TWI376418B - Phage of acinetobacter baumannii - Google Patents

Description

1376418 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a novel phage, and more particularly to a bacteriophage of Acinetobacter baumannii. [Prior Art]

Nosocomial infection is the most difficult problem in hospitals. According to statistics, the hospital's in-hospital infection rate is generally about 3% to 5%. The fine side of the nosocomial infection is usually a 〇pp〇rtunistic pathogen, which means that for a host with normal immunity, the bacteria are harmless, some fine edges and even 疋 _ normal bacteria on the surface of the human body Clumps (n〇rmal £J〇ra); but when the main immunity declines, these bacteria are prone to cause infection and cause disease. Bacteria that cause nosocomial infections may survive in stethoscopes, medical records, tourniquets, gloves, needles, respirators, wet bottles, furniture, floors, vents, monitors, etc.; or in water, soil, and food (fruits, vegetables) And sewerage; or in the human body, such as the skin, underarms, conjunctiva, mouth, upper respiratory tract, nasopharynx and gastrointestinal tract. The occurrence of nosocomial infections is based on the intensive care unit. Because the patients are mostly critically ill, 'human immunity is poor' and often require invasive treatment, such as intubation and vascular devices, which greatly increases the possibility of nosocomial infection. According to statistics, The infection rate in the intensive care unit is about 20 to 3 thousand. Miyagang's most common nosocomial infections include Pseudomonas aeruginosa, Helong, and sputum g (10) (10) (4) and Acinetobacter baumannii (a- baumannii). 111296 3 1376418 The treatment of common bacterial infections is the use of antibiotics. However, due to the abuse of antibiotics, bacteria have been screened to evolve more resistance, and there are more and more bacteria that are resistant to antibiotics in the hospital. To treat patients infected with this bacterium, expensive new anti-tai's must be used and if bacterial resistance continues to develop, it will result in the availability of no effective antibiotics. Acinetobacter baumannii in this description Yuan: sometimes referred to as sputum bacteria is a gram-negative bacterium. Generally, the presence of Bow's fixed rod can be found in the skin, respiratory tract, gastrointestinal tract, etc. of about 1% of people. Acinetobacter baumannii prefers to grow in a humid and warm environment, so it can be used in hospital carts, medical equipment, sinks, hospital beds, mattresses, respirators, and even in the air. Clinically, sputum bacteria with multiple antisense properties have been isolated. They are for gentamicin, amikacin, piperacillin/tazobactam, and ticarcillin/clavulanate. ), ceftazidime, cefepime, cefpirome, aztreonam, imipenem, meropenem, ciprofloxacin and Levofloxacin is resistant. Because Acinetobacter baumannii is easy to form multiple drug resistance and can survive on the surface of the object for a period of time, it becomes a difficult problem in the prevention and treatment of nosocomial infection. The phage (bacteriophage) is a kind of virus characterized by the fact that the host of the 1& corpuscle is fine 囷' must be able to grow and replicate in the corpus callosum. The bacillus can be divided into lytic and lysogenic, and the lytic bacterium can infect the host bacteria. After replication in the host, 嗤4 111296 1376418, 'the bacteria will lyse and release the bacteria. Out, the bacteria ruptured and died. Solvent-producing bacteria The milder sputum bacteria can undergo a history of lytic or prototyping, and coexist with the host in the path of the protoplast. In the case of the use of bacteriophage for the treatment of bacterial diseases, for example, U.S. Patent Nos. 5,688,501, 5,997,862, etc., 6,248,324, 6,4S5,902, respectively, disclose the use of pharmaceutical compositions including phage Treatment of bacterial diseases, diseases caused by Streptococcus type A (you _ 〇 (3) c (10) A), bacteria causing skin infection, E. coli 〇 15 % strain, etc.; US Patent No. 6,121, 〇 36 discloses more than one The pharmaceutical composition of the bacteriophage; U.S. Patent No. 6,699,7,1 discloses a method for food packaging using Salmonella typhimurium (10)/7_Taga (7). On the packaging material, the food (such as fruits and vegetables) is covered with packaging materials. None of the above cases reveals the bacillus of Acinetobacter baumannii, nor does it disclose the use of bacteriophage of Acinetobacter baumannii to reduce the number of Acinetobacter baumannii and reduce the infection of the hospital. SUMMARY OF THE INVENTION To overcome the above and other problems, the present invention provides an isolated Acinetobacter baumannii 6 (10) visceral (10) (1) phage having a sequence selected from SEQ ID NO. 1, 1, 2, 3, and with SEq ID N〇", 2, 3, 4 sequence sequence similarity of more than 80% of the sequence of the genome sequence of one or more sequences; SEQ ID NO, 1, 2, 3, 4 of the sequence is The sequence listing attached to the manual is shown. The RN A polymerase gene sequence is known in the art as a viral genetic material. 5 111296 ΐ i 1376418 in a highly conserved region, the species can be judged by comparing the sequence similarity of the RN A polymerase gene between species Prosociality. The sequence of SEQ ID NO, 1 to 2 of the present invention is a deoxyribonucleic acid (1) > ^) sequence ', an RNA polymerase polymerase encoding the AB bacillus cell line). In the present invention, the sequences of SEQ ID NOS. 1 to 2 are aligned with the gene library, and the same or similar viral sequences are not obtained. The Acinetobacter baumannii phage system of the present invention is deposited in the Food Industry Development Research Institute (Hsinchu City, Taiwan) under the registration numbers BCRC97〇〇46 and BCRC970047. In one aspect, the AB bacteriophage of the present invention may also be a mutant strain of the above-mentioned registered number, wherein the mutant strain has a sequence of 8 % or more with any of the phage of the accession number BCRC970046 or BCRC970047. Similarity. The AB bacteriophage system of the present invention specifically infects Acinetobacter baumannii, and is a lytic (four) body, that is, the body of the present invention can infect the host bacteria AB bacteria, and can be lysed after replication and proliferation in the bacterial cells are completed. The cell wall of Acinetobacter baumannii releases and proliferates, and the body, Acinetobacter baumannii ruptures and dies. Thereby, the phage of the invention can reduce the number of ab bacteria, and can be applied to environmental disinfection, especially the nosocomial infection of AB bacteria. In the aspect, the AB 8(9) body of the present invention has the ability to rapidly adsorb Acinetobacter sphaeroides, has a short incubation period, and has a large burst size of the stalked body released by the lysis of Acinetobacter baumannii. The sputum system of the present invention uses double-stranded DNA as a genetic material, and the DNA has a total length of about 35 to 40 Kb, and the virion particle shape is as shown in the figure, and has a head and a tail, and the head has a 2-faceted structure. The tail has a filamentous structure]] 296 1376418 attached to the surface of the host cell, the size of the virus particle is about 60 nm in the head, and the tail is about 9 to 11 nm. In one aspect, the AB bacteriophage of the present invention has acid. Tolerance and alkali tolerance The biological activity of the phage is maintained in an environmental condition above pH 4 to below pH 12. The biological activity of the phage contained in the present specification means that the phage retains the infectivity to the host Acinetobacter baumannii in the environment, and is capable of infecting the host. The ability to proliferate in a host cell and/or to lyse host cells.

