Description
Title of Invention: NOVEL BACTERIOPHAGE AND AN¬
TIBACTERIAL COMPOSITION COMPRISING THE SAME
Technical Field
[1] The present invention relates to a novel bacteriophage and an antibacterial com
position comprising the same.
[2]
Background Art
[3] Salmonella has an average genomic GC content of 50-52%, which is similar to that
of Escherichia coli and Shigella. The genus Salmonella is a pathogenic microorganism
that causes infections in livestock as well as in humans. Serological division has it that
Salmonella enterica, a species of Salmonella bacterium, has a variety of serovars
including Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium (ST),
Salmonella enteritidis (SE), Salmonella typhi, Salmonella choleraesuis (SC),
Salmonella derby (SD). Of them, choleraesuis and derby are swine-adapted pathogens,
gallinarum and pullorum are fowl-adapted pathogens, typhimurium and enteritis are
pathogenic for humans and animals, and typhi is a human-adapted pathogen, all of
which cause illness in their respective species, resulting in tremendous damage to
farmers and consumers (Zoobises Report; United Kingdom 2003).
[4] Recently, implementation of HACCP (Hazard analysis and critical control points)
has become a mandatory requirement for all slaughterhouses in Korea as of July 1,
2003, because of the high risk of contamination in the course of manufacturing
livestock products with salmonella, which causes direct damage to pigs, and meat
hygiene and is found in the digestive tract of pigs (Jae-gil Yeh. Characterization and
Counterplan of Salmonellosis in Pigs. Monthly Magazine of Pig Husbandry. 2004).
[5] Paratyphoid, the acute or chronic infectious disease in the digestive tract of pig
caused by salmonella infection, is characterized by gastroenteritis and septicaemia and
mainly occurs during the fatting period. In particular, some of pathogenic bacteria that
cause this disease can cause food poisoning in humans through meat ingestion, and
thus it is a disease having major public health importance. A variety of types of
salmonella bacteria can be pathogenic. Among them, Salmonella choleraesuis and
Salmonella typhisuis known to cause hog cholera are the major causes of acute
salmonella septicaemia. Acute enteritis occurs during the fattening period, and is ac
companied by irregular appetite, severe watery diarrhea, high fever, loss of vitality,
pneumonia, and nervous signs. Discoloration of the skin may occur in some severe
cases. Salmonella typhimurium, Salmonella enteritidis, and Salmonella derby are the
major causes of chronic enteritis.
[6] Salmonellosis is caused by oral route through feed or water contaminated with
salmonella, and thus these routes should be prevented. Contaminated feed, raw
materials or water, or adult pigs carrying the pathogen can be major sources of
infection. During the acute period of infection, pigs shed up to l 6Salmonella
choleraesuis or 10''Salmonella typhimurium per gram of feces. However, many ex
perimental infections reported successful disease reproduction with a dose of 108 to 10
Salmonella. In an experiment injecting 10 Salmonella into pigs, the injected pigs
showed no symptoms of the disease, but other pigs raised in the same pen showed
typical clinical symptoms. These results indicate that a large amount of salmonella
grow in naturally infected pigs, resulting in the infection of other pigs (Jung-Bok Lee.
Control of the Recent Outbreaks of Porcine Salmonella and Proliferative Enteropathy.
Korea Swine Association. 2009).
[7] At present, severe viral infections such as Porcine Reproductive and Respiratory
Syndrome (PRRS) and Porcine Circovirus (PCV2) have been causing tremendous
economic losses to the swine industry in Korea, and thus disease management has been
focused on these diseases. Since these bacterial diseases may cause tremendous
damage comparable to that caused by viral diseases beginning with a ban on the use of
in-feed antibiotics and an investigation of disease occurrence or disease management
should be performed in advance (Jung-Bok Lee. Control of the Recent Outbreaks of
Porcine Salmonella and Proliferative Enteropathy, Infectious Disease Laboratory,
College of Veterinary Medicine, Konkuk University, Livestock Product Safety, 2010)
(Robert W. Wills, Veterinary Microbiology, 1999). Meanwhile, bacteriophage, also
called phage, is a specialized type of virus that infects only particular bacteria and
controls the growth of bacteria, and can self-replicate only inside the host bacteria.
After the discovery of bacteriophages, a great deal of faith was initially placed in their
use for infectious-disease therapy. However, when broad spectrum antibiotics came
into common use, bacteriophages were seen as unnecessary due to a specific target
spectrum. Antibiotics or antimicrobial agents have been widely used for the treatment
of infectious diseases caused by bacterial infection. Nevertheless, the misuse and
overuse of antibiotics resulted in rising concerns about antibiotic resistance and the
harmful effects of residual antibiotics in foods. However, the removal of current infeed
antibiotics might increase occurrence of bacterial diseases including salmonellosis
that have been controlled by antibiotics, as expected in the experiment data or from
other countries. Thus, there is an urgent need to establish a detailed practical guideline
for salmonella management (Jung-Bok Lee. Control of the Recent Outbreaks of
Porcine Salmonella and Proliferative Enteropathy, Infectious Disease Laboratory,
College of Veterinary Medicine, Konkuk University, Livestock Product Safety. 2010).
