FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“NOVEL BACTERIOPHAGE AND ANTIBACTERIAL
COMPOSITION COMPRISING THE SAME”
CJ CHEILJEDANG CORPORATION of 500,
Namdaemunno 5-ga, Jung-gu, Seoul 100-749,
Republic of Korea
The following specification particularly describes the invention and the manner
in which it is to be performed.
- 1 -
[DESCRIPTION]
[Invention Title]
Novel bacteriophage and antibacterial composition
comprising the same
[Technical Field]
The present invention relates to a novel bacteriophage,
more particularly, a bacteriophage that has a specific
bactericidal activity against one or more Salmonella
bacteria selected from the group consisting of Salmonella
Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,
and Salmonella Pullorum. Further, the present invention
relates to a composition for the prevention or treatment of
infectious diseases including salmonellosis and Salmonella
food poisoning caused by Salmonella enteritidis or
Salmonella Typhimurium, Fowl Typhoid caused by Salmonella
Gallinarum, and Pullorum disease caused by Salmonella
Pullorum, comprising the bacteriophage as an active
ingredient. Furthermore, the present invention relates to a
feed additive, drinking water, a cleaner and a sanitizer,
comprising the bacteriophage as an active ingredient.
[Background Art]
- 2 -
Salmonella is a genus of the family Enterobacteriaceae,
characterized as Gram-negative, facultatively anaerobic, non
spore-forming, rod-shaped bacteria, and most strains are
motile by flagella. Salmonella has an average genome GC
content of 50-52%, which is similar to those of Escherichia
coli and Shigella. The genus Salmonella is a pathogenic
microorganism that causes infections in livestock as well as
in human. Salmonella enterica, a species of Salmonella
bacterium, has a variety of serovars including Gallinarum,
Pullorum, Typhimurium, Enteritidis, Typhi, Choleraesuis, and
derby (Bopp CA, Brenner FW, Wells JG, Strokebine NA.
Escherichia, Shigella, Salmonella. In Murry PR, Baron EJ, et
al eds Manual of Clinical Microbiology. 7th ed. Washington
DC American Society for Microbiology 1999; 467-74; Ryan KJ.
Ray CG (editors) (2004). Sherris Medical Microbiology (4th
ed). McGraw Hill. ISBN 0-8385-8529-9.). Of them, Salmonella
Gallinarum and Pullorum are fowl-adapted pathogens,
Salmonella Typhi is a human-adapted pathogen, Salmonella
Choleraesuis and Salmonella derby are swine-adapted
pathogens, and Salmonella Enteritis and Salmonella
Typhimurium are pathogenic for human and animals. Each
serovar causes illness in the respective species, resulting
in tremendous damage to farmers or consumers.
- 3 -
A disease of domestic birds caused by Salmonella
bacterium is Fowl Typhoid (FT), which is caused by a
pathogen, Salmonella Gallinarum (hereinbelow, designated as
SG). Fowl Typhoid (FT) is a septicemic disease of domestic
birds such as chicken and turkey, and the course may be
acute or chronic with high mortality. Recently, it has been
reported that Fowl Typhoid frequently occurs in Europe,
South America, Africa, and South-East Asia, and damages are
increasing. Outbreaks of FT in Korea have been reported
since 1992 and economic losses caused by FT in brown, egglaying
chickens are very serious (Kwon Yong-Kook. 2000
annual report on avian diseases. Information publication by
National Veterinary Research & Quarantine Service. March,
2001; Kim Ae-Ran et al., The prevalence of pullorum diseasefowl
typhoid in grandparent stock and parent stock in Korea,
2003, Korean J Vet Res(2006) 46(4): 347~353).
Pullorum Disease is also caused by one of Salmonella
bacteria, Salmonella Pullorum (hereinbelow, designated as
SP). Pullorum disease occurs in any age or season, but
young chickens are particularly susceptible to the disease.
During the past century, it has been a serious disease among
- 4 -
young chickens at 1-2 weeks of age or younger. Since the
1980s, the occurrence has greatly decreased. However, it
has been growing since the middle of the 1990s (Kwon Yong-
Kook. 2000 annual report on avian diseases. Information
publication by National Veterinary Research & Quarantine
Service. March, 2001; Kim Ae-Ran et al., The prevalence of
pullorum disease-fowl typhoid in grandparent stock and
parent stock in Korea, 2003, Korean J Vet Res(2006) 46(4):
347~353).
In Korea, outbreaks of Fowl Typhoid and Pullorum
disease have been increasing since the 1990s, inflicting
economic damages upon farmers. For this reason, a live
attenuated SG vaccine has been used in broilers for the
prevention of Fowl Typhoid from 2004 (Kim Ae-Ran et al., The
prevalence of pullorum disease-fowl typhoid in grandparent
stock and parent stock in Korea, 2003, Korean J Vet
Res(2006) 46(4): 347~353). Its efficacy is doubtful, and the
live vaccine is not allowed to be used for layers because of
the risk of egg-transmitted infections. Unfortunately,
there are still no commercially available preventive
strategies against Pullorum disease, unlike Fowl Typhoid.
Thus, there is an urgent need for new ways to prevent Fowl
- 5 -
Typhoid and Pullorum disease.
Meanwhile, Salmonella Enteritidis (hereinbelow,
designated as SE) and Salmonella Typhimurium (hereinbelow,
designated as ST) are zoonotic pathogens, which show no host
specificity, unlike SG or SP (Zoobises Report; United
Kingdom 2003).
SE and ST are a cause of salmonellosis in poultry, pigs,
and cattle. Salmonellosis, caused by Salmonella bacteria,
is an acute or chronic infection of the digestive tract in
livestock, and shows the major symptoms of fever, enteritis,
and septicemia, occasionally pneumonia, arthritis, abortion,
and mastitis. Salmonellosis occurs worldwide, and most
frequently during the summer months (T.R. Callaway et al.
Gastrointestinal microbial ecology and the safety of the
food supply as related to Salmonella. J Anim Sci
2008.86:E163-E172). In cattle, typical symptoms include
loss of appetite, fever, dark brown diarrhea or bloody
mucous stool. The acute infection in calves leads to rapid
death, and the infection during pregnancy leads to fetal
death due to septicemia, resulting in premature abortion
(www.livestock.co.kr). In pigs, salmonellosis is
characterized clinically by three major syndromes-acute
- 6 -
septicemia, acute enteritis, and chronic enteritis. Acute
septicemia occurs in 2~4 month-old piglets, and death
usually occurs within 2~4 days after onset of symptoms.
Acute enteritis occurs during the fattening period, and is
accompanied by diarrhea, high fever, pneumonia, and nervous
signs. Discoloration of the skin may occur in some severe
cases. Chronic enteritis is accompanied by continuing
diarrhea (www.livestock.co.kr).
Once an outbreak of salmonellosis by SE and ST occurs
in poultry, pigs, and cattle, it is difficult to cure by
therapeutic agents. The reasons are that Salmonella
bacteria exhibit a strong resistance to various drugs and
live in cells being impermeable to antibiotics upon the
occurrence of clinical symptoms. Up to now, there have been
no methods for effectively treating salmonellosis caused by
SE and ST, including antibiotics (www.lhca.or.kr).
As in livestock, SE and ST cause infections in human
via livestock products, leading to salmonella food poisoning.
Consumption of infected, improperly cooked livestock
products (e.g., meat products, poultry products, eggs and
by-products) infects human. Salmonella food poisoning in
human usually involves the prompt onset of headache, fever,
abdominal pain, diarrhea, nausea, and vomiting. The
- 7 -
symptoms commonly appear within 6-72 hours after the
ingestion of the organism, and may persist for as long as 4-
7 days or even longer (NSW+HEALTH. 2008.01.14.).
According to a report by the CDC (The Centers for
Disease Control and Prevention, USA), 16% of human food
poisoning outbreaks between 2005 and 2008 were attributed to
Salmonella bacteria, and 20% and 18% of that total was
caused by SE and ST, respectively. With respect to
salmonella food poisoning in human between 1973 and 1984,
the implicated food vehicles of transmission were reportedly
chicken (5%), beef (19%), pork (7%), dairy products (6%),
and turkey (9%). In 1974~1984, the bacterial contamination
test on broilers during the slaughter process showed 35% or
more of salmonella incidence. In 1983, salmonella was
isolated in 50.6% of chicken, 68.8% of turkey, 60% of goose,
11.6% of pork, and 1.5% of beef. Further, a survey carried
out in 2007 reported that salmonella was found in 5.5% of
raw poultry meat and 1.1% of raw pork. In particular, it
was revealed that SE commonly originated from contaminated
egg or poultry meat, and ST from contaminated pork, poultry
meat, and beef (www.cdc.gov). For example, food poisoning
caused by SE has rapidly increased in the US, Canada, and
- 8 -
Europe since 1988, and epidemiological studies demonstrated
that it was attributed to eggs or egg-containing foods
(Agre-Food Safety Information Service(AGROS). Domestic and
foreign food poisoning occurrence and management trend. 2008.
02). A risk assessment conducted by FAO and WHO in 2002
noted that the human incidence of salmonellosis transmitted
through eggs and poultry meat appeared to have a linear
relationship to the observed Salmonella prevalence in
poultry. This means that, when reducing the prevalence of
Salmonella in poultry, the incidence of salmonellosis in
humans will fall (Salmonella control at the source; World
Health Organization. International Food Safety Authorities
Network (INFOSAN) Information Note No. 03/2007). Recently,
fears about food safety have been spurred by outbreaks of
salmonella from products as varied as peanuts, spinach,
tomatoes, pistachios, peppers and, most recently, cookie
dough (Jane Black and Ed O’Keefe. Overhaul of Food Safety
Rules in the Works. Washington Post Staff Writers Wednesday,
July 8, 2009).
For these reasons, Salmonella infections must be
reported in Germany (§6 and §7 of the German law on
infectious disease prevention, Infektionsschutzgesetz).
