Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-06-05
2002-11-26
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C424S093600
Reexamination Certificate
active
06485902
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to the use of bacteriophages for reducing levels of
Escherichia coli
O157, and/or treating or preventing
Escherichia coli
O157 infection or diseases caused thereby, as well as methods for producing said bacteriophages.
BACKGROUND OF THE INVENTION
Human enteric infections with enterohemorrhagic
E. coli
O157 are a significant public health problem in many countries. They lead to diarrhea and to serious complications that include hemorrhagic colitis, the hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and death.
Healthy cattle carry
E. coli
O157 and humans acquire
E. coli
O157 infections most often by consuming food and water that has been contaminated with faeces from these animals. The demand for effective control of
E. coli
O157 infections at the pre-harvest level is high because it is generally believed that reducing, or eliminating
E. coli
O157 infections in cattle and other food animals will dramatically reduce the number of
E. coli
O157 infections in humans. Benefits occur not only through production of safer food, but through reduced transmission of
E. coli
O157 among cattle and dissemination in water and the environment. Probiotic bacteria, dietary management, and vaccination with relevant antigens could reduce shedding of
E. coli
O157 by cattle, however these approaches are poorly developed, or only marginally effective at this time.
Zhao et al. (
Journal of Clinical Microbiology
36, 641-7, 1998; U.S. Pat. No. 5,965,128) showed that a mixture of probiotic bacteria, comprised of 18 isolates of
E. coli
and one
Proteus mirabilis
strain, reduced shedding of
E. coli
O157 by cattle. The mechanism by which the probiotic bacteria reduced the shedding of
E. coli
O157 is not known and the probiotic bacteria are not available commercially.
Hovde et al. (
Applied and Environmental Microbiology
65, 3233-5, 1999) showed that higher energy, lower fiber diets reduced the duration of shedding of
E. coli
O157 by experimentally infected animals.
To date there are no reports of successful vaccination of cattle against carriage of
E. coli
O157.
Barrow et al. (Barrow et al., 1998,
Clin Diag Lab Imm
5:294-298) demonstrated that bacteriophages can control septicemia and meningitis, both non-enteric infections, in a variety of animals. Both of these infections are systemic, rather than intestinal infections leading to diarrheal disease. The bacteriophages were not used to control
E. coli
O157, or other zoonotic enteric pathogens affecting humans.
Alisky et al. (Alisky et al., 1998,
J Infect
36: 5-15) presented this recent review article that provides an historical perspective on the use of bacteriophages for controlling infections and current research in this area, which has been spurred by the emergence of antibiotic-resistant bacterial pathogens.
Bacteriophages have not been used to control
E. coli
O157 infections in cattle. Kudva et al. (
Applied and Environmental Microbiology
65, 3767-73, 1999) recently reported that bacteriophages could kill
E. coli
O157 in pure culture, and speculated that bacteriophages could be used to control
E. coli
O157 in animals. However, the ability of phages to kill
E. coli
O157 in pure culture has been known for some time and forms the basis for a phage typing scheme for
E. coli
O157. It is of note that this in vitro effect of bacteriophages cannot be extended to therapeutic use in cattle without further research to establish that candidate bacteriophages survive in the bovine gastro-intestinal tract and retain the ability to infect and kill
E. coli
O157 in vivo. Moreover, bacteriophages for use in vivo must not adversely affect the health of treated animals.
Smith et al. (Smith et al., 1987,
J. Gen. Micro.
133: 1111-1126) showed that experimental diarrhea in 6-12 hour old calves due to infection with certain enterotoxigenic strains of
E. coli
could be controlled by specific bacteriophages infecting those strains of bacteria. As will be appreciated by one knowledgeable of the art, serotypes of
E. coli
are defined by the presence of a combination of three known antigens on the surface of the cells and are a form of classification of different strains of
E. coli.
The antigens are O-antigens (somatic carbohydrate component of the cell wall lipopolysaccharide), H-antigens (flagella—organelles involved with cell locomotion), and K antigens (polysaccharide capsules or pill). The identity of these antigens on individual
E. coli
is determined by agglutionation tests using specific, highly cross-adsorbed anti-sera. The O157:H7 serotype is one of many different known serotypes of
E. coli.
The strains used by Williams Smith are of different serotypes than O157:H7 (ie. different O, K, and H antigens). That is, these strains belong to the group of pathogens called enterotoxigenic
E. coli,
which cause disease by a mechanism distinct from that of
E. coli
O157. Furthermore, it is of note that the phages used by Williams Smith would not work on
E. coli
O157:H7 because it lacks the appropriate K antigen receptor. Furthermore, Williams Smith did not show that bacteriophages could be used to eliminate human zoonotic pathogens carried asymptomatically by cattle. It is also of note that the activity of the bacteriophages towards target organisms in host matrices and their stability in host matrices were not evaluated. Finally, the bacteriophages were not used to control
E. coli
O157, or other zoonotic enteric pathogens affecting humans.
U.S. Pat. Nos. 5,766,892 and 5,688,501 describe methods to treat bacterial infections in animals using bacteriophages that have been altered to resist host defense mechanisms. The bacteriophages were not used to control
E. coli
O157, or other zoonotic enteric pathogens affecting humans, but rather were serially administered to an animal's circulatory system and recovered so as to enrich for phage capable of surviving in the host circulatory system. Thus, these patents teach that phage must be modified to avoid the host immune system in order to be effective treatments.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a pharmaceutical composition comprising at least one bacteriophage selected from the group consisting y of: V4, V5, V5-re-isolated, V7, V8, V11 and V14.
According to a second aspect of the invention, there is provided a method of reducing levels of
E. coli
O157 in a ruminant animal comprising:
administering to a ruminant animal having
E. coli
O157 within its gastrointestinal tract at least one bacteriophage selected from the group consisting of: V4, V5, V5-re-isolated, V7, V8, V11, and V14; and
retaining said ruminant animal under conditions such that said bacteriophage infect and lyse said
E. coli
O157,
thereby reducing levels of
E. coli
O157 within the gastrointestinal tract of said ruminant animal.
According to a third aspect of the invention, there is provided a method of selecting and isolating bacteriophages capable of lysing
E. coli
O157 comprising:
administering to a ruminant animal having
E. coli
O157 within its gastrointestinal tract at least one bacteriophage selected from the group consisting of: V4, V5, V5-re-isolated, V7, V8, V11, and V14;
recovering gastrointestinal content from said ruminant animal;
isolating bacteriophage from said gastrointestinal content;
plating said isolated bacteriophage onto a suitable host; and
purifying the resulting plaques.
According to a fourth aspect of the invention, there is provided a method of reducing levels of
E. coli
O157 in a matrix comprising:
administering to the matrix containing
E. coli
O157 therein at least one bacteriophage selected from the group consisting of: V4, V5, re-isolated V5, V7, V8, V11 and V14; and
retaining said matrix under conditions such that said bacteriophage infect and lyse said
E. coli
O157,
thereby reducing levels of
E. coli
O157 within the matrix.
According to
Ahmed Rafig
Johnson Roger
Khakhria Rasik
Mazzocco Amanda
Pacan Jennifer
Battison Adrian D.
Dupuis Ryan W.
Williams Michael R.
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