[8] These growing concerns have led to a resurgence of interest in bacteriophage. Seven
bacteriophages for control of E.coli 0157:H7 are disclosed in U.S. Pat. No. 6,485,902
(2002) and two bacteriophages for control of various microorganisms are disclosed in
U.S. Pat. No. 6,942,858 (issued to 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 Listerix-PlOO, which is the first bacteriophage product
approved by the USFDA. 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 0157 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
feed preservative against Clostridium contamination of feed in 2003 and 2005, re
spectively. Such studies show that research into bacteriophages for the control of an
tibiotic-unsusceptible bacteria and contamination of livestock products by zoonotic
pathogens is presently ongoing.
[9] 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 Salmonella exhibits multiple
drug resistances, nationwide antimicrobial resistance surveillance has been conducted
in Korea under the 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.
[10]
Disclosure of Invention
Technical Problem
[11] In order to overcome problems occurring upon the misuse or overuse of broad
spectrum antibiotics, such as drug resistant bacteria and drug residues in foods, the
present inventors isolated a bacteriophage from natural sources, in which the bacte
riophage has a specific bactericidal activity against salmonella causing major diseases
in livestock. As a result, they found that the bacteriophage has a specific bactericidal
activity against Salmonella choleraesuis (SC), Salmonella typhimurium (ST),
Salmonella derby (SD), Salmonella infantis (SI) and Salmonella newport (SN) with no
influences on beneficial bacteria, in addition to showing excellent acid- and heatresistance,
as identified for the morphological, biochemical and genetic properties
thereof. Further, they found that the bacteriophage can be applied to compositions for
the prevention or treatment of Salmonella typhimurium-medi&ted diseases, such as
livestock salmonellosis and Salmonella food poisoning, and to various products for the
effective prevention and control of Salmonella bacteria proliferation, including
livestock feed additives, drinking water for livestock, barn sanitizers, and cleaners for
meat products, thereby completing the present invention.
Solution to Problem
An object of the present invention is to provide a novel bacteriophage having a b ac
tericidal activity against Salmonella choleraesuis.
Another object of the present invention is to provide a composition for the prevention
or treatment of infectious diseases caused by Salmonella choleraesuis, Salmonella typhimurium,
Salmonella derby, Salmonella injantis or Salmonella newport, comprising
the bacteriophage as an active ingredient.
Still another object of the present invention is to provide an antibiotic, comprising the
bacteriophage as an active ingredient.
Still another object of the present invention is to provide an animal feed or drinking
water, comprising the bacteriophage as an active ingredient.
Still another object of the present invention is to provide a sanitizer or cleaner,
comprising the bacteriophage as an active ingredient.
Still another object of the present invention is to provide a method for preventing or
treating of infectious diseases caused by Salmonella choleraesuis, Salmonella typhimurium,
Salmonella derby, Salmonella injantis or Salmonella newport, using the
bacteriophage or the composition.
Advantageous Effects of Invention
The novel bacteriophage of the present invention has a specific bactericidal activity
against Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby,
Salmonella injantis or Salmonella newport with no influences on beneficial bacteria,
and excellent acid- and heat-resistance and desiccation tolerance. Therefore, the novel
bacteriophage can be used for the prevention or treatment of salmonellosis or
salmonella food poisoning, which is an infectious disease caused by Salmonella
choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella injantis or
Salmonella newport, and also widely used in animal feeds, drinking water for
livestock, sanitizers, and cleaners.
