U.S. patent application number 12/854542 was filed with the patent office on 2012-11-01 for phage of acinetobacter baumannii.
This patent application is currently assigned to TZU CHI BUDDHIST GENERAL HOSPITAL. Invention is credited to Li-Kuang Chen, Nien-Tsung Lin.
Application Number | 20120276612 12/854542 |
Document ID | / |
Family ID | 43530842 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120276612 |
Kind Code |
A1 |
Chen; Li-Kuang ; et
al. |
November 1, 2012 |
PHAGE OF ACINETOBACTER BAUMANNII
Abstract
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, and sequences having more than 80% homology thereof. The phages
of the present invention infect Acinetobacter baumannii
specifically, and can be applied to reduce the amount of
Acinetobacter baumannii.
Inventors: |
Chen; Li-Kuang; (Hualien,
TW) ; Lin; Nien-Tsung; (Hualien, TW) |
Assignee: |
TZU CHI BUDDHIST GENERAL
HOSPITAL
Hualien
TW
|
Family ID: |
43530842 |
Appl. No.: |
12/854542 |
Filed: |
August 11, 2010 |
Current U.S.
Class: |
435/235.1 |
Current CPC
Class: |
C12N 2795/10131
20130101; C12N 2795/10231 20130101; C12N 7/00 20130101; A61P 31/02
20180101 |
Class at
Publication: |
435/235.1 |
International
Class: |
C12N 7/00 20060101
C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 2009 |
TW |
098127069 |
Aug 12, 2009 |
TW |
098127070 |
Claims
1. 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, and sequences having more
than 80% homology thereof.
2. The isolated Acinetobacter baumannii phage of claim 1, being an
Acinetobacter baumanni-specific phage.
3. The isolated Acinetobacter baumannii phage of claim 1, being a
lytic phage.
4. The isolated Acinetobacter baumannii phage of claim 1, having
bioactivity at pH 4 to 12.
5. The isolated Acinetobacter baumannii phage of claim 1, being
selected from the group consisting of DSM 23599 phage, DSM 23600
phage and variants thereof.
6. The isolated Acinetobacter baumannii phage of claim 5, wherein
the DSM 23599 phage and variants thereof or DSM 23600 phage and
variants thereof have more than 80% homology.
7. The isolated Acinetobacter baumannii phage of claim 1, having
bioactivity in a surfactant.
8. The isolated Acinetobacter baumannii of claim 7, wherein the
surfactant is one selected from the group consisting of an anionic
surfactant, a cationic surfactant, an amphoteric surfactant and a
non-ionic surfactant.
9. The isolated Acinetobacter baumannii phage of claim 8, wherein
the surfactant is a non-ionic surfactant.
Description
FIELD OF INVENTION
[0001] The present invention relates to a novel phage, and more
particularly, to a phage of Acinetobacter baumannii.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] 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%.
[0005] Currently, the most common bacteria causing nosocomial
infections include Pseudomonas aeruginosa, Staphylococcus aureus,
Acinetobacter baumannii, etc.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] There are no publications disclosing Acinetobacter
baumannii-specific phages, which are used for reducing the
population of Acinetobacter baumannii and further reducing
nosocomial infections.
SUMMARY OF THE INVENTION
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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
[0017] 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.
[0018] In an aspect of the present invention, the phages of the
present invention have bioactivity in a surfactant.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] In a preferred embodiment of the present invention, the
surfactant is a non-ionic surfactant.
[0025] In a preferred embodiment, the surfactant is a commercial
product, especially a detergent.
[0026] 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.
[0027] 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.
[0028] 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
[0029] FIG. 1 shows SEM images of Acinetobacter baumanni phages
according to the present invention;
[0030] 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;
[0031] FIG. 2B shows the restriction enzyme map of DNA of
Acinetobacter baumannii phage according to one embodiment of the
present invention;
[0032] 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;
[0033] FIG. 4 shows the absorption of Acinetobacter baumannii phage
according to the present invention to Acinetobacter baumannii ATCC
17978;
[0034] FIG. 5 shows the one-step growth curve of Acinetobacter
baumannii phages according to the present invention on
Acinetobacter baumannii ATCC 17978;
[0035] FIG. 6 shows the viability of Acinetobacter baumannii phages
according to the present invention in surfactants;
[0036] FIG. 7A shows the viability of Acinetobacter baumannii
phages according to the present invention at different
temperatures;
[0037] FIG. 7B shows the viability of Acinetobacter baumannii
phages according to the present invention at different temperatures
and thaw conditions;
[0038] FIG. 8 shows the viability of Acinetobacter baumannii phages
according to the present invention at different pH; and
[0039] FIG. 9 shows the viability of Acinetobacter baumannii phages
according to the present invention in chemicals.