Between 1990 and 2005 the number of officially recorded
- 9 -
cases decreased from approximately 200,000 cases to
approximately 50,000. It is estimated that every fifth
person in Germany is a carrier of Salmonella. In the USA,
there are approximately 40,000 cases of Salmonella infection
reported each year
(en.wikipedia.org/wiki/Salmonella#cite_note-2).
Therefore, there is an urgent need to control SE and ST,
which cause salmonellosis in livestock and human. The
collaborative efforts of USDA and FDA have developed a
number of effective strategies to prevent salmonellosis that
causes over 1 million cases of food-borne illness in the
United States. Among them is a final rule, issued by the
FDA, to reduce the contamination in eggs. The FDA will now
require that egg producers test regularly for lethal
salmonella during egg production, storage and shipment. As
a result, an estimated 79,000 illnesses and 30 deaths due to
contaminated eggs will be avoided each year (Jane Black and
Ed O’Keefe. Overhaul of Food Safety Rules in the Works.
Washington Post Staff Writers Wednesday, July 8, 2009). In
Denmark, conservative estimates from a cost benefit analysis
comparing Salmonella control costs in the production sector
with the overall public health costs of salmonellosis
suggest that Salmonella control measures saved the Danish
- 10 -
economy US$ 14.1 million in the year 2001 (Salmonella
control at the source. World Health Organization.
International Food Safety Authorities Network(INFOSAN)
Information Note No. 03/2007).
Meanwhile, bacteriophage is a specialized type of virus
that only infects and destroys bacteria, and can selfreplicate
only inside host bacteria. Bacteriophage consists
of genetic material in the form of single or double stranded
DNA or RNA surrounded by a protein shell. Bacteriophages
are classified based on their morphological structure and
genetic material. There are three basic structural forms of
bacteriophage according to morphological structure: an
icosahedral (twenty-sided) head with a tail, an icosahedral
head without a tail, and a filamentous form. Based on their
tail structure, bacteriophages having icosahedral head and
double-stranded, linear DNA as their genetic material are
divided into three families: Myoviridae, Siphoviridae, and
Podoviridae, which are characterized by contractile, long
noncontractile, and short noncontractile tails, respectively.
Bacteriophages having icosahedral head without a tail and
RNA or DNA as their genetic material are divided based on
their head shape and components, and the presence of shell.
- 11 -
Filamentous bacteriophages having DNA as their genetic
material are divided based on their size, shape, shell, and
filament components (H.W.Ackermann. Frequency of
morphological phage descriptions in the year 2000; Arch
Virol (2001) 146:843-857; Elizabeth Kutter et al.
Bacteriophages biology and application; CRC press).
During infection, a bacteriophage attaches to a
bacterium and inserts its genetic material into the cell.
After this a bacteriophage follows one of two life cycles,
lytic or lysogenic. Lytic bacteriophages take over the
machinery of the cell to make phage components. They then
destroy or lyse the cell, releasing new phage particles.
Lysogenic bacteriophages incorporate their nucleic acid into
the chromosome of the host cell and replicate with it as a
unit without destroying the cell. Under certain conditions,
lysogenic phages can be induced to follow a lytic cycle
(Elizabeth Kutter et al. Bacteriophages biology and
application. CRC press).
After the discovery of bacteriophages, a great deal of
faith was initially placed in their use for infectiousdisease
therapy. However, when broad spectrum antibiotics
- 12 -
came into common use, bacteriophages were seen as
unnecessary because of having a specific target spectrum.
Nevertheless, the misuse and overuse of antibiotics resulted
in rising concerns about antibiotic resistance and harmful
effects of residual antibiotics in foods (Cislo, M et al.
Bacteriophage treatment of suppurative skin infections. Arch
Immunol.Ther.Exp. 1987.2:175-183; Kim sung-hun et al.,
Bacteriophage; New Alternative Antibiotics. Biological
research information center (BRIC)). In particular,
antimicrobial growth promoter (AGP), added to animal feed to
enhance growth, is known to induce antibiotic resistance,
and therefore, the ban of using antimicrobial growth
promoter (AGP) has been recently introduced. In the
European Union, the use of all antimicrobial growth
promoters (AGPs) was banned from 2006. Korea has banned the
use of some AGPs from 2009, and is considering restrictions
on the use of all AGPs at 2013~2015.
These growing concerns about the use of antibiotics
have led to a resurgence of interest in bacteriophage as an
alternative to antibiotics. 7 bacteriophages for control of
E.coli O157:H are disclosed in US Patent 6,485,902 (granted
in 2002 - Use of bacteriophages for control of Escherichia
coli O157). 2 bacteriophages for control of various
- 13 -
microorganisms are disclosed in US Patent 6,942,858 (granted
by Nymox in 2005). Many companies have been actively trying
to develop various products using bacteriophages. EBI food
system (Europe) developed a food additive for preventing
food poisoning caused by Listeria monocytogenes, named
Listex-P100, which is the first bacteriophage product
approved by the US FDA. A phage-based product, LMP-102 was
also developed as a food additive against Listeria
monocytogenes, approved as GRAS (Generally regarded as safe).
In 2007, a phage-based wash produced by OmniLytics was
developed to prevent E.coli O157 contamination of beef
during slaughter, approved by USDA’s Food Safety and
Inspection Service (FSIS). In Europe, Clostridium
sporogenes phage NCIMB 30008 and Clostridium tyrobutiricum
phage NCIMB 30008 were registered as a feed preservative
against Clostridium contamination of feed in 2003 and 2005,
respectively. Such studies show that research into
bacteriophages for use as antibiotics against zoonotic
pathogens in livestock products is presently ongoing.
However, most of the phage biocontrol studies have
focused on the control of E.coli, Listeria, and Clostridium.
Salmonella is also a zoonotic pathogen, and damages due to
this pathogen are not reduced. As mentioned above, since SE
- 14 -
and ST exhibit multiple drug resistance, nationwide
antimicrobial resistance surveillance has been conducted in
Korea under Enforcement Decree of the Act on the Prevention
of Contagious Disease (Executive Order 16961), Enforcement
ordinance of the Act on the Prevention of Contagious Disease
(Ministry of Health and Welfare’s Order 179), and
Organization of the National Institute of Health (Executive
Order 17164). Accordingly, there is a need for the
development of bacteriophages to control Salmonella.
[Disclosure]
[Technical Problem]
In order to solve the problems generated by the use of
broad spectrum antibiotics, the present inventors isolated a
novel Salmonella bacteriophage from natural sources, and
identified its morphological, biochemical, and genetic
properties. The present inventors found that the
bacteriophage has a specific bactericidal activity against
Salmonella Enteritidis (SE), Salmonella Typhimurium (ST),
Salmonella Gallinarum (SG), and Salmonella Pullorum (SP)
without affecting beneficial bacteria, and excellent acid-,
heat- and dry-resistance, and thus can be used for the
prevention and treatment of livestock salmonellosis and
- 15 -
Salmonella food poisoning that are caused by Salmonella
Enteritidis or Salmonella Typhimurium, and Fowl Typhoid and
Pullorum disease that are caused by Salmonella Gallinarum
and Salmonella Pullorum. Also, the bacteriophage according
to the present invention can be applied to various products
for the control of Salmonella bacteria, including feed
additive and drinking water for livestock, barn sanitizers,
and cleaners for meat products, thereby completing the
present invention.
[Technical Solution]
It is an object of the present invention to provide a
novel bacteriophage having a specific bactericidal activity
against one or more Salmonella bacteria selected from the
group consisting of Salmonella Enteritidis, Salmonella
Typhimurium, Salmonella Gallinarum, and Salmonella Pullorum.
It is another object of the present invention to
provide a composition for the prevention or treatment of
infectious diseases caused by one or more Salmonella
bacteria selected from the group consisting of Salmonella
Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,
and Salmonella Pullorum, comprising the bacteriophage as an
active ingredient.
- 16 -
It is still another object of the present invention to
provide a feed additive or drinking water for livestock,
comprising the bacteriophage as an active ingredient.
It is still another object of the present invention to
provide a sanitizer or cleaner, comprising the bacteriophage
as an active ingredient.
It is still another object of the present invention to
provide a method for preventing or treating infectious
diseases caused by one or more Salmonella bacteria selected
from the group consisting of Salmonella Enteritidis,
Salmonella Typhimurium, Salmonella Gallinarum, and
Salmonella Pullorum using the bacteriophage or the
composition comprising the bacteriophage as an active
ingredient.
[Advantageous Effects]
The novel bacteriophage of the present invention has a
specific bactericidal activity against one or more
Salmonella bacteria selected from the group consisting of
Salmonella Enteritidis, Salmonella Typhimurium, Salmonella
Gallinarum, and Salmonella Pullorum, and excellent acid-,
heat- and dry-resistance. Therefore, the novel
bacteriophage can be used for the prevention and treatment
- 17 -
of infectious diseases caused by Salmonella Enteritidis,
Salmonella Typhimurium, Salmonella Gallinarum, or Salmonella
Pullorum, including salmonellosis, Salmonella food poisoning,
Fowl Typhoid and Pullorum disease, and also used for the
control of Salmonella Enteritidis, Salmonella Typhimurium,
Salmonella Gallinarum, and Salmonella Pullorum.