Brief Description of Drawings
[22] FIG. 1 is an electron microscopy photograph of OCJl 1, which belongs to the family
Siphoviridae of morphotype Bl, characterized by an isometric capsid and a long noncontractile
tail;
[23] FIG. 2 is a photograph showing the formation of OCJl 1 plaques in a lawn of
salmonella bacteria, in which (A,B;SC, C;SG, D;ST, E;SI, F,G;SD, H;SN), plaque
formation was observed in lawns of SC, ST, SI, SD and SN, but not in lawns of SG;
[24] FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage OCJl 1, in which the
major proteins were detected at 33 kDa, 55 kDa and 69.5 kDa, and Precision plus
protein standard (BIO-RAD) was used as a marker;
[25] FIG. 4 is the result of PFGE of the isolated bacteriophage OCJl 1, in which a total
genome size of 1 was approximately 140 kbp, and the CHEF DNA Size Standard
Lambda Ladder (Bio-Rad) was used as a DNA size marker;
[26] FIG. 5 is the result of PCR, performed using each primer set for the OCJl 1 genomic
DNA, in which A; a primer set of SEQ ID NOs. 5 and 6, B; a primer set of SEQ ID
NOs. 7 and 8, C; a primer set of SEQ ID NOs. 9 and 10, and D; a primer set of SEQ ID
NOs. 11 and 12, and all of A, B, C and D lanes had PCR products of approximately 1
kbp or more to 2 kbp or less;
[27] FIG. 6 is the result of acid-resistance assay on the bacteriophage OCJl 1, 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, 9.8 and 11.0, in which the bacteriophage OCJl 1 did not lose its activity until pH
5.5, but the bacteriophage 1 showed reduced activity at pH 4 and pH 3.5, and
completely lost its activity at pH 3.0 or lower, as compared to a control;
[28] FIG. 7 is the result of heat-resistance assay on the bacteriophage OCJl 1, showing the
number of surviving bacteriophage at 37, 45, 53, 60, and 70°C for 0, 10, 30, 60 and
120 minutes, in which the bacteriophage OCJl 1 maintained its activity even though
exposed to 60°C for up to 2 hours;
[29] FIG. 8 is the result of desiccation tolerance assay on the bacteriophage OCJl 1 dried
with the aid of a SpeedVac concentrator, in which when titer changes under the dry
condition were measured in comparison with pre-drying titers, the activity was
maintained at 60°C for up to 1 hour; and
[30] FIG. 9 is the results of body weight changes due to toxicity after single oral admin
istration of Sprague-Dawley rats with , in which observation of body weight
changes before and 1, 3, 7, 10 and 14 days after administration with OCJl 1 showed no
significant changes in comparison with the control group
[31]
Best Mode for Carrying out the Invention
[32] In one aspect to achieve the above objects, the present invention provides a novel
bacteriophage having a specific bactericidal activity against Salmonella choleraesuis,
Salmonella typhimurium, Salmonella derby, Salmonella injantis or Salmonella newport
[33]
[34] The present inventors collected fecal and sewage samples from swinery, and isolated
therefrom bacteriophages that can lyse the host cell SC. They were also found that
these bacteriophages can lyse ST, SD, SI and SN (FIG. 2 and Table 1). A mor
phological examination under an electron microscope confirmed that the bacteriophage
(OCJll) belongs to the family Siphoviridae of morphotype Bl (FIG. 1). Further, the
bacteriophage OCJl 1 was found to have major structural proteins of approximately
69.5 kDa, 55 kDa and 33 kDa, as measured by a protein pattern analysis (FIG. 3), and
a genome analysis showed that OCJl 1 has a total genome size of approximately
97-145.5 kbp (FIG. 4). Furthermore, 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 (Example 6). Based on SEQ ID NOs. 1 to 4,
genetic similarity with other species was compared. It was found that the bacte
riophage showed very low genetic similarity with the known bacteriophages, in
dicating that the bacteriophage is a novel bacteriophage (Table 2). For more detail
analysis of genetic features, the 1 1-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 1.4 kbp, 1.2
kbp, 1.25 kbp and 1.5 kbp (FIG. 5).
[35] Meanwhile, when SC, ST, SD, SI and SN were infected with OCJl 1, the phage
plaques (clear zone on soft agar created by host cell lysis of one bacteriophage) were
observed (FIG. 2). The stability of 1 was examined under various temperature
and pH conditions, resulting in that OCJl 1 stably maintains in a wide range of pH en
vironments from pH 3.5 to pH 11.0 (FIG. 6) and in high temperature environments
from 37°C to 70°C (FIG. 7), and even after desiccation (FIG. 8). Also, the wild-type
strains SC, ST, SD, SI and SN were also found to fall within the host cell range of
OCJll (Table 3).
[36] Finally, the results of dermal and ocular irritation tests on 1 in specificpathogen-
free (SPF) New Zealand White rabbits showed that the primary irritation
index (PII) was 0.33, indicating no irritant, and the index of acute ocular irritation
(IAOI) was 0 in washing and non-washing groups during the whole experimental
periods, indicating no irritant. The results of oral administration of OCJl 1 showed no
changes in weight gain (FIG. 9). As well, mortality, general symptoms (Table 4) and
organ abnormality (Table 5) were not observed, indicating no toxicity.
[37] Accordingly, the present inventors designated the bacteriophage as "Bacteriophage
CJ ", in which the bacteriophage was isolated from fecal and sewage samples from
swinery and has a specific bactericidal activity against SC, ST, SD, SI and SN and the
above characteristics, and deposited at the Korean Culture Center of Microorganisms
(361-221, Honje 1, Seodaemun, Seoul) on Sep. 9, 2011 under accession number
KCCM11208P.
[38]
[39] In another aspect to achieve the above objects, the present invention provides a com
position for the prevention or treatment of infectious disease caused by one or more
Salmonella bacteria selected from the group consisting of Salmonella choleraesuis,
Salmonella typhimurium, Salmonella derby, Salmonella injantis and Salmonella
newport, comprising the bacteriophage as an active ingredient.
[40]
[41] Having specific bactericidal activity against Salmonella choleraesuis, Salmonella ty
phimurium, Salmonella derby, Salmonella injantis and Salmonella newport, the bacte
riophage of the present invention may be used for the purpose of preventing or treating
the diseases caused by these bacteria. Preferably, examples of the infectious diseases
include porcine salmonellosis and Salmonella food poisoning caused by Salmonella
choleraesuis or Salmonella typhimurium, and acute or chronic porcine enteritis caused
by Salmonella derby, Salmonella injantis, Salmonella newport, but are not limited
thereto.
