FIELD OF INVENTION
The present invention relates to a novel phage, and more particularly, to a phage of Acinetobacter baumannii.
BACKGROUND OF THE INVENTION
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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.
For example, 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%.
Currently, the most common bacteria causing 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. Currently, 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 (bacteriophages) 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.
It has been disclosed that bacterial diseases are treated with phages. For example, 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.
There are no publications disclosing Acinetobacter baumannii-specific phages, which are used for reducing the population of Acinetobacter baumannii and further reducing nosocomial infections.
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OF THE INVENTION
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.
It is known that the nucleotide sequence of RNA polymerase is a highly conserved region in viral genome, and thus homology among species can be determined by identifying homology of RNA polymerase. In the present invention, 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.
The 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. In one embodiment of the present invention, 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. In other words, after the pages of the present invention infect host cells, 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. Accordingly, 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.
In an aspect of the present invention, 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
In an aspect of the present invention, the Acinetobacter baumannii phages have acid tolerance and alkali tolerance, and have bioactivity in the environment at pH 4 to 12. In the present invention, the term “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.
In an aspect of the present invention, the phages of the present invention have bioactivity in a surfactant.
In one embodiment of the present invention, the surfactant is one selected from the group consisting of an anionic surfactant, a cationic surfactant, an amphoteric surfactant and a non-ionic surfactant.
In one embodiment of the present invention, 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.
In a preferred embodiment of the present invention, 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.
In a preferred embodiment of the present invention, 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.
In a preferred embodiment of the present invention, 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.
In a preferred embodiment of the present invention, the surfactant is a non-ionic surfactant.
In a preferred embodiment, 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. In an aspect of the present invention, 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.
In a preferred embodiment of the present invention, Acinetobacter baumannii phages of the present invention can be directly or indirectly sprayed or applied on the objects (such as lotion for human skin). Alternatively, the objects can be immersed in the composition having Acinetobacter baumannii phages of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
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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; and
FIG. 9 shows the viability of Acinetobacter baumannii phages according to the present invention in chemicals.
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OF THE PREFERED EMBODIMENTS
The detailed description of the present invention is illustrated by the following specific examples. Persons skilled in the art can conceive the other advantages and effects of the present invention based on the disclosure contained in the specification of the present invention.
Isolation of Acinetobacter baumannii Phages
There were 87 samples collected from washing solution of catheter, waste water from drainage systems and untreated waste water in Buddhist Tzu Chi General Hospital, Hualien (Taiwan). The samples were respectively centrifuged at 5,000×g (4° C.) for 10 minutes, and then the supernatants were filtered via filters of 0.45 μm for plaque tests.
10 μl of each filtrate was dropped to bacterial lawns (preparation method described in Example 2) of Acinetobacter baumannii. If there were phages in the filtrate, there would be clear zones on the bacterial lawns. Then, the clear zones were picked up and immersed in LB medium, which was filtered to remove bacteria, so as to obtain high concentrated phage solution. Subsequently, the concentrated phage solution was diluted, and plated on the LB plate to form plaques. Single plaque isolation process was performed for at least twice to obtain pure phages.
After identification, there were four strains of Acinetobacter baumannii phages obtained in the present invention, which were named as ψAB1 (deposition number: DSM 23599), ψAB2 (deposition number: DSM 23600), ψAB3 (a variant of ψAB2) and ψAB4 (a variant of ψAB2), wherein ψAB3 and ψAB4 respectively have more than 80% of homology to ψAB2. The four strains of phages were all capable of infecting Acinetobacter baumannii with different infectivity.
Tests of Host Cell Specificity
In order to test the Acinetobacter baumannii specificity of phages obtained in the present invention, 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).
The bacteria were cultured in the LB medium (Difco Laboratories, Detroit, Mich., USA) at 37° C., and the bacterial growth was monitored by turbidity at OD.600. When OD unit was 1, the bacterial concentration was 3×108 cells/ml. 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).
10 μl of phage solution (1010 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.
ATCC standard strains
M495, M1094, M2472, M2477,
Clinical strains, MDRAB