U.S. patent application number 11/831813 was filed with the patent office on 2007-12-20 for use of viruses and virus-resistant microorganisms for controlling microorganism populations.
This patent application is currently assigned to OMNILYTICS INCORPORATED. Invention is credited to Lee E. Jackson, Rex S. Spendlove.
Application Number | 20070292395 11/831813 |
Document ID | / |
Family ID | 36648261 |
Filed Date | 2007-12-20 |
United States Patent
Application |
20070292395 |
Kind Code |
A1 |
Jackson; Lee E. ; et
al. |
December 20, 2007 |
USE OF VIRUSES AND VIRUS-RESISTANT MICROORGANISMS FOR CONTROLLING
MICROORGANISM POPULATIONS
Abstract
A lytic virus specific for a target strain of a microorganism
and substantially free of undesirable genes may be utilized in
processes including control of populations of microorganisms. The
virus may include a host-range mutant, or "h-mutant." A method for
generating virus includes growing virus-resistant variants of a
target strain of a microorganism in the presence of viruses that
are specific for the target strain. Only h-mutant viruses will
proliferate. Wild-type virus-resistant and virus-resistant variants
of a microorganism are also disclosed, as are methods generating
such variants. Methods for controlling target strain microorganisms
include introducing virus into a treatment site where control of a
population of a target strain microorganism is desired or
introducing virus-resistant variants of a microorganism into
treatment sites where the presence of the microorganism is
desired.
Inventors: |
Jackson; Lee E.; (Layton,
UT) ; Spendlove; Rex S.; (Millville, UT) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
OMNILYTICS INCORPORATED
5450 West Wiley Post Way
Salt Lake City
UT
84116
|
Family ID: |
36648261 |
Appl. No.: |
11/831813 |
Filed: |
July 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11033022 |
Jan 10, 2005 |
|
|
|
11831813 |
Jul 31, 2007 |
|
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|
Current U.S.
Class: |
424/93.4 ;
435/235.1; 435/245; 435/252.1; 435/252.5; 435/262.5; 435/267;
435/281 |
Current CPC
Class: |
A23B 4/20 20130101; A01N
63/20 20200101; A01N 63/40 20200101; C12N 7/00 20130101; A61K 35/76
20130101; C12N 2795/00032 20130101; A61K 35/741 20130101; Y02A
50/30 20180101; A61K 35/741 20130101; A61K 2300/00 20130101; A61K
35/76 20130101; A61K 2300/00 20130101; A01N 63/40 20200101; A01N
2300/00 20130101 |
Class at
Publication: |
424/093.4 ;
435/235.1; 435/245; 435/252.1; 435/252.5; 435/262.5; 435/267;
435/281 |
International
Class: |
C12N 1/20 20060101
C12N001/20; A61K 35/00 20060101 A61K035/00; C12N 7/00 20060101
C12N007/00 |
Claims
1. A composition for controlling a population of a beneficial
microorganism, comprising a virus-resistant variation of the
beneficial microorganism.
2. The composition of claim 1, wherein the virus-resistant
variation comprises resistance to a wild-type of a virus specific
for the beneficial microorganism.
3. The composition of claim 1, wherein the virus-resistant
variation comprises resistance to an h-mutant virus specific for
the beneficial microorganism.
4. The composition of claim 1, further comprising: an h-mutant
virus specific for an undesirable microorganism.
5. The composition of claim 1, wherein the beneficial microorganism
comprises at least one of Pantoea annanus, Serratia entomophilia,
Enterobacter aerogenes, T. ferroxidans, a member of the genus
Acidiphilium, a member of the genus Rhodococcus, a member of the
genus Sulfobus, and a member of the genus Thiobacillus.
6. A method of controlling a population of a beneficial
microorganism, comprising introducing a virus-resistant variation
of the beneficial microorganism into a treatment site.
7. The method according to claim 6, wherein introducing comprises
exposing a toxic chemical or other pollutant to the virus-resistant
variation of the beneficial microorganism.
8. The method according to claim 6, wherein introducing comprises
introducing at least one of Pantoea ananus, Serratia entomophilia,
Enterobacter aerogenes, T. ferroxidans, a member of the genus
Acidiphilium, a member of the genus Rhodococcus, a member of the
genus Sulfobus, and a member of the genus Thiobacillus into the
treatment site.