In one aspect, the AB bacteriophage of the present invention retains the biological activity of the phage in the surfactant. In one aspect, the surfactant is selected from the group consisting of an anionic interface active agent, a cationic surfactant, a zwitterionic surfactant, and a nonionic surfactant. In a preferred embodiment, the anionic surfactant is, by way of example and not limitation, ammonium lauryl sulfate, disodium laureth sulfosuccinate, disodium octylsulfonyl succinate, soft Dodecyl sulfonic acid, dodecyl phosphate (MAP), secondary alkyl phosphate (SAS), sodium cocoyl hydroxyethyl sulfonate (SCID), sodium laureth sulfate (SLES) ), laurel, sarcosine, sodium sulphate, sodium sulphate, sodium sulphate, sodium sulphate, sodium sulphate In a preferred embodiment, the cationic surfactant is, by way of example and not limitation, Cetyl tridecyl ammonium chloride, dicocoyl dimercapto ammonium chloride, dimercapto Mercapto ammonium chloride, diester quaternary ammonium salt, alkylbenzyl diammonium ammonium chloride, tallow alkyl diammonium ammonium chloride (DTDMAC), imidazole 7 111296 x 1376418 Linde salt and the like. In the preferred embodiment, the zwitterionic surfactant is, by way of example but not limited to, cocoyl lmidazolinium betaine, cocoamine hydroxyglycoline betaine, coconut oil Amidoxime bis-indenyl betaine, cocoyl amphoteric dipropionate di-n-lauric acid propyl hydrazide-based sweet test, s〇diUm alkylamphopropionate, tallow base II Hydroxyethyl betaine and the like. In a preferred embodiment, the nonionic surfactant is, by way of example and not limitation, alkyl polyglucoside (APG), cocamide (c〇c〇amide DEA), laurylamine oxide, laurel Carboxyl carboxylate, Trit〇n χ (eg TX-100, TX-405, etc.), PFG-ΐα a Μ , 150 di_stearate, Tween (eg

Tween-40, Tween-80, etc.), 0#', Span (such as Span-2〇, Span-80, etc.).

In a preferred embodiment, the 5 " surfactant is a nonionic interfacial activity. In a preferred embodiment, the interface is a cleaner product. " ^Products, particularly because of the use of the present invention for the purpose of sterilizing _specificity + sterilization or for the preparation of a " 囷, which can be applied to pharmaceutical uses. In the case of the disease, the place of care of the disease caused by Acinetobacter, the medical institution or the doctor: < AB囷1 is suitable for the way, and the effective amount of the pathogen is expanded and the research is carried out. The research institute for the sterilization of the institution or the medical research related to the medical equipment, such as the care place, the medical machine, to reduce the care place, the home care, the hospital or the treatment of the machine and the medical vision of the research machine 111296 8 1376418, the number of Acinetobacter baumannii in the structure. For example, the AB bacteriophage of the present invention can be administered to the environment of the home care, hospital or nursing home, for example, but not limited to, an intensive care unit, an operating room, a recovery room, a treatment room, a reception room, etc.; or in the hospital or Equipment for nursing homes, such as, for example, but not limited to, intubation, vascular devices, stethoscopes, medical records, tourniquets, gloves, respirators, wet bottles, furniture, floors, vents, monitors, and the like.

