of Acinetobacter baumannii.
  • the phages of the present invention specifically infected Acinetobacter baumannii.
  • the phages obtained from example 1 formed plaques on the bacterial lawn of Acinetobacter baumannii strains listed on Table 1.
  • the pages of the present invention are capable infecting clinically separated Acinetobacter baumannii having multiple resistances, wherein ⁇ AB2 also infect two standard strains obtained from ATCC, in addition to infect clinically separated Acinetobacter baumannii having multiple resistances.
  • the isolated ⁇ AB2 (10 12 PFU/ml) was dropped on formvar-coated copper grid (200 mesh copper grids), negatively stained by 2% uranyl acetate, and placed on TEM (Hitachi Company, Japan; mold: H-7500, operation condition: 80 kV). The image obtained is shown in FIG. 1 .
  • the viral particle of ⁇ AB2 has a head portion having a size of 60 nm and 20 faces, and a tail portion having a size of about 9-11 nm and filament structures.
  • AB culture solution 200 ml of AB culture solution at early stage of log phase was infected with ⁇ AB2 (MOI being about 1.0), and incubated under aeration until AB were completely lysed. Then, the culture solution was centrifuged, the supernatant was filtered with 0.45 ⁇ m film, the filtrate was centrifuged at 18,000 rpm for 2 hours (Beckman Avanti J-251), and the precipitants obtained were viral particles of phages. Then, the precipitants were dissolved with 1.0 ml of TE buffer (10 mM Tris-HCl, pH7.0 including 1.0 mM EDTA), and then super-centrifuged at 25,000 rpm, 4° C. for 2 hours, so as to purify the band of phages. The purified phages band were dialyzed to remove TE buffer, and then storage at 4° C.
  • MOI being about 1.0
  • the phage particles were concentrated with 20% polyethylene glycol 6000, extracted with phenol/chloroform, and then precipitated with ethanol, so as to obtain DNA of phages.
  • the DNA was treated with ApaI, BamHI, BanII, BglII, EcoRI, EcoRV, HincII, HindIII, KpnI, MluI, PstI, PvuII, SacI, SmaI, SnaBI, SphI, SspI, StuI and XbaI, respectively, and analyzed by 0.8% and 1.0% agarose gel with pulse field electrophoresis in TAE buffer.
  • DNA of ⁇ AB2 was digested by BglII, EcoRI, EcoRV, HincII, HindIII, MluI, SnaBI, SphI, SspI and XbaI.
  • the standard molecule (M) was 1-kb plus DNA Ladder (Invitrogen, CA).
  • the restriction enzyme map of the phage DNA is shown in FIG. 2B , in which the cutting sites of BglII, EcoRI, EcoRV, MluI and XbaI are indicated.
  • the purified phage particles and the sample buffer solution (62.5 mM Tris-HCl including 5% 2-mercaptoethanol, 2% sodium dodecylsulfate, 10% glycerol and 0.01% phenol blue, pH 6.8) were mixed, heated in boiled water bath for 3 minutes, and then analyzed in 12.5% SDS-PAGE.
  • FIG. 3 shows protein electrophoresis patterns of ⁇ AB2.
  • the phage has at least 10 different protein bands in the range of 21 and 140 kDa, in which the protein of 33 kDa is the most abundant and could be the coat protein of the phage.
  • the Sau3A1-partial fragments (ca. 15 kb) of the phage genome was cloned to pUC18, and DNA inserts from six clones were sequenced.
  • the sequence analysis was performed by NCBI package.
  • sequences of SEQ. ID NO. 1 to 4 were obtained.
  • the sequences of SEQ ID NO. 1 and SEQ ID NO. 2 are DNA sequence encoding RNA polymerase of the Acinetobacter baumannii phage.
  • the sequences of SEQ ID NO. 3 and SEQ ID NO. 4 are DNA sequence encoding the head-tail connector of the Acinetobacter baumannii phage.