[42] As used herein, the term "salmonellosis" refers to symptoms following salmonella
infection, such as fever, headache, diarrhea, and vomiting. That is, salmonellosis is an
infection with bacteria of the genus Salmonella, which is defined with two clinical
forms: an acute septicemic form that resembles typhoid fever and an acute gas
troenteritis, including enteritis, food poisoning, and acute septicemia.
[43] 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.
[44] As used herein, the term "treatment" means all of the actions in which the condition
has taken a turn for the better or been restrained or modified favorably by the admin
istration of the composition.
[45] The composition of the present invention includes OCJl 1 in an amount of lxlO 2 to
lxlO 2 PFU/mL, and preferably in an amount of lxlO 6 to lxlO 10 PFU/mL.
[46] On the other hand, the composition of the present invention may further include a
pharmaceutically acceptable carrier.
[47] 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, ethanol, and mixtures of one or more thereof. If
necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic
agents may be added. 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 pills,
capsules, granules, or tablets.
[48] 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, sub
cutaneous or topical administration.
[49] 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.
[50] Examples of the oral dosage forms including the composition of the present
invention as an active ingredient 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, the following are useful: a binder
such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or
gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or
sweet potato starch; and 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 above-mentioned compounds.
[51] The parenteral dosage forms including the composition of the present invention as an
active ingredient may be formulated into injections via subcutaneous, intravenous, or
intramuscular routes, suppositories, or sprays inhalable via the respiratory tract, such as
aerosols. Injection forms may be prepared by dissolving or suspending the composition
of the present invention, together with a stabilizer or a buffer, in water and loading the
solution or suspension onto ampules or vial unit forms. For sprays, such as aerosols, a
propellant for spraying a water-dispersed concentrate or wetting powder may be used
in combination with an additive.
[52]
[53] In still another aspect to achieve the above objects, the present invention provides an
antibiotic comprising the bacteriophage as an active ingredient.
[54]
[55] 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 an
tibacterial agents. The animals are mammals including human. The bacteriophage 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 drug resistance so that it can be provided as a novel antibiotic with
a comparatively long life cycle.
[56]
[57] In still another aspect to achieve the above objects, the present invention provides an
animal feed or drinking water comprising the bacteriophage as an active ingredient.
[58]
[59] In-feed antibiotics used in the livestock and fishery industries are intended to prevent
infections. However, most of the currently available in-feed antibiotics are problematic
in that they are apt to induce the occurrence of resistant strains and may be transferred
to humans, due to remaining in livestock products. The uptake of such residual an
tibiotics may make human pathogens resistant to antibiotics, resulting in the spread of
diseases. In addition, since there are a variety of in-feed antibiotics, the increasing
global emergence of multidrug-resistant strain is a serious concern. Therefore, the bac
teriophage of the present invention can be used as an in-feed antibiotic that is more
eco-friendly and able to solve the above problems.
[60] The animal feed of the present invention may be prepared by adding the bacte
riophage directly or in separate feed additive form to an animal feed. The bacte
riophage of the present invention may be contained in the animal feed as a liquid or in
a solid 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
long-term preservation, in addition to the bacteriophage of the present invention.
[61] The feed additive of the present invention may additionally include other non
pathogenic microorganisms. The available additional microorganism may be selected
from the group consisting of Bacillus subtilis that can produce protease, lipase and
invertase, Lactobacillus sp. strain that can exert physiological activity and a function of
decomposing under anaerobic conditions, such as in the stomach of cattle, filamentous
fungi including Aspergillus oryzae (J Animal Sci 43:910-926, 1976) that increases the
weight of domestic animals, enhances milk production and helps the digestion and absorptiveness
of feeds, and yeast including Saccharomyce scerevisiae (J Anim
Sci56:735-739, 1983).
[62] The feed including OCJl 1 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, inorganic matter, fat,
mineral, fat, single cell protein, zooplankton, and food waste, but is not limited thereto.
[63] The feed additive including OCJl 1 of the present invention may include binders,
emulsifiers, and preservatives for the prevention of quality deterioration, amino acids,
vitamins, enzymes, probiotics, flavorings, non-protein nitrogen, silicates, buffering
agents, coloring agents, extracts, and oligosaccharides for efficiency improvement, and
other feed premixtures, but is not limited thereto.
[64] 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.
[65]
[66] In still another aspect to achieve the above objects, the present invention provides a
sanitizer or cleaner comprising the bacteriophage as an active ingredient.
[67]
[68] In still another aspect to achieve the above objects, the present invention provides a
method for treating infectious diseases caused by Salmonella choleraesuis, Salmonella
typhimurium, Salmonella derby, Salmonella injantis or Salmonella newport using the
bacteriophage or the composition.
[69]
[70] In detail, the therapeutic method of the present invention comprises the step of ad
ministering a pharmaceutically effective amount of the bacteriophage or the com
position to an individual having infectious diseases caused by Salmonella choleraesuis,
Salmonella typhimurium, Salmonella derby, Salmonella injantis or Salmonella newport
[71] The bacteriophage or the composition of the present invention may be administered
in the form of a pharmaceutical formulation into animals or may be ingested as a
mixture with animal feed or drinking water by animals and preferably as a mixture
with animal feed.