In a preferred embodiment, depending on the subject matter to be administered, it may be selected for direct spraying, indirect spraying, soaking or smearing. The embodiments of the present invention will be described by way of specific examples, and those skilled in the art can understand the other advantages and effects of the present invention from the disclosure. Example 1 (Inseparation of Acinetobacter baumannii phage) A total of 87 samples from catheter washing liquid, drainage system wastewater, untreated sewage collected from Hualien Tzu Chi Hospital, and samples were taken at 4,000 X at 4 ° C. The mixture was centrifuged for 10 minutes, and the supernatant was filtered through a membrane having a pore size of 0.45 // m, followed by a plaque test. The sample of 10' // 1 is dropped to the bacterial lawns of AB bacteria (the preparation method is detailed in Example 2), and if the sample filtrate contains phage, a clear zone is generated on the bacterial layer (clear zone) ), pick it up and immerse it in LB medium, and filter to remove bacteria to obtain a high concentration of phage. After the phage was diluted, it was applied flat on LB medium to form plaques. Perform a single plaque detachment step at least twice to obtain sputum pure 9 111296 1376418 ' ι. Four strains of AB phage were obtained after identification of the Acinetobacter baumannii phage isolated from the sample, and were named 0 AB1 (registered number BCRC970046), 0 AB2 (registered number BCRC970047), 0 AB3 (variant of AB 2) 0 AB4 (variant of AB 2), wherein 0 AB3 and 0 AB4 are variants of ψ AB2, which have 80% or more sequence identity with 0 AB2, respectively. All of the above 4 phages can be infected with AB bacteria, and the infectivity of different strains of AB bacteria is slightly different. Example 2 (Host Specificity Test) To test the host specificity of the Acinetobacter baumannii phage of the present invention, a strain as shown in Table 1 was selected, wherein Acinetobacter baumannii (10), sometimes referred to as abbreviated in the present specification AB strain) Source: 35 strains were collected from Hualien Tzu Chi Hospital and 2 strains were from ATCC (American Type Culture Collection). The bacteria were cultured in LB medium (Difco Laboratories, Detroit, MI, USA) at 37 ° C, and the growth of the bacteria was monitored by turbidity, and the absorbance was measured at 600 nm (〇D_). When the 〇D was 1, the bacterial concentration was 3x10. Cells / ml. The host bacterial bacterias were prepared by coating a 1.8% LB agar medium containing 1.8% of the host bacteria (e.g., the strains in Table 1) on 1.8% of the LB agar medium. The culture medium (the concentration of sputum bacteria is 10 FU 10 PFU/m) of the sputum cells isolated from the 1 〇//1 implementation 1 was dropped into the bacterial layer, and the culture medium was dried in a sterile laminar flow station. After 10 minutes, culture at 37 ° C for 18 to 20 hours, and observe whether plaque production or not. 10 1Π296 1376418 ,

Table 1. Bacterial name Characteristics Sources Strain _ Acinetobacter baumannii 19606,17987 ATCC Standard strain ATCC

M495, Ml 094, M2472, M2477, M2641, M2835, M3069, M3237, M3739, M3982, M4666, M5473, M67329, M67649, M67777, M68092, M68282, M68316, M68630, M68651, M68653, M6B661, 45530, 46709, 47538 , 47543, 48393, 48465, 48829, 49575, 50064, 50068, 50913,

Clinical strain, MDRAB Hualien Tzu Chi Hospital 51360 M2383 Clinical strain Abl-Ab9 Imir Merr Ampr Acinetobacter Hualien Tzu Chi Hospital Wu, et al. (2007) calcoaceticus 33305 ATCC standard strain

ATCC π 111296 1376418

Escherichia coli DH5a endAl hsdRl 7 (rk- mk-l·) supE44 thi-1 recAl gyrA relAl <p80d lacZAMl 5 A (lacZYA-argF)Ul 69 Hanahan D. (1983) G0003, G0004, G0008, G0010, G0012, G0070, G0071, G0072, G0081 Clinical strain Hualien Tzu Chi Hospital Klebsiella pneumoniae Kp2, Kp50, Kp53, Kp90, Kpl20, Kpl21 Clinical strain Wu, et al. (2007) Pseudomonas aeruginosa Pa79, Pa81, Pa86 clinical strain Wu, et al (2007 MDRAB: Multi-drug resistant AB bacteria, for gentamicin, Anhui (811111^(:111), dabinimycin (?1?6"&(^11111/1320^&( ^&111), special, ticarcillin/clavulanate, ceftazidime, cefepime, cefpirome, aztreonam, imipenem Meropenem, ciprofloxacin, and levofloxacin are resistant. Amp: ampicillin; Imi: imipenem; Mer: meropenem; : drug resistance; s: sensitivity 〇 results show that the example 1 is separated The strain of AB strain is not found in the strain of '1〇5.co", 6 strains of [houseway (10)_face-like strain, and 3 strains/>. (3) laughter/(10) strains Producing plaques and producing plaques only on the bacterial layer of AB bacteria, it is true that the phage of the present invention has host specificity for AB bacteria. The AB bacteria phage isolated in Example t are all AB strains of Table i The bacterial layer produced 111296 12 1376418. Not only for the two labeled multi-drug resistant AB bacteria lytic plaques of ATCC, it was confirmed that the phage quasi-strain of the present invention has infectivity and is also infectious to clinical ramets. 3 (observing the morphology of AB bacteria by a transmission electron microscope) The obtained 4AB2 (concentration of 1Gl2pFU/ml) was dropped on a polyethylene-coated carboxylic acid coated grid (200 mesh copper grid). On the upper, the negative staining was carried out with the acetic acid shop (the ^ acetate), and then placed in a transmissive electronic display (purchased from Hitachi Company, Japan; model H_75 〇〇, operating conditions: 80 kV). } The picture shows. The virus particles of 0AB2 have a head and a tail, and the head has a 2〇 facet structure with a size of about 60 nm. The tail has a filamentous structure for attachment to the surface of the host cell and has a size of about 9 to n nm. Example 4 (PFGE electrophoresis analysis) 200 ml of the ab culture medium in the early phase of the logarithmic growth phase was subjected to 0-staining with an MOI (Multiplicity Of infecti〇n) of about 1 ,, and aerobically cultured to AB bacteria. Completely lysed. The culture was centrifuged, and the solution was filtered through a 0.45/m pore-controlled membrane, followed by sputum by Beckman.