[Description of Drawings]
FIG. 1 is an electron microscopy photograph of ΦCJ3, in
which ΦCJ3 belongs to the morphotype group of the family
Myoviridae, characterized by an isometric capsid and a long
contractile tail;
FIG. 2 is the result of SDS-PAGE of the isolated
bacteriophage ΦCJ3, in which protein patterns of the
bacteriophage are shown, major proteins of 45 kDa, 62 kDa
and 80 kDa and other proteins of 17 kDa, 28 kDa, 110 kDa,
and 170 kDa (See-blue plus 2 prestained-standard
(Invitrogen) used as marker);
FIG. 3 is the result of PFGE of the isolated
bacteriophage ΦCJ3, showing the total genome size of
approximately 158 kbp (5 kbp DNA size standard (Bio-rad) as
size marker);
FIG. 4 is the result of PCR, performed by using each
- 18 -
primer set of ΦCJ3 genomic DNA, in which (A; PCR
amplification using the primer set of SEQ ID NOs. 5 and 6,
B; PCR amplification using primer set of SEQ ID NOs. 7 and 8,
C; PCR amplification using primer set of SEQ ID NOs. 9 and
10, D; PCR amplification using primer set of SEQ ID NOs. 11
and 12) all of A, B, C and D lanes have a PCR product of
approximately 1.0 kbp;
FIGs. 5 to 8 are the result of one-step growth
experiment of the bacteriophage ΦCJ3, in which the
bacteriophage had the burst size of 2 X 102 pfu or more in
Salmonella Gallinarum, Salmonella Pullorum, Salmonella
Typhimurium, and Salmonella enteritidis;
FIG. 9 is the result of acid-resistance test on the
bacteriophage ΦCJ3, showing the number of surviving
bacteriophage at pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9,
7.4, 8.0, 9.0, in which the bacteriophage ΦCJ3 did not lose
its activity until pH 3.5, but completely lost its activity
at pH 3.0 or lower, as compared to control;
FIG. 10 is the result of heat-resistance test on the
bacteriophage ΦCJ3, showing the number of surviving
bacteriophage at 37, 45, 53, 60, 70, 80°C and a time point of
0, 10, 30, 60, 120 min, in which the bacteriophage ΦCJ3 did
not lose its activity even after exposure at 60°C for 2 hrs,
- 19 -
and completely lost its activity after exposure at 70°C or
higher for 10 min; and
FIG. 11 is the result of dry-resistance test on the
bacteriophage ΦCJ3, performed by using a spray dryer and
adding dextrin and sugar as a stabilizer, in which changes
in the titers before and after drying were compared to
examine the relative stability, and its activity was
decreased to approximately 5 X 103.
[Best Mode]
In accordance with an aspect, the present invention
relates to a novel bacteriophage having a specific
bactericidal activity against one or more Salmonella
bacteria selected from the group consisting of Salmonella
Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,
and Salmonella Pullorum, in which the bacteriophage belongs
to the morphotype group of the family Myoviridae, and has a
total genome size of 157-159 kbp and major structural
proteins with the size of 44-46 kDa, 61-63 kDa and 79-81 kDa.
Specifically, the bacteriophage of the present
invention has the capability of selectively infecting
Salmonella gallinarum, Salmonella pullorum, Salmonella
typhimurium, and Salmonella Enteritidis, namely, species
- 20 -
specificity.
The bacteriophage of the present invention genetically
has a total genome size of 157-159 kbp, preferably about 158
kbp, and may include one or more nucleic acid molecules
selected from the group consisting of SEQ ID NOs. 1, 2, 3
and 4 within the entire genome, preferably nucleic acid
molecules represented by SEQ ID NOs. 1 to 4 within the
entire genome.
When the bacteriophage of the present invention is
subjected to PCR using one or more primer sets selected from
the group consisting of SEQ ID NOs. 5 and 6, SEQ ID NOs. 7
and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12, each
PCR product of approximately 1 kbp is given. Preferably,
when PCR is performed using all of the primer sets, each PCR
product of approximately 1 kbp is given.
The bacteriophage of the present invention belongs to
the morphotype group of the family Myoviridae, characterized
by an isometric capsid and a long contractile tail, and
preferably the morphology depicted in FIG. 1.
As used herein, the term “nucleic acid molecule”
encompasses DNA (gDNA and cDNA) and RNA molecules, and the
term nucleotide, as the basic structural unit of nucleic
- 21 -
acids, encompasses natural nucleotides and sugar or basemodified
analogues thereof.
The bacteriophage of the present invention genetically
has major structural proteins with the size of 44-46 kDa,
61-63 kDa, and 79-81 kDa, and preferably the size of
approximately 45 kDa, 62 kDa, and 80 kDa.
Further, the bacteriophage of the present invention has
one or more biochemical properties of acid-, heat-, and dryresistance.
More specifically, the bacteriophage of the present
invention can stably survive in a wide range of pH
environment from pH 3.5 to pH 9.0, and in a high temperature
environment from 37°C to 60°C. In addition, the bacteriophage
of the present invention is resistant to dessication to
remain viable even after high-temperature drying (e.g., at
60°C for 120 min). Such properties of acid-, heat-, and
drying-resistance allow application of the bacteriophage of
the present invention under various temperature and pH
conditions upon the production of prophylactic or
therapeutic compositions for livestock diseases or human
diseases caused by the contaminated livestock.
- 22 -
The present inventors collected sewage samples at
chicken slaughterhouses, and isolated the bacteriophage of
the present invention that has a specific bactericidal
activity against SE, ST, SG and SP and the above
characteristics, which was designated as ΦCJ3 and deposited
at the Korean Culture Center of Microorganisms (361-221,
Honje 1, Seodaemun, Seoul) on December 17, 2008 under
accession number KCCM10977P.
In accordance with the specific Example of the present
invention, the present inventors collected sewage samples at
chicken slaughterhouses to isolate bacteriophages that lyse
the host cell ST, and they confirmed that the bacteriophages
are able to lyse SE, ST, SG and SP. Further, they examined
the bacteriophage (ΦCJ3) under electron microscope, and
found that it belongs to the morphotype of the family
Myoviridae (FIG. 1).
Further, the protein patterns of the bacteriophage ΦCJ3
were also analyzed, resulting in that it has major
structural proteins with the size of 45 kDa, 62 kDa, and 80
kDa (FIG. 2).
Furthermore, the total genome size of the bacteriophage
ΦCJ3 was also analyzed, resulting in that it has a total
- 23 -
genome size of approximately 158 kbp (FIG. 3). The results
of analyzing its genetic features showed that the
bacteriophage includes nucleic acid molecules represented by
SEQ ID NOs. 1 to 4 within the total genome. Based on these
results, genetic similarity with other species was compared.
It was found that the bacteriophage showed very low genetic
similarity with the known bacteriophages, indicating that
the bacteriophage is a novel bacteriophage (Table 2). More
particularly, the ΦCJ3-specific primer sets, namely, SEQ ID
NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and
SEQ ID NOs. 11 and 12 were used to perform PCR. Each PCR
product was found to have a size of approximately 1 kbp (FIG.
4).
Further, when SE, ST, SG and SP were infected with ΦCJ3,
the phage plaques (clear zone on soft agar created by host
cell lysis of one bacteriophage) showed the same size and
turbidity.
Furthermore, the stability of ΦCJ3 was examined under
various temperature and pH conditions, resulting in that
ΦCJ3 stably maintains in a wide range of pH environments
from pH 3.5 to pH 9.0 (FIG. 9) and in high temperature
environments from 37°C to 60°C (FIG. 10), and even after hightemperature
drying (FIG. 11). These results indicate that
- 24 -
the bacteriophage ΦCJ3 of the present invention can be
applied to various products for the control of salmonella
bacteria.
In accordance with another aspect, the present
invention relates to a composition for the prevention or
treatment of infectious diseases caused by one or more
selected from the group consisting of Salmonella Gallinarum,
Salmonella Pullorum, Salmonella Typhimurium, and Salmonella
enteritidis, comprising the bacteriophage as an active
ingredient.
Preferably, the infectious diseases caused by
Salmonella enteritidis or Salmonella Typhimurium include
salmonellosis and Salmonella food poisoning, the infectious
diseases caused by Salmonella Gallinarum include Fowl
Typhoid, and the infectious diseases caused by Salmonella
Pullorum include Pullorum disease, but are not limited
thereto.
The bacteriophage of the present invention has a
specific bactericidal activity against Salmonella Gallinarum,
Salmonella Pullorum, Salmonella Typhimurium, and Salmonella
enteritidis, and thus can be used for the purpose of
- 25 -
preventing or treating diseases that are caused by these
bacteria. Specifically, in the preferred embodiment, an
antibiotic may be included.
As used herein, the term “prevention” means all of the
actions in which disease progress is restrained or retarded
by the administration of the composition. As used herein,
the term “treatment” means all of the actions in which the
patient’s condition has taken a turn for the better or been
modified favorably by the administration of the composition.
The composition of the present invention comprises ΦCJ3
of 5 x 102 to 5 x 1012 pfu/ml, and preferably 1 x 106 to 1 x
1010 pfu/ml.
Preferred examples of infectious diseases, to which the
composition of the present invention can be applied, include
Fowl Typhoid caused by Salmonella Gallinarum, Pullorum
disease caused by Salmonella Pullorum, and salmonellosis or
Salmonella food poisoning caused by Salmonella enteritidis
or Salmonella Typhimurium, but are not limited thereto.
As used herein, the term “salmonellosis” refers to
symptoms caused by salmonella infection, including fever,
headache, diarrhea, and vomiting, namely, diseases caused by
bacteria of the genus Salmonella, which is defined two
clinical forms - an acute septicemic form that resembles
- 26 -
typhoid fever and an acute gastroenteritis, including
enteritis, food poisoning, and acute septicemia.
The composition of the present invention may
additionally include a pharmaceutically acceptable carrier,
and formulated together with the carrier to provide foods,
medicines, and feed additives.
As used herein, the term “pharmaceutically acceptable
carrier” refers to a carrier or diluent that does not cause
significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. For formulation of the composition into a liquid
preparation, a pharmaceutically acceptable carrier which is
sterile and biocompatible may be used such as saline,
sterile water, Ringer’s solution, buffered physiological
saline, albumin infusion solution, dextrose solution,
maltodextrin solution, glycerol, and ethanol. These
materials may be used alone or in any combination thereof.
If necessary, other conventional additives may be added such
as antioxidants, buffers, bacteriostatic agents, and the
like. Further, diluents, dispersants, surfactants, binders
and lubricants may be additionally added to the composition
to prepare injectable formulations such as aqueous solutions,
suspensions, and emulsions, or oral formulations such as
- 27 -
pills, capsules, granules, or tablets.