[72] As long as it reaches target tissues, any route, whether oral or parenteral, may be
taken for administering the bacteriophage or the composition of the present invention.
In detail, the composition of the present invention may be administered in a typical
manner via any route such as oral, rectal, topical, intravenous, intraperitoneal, intra
muscular, intraarterial, transdermal, intranasal, and inhalation routes.
[73] It will be obvious to those skilled in the art that the total daily dose of the bacte
riophage or the composition of the present invention to be administered by the
therapeutic method should be determined through appropriate medical judgment by a
physician. Preferably, the therapeutically effective amount for given patients may vary
depending on various factors well known in the medical art, including the kind 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.
[74]
Mode for the Invention
[75] Hereinafter, the present invention will be described in more detail with reference to
Examples. However, these Examples are for illustrative purposes only, and the
invention is not intended to be limited by these Examples.
[76]
[77] Example 1: Salmonella Bacteriophage Isolation
[78]
[79] Example 1-1: Bacteriophage Screening and Single Bacteriophage Isolation
[80] 50 mL of sample from swinery 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 ΐ of Salmonella
choleraesuis ("SC") shaking culture medium (OD oo=2) and 2 mL of lOx Luria-Bertani
medium (tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L: LB medium). 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 ΐ of SC shaking culture medium (OD oo=2) were mixed, and
plated onto LB plate ("top-agar"), and allowed to solidify. 10 ΐ of the culture filtrate
was spread thereon, and cultured for 18 hours at 37°C, and the titration of phage lysate
was performed on the top-agar, called soft agar overlay method.
[81] The sample culture medium containing the phage lysate was properly diluted, and
mixed with 150 ΐ of SC shaking culture medium (OD600=2), followed by soft agar
overlay method, so that single plaques were obtained. A single plaque represents one
bacteriophage and thus, for isolation of single bacteriophages, one phage plaque was
added to 400 ΐ of SM solution (NaCl, 5.8 g/L, MgS0 47H20 , 2 g/L, 1M Tris-Cl (pH
7.5) 50 mL/L), and left for 4 hours at room temperature to isolate single bacteriophages.
To purify the bacteriophage in large quantities, 100 ΐ of supernatant was
taken from the single bacteriophage solution, and mixed with 12 mL of 0.7% agar and
500 ΐ of SC shaking culture medium, followed by soft agar overlay method on LB
plate having a diameter of 150 mm. 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 b ac
teriophages was recovered, chloroform was added to a final volume of 1%, and mixed
well for 10 minutes. The solution was centrifuged at 4000 rpm for 10 minutes. The
obtained supernatant was filtered using a 0.45 filter, and stored in the refrigerator.
[82]
[83] Example 1-2: Large-Scale Batches of Bacteriophage
[84] The selected bacteriophages were cultured in large quantities using SC. SC was
shaking-cultured, and an aliquot of 1.5xl0 10 cfu (colony forming units) was cen
trifuged at 4000 rpm for 10 minutes, and the pellet was resuspended in 4 ml of SM
solution. The bacteriophage of 9.0xl0 8 PFU (plaque forming unit) was inoculated
thereto (MOI: multiplicity of infection=0.001), 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
for 20 minutes. DNase I and RNase A were added to a final concentration of 1 g/ml,
respectively. The solution was left at 37°C for 30 minutes. NaCl and PEG
(polyethylene glycol) were added to a final concentration of 1M and 10% (w/v), re
spectively and left at 4°C for an additional 3 hours. The solution was centrifuged at
4°C and 12,000 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
the bacteriophage was purified by glycerol density gradient ultracentrifugation
(density: 40%, 5% glycerol at 35,000 rpm and 4°C for 1 hour). The purified bacte
riophage was designated as "Bacteriophage CJ ", and resuspended in 300 ΐ of SM
solution, followed by titration. The bacteriophage OCJl 1 was deposited at the Korean
Culture Center of Microorganisms (361-221, Honje 1, Seodaemun, Seoul) on Sep. 9,
201 1 under accession number KCCM1 1208P.
[85]
[86] Example 2 : Examination on C.Tl l Infection of Salmonella
[87] To analyze the selected bacteriophage for lytic activity on Salmonella species other
than SC, attempts were made of cross infection with other Salmonella species. As a
result, OCJll infected SC (Salmonella choleraesuis), ST (Salmonella typhimurium),
SD (Salmonella derby), SN (Salmonella newport), SI (Salmonella infantis), SA (
Salmonella arizonae) and SB (Salmonella bongori), but did not infect SE (Salmonella
enteritidis), SG (Salmonella gallinarum), and SP (Salmonella pullorum) (Table 1 and
FIG. 2).