Avanti J-251 Green~ machine was centrifuged at 18, 2 for 2 hours, the sputum was phage particles, and the precipitate was reconstituted with 1.0 ml of TE buffer solution (10 mM Tris_Hcl, pH 7 ED containing 1.0 mM EDTA). ,to

The phage band was purified by ultracentrifugation at 25,000 rpm for 2 hours at 25,000 rpm in a Beckman LE-80K centrifuge and a sw41Ti turntable. The purified phage body band was dialyzed to remove the TE buffer solution and stored at 4. (: standby. Concentrate the phage particles with 20% polyethylene glycol. 6000, extract with phenol / 13 111296 1376418 and precipitate with ethanol to obtain phage DNA to limit the enzyme Apal,

BamHI, Banll, Bglll, EcoRI, EcoRV, HincII, Hindlll, Kpnl, Mlul, PstI, PvuII, SacI, Smal, SnaBI, SphI, SspI,

After StuI and Xbal were treated separately, pulsed-field gel electrophoresis (PFGE) was performed in 0.8% and 1%% colloid and TAE buffer solution. As shown in Figure 2A, the phage DNA of AB2 can only be Bglll, EcoRI, EcoRV, HincII, Hindlll, Mlul, SnaBI,

The action of SphI, SspI and Xbal, the molecular weight standard (M) was 1-kb plus DNA Ladder (purchased from Invitrogen, CA). The phage DNA is about 35 to 40 kb in length by restriction enzyme fragment detection. The restriction enzyme map of the phage DNA is shown in Fig. 2B, and the Bglll, EcoRI, EcoRV, Mlul and Xbal cleavage sites are indicated. Example 5 (SDS-PAGE electrophoresis analysis) Purified phage particles and sample buffer solution (containing 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate, 1% glycerol, and 〇.〇1%) The bromide blue 62.5 mM Tris-HCl, pH 6.8) was mixed and heated in a boiling water bath for 3 minutes and subjected to 12.5% SDS-PAGE electrophoresis. For example, Figure 3 shows a protein electropherogram of 0AB2 with at least 10 different protein bands with molecular weights ranging from 21 to 140 kDa, where '33 kDa protein is the most abundant, most likely to be a bacterium The main coat protein of the body. Example 6 (sequence analysis) The Sau3Al-partial fragment (Ca. 15 kb) of the phage gene was cloned to pUC18 and sequenced with DNA from 6 inserts. 14 Π 129^0) 1376418, sequence analysis with NCBI package. The sequence of SEQs NO. 1 to 4 as shown in the Sequence Listing was obtained by DNA sequencing and alignment. Wherein, the sequence of SEQ ID N0.1 and SEQ ID NO. 2 is an RNA polymerase-based immobilization sequence, and the RNA polymerase of the AB phage can be edited. The sequences of SEQ ID N0.3 and SEQ N0.4 encode the head-tail connector of the AB bacteriophage.