The prophylactic or therapeutic compositions of the
present invention may be applied or sprayed to the afflicted
area, or administered by oral or parenteral routes. The
parenteral administration may include intravenous,
intraperitoneal, intramuscular, subcutaneous or topical
administration.
The dosage suitable for applying, spraying, or
administrating the composition of the present invention will
depend upon a variety of factors including formulation
method, the mode of administration, the age, weight, sex,
condition, and diet of the patient or animal being treated,
the time of administration, the route of administration, the
rate of excretion, and reaction sensitivity. A physician or
veterinarian having ordinary skill in the art can readily
determine and prescribe the effective amount of the
composition required.
Examples of the oral dosage forms suitable for the
composition of the present invention include tablets,
troches, lozenges, aqueous or emulsive suspensions, powder
or granules, emulsions, hard or soft capsules, syrups, or
elixirs. For formulation such as tablets and capsules,
useful are a binder such as lactose, saccharose, sorbitol,
- 28 -
mannitol, starch, amylopectin, cellulose or gelatin, an
excipient such as dicalcium phosphate, a disintegrant such
as corn starch or sweet potato starch, a lubricant such as
magnesium stearate, calcium stearate, sodium stearylfumarate,
or polyethylene glycol wax. For capsules, a liquid carrier
such as lipid may be further used in addition to the abovementioned
compounds.
For non-oral administration, the composition of the
present invention may be formulated into injections for
subcutaneous, intravenous, or intramuscular routes,
suppositories, or sprays inhalable via the respiratory tract,
such as aerosols. Injection preparations may be obtained by
dissolving or suspending the composition of the present
invention, together with a stabilizer or a buffer, in water
and packaging the solution or suspension in ampules or vial
units. For sprays, such as aerosol, a propellant for
spraying a water-dispersed concentrate or wetting powder may
be used in combination with an additive.
As used herein, the term “antibiotic” means any drug
that is applied to animals to kill pathogens, and used
herein as a general term for antiseptics, bactericidal
agents and antibacterial agents. The animals are mammals
including human, and preferably poultry. The bacteriophage
- 29 -
of the present invention, unlike the conventional
antibiotics, has a high specificity to Salmonella so as to
kill the specific pathogens without affecting beneficial
bacteria, and does not induce resistance so that its life
cycling is comparatively long.
In accordance with one specific embodiment of the
present invention, toxicity test on the bacteriophage ΦCJ3
for the prevention of Fowl Typhoid was performed by
evaluating its safety and effect on egg production in layer
chickens, including its residual amount in chicken flesh and
eggs. It was found that there was no difference in the egg
production rate between the control and ΦCJ3-treated groups
(Table 4), no ΦCJ3 was isolated in the collected eggs (Table
5), and upon treating the SG-infected chickens with ΦCJ3,
the ΦCJ3-treated group showed a significantly higher
protection rate than the non-treated group (Table 7),
indicating its preventive and therapeutic effects.
In accordance with still another aspect, the present
invention relates to an animal feed or drinking water,
comprising the bacteriophage as an active ingredient.
Feed additive antibiotics used in fishery and livestock
- 30 -
industry are used for the purpose of preventing infections,
but lead to an increase in resistant strains of bacteria and
the residual antibiotics in livestock products may be
ingested by humans, contributing to antibiotic resistance in
human pathogens and the spread of diseases. In addition,
since there are a variety of feed additive antibiotics, the
increasing global emergence of multidrug-resistant strain is
a serious concern. Therefore, the bacteriophage of the
present invention can be used as a feed additive antibiotic
that is more eco-friendly and able to solve the above
problems.
The bacteriophage of the present invention may be
separately prepared as a feed additive, and then added to
the animal feed, or directly added to the animal feed. The
bacteriophage of the present invention may be contained in
the animal feed as a liquid or in a dried form, preferably
in a dried powder. The drying process may be performed by
air drying, natural drying, spray drying, and freeze-drying,
but is not limited thereto. The bacteriophage of the
present invention may be added as a powder form in an amount
of 0.05 to 10% by weight, preferably 0.1 to 2% by weight,
based on the weight of animal feed. The animal feed may
also include other conventional additives for the long-term
- 31 -
preservation, in addition to the bacteriophage of the
present invention.
The feed additive of the present invention can
additionally include other non-pathogenic microorganisms.
The additional microorganism can be selected from a group
consisting of Bacillus subtilis that can produce protease,
lipase and invertase, Lactobacillus sp. strain having an
ability to decompose organic compounds and physiological
activity under anaerobic conditions, filamentous fungi like
Aspergillus oryzae (J AnimalSci 43: 910-926, 1976) that
increases the weight of domestic animals, enhances milk
production and helps digestion and absorptiveness of feeds,
and yeast like Saccharomyces cerevisiae (J Anim Sci 56:735-
739, 1983).
The feed comprising ΦCJ3 of the present invention may
include plant-based feeds, such as grain, nut, food
byproduct, seaweed, fiber, drug byproduct, oil, starch, meal,
and grain byproduct, and animal-based feeds such as protein,
mineral, fat, single cell protein, zooplankton, and food
waste, but is not limited thereto.
The feed additive comprising ΦCJ3 of the present
invention may include binders, emulsifiers, and
preservatives for the prevention of quality deterioration,
- 32 -
amino acids, vitamins, enzymes, probiotics, flavorings, nonprotein
nitrogen, silicates, buffering agents, coloring
agents, extracts, and oligosaccharides for the efficiency
improvement, and other feed premixtures, but is not limited
thereto.
Further, the supply of drinking water mixed with the
bacteriophage of the present invention can reduce the number
of Salmonella bacteria in the intestine of livestock,
thereby obtaining Salmonella-free livestock.
In accordance with still another aspect, the present
invention relates to a sanitizer and a cleaner, comprising
the bacteriophage as an active ingredient.
In order to remove Salmonella, the sanitizer comprising
the bacteriophage as an active ingredient can be used in the
poultry barns, slaughterhouses, contaminated areas, and
other production facilities, but is not limited thereto.
Further, the cleaner comprising the bacteriophage as an
active ingredient can be applied to the contaminated skin,
feather, and other contaminated body parts of living animals,
in order to remove Salmonella.
In accordance with still another aspect, the present
invention relates to a method for preventing or treating
- 33 -
infectious diseases caused by one or more Salmonella
bacteria selected from the group consisting of Salmonella
Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum,
and Salmonella Pullorum using the bacteriophage or the
composition.
The composition of the present invention may be
administered into animals in a pharmaceutical formulation or
as a component of the animal feed or in their drinking water,
preferably administered by mixing into the animal feed as a
feed additive.
The composition of the present invention may be
administered in a typical manner via any route such as oral
or parenteral routes, in particular, oral, rectal, topical,
intravenous, intraperitoneal, intramuscular, intraarterial,
transdermal, intranasal, and inhalation routes.
The method for treating the diseases of the present
invention includes administration of a pharmaceutically
effective amount of the composition of the present invention.
It will be obvious to those skilled in the art that the
total daily dose should be determined through appropriate
medical judgment by a physician. The therapeutically
effective amount for patients may vary depending on various
factors well known in the medical art, including the kind
- 34 -
and degree of the response to be achieved, the patient’s
condition such as age, body weight, state of health, sex,
and diet, time and route of administration, the secretion
rate of the composition, the time period of therapy,
concrete compositions according to whether other agents are
used therewith or not, etc.
Hereinafter, the present invention will be described in
more detail with reference to the following Examples.
However, these Examples are for illustrative purposes only,
and the invention is not intended to be limited by these
Examples.
[Mode for Invention]
Example 1: Salmonella Bacteriophage Isolation
1-1. Bacteriophage screening and Single bacteriophage
isolation
50 ml of sample from chicken slaughterhouse and sewage
effluent was transferred to a centrifuge tube, and
centrifuged at 4000 rpm for 10 minutes. Then, the
supernatant was filtered using a 0.45 ㎛ filter. 18 ml of
sample filtrate was mixed with 150 ㎕ of ST shaking culture
- 35 -
medium (OD600=2) and 2 ml of 10 X Luria-Bertani medium
(Hereinbelow, designated as LB medium, tryptone 10 g; yeast
extract 5 g; NaCl 10 g; final volume to 1 L). The mixture
was cultured at 37°C for 18 hours, and the culture medium was
centrifuged at 4000 rpm for 10 minutes. The supernatant was
filtered using a 0.2 ㎛ filter. 3 ml of 0.7% agar (w/v) and
150 ㎕ of ST shaking culture medium (OD600=2) were mixed, and
plated onto LB plate, changed to a solid medium. 10 ㎕ of
culture filtrate was spread thereon, and cultured for 18
hours at 37°C (0.7% agar was used as “top-agar” and the
titration of phage lysate was performed on the top-agar,
called soft agar overlay method).
The sample culture medium containing the phage lysate
was diluted, and mixed with 150 ㎕ of ST shaking culture
medium (OD600=2), followed by soft agar overlay method, so
that single plaques were obtained. Since a single plaque
represents one bacteriophage, to isolate single
bacteriophages, one plaque was added to 400 ㎕ of SM solution
(NaCl, 5.8 g; MgSO47H2O, 2 g; 1 M Tris-Cl (pH7.5), 50 ml; H2O,
final volume to 1 L), and left for 4 hours at room
temperature to isolate single bacteriophages. To purify the
bacteriophage in large quantities, 100 ㎕ of supernatant was
taken from the single bacteriophage solution, and mixed with
- 36 -
12 ml of 0.7% agar and 500 ㎕ of ST shaking culture medium,
followed by soft agar overlay method on LB plate (150 mm
diameter). When lysis was completed, 15 ml of SM solution
was added to the plate. The plate was gently shaken for 4
hours at room temperature to elute the bacteriophages from
the top-agar. The SM solution containing the eluted
bacteriophages was recovered, and chloroform was added to a
final volume of 1%, mixed well for 10 minutes. The solution
was centrifuged at 4000 rpm for 10 minutes. The obtained
supernatant was filtered using a 0.2 ㎛ filter, and stored in
the refrigerator.