[88]
[89] Table 1
[90]
[91]
[92]
[93] Moreover, FIG. 2 is a photograph showing the formation of OCJl 1 plaques in a lawn
of salmonella bacteria. As shown in FIG. 2 (A,B;SC, C;SG, D;ST, E;SI, F,G;SD,
H;SN), plaque formation was observed in lawns of SC, ST, SI, SD and SN, but not in
lawns of SG.
[94]
[95] Example 3 : Morphology of CJ11
[96] The purified OCJl 1 was diluted in the SM buffer solution, and then mounted on a
copper grid, stained with 2% uranyl acetate for 3 to 5 seconds, and dried. Examination
under a transmission electron microscope (LIBRA 120, Carl Zeiss transmission
electron Microscope, 80 kV, magnification of xl20,000~x200,000) was performed
(FIG. 1). FIG. 1 is an electron microscopy photograph of OCJll. As shown in FIG. 1,
it was found that the purified 1 belongs to the family Siphoviridae of morphotype
Bl, characterized by an isometric capsid and a long non-contractile tail.
[97]
[98] Example 4 : Protein Pattern Analysis of CJ11
[99] 15 of a OCJl 1 solution purified at a titer of 10 PFU/mL was mixed with 3 of
a 5xSDS sample solution, and heated for 5 minutes. 12% SDS-PAGE was performed,
and then the gel was stained with Coomassie blue for 1 hour at room temperature (FIG.
3). FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage OCJl 1, in which
Precision plus protein standard (BIO-RAD) was used as a marker. As shown in FIG. 3,
the major proteins were detected at 33 kDa, 55 kDa and 69.5 kDa.
[100]
[101] Example 5 : Analysis of Total Genomic DNA Size of 
[102] Genomic DNA of the purified 1 was isolated using ultracentrifugation. In
detail, to the purified OCJl 1 culture medium were added EDTA
(ethylenediaminetetraacetic acid (pH 8.0)), proteinase K, and SDS (sodium dodecyl
sulfate) at a final concentration of 20 mM, 50 ug/mL, and 0.5% (w/v), respectively,
followed by incubation at 50°C for 1 hour. An equal volume of phenol (pH 8.0) was
added and mixed well. After centrifugation at room temperature and 12,000 rpm for 10
minutes, the supernatant was mixed well with an equal volume of PC
(phenol:chloroform = 1:1). Another centrifugation was performed at room temperature
and 12,000 rpm for 10 minutes. Then, a supernatant was obtained, and mixed with an
equal volume of chloroform, followed by centrifugation at room temperature and
12,000 rpm for 10 minutes. The obtained supernatant was mixed with 1/10 volume of
3 M sodium acetate and two volumes of cold 95% ethanol, and left at -20°C for 1 hour.
After centrifugation at 0°C and 12,000 rpm for 10 minutes, the supernatant was
completely removed, and the DNA pellet was dissolved in 50 of TE (Tris-EDTA
(pH 8.0)). The extracted DNA was diluted 10-fold, and measured for absorbance at OD
260 to determine its concentration. 1 g of the total genomic DNA was loaded onto 1%
PFGE (pulse-field gel electrophoresis) agarose gel, and electrophoresed at 14°C for 22
hours in a BIORAD CHEF DR II PFGE system under the conditions of switch time
ramp for 50-90 seconds, 6 V/cm (200V). The CHEF DNA Size Standard Lambda
Ladder (Bio-Rad) was used as a DNA size marker (FIG. 4). FIG. 4 is the result of
PFGE of the isolated bacteriophage 1. As shown in FIG. 4, DNA of ap
proximately 140 kbp present between 48.5 to 1,000 kbp was observed.
[103]
[104] Example 6 : Genetic Analysis of C.Tl l
[105] For the genetic analysis of the purified , 5 g of the genomic DNA of OCJl 1
was double digested with the restriction enzymes Pstl, Xbal and BamHI, EcoRI and
Sail. The vector pCL1920 (Promega) was digested with the restriction enzymes Pstl,
Xbal and BamHI, EcoRI and Sail, and then treated with CIP (calf intestinal alkaline
phosphatase). The digested genomic DNA was mixed at a ratio of 3:1 with the vector,
and ligated at 16°C for 2 hours. The resulting recombinant vector was transformed into
E. coli DH5a which was then plated on an LB plate containing specinomycin and Xgal
(5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) for selection of blue/white
colonies. The selected colonies were cultured for 16 hours in a culture medium
containing the antibiotic with shaking. Then, plasmids were extracted using a Plasmid
purification kit (Promega).
[106]
[107] The cloning of the plasmids was confirmed by PCR using primer sets of FTR135 and
FTR136 (SEQ ID NOs. 13 and 14) and selection was made only of insert fragments
having a size of 1 kb or longer. Their nucleotide sequences were analyzed using the
primer sets. The nucleotide sequences thus obtained were given in SEQ ID NOs. 1 to
4, respectively, each having a size of 1 to 2 kbp or less, and analyzed for sequence
similarity with the aid of NCBI blastx and blastn program, and the results are
summarized in Table 2, below.