DETAILED DESCRIPTION OF THE PREFERED EMBODIMENTS
[0040] 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.
EXAMPLE 1
Isolation of Acinetobacter baumannii Phages
[0041] 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.times.g (4.degree.
C.) for 10 minutes, and then the supernatants were filtered via
filters of 0.45 .mu.m for plaque tests.
[0042] 10 .mu.l 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.
[0043] After identification, there were four strains of
Acinetobacter baumannii phages obtained in the present invention,
which were named as .psi.AB1 (deposition number: DSM 23599),
.psi.AB2 (deposition number: DSM 23600), .psi.AB3 (a variant of
.psi.AB2) and .psi.AB4 (a variant of .psi.AB2), wherein .psi.AB3
and .psi.AB4 respectively have more than 80% of homology to
.psi.AB2. The four strains of phages were all capable of infecting
Acinetobacter baumannii with different infectivity.
EXAMPLE 2
Tests of Host Cell Specificity
[0044] 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).
[0045] The bacteria were cultured in the LB medium (Difco
Laboratories, Detroit, Mich., USA) at 37.degree. C., and the
bacterial growth was monitored by turbidity at OD.600. When OD unit
was 1, the bacterial concentration was 3.times.10.sup.8 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).
[0046] 10 .mu.l of phage solution (10.sup.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.degree. C. for 18-20 hours. Subsequently, the production of
plaques was observed.
TABLE-US-00001 TABLE 1 Strain Features Source Acinetobacter
baumannii 19606, 17978 ATCC standard strains ATCC M495, M1094,
M2472, M2477, Clinical strains, MDRAB Buddhist Tzu Chi M2641,
M2835, M3069, M3237, General Hospital, M3739, M3982, M4666, M5473,
Hualien M67329, M67649, M67777, M68092, M68282, M68316, M68630
(deposition number: DSM 23587, a host of .psi.AB1), M68651, M68653,
M68661, 45530, 46709, 47538, 47543, 48393, 48465, 48829, 49575,
50064, 50068, 50913, 51360 M2383.sup.s Clinical Strain Buddhist Tzu
Chi General Hospital, Hualien Ab1-Ab9 Imi.sup.r Mer.sup.r Amp.sup.r
Wu et al. (2007) Acinetobacter calcoaceticus 33305 ATCC standard
strain ATCC Escherichia coli DH5.alpha. endA1 hsdR17 (rk- mk+)
Hanahan D. supE44 thi-1 recA1 gyrA (1983) relA1.phi.80d
lacZ.DELTA.M15.DELTA.(lacZYA-argF)U169 G0003, G0004, G0008, G0010,
Clinical strains Buddhist Tzu Chi G0012, G0070, G0071, G0072,
General Hospital, G0081 Hualien Klebsiella pneumoniae Kp2, Kp50,
Kp53, Kp90, Clinical strains Wu et al. (2007) Kp120, Kp121
Pseudomonas aeruginosa Pa79, Pa81, Pa86 Clinical strains Wu et al.
(2007) 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; .sup.rresistant;
.sup.ssensitive
[0047] 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
.psi.AB2 also infect two standard strains obtained from ATCC, in
addition to infect clinically separated Acinetobacter baumannii
having multiple resistances.
EXAMPLE 3
Morphology of Acinetobacter baumannii Phages under TEM
[0048] The isolated .psi.AB2 (10.sup.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.
[0049] The viral particle of .psi.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.
EXAMPLE 4
PAGE Electrophoresis Analysis
[0050] 200 ml of AB culture solution at early stage of log phase
was infected with .psi.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
.mu.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.degree. 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.degree. C.
[0051] 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.
[0052] As shown in FIG. 2A, DNA of .psi.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). Upon restriction enzyme digestion analysis, the full length of
phage DNA was about 35-40 kb. 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.
EXAMPLE 5
SDS-PAGE Analysis
[0053] 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.
[0054] FIG. 3 shows protein electrophoresis patterns of .psi.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.
EXAMPLE 6
Sequence Analysis
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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).
EXAMPLE 7
Efficiency of Sterilization
[0061] The AB culture (host cell) was incubated to OD.sub.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
.mu.l of culture was sampled and diluted with 0.9 ml of cold LB,
and then centrifuged at 12,000.times.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
.psi.AB2 to ATCC 17978 is shown in FIG. 4.