1-2. Large-scale batches of bacteriophage
The selected bacteriophages were cultured in large
quantities using ST. ST was shaking-cultured, and an
aliquot of 1.5 X 1010 cfu (colony forming units) was
centrifuged at 4000 rpm for 10 minutes, and the pellet was
resuspended in 4 ml of SM solution. The bacteriophage of
7.5 X 107 pfu (plaque forming unit) was inoculated thereto
(MOI: multiplicity of infection=0.005), and left at 37°C for
20 minutes. The solution was inoculated into 150 ml of LB
media, and cultured at 37°C for 5 hours. Chloroform was added
to a final volume of 1%, and the culture solution was shaken
- 37 -
for 20 minutes. DNase I and RNase A were added to a final
concentration of 1 ㎍/ml, respectively. The solution was
left at 37°C for 30 minutes. NaCl and PEG (polyethylene
glycol) were added to a final concentration of 1 M and 10%
(w/v), respectively and left at 37°C for additional 3 hours.
The solution was centrifuged at 4°C and 12000 rpm for 20
minutes to discard the supernatant. The pellet was
resuspended in 5 ml of SM solution, and left at room
temperature for 20 minutes. 4 ml of chloroform was added
thereto and mixed well, followed by centrifugation at 4°C and
4000 rpm for 20 minutes. The supernatant was filtered using
a 0.2 ㎛ filter, and ΦCJ3 was purified by glycerol density
gradient ultracentrifugation (density: 40%, 5% glycerol at
35,000 rpm and 4°C for 1 hour). The purified bacteriophge was
designated as bacteriophage ΦCJ3, and resuspended in 300 ㎕
of SM solution, followed by titration. The bacteriophage
ΦCJ3 was deposited at the Korean Culture Center of
Microorganisms (361-221, Honje 1, Seodaemun, Seoul) on
December 17, 2008 under accession number KCCM10977P.
Example 2: Examination on ΦCJ3 Infection of Salmonella
To examine the lytic activity of the selected
bacteriophages on other Salmonella species as well as ST,
- 38 -
cross-infection attempts with other Salmonella species were
made. As a result, ΦCJ3 did not infect SC (Salmonella
enterica Serotype Choleraesuis), SD (Salmonella enterica
Serotype Derby), SA (Salmonella enterica subsp. Arizonae),
SB (Salmonella enterica subsp. Bongori), but specifically
infected SG, SP, ST, and SE (see Example 12). The results
are shown in the following Table 1. The bacteriophages ΦCJ3
that were produced using SG as a host cell showed the same
plaque size and plaque turbidity, and the same protein
patterns and genome size as those produced using ST as a
host cell.
[Table 1]
<ΦCJ3 infection of Salmonella>
Sero
type
Strain
name
Plaque
formation
Sero
type
Strain
name
Plaque
formation
SE SGSC 2282 O SA
ATCC
13314
X
ST
ATCC
14028
O SB
ATCC
43975
X
SG SGSC 2293 O SC
ATCC
10708
X
SP SGSC 2295 O SD ATCC X
- 39 -
6960
* ATCC: The Global Bioresource Center
* SGSC: salmonella genetic stock center
Example 3: Morphology Examination of Bacteriophage ΦCJ3
The purified ΦCJ3 was diluted in 0.01% gelatin solution,
and then fixed in 2.5% glutaraldehyde solution. After the
sample was dropped onto a carbon-coated mica plate (ca.2.5 X
2.5 mm) and adapted for 10 minutes, it was washed with
sterile distilled water. Carbon film was mounted on a
copper grid, and stained with 4% uranyl acetate for 30-60
seconds, dried, and examined under JEM-1011 transmission
electron microscope (80kV, magnification of X 120,000 ~ X
200,000). As a result, the purified ΦCJ3 had morphological
characteristics including an isometric capsid and a long
contractile tail, as shown in , indicating that it
belongs to the morphotype group of the family Myoviridae.
Example 4: Protein Pattern Analysis of Bacteriophage
ΦCJ3
15 ㎕ of purified ΦCJ3 solution (1011 pfu/ml titer) was
treated with 3 ㎕ of 5X SDS sample solution, and heated for 5
minutes. The total protein of ΦCJ3 was run in 4-12% NuPAGE
- 40 -
Bis-Tris gel (Invitrogen), and then the gel was stained with
Coomassie blue for 1 hour at room temperature. As shown in
FIG. 2, the protein patterns showed that 45 kDa, 62 kDa, and
80 kDa bands were observed as major proteins, and 17 kDa, 28
kDa, 110 kDa, and 170 kDa bands were also observed.
Example 5: Total Genomic DNA Size Analysis of
Bacteriophage ΦCJ3
Genomic DNA was isolated from the purified ΦCJ3 by
ultracentrifugation. Specifically, to the purified ΦCJ3
culture medium, EDTA (ethylenediaminetetraacetic acid
(pH8.0)), proteinase K, and SDS (sodium dodecyl sulfate)
were added to a final concentration of 20 mM, 50 ug/ml, and
0.5% (w/v), respectively and left at 50°C for 1 hour. An
equal amount of phenol (pH8.0) was added and mixed well,
followed by centrifugation at 12000 rpm and room temperature
for 10 minutes. The supernatant was mixed well with an
equal amount of PC (phenol:chloroform=1:1), followed by
centrifugation at 12000 rpm and room temperature for 10
minutes. The supernatant was mixed well with an equal
amount of chloroform, followed by centrifugation at 12000
rpm and room temperature for 10 minutes. Again, to the
supernatant, added were 1/10 volume of 3 M sodium acetate
- 41 -
and two volumes of cold 95% ethanol, and left at -20°C for 1
hour. After centrifugation at 0°C and 12000 rpm for 10
minutes, the supernatant was completely removed, and the DNA
pellet was dissolved in 50 ㎕ TE (Tris-EDTA (pH 8.0)). The
extracted DNA was diluted 10-fold, and its absorbance was
measured at OD260. After loading 1 ㎍ of total genomic DNA in
1% PFGE (pulse-field gel electrophoresis) agarose gel,
electrophoresis was performed using a BIORAD PFGE system
program 7 (size range 25-100 kbp; switch time ramp 0.4-2.0
seconds, linear shape; forward voltage 180 V; reverse
voltage 120 V) at room temperature for 20 hours. As shown
in FIG. 3, ΦCJ3 had a genomic DNA size of approximately 158
kbp.
Example 6: Genetic Analysis of Bacteriophage ΦCJ3
To analyze genetic features of the purified ΦCJ3, 5 ㎍
of genomic DNA of ΦCJ3 was treated with the restriction
enzymes, EcoR V and Sca I. The vector, pBluescript SK+
(Promega) was digested with EcoR V, and treated with CIP
(calf intestinal alkaline phosphatase). The digested
genomic DNA and vector were mixed in a ratio of 3:1, and
ligated at 16°C for 5 hours. The ligation product was
transformed into E.coli DH5α. The transformed cells were
- 42 -
plated on LB plate containing ampicillin and X-gal (5-bromo-
4-chloro-3-indolyl-beta-D-galactopyranoside) for blue/white
selection, so as to select four colonies. The selected
colony was shaking-cultured in a culture medium containing
ampicillin for 16 hours. Then, plasmids were extracted
using a plasmid purification kit (Promega).
The cloning of the plasmids was confirmed by PCR using
a primer set of M13 forward and M13 reverse, and insert
fragments of 1 kbp or more were selected, and their base
sequence was analyzed using the primer set of M13 forward
and M13 reverse. As shown in SEQ ID NOs. 1 and 4, all have
the size of 1 kbp. Sequence similarity was analyzed using a
NCBI blastx program, and the results are shown in Table 2.
As shown in Table 2, ΦCJ3 showed no sequence similarity
with other proteins in the upstream region of SEQ ID NO. 1,
and about 40% sequence similarity with the single-stranded
DNA binding protein of synechococcus phage in the downstream
region of the sequence in a backward direction. The
upstream region of SEQ ID NO. 2 showed about 32% sequence
similarity with the sliding clamp protein of synechococcus
phage in a backward direction. The downstream region also
showed about 32% sequence similarity with the UvsW RNA-DNA
and DNA-DNA helicase ATPase of ecterobacteria phage Phi1 in
- 43 -
a backward direction. The sequence of SEQ ID NO. 3 showed
no sequence similarity with the proteins of bacteriophage.
The downstream region of SEQ ID NO. 3 showed about 29%
sequence similarity with ATP-dependent DNA helicase RecG of
psychroflexus torques, and the upstream region of SEQ ID NO.
3 showed about 38% sequence similarity with the conserved
protein of leishmania major. The upstream region of SEQ ID
NO. 4 showed about 46% sequence similarity with the UvsX
RecA-like recombination protein of enterobacteria phage in a
backward direction.
Further, SEQ ID NOs. 2 and 3 of ΦCJ3 showed high evalue,
and the sequence of SEQ ID NO. 3 showed no sequence
similarity with the proteins of bacteriophage. Furthermore,
as a result of the base sequence analysis of SEQ ID NOs. 1
to 4 by NCBI blastn program, no sequence similarity was
observed. These results indicate that ΦCJ3 is a novel
bacteriophage.
[Table 2]

Organism Protein
Accessio
n number
Subject
locatio
Query
location
Identi
ties
E
value
- 44 -
n
1
Synechoc
occus
phage
syn9
singlestrande
d DNA
binding
protein
NC_00829
6.2
68-247 995-429
77/192
(40%)
2e-30
Synechoc
occus
phage SPM2
ssDNA
binding
protein
gp32
NC_00682
0.1
63-242 995-423
79/202
(39%)
3e-29
2
Enteroba
cteria
phage
Phi1
UvsW
RNA-DNA
and
DNA-DNA
helicas
e
ATPase
NC_00982
1.1
443-500 998-855
19/58
(32%)
1.9
Synechoc
occus
phage
syn9
sliding
clamp
NC_00829
6.2
4-45 126-1
19/58
(32%)
4.5
3
Psychrof
lexus
ATPdepende
NZ_AAPR0
1000001.