The sequences of SEQ ID NOS. 1 to 4 were aligned with the NCBI gene library 'The same or similar viral sequences were not obtained. For example, the DNA sequence of SEQ ID N0.1 is compared with the NCBI gene database, and the degree of identity with phiAB1-LKAl is 39.4%, the degree of identity with phiKMV is 41.3%, and the degree of identity with phiPT5 is 41.3. %, the degree of identity with phiPT2 is 41.5%, and the degree of identity with phiLKD16 is 41.5%. Overall, 3 £(^10 1^0.1 is aligned with the DNA sequence of the gene database, and the degree of identity is only about 40% identical. In addition, the amino acid sequence encoded by SEQ ID N0.1 and phiABl-LKAl The degree of identity is 30.6%, the degree of identity with phiKMV is 29.4%, the degree of identity with phiPT5 is 29.4%, the degree of identity with phiPT2 is 29.3%, and the degree of identity with phiLKD16 is 29.2%. Overall, with SEQ ID N0 The amino acid sequence encoded by .1 is aligned with the protein of the gene database, and the degree of identity is only about 30% identical. It is known in the art that the RNA polymerase gene sequence is a highly retained segment of the viral genetic material, by comparison The RNA polymerase gene sequence similarity between species can determine the species affinity. Therefore, it can be seen from the above-mentioned alignment result 111296 15 1376418 that the AB bacteriophage of the present invention is similar to the rnA polymerase gene sequence of the existing sputum bacterium. The gene is extremely low and is indeed a novel phage. In addition, the inventors registered the sequences of SEQ ID N0.1 and SEQ ID N0.2 in the NCBI gene database, and obtained the registration codes of bankitl 192576 FJ809932 and bankitl 192679 FJ809933 respectively. Application date of the case Example 7 (Bactericidal efficiency) When the AB strain is cultured to a D_ value of 〇.6 U, an AB phage having an M〇I of 0.0005 is added to the host bacterial culture solution, Under the culture, sample 100//1 at the time of culture, 1, 2, 3, 4, 5, 1 , 20, 30 minutes, and dilute with mL 9 mL of cold LB, centrifuge at 12,000 X g 5 Minutes, the supernatant was taken, and the amount of phage that was not adsorbed to the host bacteria was measured. The result of 0AB2 is shown in Fig. 4. The phage-immobilized host bacterial culture solution was observed, and it was found that the culture solution was changed from turbidity to 100 minutes. Clarification, showing that the phage completely lysed the host bacteria, and confirmed that the bactericidal composition of the present invention can achieve the bactericidal effect. As can be seen from Fig. 4, about 75% of the phage particles are adsorbed to the host bacteria within 2 minutes, about 95% The phage particle adsorbed to the host bacterium in 4 minutes reached 1% adsorption at 10 minutes. In addition, the phage replication curve was determined by a 〇ne-step growth curve to OD6 〇81; After collecting the AB bacteria culture solution, collect the f house Then re-dissolve in 8 ml of LB medium to a concentration of 1〇9 CFU/ml. Add the AB strain of _1 to the host bacterial culture solution, and place it under 4ΐ3Q. Minutes, the sputum bacteria were adsorbed to the lodge 111296 .16 1376418 - the main bacteria. The mixture was centrifuged at 12,000 x g for 10 minutes, and the lysate containing the infected bacteria was re-dissolved in 20 ml of LB medium, cultured at 37 C to sample every 5 minutes, and the sample was immediately diluted and fixed. Among them, the result of 0AB2 is shown in Figure 5. The incubation period is defined as the first incubation period (10 minutes without pretreatment) and the first burst (burst, release of bacteria lysing bacteria), as shown in Figure 5 with an incubation period of 15 minutes. The average burst volume was approximately 200 PFU/% of cells, based on the ratio of the final phage particle size to the initial amount of susceptible bacteria. The infectivity of 4AB1 to 0AB4 of the present invention was tested in the same manner as above, and as a result, it was revealed that the bacteriophage AB1 to φ AB4 of the present invention have the advantages of rapid infection, short latency, large burst, and rapid bactericidal effect. Example 8 (compatibility)

The compatibility of the AB strains isolated from Example 1 was tested with the surfactants TWEEN 20 'TWEEN 80 and Triton X-100 (available from Sigma-Aldrich Biotechnology, USA). The common concentration of the surfactant is usually between 0.1 and 1 Wt%. Therefore, 1 wt% of the above surfactant is mixed with the AB phage at a concentration of 5×10 PFU/ml, and cultured at room temperature every The phage concentration was determined by 24-hour sampling, and the survival fraction was calculated as follows: Phage survival rate = sampled phage concentration / phage original concentration to determine the effect of the surfactant. It has been determined that the inclusion of 0.1 to 1 wt% of the surfactant does not affect the activity of AB bacteria of 0 AB1 to (/) AB4. Among them, the result of 0AB2 is shown in Fig. 6. In 17 111296 1376418, Figure 6 shows that 'AB bacteria are most stable in TritGnX, Tw £EN 20 times; while in TWEEN 80, although the survival rate of AB phage changes greatly, it still maintains sufficient infection. The activity of the host bacteria. And as time progresses, the trend of decreasing phage concentration tends to moderate and then gradually increase. According to the coefficient of Variati〇n, the CV values of the three surfactants were all lower than 20%. Therefore, it was confirmed that the phage was very stable in all three surfactants and could maintain the biological activity of the body. 1 π * Pan Yun 2 to ^ species ... to 0AB4 ΑΒ Πΐ Πΐ 纯 pure strain can be mixed with a carrier (such as water, surfactants (such as Wt〇n χ · i 〇〇 ' butyl formation of bactericidal composition, for Environment or instrument =; Γ, Γ composition of the 起始 (4) the initial content of the bacteria: into (3) A n PFU / m ΑΒ carcass, the content of the surfactant is 0.1 to 2 Wt%. Example 9 (biological activity of cells under different environmental conditions), under the conditions of a single detachment from a case 1 and a dilution of turmeric cells with sterile water at a temperature of 8

