Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Virus or bacteriophage
Reexamination Certificate
1999-02-26
2001-11-27
Wortman, Donna C. (Department: 1645)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Virus or bacteriophage
C424S543000, C435S235100
Reexamination Certificate
active
06322783
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel bacteriophage, a method for screening the same as well as a novel bactericidal composition using the same and a detection kit using the same. More particularly, the present invention relates to a novel bacteriophage having a high specificity against a variety of pathogenic bacteria, including the pathogenic
Escherichia coli,
a method for screening such pathogenic bacteria as well as a novel bactericidal composition using such a bacteriophage singly or in combination of two or more different kinds of bacteriophages as a cocktail, a stabilizer for storing the same in a stable manner, and a reagent and a detection kit for detecting the pathogenic bacteria by the bacteriophage.
BACKGROUND TECHNOLOGY
A variety of bacteria are distributed widely in nature, and many out of such bacteria are playing a significant role in natural phenomena including, e.g., decomposition, fermentation, etc. On the other hand, such bacteria also include so-called pathogenic bacteria with which animals including, for example, human beings, pets such as dogs, cats, etc. and plants are infected and which cause diseases and blights. Many bacteria may also invade the tissues of animals and plants and propagate therein, thereby causing diseases and blights.
Escherichia coli
is one of such pathogenic bacteria, and exists everywhere in the nature.
E. coli
is always present in the intestines of human beings and other mammals. Under regular circumstances,
E. coli
is not hazardous to human beings and other mammals. However, among the various strains of
E. coli,
pathogenic bacteria such as intestinal hemolytic
E. coli
may cause serious infection under some conditions in human beings and other mammals.
In order to prevent infection from such pathogenic bacteria such as, e.g., pathogenic
E. coli
or to treat the infection from such bacteria, a number of bactericides have been used. The bactericides may include iodo tincture solution, merculon chlorhexizine and the like. These have been extensively employed for the disinfection of wounds. Further, preservatives such as, e.g., benzoic acid are employed for food. Moreover, a variety of effective antibiotics have been employed for the prevention and treatment of infection from bacteria.
When there is a risk of infection from a pathogenic
E. coli,
such as intestinal hemolytic
E. coli,
it occurs through materials such as raw ox liver or vegetables such as radish shoots, lettuce, etc. In cases where infection from pathogenic bacterium, i.e. intestinal hemolytic
E. coli,
has recently been occurred, it is also suspected that the infection has been caused via sashimi (raw fish slices) or rare steak, etc. It will be noted that iodo tincture solution or merculon could not be employed as bactericides for controlling infection through food such as fresh food, etc., due to their toxicity. Further, it is also impossible to use antibiotics in the prevention or treatment of bacterial infections of food.
Even if a bactericide existed which were safe for use in food, it would be very difficult to spray the food with it uniformly. If the bactericide were sprayed irregularly on the food, there would be a difference in the concentration of the bactericide, and the bactericidal effects would be localized, leaving areas where bacteria would still be alive, thereby resulting in serious problems with infection.
Substances such as, e.g. polylysine, etc., are known to control the multiplication of bacteria. They have bacteriostatic action, although they do not have the bactericidal action. When polylysine is applied for the purpose of controlling the multiplication of bacteria, it has to be used in concentrations as high as 0.5% to 1%. If it would be used with food in such high concentrations, it is readily anticipated that taste of the food may be changed. Further, it is expensive and irregularities in the level of concentrations may be anticipated.
Currently, there is no known bactericidal substance that is non-toxic to human beings when applied to food such as fresh food. Therefore, the current situation is that food such as fresh vegetables, particularly those to be used for school meals, etc., are disinfected with hot water. There needs to be a bactericidal material that can be applied to food, such as fresh food, etc. and that is safe to human beings.
It should be noted that a bacteriophage (hereinafter referred to sometimes as a “phage”) consists only of protein and nucleic acids, and may be classified as an organism which can propagate only in a specific bacterium as a specific host bacterium. It is an abiotic material that it is so minute that it can be observed only through an electron microscope. It is known that such a bacteriophage can infect only a specific bacterium as a host bacterium and it can propagate by eating up the infected bacterium. Presently, there are a large number of bacteriophages which are known to infect a variety of bacteria as hosts. Such bacteriophages may include, for example, phages peculiar to Salmonella as a host and phages peculiar to vibrio as a host. Further, there are known phages which utilize
E. coli
as a host and have destructive phagocytic action against
E. coli.
About three hundred kinds of such phages are known and a great number are present in the air. Each of the bacteriophages causes the infection of
E. coli
but it is non-toxic to human beings and other mammals. Likewise, there are known phages which use bacteria other than
E. coli
as hosts. These do not cause infection, and are non-toxic to human beings and other mammals.
It is also known that bacteriophages infect bacteria only, or a very small number of specific bacteria, and it has a high specificity of bacteria hosts. For instance, phages which infect one type of
E. coli
only may not infect any other different kinds of
E. coli.
For instance, it is known that a well known species, i.e. T2 phage, cannot infect C-type
E. coli.
Such bacteriophages have so far been used only as objects of research, and they have greatly served to develop genetic research. However, they have yet to attract an attention for their application to industry, due to their high specificity. Therefore, such bacteriophages have never been used as a bactericide.
Although many bacteriophages are known to utilize a variety of bacteria as hosts in the manner as described above, it is not yet known if there is any bacteriophage which utilizes a pathogenic bacterium such as, e.g. intestinal hemolytic
E. coli,
as a host.
Of the bacteria which act as hosts for bacteriophages, particularly
E. coli
is extremely high in multiplication, and its cells divide once every 20 to 30 minutes. If conditions permit, one cell of
E. coli
propagates up to 1 gram for one day. However,
E. coli
is present everywhere in nature and exists in the intestine of human beings and the other mammals, and yet it does not usually harm human beings or other mammals. Therefore, many so-called biomedicines are produced from
E. coli
cultures because of its properties.
Although there has not yet been discovered a bacteriophage which can utilize pathogenic bacterium such as intestinal hemolytic
E. coli
as a host, nor potential bacteriophages been found by conventional screening methods. A method for screening such bacteriophages with high efficiency and certainty needs to be established yet. Specifically, a screening method that can select such bacteriophages efficiently and surely must be developed.
Further, no bactericide effective for direct application to food, such as fresh food, etc. is currently available commercially. It has also been demanded to develop a product that can be applied directly to food, such as fresh ones, etc.
In order to develop such a bactericide, it is also necessary to develop a stabilizer or a preservative that can ensure safety when the such bactericide is applied directly to food, such as fresh food, etc. and allow for storage over a long period of time.
To date, buffers such as, e.g. tris-HCl, phosphate, etc., have been used as an ef
Shanks & Herbert
Wortman Donna C.
Zeman Robert A.
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