12-136 541-873
38/129
(29%)
0.045
- 45 -
torquis
ATCC
700755
nt DNA
helicas
e RecG
1
Leishman
ia major
hypothe
tical
protein
,
conserv
ed
XM_00168
1761.1
58-115 121-297
23/59
(38%)
0.22
4
Enteroba
cteria
phage
RB69
UvsX
RecAlike
recombi
nation
protein
NC_00492
8.1
34-225 578-3
89/192
(46%)
2e-41
Enteroba
cteria
phage
JS98
UvsX
RecAlike
recombi
nation
protein
NC_01010
5.1
34-225 578-3
87/192
(45%)
3e-41
Example 7: Construction of ΦCJ3-specific primer
- 46 -
sequence
To identify ΦCJ3, ΦCJ3-specific primers were
constructed on the basis of SEQ ID NOs. 1 and 4. PCR was
performed using each primer set of SEQ ID NOs. 5 and 6, SEQ
ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11
and 12. 0.1 ㎍ of genomic DNA of bacteriophage and 0.5 pmol
of primer were added to pre-mix (Bioneer), and the final
volume was adjusted to 20 ㎕. PCR was performed with 30
cycles of denaturation; 94°C 30 sec, annealing; 60°C 30 sec,
and polymerization; 72°C, 1 min. When SEQ ID NOs. 5 and 6,
SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs.
11 and 12 were used as primer set, all PCR products of
approximately 1 kbp were obtained. The results are shown in
FIG. 4.
Example 8: Test on Infection Efficiency of
Bacteriophage
To test the infection efficiency of the bacteriophage
ΦCJ3, a one-step growth experiment was performed.
50 ml of SG culture medium (OD600=0.5) was centrifuged
at 4000 rpm for 10 minutes, and resuspended in 25 ml of
fresh LB medium. The purified bacteriophage (MOI=0.0005)
was inoculated thereto, and left for 5 minutes. The
- 47 -
reaction solution was centrifuged at 4000 rpm for 10 minutes,
and the cell pellet was resuspended in fresh LB medium.
While the cells were cultured at 37°C , two samples of cell
culture medium were collected every 10 minutes, and
centrifuged at 12000 rpm for 3 minutes. The obtained
supernatant was serially diluted, and 10 ㎕ of each diluted
sample was cultured at 37°C for 18 hours by soft agar overlay
method, and the titration of phage lysates was performed.
Thus, the same experiment was performed on SP, ST, and SE.
The result of one-step growth experiment on SG, SP, ST, and
SE showed the burst size of 2 X 102 or more. The results are
shown in FIGs. 5 to 8.
Example 9: pH Stability Test on Bacteriophage
To test the stability of ΦCJ3 in a low-pH environment
like the livestock stomach, the stability test was performed
in a wide range of pH (pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4,
6.9, 7.4, 8.2, 9.0). Various pH solutions (Sodium acetate
buffer (pH 2.1, pH 4.0, pH 5.5, pH 6.4)), Sodium citrate
buffer (pH 2.5, pH 3.0, pH 3.5), Sodium phosphate buffer (pH
6.9, pH 7.4), Tris-HCl (pH 8.2, pH 9.0)) were prepared at a
concentration of 2 M. 100 ㎕ of pH solution was mixed with
an equal amount of bacteriophage solution (1.0 X 1010 pfu/ml)
- 48 -
to the concentration of each pH solution to 1 M, and left at
room temperature for 1 hour. The reaction solution was
serially diluted, and 10 ㎕ of each diluted sample was
cultured at 37°C for 18 hours by soft agar overlay method,
and the titration of phage lysates was performed. Changes
in the titers according to pH difference were compared to
examine the relative stability. The results showed that the
bacteriophage did not lose its activity and maintained
stability until pH 3.5. However, it lost its activity at pH
3.0 or lower. The results are shown in FIG. 9.
Example 10: Heat Stability Test on Bacteriophage
To test stability of bacteriophage to heat generated
during formulation process when used as a feed additive, the
following experiment was performed. 200 ㎕ of ΦCJ3 solution
(1.0 X 1010 pfu/ml) was left at 37°C, 45°C, 53°C, 60°C, 70°C, and 80°C,
for 0 min, 10 min, 30 min, 60 min, and 120 min, respectively.
The solution was serially diluted, and 10 ㎕ of each diluted
sample was cultured at 37°C for 18 hours by soft agar overlay
method, and the titration of phage lysates was performed.
Changes in the titers according to temperature and exposure
time were compared to examine the relative stability. The
results showed that the bacteriophage did not lose its
- 49 -
activity at 60°C until the exposure time reached 2 hours.
However, the bacteriophage lost its activity at 70°C or
higher. The results are shown in FIG. 10.
Example 11: Dry Stability Test on Bacteriophage
To test stability of bacteriophage under the dry
condition used during the formulation process for feed
additive, the following experiment was performed. On the
basis of the results of heat stability test, the experiment
was performed under high-temperature drying conditions (60°C
for 120 min). 200 ㎕ of ΦCJ3 solution (1.0 X 1011 pfu/ml) was
dried using a Speed vacuum (Speed - Vacuum Concentrator 5301,
Eppendorf). The obtained pellet was completely resuspended
in an equal amount of SM solution at 4°C for one day. The
solution was serially diluted, and 10 ㎕ of each diluted
sample was cultured at 37°C for 18 hours by soft agar overlay
method, and the titration of phage lysates was performed.
Changes in the titers before and after drying were compared
to examine the relative stability. The results showed that
its activity was decreased to 5 x 103. The results are shown
in FIG. 11.
Example 12: Examination on Bacteriophage Infection of
- 50 -
Wild-Type strain
The lytic activity of bacteriophage ΦCJ3 was tested for
the Korean wild-type SE (38 strains), ST (22 strains), SG
(56 strains) and SP (19 strains), isolated by Laboratory of
Avian Diseases, College of Veterinary Medicine, Seoul
National University, and National Veterinary Research and
Quarantine Service and the Korea Centers for Disease Control
and Prevention, in addition to SG (SG SGSC2293), SP (SP
SGSC2295), ST (ST ATCC14028), and SE (SE SCSG 2282) used in
the present invention. 150 ㎕ of each strain shaking culture
medium (OD600=2) was mixed, and 10 ㎕ of ΦCJ3 solution (1010
pfu/ml) was cultured at 37°C for 18 hours by soft agar
overlay method, and the plaque formation was examined. It
was found that the bacteriophage ΦCJ3 showed lytic activity
of 95%, 58%, 100% and 81% on the wild type SE, ST, SG, and
SP, respectively. The results are shown in the following
Table 3.
[Table 3]

Sero
type
Strain
name
Plaque
formation
Sero
type
Strain name
Plaque
formation
SG SNU SG1 O ST SNU ST1 X
- 51 -
SNU SG2 O SNU ST2 O
SNU SG3 O SNU ST3 O
SNU SG4 O SNU ST4 O
SNU SG5 O SNU ST7 O
SNU SG6 O SNU ST8 O
SNU SG7 O SNU ST11 O
SNU SG8 O SNU ST12 O
SNU SG9 O SNU ST13 X
SNU SG10 O SNU ST14 X
SNU SG11 O SNU ST17 O
SNU SG12 O SNU ST18 X
SNU SG13 O SNU ST19 X
SNU SG14 O SNU ST20 X
SNU SG15 O SNU ST25 X
SNU SG16 O SNU ST26 X
SNU SG17 O SNU ST37 O
SNU SG18 O SNU ST38 O
SNU SG19 O SNU ST41 O
SNU SG20 O SNU ST42 O
SNU SG21 O ATCC UK1 O
SNU SG22 O ATCC 14028S O
SNU SG23 O SGSC STM1412 O
SNU SG24 O SGSC STM260 O
- 52 -
SNU SG25 O
SGSC STM
SA2197
X
SNU SG26 O
SE
SGSC SE2282 O
SNU SG27 O SGSC SE2377 O
SNU SG28 O PT4 S1400194 X
SNU SG30 O PT4 LA52 O
SNU SG31 O NVRQS SE004 O
SNU SG32 O NVRQS SE005 O
SNU SG33 O KCDC SE008 O
SNU SG34 O KCDC SE009 O
SNU SG36 O KCDC SE010 O
SNU SG37 O KCDC SE011 X
SNU SG38 O KCDC SE012 O
SNU SG39 O KCDC SE013 O
SNU SG40 O KCDC SE014 O
SNU SG41 O KCDC SE015 O
SNU SG42 O KCDC SE018 O
SNU SG43 O KCDC SE019 O
SNU SG44 O KCDC SE020 O
SNU SG45 O KCDC SE021 O
SNU SG46 O KCDC SE024 O
SNU SG47 O KCDC SE025 O
SNU SG48 O KCDC SE026 O
- 53 -
SNU SG49 O KCDC SE027 O
SNU SG50 O KCDC SE028 O
SGSC
SG9184
O KCDC SE029 O
SGSC
SG2292
O KCDC SE030 O
SGSC
SG2293
O KCDC SE031 O
SGSC
SG2744
O KCDC SE032 O
SGSC
SG2796
O KCDC SE033 O
SP
SNU SP1 O KCDC SE034 O
SNU SP4 O KCDC SE035 O
SNU SP5 O KCDC SE036 O
SNU SP8 O KCDC SE037 O
SNU SP11 O KCDC SE039 O
SGSC
SP2294
O KCDC SE040 O
SGSC
SP2295
O KCDC SE041 O
SGSC
SP2737
X KCDC SE042 O
- 54 -
SGSC
SP2739
X KCDC SE043 O
SGSC
SP2742
O KCDC SE044 O
SGSC
SP2743
O KCDC SE045 O
SGSC
SP2745
O
SC
ATCC SC10708 X
SGSC
SP2751
O ATCC SC2929 X
SGSC
SP4663
X
SD
ATCC SD6960 X
SGSC
SP4664
O ATCC SD2466 O
SGSC
SP4665
O ATCC SD2467 X
SGSC
SP4666
O ATCC SD2468 O
SGSC
SP4667
X SA ATCC SA13314 X
SGSC
SA1684
O SB ATCC SB43975 X
* SNU: Laboratory of Avian Diseases, College of
- 55 -
Veterinary Medicine, Seoul National University
* SGSC: salmonella genetic stock center
Example 13: Toxicity test on Bacteriophage
Toxicity test on the bacteriophage ΦCJ3 for the
prevention of Fowl Typhoid was performed by evaluation of
its safety, residual amount, and eggs in layer chickens.