[0062] 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.
[0063] As shown in FIG. 4, about 75% of the phage particles
attached to the host cells in 2 minutes, about 95% of the phage
particles attached to the host cells in 4 minutes, and 100% of the
phage particle attached to the host cells in 10 minutes.
[0064] Further, the replication curve of the phages was determined
by one-step growth curve. The AB culture solution (OD.sub.600:
0.8U) was centrifuged, and the precipitant was collected and
resuspended in 0.8 ml of LB medium to a concentration of 10.sup.9
CFU/ml. The AB-specific phages (MOI: 0.0001) were added to the host
cell culture solution, and placed at 4.degree. C. for 30 minutes,
such that the phages attached to the host cells. The mixture was
centrifuged at 12,000.times.g for 10 minutes, and the precipitant
including the infected bacteria was re-suspended with 20 ml of LB
medium, and incubated at 37.degree. C. The culture was sampled
every 5 minutes, and the samples were immediately diluted and
quantified. For example, the result of .psi.AB2 to ATCC 17978 is
shown in FIG. 5.
[0065] 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.
[0066] Similarly, the infectivity of .psi.AB1 to .psi.AB4 was
determined The results showed that all the phages .psi.AB1 to
.psi.AB4 had strong infectivity, short latent period, big burst and
immediate sterilization.
EXAMPLE 8
Compatibility
[0067] 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.times.10.sup.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
[0068] Upon determination, the activities of .psi.AB1 to .psi.AB4
phages were not influenced by 0.1-1 wt % of surfactants. For
example, FIG. 6 shows the result of .psi.AB2. As shown in FIG. 6,
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. As shown in FIG. 6, 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.
[0069] Hence, at least one of .psi.AB1 to .psi.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. Preferably, in the composition, the
initial content of the phage is 1.times.10.sup.7 to
1.times.10.sup.9 PFU/ml, and the content of the surfactant is 0.1
to 2 wt %.
EXAMPLE 9
Bioactivity of the Phages Isolated in Example 1 Under Different
Conditions
[0070] 1. Temperature
[0071] The phages were diluted with autoclaved water to 10.sup.8
PFU/ml, and then placed at different temperatures, 4.quadrature.,
25.quadrature., 37.quadrature., 42.quadrature., -20.quadrature. and
-80.quadrature.. For the tests at 4.quadrature., 25.quadrature. and
37.quadrature., 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 FIG. 7A, there were respective two
groups at -20.quadrature. and -80.quadrature., 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.
[0072] 2. pH
[0073] The phages were diluted with acidic solution (pH 4) or basic
solution (pH 11) to 10.sup.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.
[0074] 3. Chemicals
[0075] The phages were added to chloroform solution (0.5% and 2%,
respectively), and the phages were diluted to 10.sup.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.
[0076] 4. Dry Treatment
[0077] 10.sup.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.
TABLE-US-00002 TABLE 2 Average concentration of Original phages
after dry concentration of Viability of treatment (PFU/ml) phages
(PFU/ml) re-dissolved phages Group A 2.18 .times. 10.sup.9 1.02
.times. 10.sup.10 21.3% Group B 2.30 .times. 10.sup.9 1.02 .times.
10.sup.10 33.4%
[0078] According to the above results, the phages of the present
invention survived for at least 8 weeks at low temperatures
(-20.quadrature., -80.quadrature., 4.quadrature.), and had the
viability more than 5%. At 25.degree. C. and 37.degree. C., the
phages survived for at least 11 weeks and had the viability more
than 14.9%. At 42.degree. 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%.
[0079] Accordingly, the phages of the present invention have
tolerance to temperatures, humidity, pH and chemicals, and maintain
good viability in these conditions.
[0080] 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.