And -80 ° C. In the generation, 25. . 7 C, 42 Ϊ, -2 (every 3 hours in the TC: then - ～37 C in the experiment 'in the culture for 24 hours and 12 weeks, the results Γ:; bacteria: _ concentration' he is divided into two groups, first The group is 'No. In the experiment of _20c and _80. The second group only solves; Dongyi\, Dongjie; East, the chase is 12 weeks. (9) The time is 5 weeks, and the result is like the Chuan map. 111296 1376418, - (2) pH, diluted phage with acidic (pH 4) and alkaline (pH 11) aqueous solution to a phage concentration of 1 〇 8 PFU / ml, at pH 4.7, 7, The concentration was measured every 3 hours for 24 hours in the experiment of 11 and then tracked once a week for 12 weeks. The results are shown in Fig. 8. (3) Chemicals added phage to chloroform solution (0.5% and 2%) After diluting the concentration to 108 PFU/ml, the concentration was measured every 3 hours for 24 hours, and then in the 0.5% chloroform solution, fixed weekly tracking for 3 weeks; 2% chloroform solution was tested. It was followed for 6 weeks, and the results are shown in Figure 9. (4) Drying treatment 101. PFU/ml phage were divided into A and B groups, group A was treated with peptone, and group B was treated with sterile water. After the cell concentration was diluted 10-fold sigh then centrifuged vacuum drying system (speed vac) drying, and dried

After the A and B groups were re-dissolved in 0.5 ml of protein gland and 0.5 ml of sterile water, the concentration changes of the phage before and after drying were observed. The results are shown in Table 2. 19 111296 1376418 Table 2. Membrane concentration (PFU/ml) of the original phage after concentrated phage drying (PFU/ml). Body viability A group 2.18xl09 1.02xl010 21.3% Β Group 2.30χ109 1.02 Xl010 33.4% From the above test results, it was found that the phage of the present invention can survive for at least 8 weeks at low temperature (-20 ° C, -80 ° C, 4 ° C), and the survival rate is over 5%. At ambient temperature (25 ° C and 37 ° C), the phage can survive for more than 11 weeks and the survival rate is above 14.9%. While being tracked for 2 weeks in a high temperature environment (42 ° C), the phage survival rate still reached 14.8%. After about 11 weeks in the alkaline (pH=11) environment, the phage survival rate of the phage of the present invention can be maintained at about 30%; while in the acidic (pH=4) environment, the survival can be measured until the 11th week. Phage. Further, the phage of the present invention can survive for more than 3 weeks in a 0.5% and 2% chloroform solution, and the survival rate is 30%. The survival rate measured after vacuum drying and reconstitution is as high as 20% or more. In summary, the phage are resistant to environmental temperature, dry humidity, pH and chemical substances, and can maintain a certain survival rate, confirming the advantages of the phage of the present invention, and facilitating subsequent applications. The above examples are merely illustrative of the phage of the present invention and the preparation method, and are not intended to limit the present invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention is as set forth in the appended claims. [Simple description of the diagram] 20 111296 1376418 , -· Figure 1 is a plot of Acinetobacter baumannii phage on a scanning electron microscope _ 'under observation; Figure 2A is a DNA electrophoresis diagram of Acinetobacter baumannii phage Among them, Μ is a molecular weight standard, and the 1 to 9 lines are DNA samples of Bglll, EcoRI, EcoRV, Hindi, Hindlll, Mlul, SnaBI, SphI, SspI and Xbal, respectively; and Figure 2B is Acinetobacter baumannii phage The restriction enzyme map of DNA; Fig. 3 is a protein electrophoresis map of Acinetobacter baumannii phage, in which Μ is a molecular weight standard; Figure 4 is the adsorption rate of Acinetobacter baumannii phage to host bacteria; Figure 5 is a step growth curve of Acinetobacter baumannii phage; Figure 6 is the survival rate of Acinetobacter baumannii phage on the surfactant; Figure 7 is the survival rate of Acinetobacter baumannii phage at different temperatures; The seventh plot is the survival rate of Acinetobacter baumannii phage at different temperatures and thawing conditions; - Figure 8 is the survival rate of Acinetobacter baumannii phage at different pH; and Figure 9 As Acinetobacter baumannii phage survival rate of chemical substances. [Description of main component symbols] -fe 〇 t 21 111296 <210> Gas <211> ^ <212><213〉

1376418 . SEQUENCE LISTING <110>Buddhist Tzu Chi General Hospital <120>Acinetobacter baumannii <130> 111296 <160> 4 <170> Patentln version 3.3 2412