The layer chickens are divided into three groups to perform
a pathogenicity test and egg test, and to examine the
presence of clinical signs and phage content in the cecal
feces.
For the pathogenicity test, 13 brown layer chickens
were divided into a ΦCJ3-treated group with 8 layers and a
control group with 5 layers. The ΦCJ3-treated group was fed
with a mixture of feed and ΦCJ3 (108 pfu or more per feed
(g)) and the control group was fed with feed only, and egg
production rate and clinical signs were examined for 3 weeks
after phage treatment. As shown in the following Table 4,
the ΦCJ3-treated group and the control group showed about
50% and 50% egg production rates, respectively. In addition,
when clinical signs were examined after phage treatment,
respiratory and digestive lesions were not observed for 24
days after ΦCJ3 treatment, and abnormal activity was not
- 56 -
observed. The results indicate that ΦCJ3 treatment does not
generate adverse effects.
For the egg test, 10 eggs were collected on day 3, 6,
and 9 after ΦCJ3 treatment, and the egg surface was washed
with 70% ethanol and broken out. Egg yolk and egg white
were mixed, and 5 ml of the mixture was diluted with 45 ml
of PBS by 10-1, 10-2, and 10-3. 106 cfu of SNUSG0197 was added
to 25 ml of each diluted solution, and incubated at 37°C for
3 hours, and cells were isolated by centrifugation. 500 ㎕
of supernatant and 100 ㎕ of SNUSG0197 (109 cfu/ml) were
mixed with each other, and plated on a tryptic soy agar
plate by top-agar overlay technique. After incubation at 37°
C for 18 hrs, the number of plaques was counted to calculate
the number of phage per 1 ml of egg. As shown in the
following Table 5, no ΦCJ3 was found in 26 eggs that were
collected on days 3, 6, and 9.
Next, the presence of clinical signs and ΦCJ3 content
in the cecal feces were examined after ΦCJ3 treatment. At 3
weeks after ΦCJ3 treatment, the test layer chickens were
euthanatized, and autopsy was performed to examine gross
lesions in the liver, spleen, kidney and ovary. The liver
sample was aseptically collected with sterile cotton swab,
and plated on a MacConkey agar plate to examine the presence
- 57 -
of Salmonella Gallinarum. The cecal feces were also
collected to measure the ΦCJ3 content in the individual
chickens. Briefly, 1 g of cecal feces was suspended in 9 ml
of PBS, and centrifuged at 15000 g for 30 min. 1 ml of
supernatant was diluted with PBS by 10-1 to 10-4, and 500 ㎕
of the dilution and 100 ㎕ of SG0197 (109 cfu/ml) were mixed
with each other, and plated on a 10x tryptic soy agar plate
by top-agar overlay technique. After incubation at 37°C for
18 hrs, the number of plaques was counted to calculate the
number of phage per cecal feces (g), taking into account the
serial dilution.
As a result, no abnormal clinical signs were observed
during the examination period, and about 3.7 x 104 pfu of
ΦCJ3 per cecal feces (g) was measured, indicating survival
of the bacteriophage in the intestine after passing through
the stomach.
Bacteriophage distribution in the organs was examined.
Briefly, 10 SPF chicks (11 day-old) were divided into two
groups with each 5 chicks. For 3 days, the treated group
was fed with feed supplemented with 108 pfu of ΦCJ3 (per g),
and the control group was fed with feed only. The chicks
were sacrificed to collect the liver, kidney and cecal feces,
and the presence of ΦCJ3 was examined. Each of the
- 58 -
collected liver, kidney and cecal feces was emulsified with
an equal volume of PBS. 1 ml of the liver and the whole
quantity of the kidney and cecal feces were transferred into
1.5 ml tubes, followed by centrifugation at 15,000 rpm for
15 min. 1 ml of supernatant was diluted with PBS by 10-1 to
10-4, and 500 ㎕ of the dilution and 100 ㎕ of SG0197 (109
cfu/ml) were mixed with each other, and plated on a 10x
tryptic soy agar plate by top-agar overlay technique. After
incubation at 37°C for 18 hrs, the number of plaques was
counted to calculate the number of bacteriophage per cecal
feces (g), taking into account the serial dilution. As
shown in the following Table 6, ΦCJ3 was not observed in the
liver and kidney, but was observed in the cecal feces.
[Table 4]

ΦCJ3 Control
Day 1 Feeding day
Day 2
Day 3 10 5
Day 6 13 7
Day 7 4 3
Day 8 8 4
- 59 -
Day 9 3 1
Day 10 2 3
Day 13 13 8
Day 14
Day 15 8 7
Day 16
Day 17 8 6
Day 20 9 7
Day 21
Day 22 9 4
Day 23
Day 24 9 3
Egg
production
rate
50.0% 50.8%
[Table 5]

Collection
day
ΦCJ3 Control
Day 3 0/10 0/5
Day 6 0/13 0/7
- 60 -
Day 9 0/3 0/1
Total 0/26 0/13
[Table 6]

Test
chicken
ΦCJ3-treated group Control group
liver kidney
cecal
feces
liver kidney
cecal
feces
1 - - + - - -
2 - - + - - -
3 - - + - - -
4 - - + - - -
5 - - + - - -
Example 14: Efficacy test on Bacteriophage
In order to evaluate the efficacy of ΦCJ3 on the
prevention and treatment of SG, efficacy test was performed
in chickens.
20 brown layers (1-day-old) were divided into 10 test
groups with 10 layers (ΦCJ3 treated group + non-treated
challenged group 1). For 1 week, the test chicks were fed
with feed supplemented with 107 pfu of ΦCJ3 (per g) and
drinking water supplemented with 107 pfu of ΦCJ3 (per ml).
- 61 -
At 1 week, 106 cfu of SG0197 (per chick) and 107 pfu (MOI=10)
of phage was mixed with 500 ㎕ of TSB, and left in ice for 1
hour or less, followed by oral administration. The
mortality rate was examined for 2 weeks. The surviving
chicks were sacrificed and subjected to autopsy and examined
for gross lesions, and the bacteria were isolated. As shown
in the following Table 7, it was found that the ΦCJ3-treated
group showed a significantly higher protection rate (P<0.05)
than the non-treated group.
[Table 7]

ΦCJ3-treated
challenged group
Non-treated
challenged
group
Survival 9 3
Mortality rate 10% 70%
Clinical signs 1/9 1/3
SG reisolation 1/9 0/3
Protection
rate 80% 20%
[Industrial Applicability]
- 62 -
The novel bacteriophage of the present inventon has a
specific bactericidal activity against one or more
Salmonella bacteria selected from the group consisting of
Salmonella Enteritidis (SE), Salmonella Typhimurium (ST),
Salmonella Gallinarum (SG), and Salmonella Pullorum (SP)
without affecting beneficial bacteria, and excellent acid-,
heat- and dry-resistance, and thus can be widely used in
therapeutic agents, animal feeds or drinking water, cleaners
and sanitizers for the purpose of preventing and treating
the infectious diseases caused by Salmonella Enteritidis,
Salmonella Typhimurium, Salmonella Gallinarum or Salmonella
Pullorum including salmonellosis, Salmonella food poisoning,
Fowl Typhoid, and Pullorum disease.
- 63 -
[CLAIMS]
[Claim 1]
A novel bacteriophage that has a specific bactericidal
activity against one or more Salmonella bacteria selected
from the group consisting of Salmonella Enteritidis,
Salmonella Typhimurium, Salmonella Gallinarum, and
Salmonella Pullorum, that belong to the morphotype group of
the family Myoviridae, and that has a total genome size of
157-159 kbp and major structural proteins with sizes of 44-
46 kDa, 61-63 kDa and 79-81 kDa.
[Claim 2]
The bacteriophage according to claim 1, wherein the
bacteriophage is identified by accession number KCCM10977P.
[Claim 3]
The bacteriophage according to claim 1, wherein the
bacteriophage has the morphology depicted in FIG. 1.
[Claim 4]
The bacteriophage according to claim 1, wherein the
bacteriophage includes one or more nucleic acid molecules
selected from the group consisting of SEQ ID NO. 1, 2, 3,
and 4 within the entire genome.
[Claim 5]
The bacteriophage according to claim 1, wherein each
- 64 -
PCR product has a size of 1 kbp, upon performing PCR using
one or more primer sets selected from the group consisting
of SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9
and 10, and SEQ ID NOs. 11 and 12.
[Claim 6]
The bacteriophage according to claim 1, wherein the
bacteriophage has one or more properties of the following
1)-3):
1) acid-resistance in a pH range from pH 3.5 to pH 9.0;
2) heat-resistance in a temperature range from 37°C to
60℃; and
3) dry-resistance at 37-60℃ for 0-120 minutes.
[Claim 7]
A composition for the prevention or treatment of
infectious diseases caused by one or more Salmonella
bacteria selected from the group consisting of Salmonella
Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, and
Salmonella Enteritidis, comprising the bacteriophage of any
one of claims 1 to 6 as an active ingredient.