Sequence CWU 1
1
412412DNAAcinetobacter baumanniigene1..1961RNA polymerase
1atgtctgatc tataccaacg ccaaatagct cttgaagaat catacagtca cgatagtatc
60 attgctggtc agaagcaagt actagatgca tatcaacaag gacgtgctgc
tgacgtaggt 120acaggtcgta tcctattagc taaagcattt gaagttggtg
tagaagcttt aaatgcagtt 180aagaaacaaa agattcgtgg tgttggcggt
aaatacttaa aattactttc tatcgctgat 240ccagaagttt tagtaatggc
tgcattacgt gatattatta atgcatgtgc tgtacctgaa 300ccagtgtcta
tgcagaaagt acttacgggt attgggcgta tgattgagtc agagtctatg
360ttggtattta tgcaggagtt aaatcctgca tacactgata agactattca
gtatttagac 420aacacaggta caaaatcagt tacccaccgt tatcgtacat
tcttagcagg ttctaagtct 480attcaactag attgggaaca gtggtcacaa
gaggagcgta taggtgtagc taagttgttg 540gtaagttgtt tatacgacgc
tacaggatta ttccaatggg ctaaactgga tagcggtatg 600taccacatta
aagcttctga acccttagcg aagcactttc aggatgcagc gagtgcagcg
660agagcagttg ttaaatatcc tcctatgttg atcaaaccta tggattggga
aggacagtat 720aacggtgggt atttaactga atggtttaaa cataactcac
ctatgtgtgg tattcgcttt 780attaagaaag agcataagca ttgggttatt
gataacttaa acaatggtgc agagctagtt 840aaggctgcaa tgaataaagc
acagtctgta ccttacagga tcaataaaga catcttagca 900atcttacgta
aagcagttgc tatgcgtgta ggtattttag gtttaccaag ctatcaacct
960gcaccgaaac ctgcatttcc atttactgat gattggttaa agtcagaagc
tactgaggaa 1020gaattagatc agttccaatt ctggaaaggt ttaatgagtt
catggtatac acaagaagct 1080aaacgtgttg gtcgtcaaca tggcatctta
agtcgtattc aagaactggt taaatatcag 1140gacgaagaac gtttgtactt
tccaacgttt attgattggc gaggtcgtct ttacttccgt 1200agtagtatta
acccacaatc gaatgattgt attaaaggtt gtcttgagtt tgcagagggt
1260aaacctctag gtaaaacagg acttaaatgg ttaaagattc atgttgcaaa
ctgttgtggt 1320tatgataaac atgatcctga tttaaaggag aaatggtgtg
atgacaactg gaactacatt 1380cagaatttta tcaataaccc gtttgatgtt
gaagcacccg aacccgacac tgcttttaca 1440ttactacaag caggtttggc
gctacaatct gcgttggaat tggaaacacc tgaatcctac 1500atatgccatg
tccccgtcgc aatggacgca acttgttcgg gtctacagca cctatctgcg
1560ctcactagag acgaagtagg tggtttatat acgaacttat tagacaacgg
tgaagaccag 1620aagtctgata tttatatgcg tgtagcgcac atagcagatg
agtctaaact agaattagct 1680gattctcctg ctgtacgtca gtattgggtg
gataaaccta ttagtcgtaa tatggcgaag 1740aaacctgtga tgacttacgt
atacggttcg aagttattat caactattca aggcttagct 1800aatgatatgt
atgaagcagg tatggatgag attcagttag atggtaagac agtctttact
1860tacaaccgat tagctaaacc agttggtaag gcattacgta aaggtgtcga
agatactgta 1920cctaaatctg ctgagatgat gaactacttg cagaacgttg
tacgtaaaaa taaagctgat 1980gctatgcgtt ggtttagtcc agtaggtgtt
cctgttgtga attgggcaga aggtatggtg 2040actaaacttg tagcaattcg
ttcgatggga atctccagaa ttgcttatag ttatccagat 2100aaccaatata
ataccttaag agcagctaac ggtattgtac ctaactttgt acatagtatg
2160gatagcagtc acttatgttt aactatctta gatttcgatg gtcaagttct
accaattcat 2220gactcattcg caacccatcc tagtgatgtg gaagctatgc
atgtatcatt acgtaagaca 2280ttcattgaaa tgtatacaca attcagtatt
gaagacttct taaagtttaa caatattgat 2340cttgaagagt acacaccacc
acttacaggt aacttagagt tatcggaaat ttctaaatcc 2400cgttatatgt tt
24122792DNAAcinetobacter baumanniigene1..669RNA polymerase
2atccagaagt tttagtaatg gctgcattac gtgatattat taatgcatgt gctgtacctg
60 aaccagtgtc tatgcagaaa gtacttacgg gtattggtcg tatgattgag
tcagagtcta 120tgttggtatt tatgcaagag ttaaaccctg catatactga