DNA Acinetobacter baumannii <220><221> gene <222> (1)..(1961) <223> RNA polymerase (RNA polymerase) <400> 1 atgtctgatc tataccaacg ccaaatagct cttgaagaat catacagtca cgatagtatc attgctggtc agaagcaagt actagatgca tatcaacaag gacgtgctgc tgacgtaggt acaggtcgta tcctattagc taaagcattt gaagttggtg tagaagcttt aaatgcagtt aagaaacaaa agattcgtgg tgttggcggt aaatacttaa aattactttc tatcgctgat ccagaagttt tagtaatggc tgcattacgt gatattatta atgcatgtgc tgtacctgaa ccagtgtcta tgcagaaagt acttacgggt attgggcgta tgattgagtc agagtctatg ttggtattta tgcaggagtt aaatcctgca tacactgata agactattca gtatttagac aacacaggta caaaatcagt tacccaccgt tatcgtacat tcttagcagg ttctaagtct 60 120 180 240 300 360 420 480 111296 1376418 attcaactag attgggaaca gtggtcacaa gaggagcgta taggtgtagc taagttgttg 540 gtaagttgtt tatacgacgc tacaggatta ttccaatggg ctaaactgga tagcggtatg 600 taccacatta aagcttctga acccttagcg aagcactttc aggatgcagc gagtgcagcg 660 agagcagttg ttaaatatcc tcctatgttg atcaaaccta tggattggga aggacagtat 720 aa cggtgggt atttaactga atggtttaaa cataactcac ctatgtgtgg tattcgcttt 780 attaagaaag agcataagca ttgggttatt gataacttaa acaatggtgc agagctagtt 840 aaggctgcaa tgaataaagc acagtctgta ccttacagga tcaataaaga catcttagca 900 atcttacgta aagcagttgc tatgcgtgta ggtattttag gtttaccaag ctatcaacct 960 gcaccgaaac ctgcatttcc atttactgat gattggttaa agtcagaagc tactgaggaa 1020 gaattagatc agttccaatt ctggaaaggt ttaatgagtt catggtatac acaagaagct 1080 aaacgtgttg gtcgtcaaca tggcatctta agtcgtattc aagaactggt taaatatcag 1140 gacgaagaac gtttgtactt tccaacgttt attgattggc gaggtcgtct ttacttccgt 1200 agtagtatta acccacaatc gaatgattgt attaaaggtt gtcttgagtt tgcagagggt 1260 aaacctctag gtaaaacagg acttaaatgg ttaaagattc atgttgcaaa ctgttgtggt 1320 tatgataaac atgatcctga tttaaaggag aaatggtgtg atgacaactg gaactacatt 1380 cagaatttta tcaataaccc gtttgatgtt gaagcacccg aacccgacac tgcttttaca 1440 ttactacaag caggtttggc gctacaatct gcgttggaat tggaaacacc tgaatcctac 1500 atatgccatg tccccgtcgc aatggacgca acttgttcgg gtctacagca cctatctgcg 1560 ctcactagag a cgaagtagg tggtttatat acgaacttat tagacaacgg tgaagaccag 1620 aagtctgata tttatatgcg tgtagcgcac atagcagatg agtctaaact agaattagct 1680 2 111296 1376418, 'gattctcctg ctgtacgtca gtattgggtg gataaaccta ttagtcgtaa tatggcgaag 1740 aaacctgtga tgacttacgt atacggttcg aagttattat caactattca aggcttagct 1800 aatgatatgt atgaagcagg tatggatgag attcagttag atggtaagac agtctttact 1860 tacaaccgat tagctaaacc agttggtaag gcattacgta aaggtgtcga agatactgta 1920 cctaaatctg ctgagatgat gaactacttg cagaacgttg tacgtaaaaa taaagctgat 1980 • gctatgcgtt ggtttagtcc agtaggtgtt cctgttgtga attgggcaga aggtatggtg 2040 actaaacttg tagcaattcg ttcgatggga atctccagaa ttgcttatag ttatccagat 2100 aaccaatata ataccttaag agcagctaac ggtattgtac ctaactttgt acatagtatg 2160 gatagcagtc acttatgttt aactatctta gatttcgatg gtcaagttct accaattcat 2220 gactcattcg caacccatcc tagtgatgtg gaagctatgc atgtatcatt acgtaagaca 2280 ttcattgaaa tgtatacaca attcagtatt gaagacttct taaagtttaa caatattgat 2340 cttgaagagt acacaccacc acttacaggt aacttagagt Tatcggaaat ttctaaatcc 2400 cgttatatgt tt 2412 t <210 2 <211〉 792

<212> DNA <213> Phage of Acinetobacter baumannii <220 <221> gene <222> (1).. (669) <22P RNA polymerase (rnA.polymerase) 2 <400 〉 Chuan 296 1376418

atccagaagt tttagtaatg gctgcattac gtgatattat taatgcatgt gctgtacctg 60 aaccagtgtc tatgcagaaa gtacttacgg gtattggtcg tatgattgag tcagagtcta 120 tgttggtatt tatgcaagag ttaaaccctg catatactga caagactatc cagtatttag 180 acaacacagg tacaaaatca gttacccacc gttatcgtac attcttagca ggttctaagt 240 ctattcaact agattgggaa cagtggtcac aagaggagcg tataggtgta gctaagttgt 300 tggtaagttg tttatacgac gctacaggat tattccaatg ggctaaactg gatagcggta 360 tgtaccacat taaagcttct gaacccttag agaagcactt tcaggatgca gcgagtgcag 420 cgagagcagt tgttaaatac ccgcctatgt tgatccaacc tatggattgg gaaggacagt 480 ataacggtgg atatttaact gaatggttta aacataactc acctatgtgt ggtattcgct 540 ttattaagaa agagcacaag cattgggtta ttgataactt aaacaatggt gcagagctag 600 ttaaggctgc aatgaataaa gcacagtctg taccttaccg tatcaataaa gacatcttag 660 caatcttacg taaagcagtt gctatacgtg taggtatttt aggtttacca agttaccaac 720 ctgcacctaa acctgcattt ccatttactg aggattggtt aaagtctgag gctactgagg 780