[108]
[109] Table 2

[HO]
[111] Example 7 : Design of OC.Tll-Specific Primer Sequences
[112] In order to identify OCJl 1, OCJl 1-specific primers were designed on the basis of
SEQ ID NOS. 1 to 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. 1 1 and 12. 0.1
g of the genomic DNA of bacteriophage and 0.5 pmol of each primer were added to a
pre-mix (Bioneer), and the final volume was adjusted to 20 . PCR was performed
with 30 cycles of denaturation; 94°C 30 seconds, annealing; 55°C 30 seconds, and
polymerization; 72°C, 1.5 minutes (FIG. 5). FIG. 5 is the result of PCR, performed
using each primer set for the OCJl 1 genomic DNA. A; a primer set of SEQ ID NOs. 5
and 6, B; a primer set of SEQ ID NOs. 7 and 8, C; a primer set of SEQ ID NOs. 9 and
10, and D; a primer set of SEQ ID NOs. 11 and 12. All of A, B, C and D lanes had
PCR products of approximately 1 to 2 kbp. As shown in FIG. 5, the PCR products thus
obtained had a size of approximately 1 kbp or more to 2 kbp or less, with the primer
sets of SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ
ID NOs. 1 1 and 12.
[113]
[114] Example 8 : pH Stability of Bacteriophage
[115] In order to determine whether OCJl 1 survives with stability under the low pH en
vironment in the stomach of pig, OCJl 1 was assayed for stability 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, 9.8 and 11.0). Various pH
solutions (sodium acetate buffer (pH 2.1, pH 4.0, pH 5.5, and pH 6.4), sodium citrate
buffer (pH 2.5, pH 3.0, and pH 3.5), sodium phosphate buffer (pH 6.9 and pH 7.4) and
Tris-HCl (pH 8.2, pH 9.0, pH 9.8 and pH 11.0)) were prepared to have a concentration
of 0.2 M. 180 of each pH solution was mixed with 20 of a bacteriophage
solution (l.OxlO 11 PFU/mL) to give each pH solution a concentration of 1M, followed
by incubation at room temperature for 2 hours. The reaction solution was serially
diluted, and 10 of each dilution was cultured at 37°C for 18 hours by a soft agar
overlay method to determine the titers of the phage lysates (FIG. 6). FIG. 6 is the result
of acid-resistance assay on the bacteriophage OCJl 1, 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, 9.8 and 11.0. The
bacteriophage OCJl 1 did not lose its activity until pH 5.5. However, the bacteriophage
1 showed reduced activity at pH 4 and pH 3.5, and completely lost its activity at
pH 3.0 or lower, as compared to a control. As shown in FIG. 6, the bacteriophage did
not lose its activity and remained stable down to pH 5.5 whereas it lost its activity at
pH 3.0 or lower.
[116]
[117] Example 9 : Heat Stability of Bacteriophage
[118] For use as a feed additive, the bacteriophage was assayed for stability to the heat
generated during a formulation process. In this regard, 200 of a OCJl 1 solution
with a titer of l.OxlO 11 PFU/mL was incubated at 37°C, 45°C, 53°C, 60°C, and 70°C
for 0 minute, 10 minutes, 30 minutes, 60 minutes and 120 minutes. The solution was
serially diluted, and 10 of each dilution was cultured at 37°C for 18 hours by a soft
agar overlay method to determine the titers of phage lysates (FIG. 7). FIG. 7 is the
result of heat-resistance assay on the bacteriophage OCJl 1, showing the number of
surviving bacteriophage at 37°C, 45°C, 53°C, 60°C, and 70°C for 0, 10, 30, 60 and
120 minutes. As shown in FIG. 7, the bacteriophage OCJl 1 maintained its activity
even though exposed at 60°C up to 2 hours.
[119]
[120] Example 10: Desiccation Tolerance of Bacteriophage
[121] For use as a feed additive, the bacteriophage OCJl 1 was assayed for tolerance to the
dry condition set for a formulation process. On the basis of the results obtained from
the heat stability assay, a desiccation assay was performed using a SpeedVac con
centrator. 200 ΐ of a OCJl 1 solution having a titer of l.OxlO PFU/mL was dried
under vacuum at 60°C for 2 hours, and the pellet thus obtained was completely resuspended
in 200 ΐ of the SM solution at 4°C for one day, and measured for titer
values (FIG. 8). FIG. 8 is the result of desiccation tolerance assay on the bacteriophage
CJ11 dried with the aid of a SpeedVac concentrator. As shown in FIG. 8, when titer
changes under the dry condition were measured in comparison with pre-drying titers,
the activity was maintained at 60°C up to 1 hour.
[122]
[123] Example 11: Infection Spectrum of Bacteriophage
[124] OCJl 1 was assayed for lytic activity against the wild-type (2 strains), Salmonella
choleraesuis (5 strains), Salmonella typhimurium (17 strains), Salmonella infantis (4
strains), Salmonella newport (6 strains), Salmonella derby (2 strains) and Salmonella
dublin (3 strains), obtained from Laboratory of Avian Diseases, College of Veterinary
Medicine, Seoul National University, in addition to SC (ATCC SC10708) used in the
experiment. 150 of each strain shaking culture medium (OD600=2) was mixed, and
10 of OCJl 1 solution having a titer of 1010 PFU/mL was cultured at 37°C for 18
hours using a soft agar overlay method to monitor the formation of plaques (Table 3).