[Claim 8]
The composition according to claim 7, wherein the
infectious disease caused by Salmonella enteritidis or
Salmonella Typhimurium is salmonellosis or Salmonella food
- 65 -
poisoning, the infectious disease caused by Salmonella
Gallinarum is Fowl Typhoid, and the infectious disease
caused by Salmonella Pullorum is Pullorum disease.
[Claim 9]
The composition according to claim 7, wherein the
composition is used as an antibiotic.
[Claim 10]
An animal feed or drinking water, comprising the
bacteriophage of any one of claims 1 to 6 as an active
ingredient.
[Claim 11]
A sanitizer or cleaner, comprising the bacteriophage of
any one of claims 1 to 6 as an active ingredient.
[Claim 12]
A method for preventing or treating infectious diseases
caused by one or more Salmonella bacteria selected from the
group consisting of Salmonella Enteritidis, Salmonella
Typhimurium, Salmonella Gallinarum, and Salmonella Pullorum,
using the bacteriophage of claims 1 to 6.
[Claim 13]
A method for preventing or treating infectious diseases
caused by one or more Salmonella bacteria selected from the
group consisting of Salmonella Enteritidis, Salmonella
- 66 -
Typhimurium, Salmonella Gallinarum, and Salmonella Pullorum,
using the composition of claim 7.
[Sequence list]
<110> CJ Cheiljedang Corporation
<120> Novel bacteriophage and antibacterial composition comprising the same
<130> OPA09112
<150> KR10-2008-0133908
<151> 2008-12-24
<160> 12
<170> KopatentIn 1.71
<210> 1
<211> 1000
<212> DNA
<213> Bacteriophage KCCM 10977P
<400> 1
ccctgctggc ttataagcaa ctcgatcagc ttgtaaacaa tctggctggt atcagcttgg 60
ctgatttcga aaggatctct atcggcatcc agagggatat attaggcaat gataacctga 120
- 80 -
cggcgtctga gaagagtagt ctgttgggat tgttacagga tgttgtcaaa aactaaaaag 180
cccccgaagg ggctttagtg aaattagtct tgcttcagga actgctcgaa ctcatcaatg 240
gaagccgtct gtttcgcatc ggcaccacca ttattggctg gaacagattg ctgtgcatta 300
gaaggctgag attgttgttg gtttagactt tcctgcgctg ttgggcgctg gggttcctga 360
gactgggtag gcgcatgtgc catagtagaa gcaccacctt caaccagagg ctgattatca 420
gggatggcca gaactttgcg caaacgtttt tccagatctt cgtacgattt gaagttggcc 480
ggattaaaga actcaaacag actgtgttct ttttcccaga tctcttcaat gtattcgtct 540
gtccccaaag gtgccggagt atcccacttc acattggtga agttggccac cagacctttc 600
cagttgccga actctttctc ttcgccaaag aggttcagaa tcaggttcgc gccttcccac 660
atatcgaacg ggtcgaattt agggtcagtt gagaacttag gattctgagc cgaatccagg 720
attttcttga cggcattacc gaactcaagc aagaagacct tgccgttgtt ttccggattg 780
ttgccatctt tgatcaccag gatgttggcg tagtatttgg tgtccggcag acgttttttg 840
- 81 -
agaactgttt tcagcttttc atcattcgtt tctttctgtt gtgcccacag aggacggtca 900
tggtcacgaa caggatcatc gttaccgaaa gtctgaggag agttttcgat ataccaacca 960
ccagcaccct ggaatgcgtg tttcatgatc atggcacacg 1000
<210> 2
<211> 1000
<212> DNA
<213> Bacteriophage KCCM 10977P
<400> 2
aatgtcggca atagcgataa ctgtactgga atcgttaaca gtgcgcaact ttttaccagg 60
tgccagaacg atagaggggc agatggtttc aaagttagcc agcagttgta aagtgcgttc 120
ggagagagtg atctcttgca ttagttgtat cctcaaaata tagtggggtt caagtcatat 180
ttgacgcaaa ttagtatcgc gtgtttgtag ttatagaaca agtgataaat tgccctacgc 240
gcgataaata aatgcctgac ggcatttata atattctgtt ttaataaaac ctttctttat 300
cagtctactc gcttcgctcg tgataatact cgttgctcgc aaagctcaca actcgtatat 360
- 82 -
tacgcacgga ttgttcaaca agaaagcgat ttttattcaa caagtaaaat attttatttg 420
gtctaaacag agcatgacat tattatgtag ccaagtttgc taacacgtga gaaataatat 480
atgaagcaat ttgttggttt atacgcagta ggggaagacc aagaagcaat tctttccata 540
gcagaacaac gttcgtcatt aaaaggcgtt tatttacaaa gccttttccg tacatcgggg 600
tttattgtgt caccgatgtt ggtgatacca ttactcccaa ataacaaagg tctgtatgtt 660
ggcattattc aacaaggcca ggcgcgggaa gtgaaagttg ttccactgct ggcatctaat 720
gaagaattgt tttctcagat tcttgagccg aaagtactac aacaatgtat tggcacgatc 780
gactgtttat ttggttccaa caaagaaggc gaggcaaccc ccgcctatgt gaatcaagat 840
atttgaaatg gttagagcgc cacttttttc attttaacag ggtggcgctc cataagataa 900
aatttatatc tctcatgaga atgcctgaga gcgtggttgt aggaaccgtt gtagcgcagg 960
ttgtctacca ggtcccagat tcgcgcaaca tccttagagg 1000
- 83 -
<210> 3
<211> 1000
<212> DNA
<213> Bacteriophage KCCM 10977P
<400> 3
aattatgcaa atggccagca gggcatcaat ggcagcaaac tccgtcaggc catctggctg 60
atggttgagc acctcaaggc cggaggctcc cctgacatca tccatggcac tgtcgttggt 120
agtccgcagt cccctatggc gacagcagtc tctcggcact tcggtggcca cacaaccact 180
gtactcggcg cgaccaaacc cacgacatgt atgaaccacg acatggtggc aatgtcggcc 240
tggtttggta gtatattcaa cttcgtcggc tcgggataca acagcaccat ccagccgcgg 300
tgtaagaagt tgatcgaaca acagaatcca aaggcgtatt atctggagta tggtatcacc 360
ctcgaccata cggcccactc ccctgagcgc attgctgggt tccatatgct gggcggggag 420
caggttgcca acatccctga ccatattacc gatctaatta tccctgctgg ctcctgcaac 480
tcatgtacaa gcatcctgac cggactggca atgcatccga aaccaaatct gaagaatgtc 540
- 84 -
tatctgatcg ggattggacc aaaccgatta gatttcattg aaagtcgttt gcgcattatc 600
ggtaagcaag caaacctccc tcacataacc gatttcactc gtcgctatca cgacaaccca 660
gattatgtgt atggtaagaa ggatcttcag catgcctcta agagcgtttc gctggctggc 720
ctcctaagtg gtatcaggca gaaggacgag ccagaggtaa cgcttcctcg ctttgaggta 780
caccattggg atcttcatac cactaattgg gttcgttaca acgacctgat ggactaccag 840
tggggtgata tcgaactgca cccgcgctat gaaggcaagg tcatgacctg gatccagcag 900
cacaagccag agatgctgaa cgagaacact ctgttctgga tcgttggtag taagccatat 960
gtcgagtcga tgaaagccgc atgtccggaa ttaggtggta 1000
<210> 4
<211> 1000
<212> DNA
<213> Bacteriophage KCCM 10977P
<400> 4
gtgatgaacc acaattggtt agaagacagg aacccctgtt taccgccttt gatgttcggc 60
- 85 -
tcggcgtatt ggttcccgat ttcatcatag tacgagttga tccataccaa aacgaatttc 120
ttttcagtga ccaacggggt gataacacgc caaaaactat tgagagcgcg agcgcgggtc 180
atatcttgtg tgtctttgcc cgcgatggca tcatcaactt ctttggtaga cggcaactgg 240
ctgattgagt caatgaatac gatgatcttg tcacctttct gagcatcatt cagaagctgt 300
gtcagcttga tcttcgtctt ttcaacgttt tcaatcggca gatacaagac acggtccatg 360
tcaataccca tagatgtcca atagttttca ttcgcaccgc cttcggaatc cgcgaagata 420
caaattgcat caggaaactt atccatgtaa gccttaacat ccaccagccc aaacatggtt 480
ttgaatgtac gagaatcccc caccaactgt ttgatgcctg atatcagacc accatcaata 540
cgaccggacc aggccaaatt cagaatagga atacccgtac tgcaaataat gtcaggcttc 600
agcgcatcgg tctttgacag cacttcggca ttcgggtcca gtttctttgc tgtcttgagc 660
atgcgagcca tcaatgaatc ggccatttcg tttcctcttg cttgttgatc gtaattaata 720
aatcggtgcc caagactttc ttggaaaata tattgattgc ttcgtgaatc gccattattg 780
- 86 -
acgggagttt ttcatcgtca atttcggaac ccccgcgttc tgttaaatac atattacgca 840
gacgattgtg ctgtgcccta ttgacacaag aaacattcaa tgcgatattc aggatataca 900
tcatttgttc agttgtaaca tcctttggaa tagcatgaac ataatatatc gcatcttcaa 960
aatagatatg ctgcaatgac tctggaattt cttccccgcc 1000
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 5
ccctgctggc ttataagcaa c 21
<210> 6
- 87 -
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 6
cgtgtgccat gatcatgaaa c 21
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 7
aatgtcggca atagcgataa c 21
- 88 -
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 8
cctctaagga tgttgcgcga a 21
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 9
- 89 -
aattatgcaa atggccagca g 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 10
taccacctaa ttccggacat g 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
- 90 -
<400> 11
gtgatgaacc acaattggtt a 21
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> primer
<400> 12
ggcggggaag aaattccaga g 21