Patent Application
20120276612
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.
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.
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.
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.
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.
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.
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.
Acinetobacter baumannii
Acinetobacter calcoaceticus
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
rresistant;
ssensitive
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. Hence, 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. It was proved that the pages of the present invention are capable infecting clinically separated Acinetobacter baumannii having multiple resistances, wherein ψAB2 also infect two standard strains obtained from ATCC, in addition to infect clinically separated Acinetobacter baumannii having multiple resistances.
The isolated ψAB2 (1012 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
The viral particle of ψAB2 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.
200 ml of AB culture solution at early stage of log phase was infected with ψAB2 (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 μm 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.
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.
As shown in
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.
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.
Upon DNA sequencing and alignment, the 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.
The 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.
For example, before the application is filed, 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.
In addition, the 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.
It is known in the art that the DNA sequence of RNA polymerase is highly conserved region in viral genome. Therefore, homology among species can be determined by identifying homology of RNA polymerase. Upon 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 OD600 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 μl 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 ψAB2 to ATCC 17978 is shown in
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.
As shown in
Further, the replication curve of the phages was determined by one-step growth curve. The AB culture solution (OD600: 0.8U) was centrifuged, and the precipitant was collected and resuspended in 0.8 ml of LB medium to a concentration of 109 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 ψAB2 to ATCC 17978 is shown in
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
Similarly, the infectivity of ψAB1 to ψAB4 was determined The results showed that all the phages ψAB1 to ψAB4 had strong infectivity, short latent period, big burst and immediate sterilization.
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 %. Thus, 1 wt % of the above surfactants was mixed with the AB phages (5×107 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
Upon determination, the activities of ψAB1 to ψAB4 phages were not influenced by 0.1-1 wt % of surfactants. For example,
Hence, at least one of ψAB1 to ψAB4 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. Preferably, in the composition, the initial content of the phage is 1×107 to 1×109 PFU/ml, and the content of the surfactant is 0.1 to 2 wt %.
1. Temperature
The phages were diluted with autoclaved water to 108 PFU/ml, and then placed at different temperatures, 4□, 25□, 37□, 42□, −20□ and −80□. For the tests at 4□, 25□ and 37□, the concentration of the phage culture was determined every 3 hours in 24 hours, and then determined every week for 12 weeks. As shown in
2. pH
The phages were diluted with acidic solution (pH 4) or basic solution (pH 11) to 108 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.
3. Chemicals
The phages were added to chloroform solution (0.5% and 2%, respectively), and the phages were diluted to 108 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.
4. Dry Treatment
1010 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.
According to the above results, 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%. At 25° C. and 37° C., the phages survived for at least 11 weeks and had the viability more than 14.9%. At 42° C. for 2 weeks, 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. In addition, 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%.
Accordingly, the phages of the present invention have tolerance to temperatures, humidity, pH and chemicals, and maintain good viability in these conditions.
The invention has been described using exemplary preferred embodiments. However, it is to be understood that the scope of the invention is not limited to the disclosed arrangements. The scope of the claims, therefore, should be accorded the broadest interpretation, so as to encompass all such modifications and similar arrangements.
| Number | Date | Country | Kind |
|---|---|---|---|
| 098127069 | Aug 2009 | TW | national |
| 098127070 | Aug 2009 | TW | national |