Aagaattaga tc 792

<210> 3 <211> 1557 <212> DNA <213>. Acinetobacter baumannii <220><221> gene <222> (1)..(1557) <; 223> phage head-tail connector 4 111296

≪ 400 3 tgaagtccaa aggaaatgat tttacaaaga ctattcgagc tttgtacgat gaatacacgg 60 acgattcttt aaaaacaaga ttagaaatgt atgcactttg gactctacct agcgtgttcc 120 cgacaggtga gattacggta gataatggaa atgccgagat tgagcatgac taccaaagtg 180 taggcgcata tctagtgaat cggttagcgt cacgtttagc gagtacgtta tttcccgtaa 240 gcacatcttt ctttagaatc gaacctagtc aagagttgaa agacttagtt gataaacgtg 300 gtacaagtac ccttatcgac ttagagaaca aagcttgtcg tcgtttattc ttcaacgcat 360 cttatgcaca gattgtgcaa gcactgcgtt tacttattat cactggtgaa gttttattac 420 ttcgtagaga taatcgccta cgtgttttta gtttaaagaa ttatgcgtta ctacgcaaca 480 atgtagggga agtacttgag atcatcacac gagaacctaa acgttatcgg gaattagatg 540 ctgagactca ggcactccta caagatcgta acgaggacga gacccttgat ctttatacta 600 gaatccgtaa gcgtaatatc aatggggtaa tctcatggaa gattacacaa gaaatagatg 660 gtgtacgctt accaaactat gaaatctacc gagataagtt atgcccatat attcctgtaa 720 cgtggagtta tatgaatggt gatgcttacg gtcgtggtta cgtagaagag tatgcaggtg 780 actttgctaa gttatctgaa ctctcacaag gtttaacaga gtaccagatc Gagtcattaa 840 ttatccgtca tgtatataat gcacagggtg gttttgatgt agaatctgct gtgaactcac 900 gtaacggtga ttggattagt ggtaacgtta atgctgtaca gaactatgaa tctggatcat 960 atcaaaagat gaatgaggtt cgattaggtt tagaagctat tatgcaacgt ctaaacgtag 1020 cgttcatgta cacaggtaat atgcgagaag gtgatcgtgt aacagcctat gagattgcac 1080 gtaatgctga tgaagcagag caagtcctcg gtggtgtgta ctcacaacta tctcagaata 1140 1376418 tgcatttacc tttagcatat ctattactct atgaagttcg taaagacttt attcaggcga 1200 ttgatagaca agaaatcgaa ttaaatattc taactggttt acaagcatta tcacgtagtt 1260 cagagaacca agctttatta gtagcagcga atgagattgc tacagttgcc caagtattct 1320 cacaagtaag taaacgattt aatcttgatg ctattgtaga taagattcta ctttctaatg 1380 gtattgatat ttcagagatt acatacagtg aagaagaaat gagagctaag gctatggaag 1440 aacaacgtgc agcagaggca cagcgacaac aagtaataca acaagctggc gcacagttag 1500 gtggtaatca attagaaaat acacaggctg ctcaattggc agcaggtatt caatagg 1557

<210> 4 <211> 360 <212> DNA <213> Phage of Acinetobacter baumannii <220><221> gene <222> (1)..(360) <223> Phage head-tail connector

≪ 400 > 4 tatactagaa tccgtaagcg taatatcaat ggagtaatct catggaagat tacacaagag 60 atagatggtg tacgtttacc aaactatgaa atctaccgag ataagttatg cccatatatt 120 cctgtaacgt ggagttatat gaatggtgat gcttacggtc gtggttacgt agaagagtat 180 gcaggtgact ttgctaagtt atctgaactc tcacaaggtt taacagagta ccagatcgag 240 tcattaatta tccgtcatgt atataatgca cagggtggtt ttgatgtaga atctgctgtg 300 aactcacgta acggtgattg gattagtggt aacgttaatg ctgtacagaa ctatgaatct 360 6 111296

Claims (1)

1376418 Announcement Seven, the scope of application for patents: Patent application No. 98,712,070, June, 2011, and the revised replacement page: 曰i year, June δ 曰 revision 1. An isolated Acinetobacter baumannii A phage having one or more genes selected from the group consisting of the sequences of SEQ ID NO. 1, 2, 3, and 4 and the sequences having a sequence similarity to SEQ ID NO. 1, 2, 3, and 4 of 80% or more The body sequence, and the phage system is at least one selected from the grouping numbers BCRC970046 and BCRC970047. 2. For example, the phage of claim 1 is specifically infected with Acinetobacter baumannii. 3. The phage of claim 1 of the patent scope, which is a lytic phage. 4. The phage of claim 1 which retains the biological activity of the phage at pH 4 to 12. 5. The phage of claim 1 of the patent application, which retains the biological activity of the bacterium in the surfactant. 6. The phage according to claim 5, wherein the surfactant is at least one selected from the group consisting of an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, and a nonionic surfactant. A group of surfactants. 7. The composition of claim 6, wherein the surfactant is a nonionic surfactant. 111296 (revision) 1