caagactatc cagtatttag 180acaacacagg tacaaaatca gttacccacc
gttatcgtac attcttagca ggttctaagt 240ctattcaact agattgggaa
cagtggtcac aagaggagcg tataggtgta gctaagttgt 300tggtaagttg
tttatacgac gctacaggat tattccaatg ggctaaactg gatagcggta
360tgtaccacat taaagcttct gaacccttag agaagcactt tcaggatgca
gcgagtgcag 420cgagagcagt tgttaaatac ccgcctatgt tgatccaacc
tatggattgg gaaggacagt 480ataacggtgg atatttaact gaatggttta
aacataactc acctatgtgt ggtattcgct 540ttattaagaa agagcacaag
cattgggtta ttgataactt aaacaatggt gcagagctag 600ttaaggctgc
aatgaataaa gcacagtctg taccttaccg tatcaataaa gacatcttag
660caatcttacg taaagcagtt gctatacgtg taggtatttt aggtttacca
agttaccaac 720ctgcacctaa acctgcattt ccatttactg aggattggtt
aaagtctgag gctactgagg 780aagaattaga tc 79231557DNAAcinetobacter
baumanniigene1..1557Phage head-tail connector 3tgaagtccaa
aggaaatgat tttacaaaga ctattcgagc tttgtacgat gaatacacgg 60
acgattcttt aaaaacaaga ttagaaatgt atgcactttg gactctacct agcgtgttcc
120cgacaggtga gattacggta gataatggaa atgccgagat tgagcatgac
taccaaagtg 180taggcgcata tctagtgaat cggttagcgt cacgtttagc
gagtacgtta tttcccgtaa 240gcacatcttt ctttagaatc gaacctagtc
aagagttgaa agacttagtt gataaacgtg 300gtacaagtac ccttatcgac
ttagagaaca aagcttgtcg tcgtttattc ttcaacgcat 360cttatgcaca
gattgtgcaa gcactgcgtt tacttattat cactggtgaa gttttattac
420ttcgtagaga taatcgccta cgtgttttta gtttaaagaa ttatgcgtta
ctacgcaaca 480atgtagggga agtacttgag atcatcacac gagaacctaa
acgttatcgg gaattagatg 540ctgagactca ggcactccta caagatcgta
acgaggacga gacccttgat ctttatacta 600gaatccgtaa gcgtaatatc
aatggggtaa tctcatggaa gattacacaa gaaatagatg 660gtgtacgctt
accaaactat gaaatctacc gagataagtt atgcccatat attcctgtaa
720cgtggagtta tatgaatggt gatgcttacg gtcgtggtta cgtagaagag
tatgcaggtg 780actttgctaa gttatctgaa ctctcacaag gtttaacaga
gtaccagatc gagtcattaa 840ttatccgtca tgtatataat gcacagggtg
gttttgatgt agaatctgct gtgaactcac 900gtaacggtga ttggattagt
ggtaacgtta atgctgtaca gaactatgaa tctggatcat 960atcaaaagat
gaatgaggtt cgattaggtt tagaagctat tatgcaacgt ctaaacgtag
1020cgttcatgta cacaggtaat atgcgagaag gtgatcgtgt aacagcctat
gagattgcac 1080gtaatgctga tgaagcagag caagtcctcg gtggtgtgta
ctcacaacta tctcagaata 1140tgcatttacc tttagcatat ctattactct
atgaagttcg taaagacttt attcaggcga 1200ttgatagaca agaaatcgaa
ttaaatattc taactggttt acaagcatta tcacgtagtt 1260cagagaacca
agctttatta gtagcagcga atgagattgc tacagttgcc caagtattct
1320cacaagtaag taaacgattt aatcttgatg ctattgtaga taagattcta
ctttctaatg 1380gtattgatat ttcagagatt acatacagtg aagaagaaat
gagagctaag gctatggaag 1440aacaacgtgc agcagaggca cagcgacaac
aagtaataca acaagctggc gcacagttag 1500gtggtaatca attagaaaat
acacaggctg ctcaattggc agcaggtatt caatagg 15574360DNAAcinetobacter
baumanniigene1..360Phage head-tail connector 4tatactagaa tccgtaagcg
taatatcaat ggagtaatct catggaagat tacacaagag 60 atagatggtg
tacgtttacc aaactatgaa atctaccgag ataagttatg cccatatatt
120cctgtaacgt ggagttatat gaatggtgat gcttacggtc gtggttacgt
agaagagtat 180gcaggtgact ttgctaagtt atctgaactc tcacaaggtt
taacagagta ccagatcgag 240tcattaatta tccgtcatgt atataatgca
cagggtggtt ttgatgtaga atctgctgtg 300aactcacgta acggtgattg
gattagtggt aacgttaatg ctgtacagaa ctatgaatct 360
* * * * *