Formation of phage plaque was observed in 8 strains of SC.
[125]
[126] Table 3
[127]
[128]
[129]
[130]
[131] Example 12: Toxicity Assay of Bacteriophage
[132] Dermal and ocular irritation tests were performed in specific-pathogen-free (SPF)
New Zealand white rabbits, which are commonly used in the toxicity test of bacte
riophage OCJl 1 for the prevention of salmonellosis and salmonella food poisoning,
and of which experimental data were accumulated to allow easy analysis of experiment
results. The normal abdominal skin (non-injured skin) and injured abdominal skin of
rabbits were covered and contacted with 2.5 cm x 2.5 cm of gauze applied with the test
substance, and each 0.5 mL/site was applied. No changes in general symptoms were
observed, and a slight weight loss was observed 1 day after application of the test
substance, which can likely to be attributed to stress due to occlusive application of the
test substance. In the dermal irritation test, the primary irritation index (PII) was 0.33,
indicating no irritant. For the ocular irritation test, the left eye of a rabbit was applied
with the test substance, and then compared to the right eye, which was not applied with
the test substance. During the experimental period, general symptoms and abnormal
changes in body weight related to application of the test substance were not observed.
After application of the test substance, the eye examination showed that the index of
acute ocular irritation (IAOI) was "0", indicating no irritant. Therefore, these results
indicate that the novel bacteriophage OCJl 1 has no toxicity.
[133] Further, toxicity assay was performed by single oral administration of Sprague-
Dawley rats with OCJH. A test substance-administered group treated with 1 x 101 1
PFU/kg of OCJl 1 and an excipient control group treated with a vehicle [20 mM Tris-
HC1 (pH 7.0) + 2mM MgCl2] as an excipient were prepared, and 10 rats of each group
(5 each of female and male sexes) were orally administered with a single dosage.
Mortality, general symptoms, changes in body weight, and autopsy findings were
monitored for 2 weeks and compared to each other. Monitoring was conducted every 6
hours, starting from 30 minutes to 1 hour after administration on the day of admin
istration. Then, general symptoms were monitored once a day for 14 days, and
recorded thereof (Tables 4 and 5).
[134]
[135] Table 4
[136]
[137] Table 5
[138]
[139]
[140]
[141] As shown in Tables 4 and 5, none of them died, and neither toxic symptoms nor no
ticeable clinical symptoms were generated by OCJl 1. The results are summarized in
Tables 4 and 5. Body weights were recorded before administration and 1, 3, 7, 10 and
14 days after administration. No significant changes were observed in body weight
compared to the control group.
[142]
[143] Meanwhile, the results of body weight changes indicate that OCJl 1 does not cause a
toxic reaction sufficient to reduce appetite or to change body weight. These results are
shown in FIG. 9. FIG. 9 is the results of body weight changes due to toxicity after
single oral administration of Sprague-Dawley rats with OCJl 1. As shown in FIG. 9,
observation of body weight changes before administration and 1, 3, 7, 10 and 14 days
after administration with OCJl 1 showed that no significant changes in body weight
were found in comparison with the control group
[144] Therefore, it was found that ADL of the novel bacteriophage OCJl 1 exceeds 1X 10
PFU/kg in both female and male rats, and thus it is non-toxic.
[145]

Claims
[Claim 1] An isolated bacteriophage having a specific bactericidal activity against
Salmonella choleraesuis, which is identified by accession number
KCCM11208P.
[Claim 2] A composition for the prevention or treatment of infectious diseases
caused by Salmonella bacteria selected from the group consisting of
Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby,
Salmonella infantis, Salmonella newport and combinations thereof,
comprising the bacteriophage of claim 1 as an active ingredient.
[Claim 3] The composition according to claim 2, wherein the infectious disease
caused by Salmonella choleraesuis or Salmonella typhimurium is
salmonellosis or Salmonella food poisoning, and the infectious disease
caused by Salmonella derby, Salmonella infantis and Salmonella
newport is bacterial infection-type Salmonella food poisoning.
[Claim 4] An antibiotic, comprising the bacteriophage of claim 1 as an active in
gredient.
[Claim 5] An animal feed or drinking water, comprising the bacteriophage of
claim 1 as an active ingredient.
[Claim 6] A sanitizer or cleaner, comprising the bacteriophage of claim 1 as an
active ingredient.
[Claim 7] A method for preventing or treating infectious diseases caused by one
or more Salmonella bacteria selected from the group consisting of
Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby,
Salmonella infantis, Salmonella newport and combinations thereof,
comprising administering the bacteriophage of claim 1 to animals in
need thereof.
[Claim 8] A method for preventing or treating infectious diseases caused by one
or more Salmonella bacteria selected from the group consisting of
Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby,
Salmonella infantis, Salmonella newport and combinations thereof,
comprising administering the composition of claim 2 to animals in need
thereof.