  • sequences of SEQ. ID NO. 1 to 4 were aligned with the gene database of NCBI. There is no identical or similar sequence in database as the sequences of SEQ. ID NO. 1 to 4 of the present invention.
  • the alignment result shows that the DNA sequence of SEQ ID NO. 1 has 39.4% homology with phiAB1-LKA1, 41.3% homology with phiKMV, 41.3% homology with phiPT5, 41.5% homology with phiPT2, and 41.5% homology with phiLKD16. Accordingly, the DNA sequence of SEQ ID NO. 1 has no more than 40% homology with the sequences in NCBI database.
  • amino acid sequence encoded by the DNA sequence of SEQ ID NO. 1 has 30.6% homology with phiAB1-LKA1, 29.4% homology with phiKMV, 29.4% homology with phiPT5, 29.3% homology with phiPT2, and 29.2% homology with phiLKD 16. Accordingly, the amino acid sequence encoded by the DNA sequence of SEQ ID NO. 1 has no more than 30% homology with the protein sequences in NCBI database.
  • RNA polymerase is highly conserved region in viral genome. Therefore, homology among species can be determined by identifying homology of RNA polymerase.
  • sequence alignment there is no identical or similar viral sequence as the sequences of the phages in the present invention. It is clear that the present invention provides novel phages. Further, the sequences of SEQ ID NO.1 and SEQ ID NO.2 have been registered in NCBI database, and had the registration numbers as bankit1192576 FJ809932 and bankit1192679 FJ809933, respectively (which are not published before the filing of the present application).
  • the AB culture (host cell) was incubated to OD 600 as 0.6 U, and then the Acinetobacter baumannii phage was added to the host cell culture (MOI: 0.0005) and incubated at room temperature. At the time points of 0, 1, 2, 3, 4, 5, 10, 20 and 30 min, 100 ⁇ l of culture was sampled and diluted with 0.9 ml of cold LB, and then centrifuged at 12,000 ⁇ g for 5 minutes. The supernatant was collected, and the amount of the phage without attaching to host cells was determined For example, the result of ⁇ AB2 to ATCC 17978 is shown in FIG. 4 .
  • the culture solution Upon observation of the host cell culture added with the phages, the culture solution turned from turbid into clear in 100 minutes. It is proved that the host cells were all lysed, and thus the composition of the present invention can be used for sterilization.
  • the replication curve of the phages was determined by one-step growth curve.
  • the AB culture solution (OD 600 : 0.8U) was centrifuged, and the precipitant was collected and resuspended in 0.8 ml of LB medium to a concentration of 10 9 CFU/ml.
  • the AB-specific phages (MOI: 0.0001) were added to the host cell culture solution, and placed at 4° C. for 30 minutes, such that the phages attached to the host cells.
  • the mixture was centrifuged at 12,000 ⁇ g for 10 minutes, and the precipitant including the infected bacteria was re-suspended with 20 ml of LB medium, and incubated at 37° C.
  • the culture was sampled every 5 minutes, and the samples were immediately diluted and quantified. For example, the result of ⁇ AB2 to ATCC 17978 is shown in FIG. 5 .
  • the definition of a latent period is from the attachment (excluding 10 minutes of the pretreatment) to the beginning of the first burst (bacteria were lysed, and phages were released). As shown in FIG. 5 , the latent period is 15 minutes. The ratio of the amount of phage particles to the initial amount of the infected bacteria was calculated. The average burst is about 200 PFU/cell.
  • the compatibility of the AB phages isolated in embodiment was determined with the surfactants TWEEN 20, TWEEN 80 and Triton X-100 (Sigma-Aldrich Biotechnology, USA).
  • the common concentration of the conventional surfactants is 0.1-1 wt %.
  • 1 wt % of the above surfactants was mixed with the AB phages (5 ⁇ 10 7 PFU/ml). The mixture was incubated at room temperature, and the concentration of phage culture was determined every 24 hours.
  • the viability of phages was calculated based on the following equation, so as to determine the effects of surfactants on the phages.
  • viability of phages concentration of sampled phage culture/original concentration of phage culture
  • FIG. 6 shows the result of ⁇ AB2.
  • the phages had excellent stability in Triton X-100 and TWEEN 20, and moreover, the phages had varied viability in TWEEN 80 but still had infectivity to host cells.
  • the concentration of phages was decreased slightly and then increased gradually. It was known that by using coefficient variation, CV values of the three surfactants were all less than 20%. Accordingly, the phages were very stable in these surfactants, and all had bioactivity.
  • At least one of ⁇ AB1 to ⁇ AB4 phages can be mixed with a carrier (such as water, a surfactant (Triton X-100, TWEEN 20, TWEEN 80, etc.) to form a composition for sterilization of equipments or environment.
  • a carrier such as water, a surfactant (Triton X-100, TWEEN 20, TWEEN 80, etc.)
  • the initial content of the phage is 1 ⁇ 10 7 to 1 ⁇ 10 9 PFU/ml
  • the content of the surfactant is 0.1 to 2 wt %.
  • the phages were diluted with autoclaved water to 10 8 PFU/ml, and then placed at different temperatures, 4 ⁇ , 25 ⁇ , 37 ⁇ , 42 ⁇ , ⁇ 20 ⁇ and ⁇ 80 ⁇ .
  • the concentration of the phage culture was determined every 3 hours in 24 hours, and then determined every week for 12 weeks.
  • FIG. 7A there were respective two groups at ⁇ 20 ⁇ and ⁇ 80 ⁇ , in which one group was repeatedly frozen and thawed and the determination was performed for 12 weeks, and the other group was thawed once and the determination was performed for 5 weeks.
  • the results were shown in FIG. 7B .
  • the phages were diluted with acidic solution (pH 4) or basic solution (pH 11) to 10 8 PFU/ml.
  • the concentration of the phage cultures at pH 4.7, 7 and 11 was determined every 3 hours in 24 hours, and then determined every week for 12 weeks.
  • FIG. 8 shows the results.
  • the phages were added to chloroform solution (0.5% and 2%, respectively), and the phages were diluted to 10 8 PFU/ml.
  • the concentration of the phage culture was determined every 3 hours in 24 hours. Then, the concentration of the phage culture in 0.5% chloroform solution was determined every week for 3 weeks, and the concentration of the phage culture in 2% chloroform solution was determined every week for 6 weeks.
  • FIG. 9 shows the results.
  • 10 10 PFU/ml of phages were grouped into groups A and B.
  • Groups A and B of the phages were diluted with peptone ad autoclaved water, respectively, for ten folds, and then dried in the speed vac system. After dry treatment, groups A and B of the phages were respectively dissolved in 0.5 ml of peptone and 0.5 ml of autoclaved water. The concentrations of the phages before and after the dry treatment were determined and shown in Table 2.
  • the phages of the present invention survived for at least 8 weeks at low temperatures ( ⁇ 20 ⁇ , ⁇ 80 ⁇ , 4 ⁇ ), and had the viability more than 5%.
  • the phages survived for at least 11 weeks and had the viability more than 14.9%.
  • the phages had the viability as 14.8%.
  • the phages of the present invention incubated at pH 11 for about 11 weeks had the viability as about 30%. There were alive phages, which were incubated at pH 4 for 11 weeks.
  • the phages of the present invention in 0.5% and 2% chloroform solution survived for at least 3 weeks and had the viability as 30%. After dry treatment and re-dissolution, the viability of phages was more than 20%.
  • the phages of the present invention have tolerance to temperatures, humidity, pH and chemicals, and maintain good viability in these conditions.

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Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
TW098127069 2009-08-12
TW098127070A TWI376418B (en) 2009-08-12 2009-08-12 Phage of acinetobacter baumannii
TW098127070 2009-08-12
TW098127069A TWI380777B (zh) 2009-08-12 2009-08-12 包含噬菌體之活體外殺菌組成物

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US8273564B2 (en) 2012-09-25
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