9. The method according to claim 6, wherein introducing is effected
to control growth of fungi.
10. The method according to claim 6, wherein introducing is
effected to control the proliferation or spread of an insect
population.
11. The method according to claim 10, wherein introducing is
effected to control the proliferation or spread of insect
populations as exemplified by New Zealand grass grub and
locusts.
12. The method according to claim 10, wherein introducing includes
applying the virus-resistant variation of the beneficial
microorganism to a plant.
13. The method according to claim 6, wherein introducing is
effected to oxidize sulfide to sulfuric acid.
14. The method according to claim 6, wherein introducing is
effected to release petroleum and petroleum-related substances from
bituminous shale.
15. The method according to claim 6, wherein introducing is
effected to acidify sulfur.
16. The method according to claim 6, wherein introducing effects at
least one of acidifying alkaline soil, decomposing rubber products,
bioremediating hazardous chemicals or pollutants, treating sewage
or wastewater, treating odors or sources of odors, fermentation
processes.
17. A method of generating a virus-resistant variant of a
microorganism, comprising: isolating an h-mutant virus by exposing
a wild-type microorganism to a virus specific for the wild-type
microorganism; isolating a virus-resistant variant of the
microorganism by exposing the wild-type microorganism to the
h-mutant virus; isolating at least one second h-mutant virus by
exposing the virus-resistant variant of the microorganism to the at
least one h-mutant virus; and isolating at least one second
virus-resistant variant of the microorganism by exposing at least
one of the wild-type microorganism and at least one type of
virus-resistant variant of the microorganism to the at least one
second h-mutant virus.
18. The method according to claim 17, wherein at least one of
isolating the virus-resistant variant and isolating the at least
one second virus-resistant variant comprises growing at least one
of the wild-type microorganism and the virus-resistant variant in
the presence of an h-mutant virus specific for the
microorganism.
19. The method according to claim 18, wherein isolating the at
least one second virus-resistant variant comprises isolating at
least one second virus-resistant variant having resistance to
infection by a plurality of h-mutant viruses.
20. A composition for controlling populations on food products of a
microorganism that is pathogenic to a mammal, comprising an
h-mutant virus specific for and capable of infecting a
virus-resistant variation of a target strain of the
microorganism.
21. The composition of claim 20, wherein the microorganism are
enteric pathogens, as exemplified by Salmonella, Campylobacter, or
E. coli.
22. The composition of claim 20, wherein the target strain
comprises E. coli 0157:H7.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of application Ser. No.
11/033,022, filed Jan. 10, 2005, pending. The disclosure of the
previously referenced U.S. patent application is hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Technical Field
[0002] The invention relates to viruses which control and prevent
growth of harmful microorganisms and to processes which employ such
viruses. It also relates to protection of helpful microorganisms
from virus attack. In particular, the viruses of the present
invention lack genes for virulence factors, toxins, antibiotic
resistance, and other undesirable genes, and include host-range
(h-mutant) viruses which are specific for wild-type virus-resistant
strains of targeted microorganisms. More specifically, viruses of
the present invention are lytic, thus they control and prevent
further growth of harmful microorganisms that infect animals or
plants by destroying these microorganisms. Such viruses may also be
employed to develop and select strains of beneficial microorganisms
which are resistant to wild-type and h-mutant viruses.
[0003] Viruses are known to alter populations of microorganisms,
such as bacteria, fungi, algae, and protozoa. It has been estimated
that, in nature, as many as one-third of all bacteria may be
attacked by viruses each day. The destruction of microorganisms by
viruses results in fluctuations of microbial populations in the
environment, which is referred to as "cycling" of microbial
populations. For example, populations of microorganisms increase in
concentration until viruses contact and infect susceptible
microorganisms, which are referred to as host microorganisms or
"hosts." Viral infections of microorganisms decrease the number of
available susceptible host microorganisms, and correspondingly
increase the number of viruses. Without hosts to infect, many
viruses are eventually destroyed by exposure to natural elements,
such as ultraviolet light from the sun and enzymes in the
environment. Thus, virus numbers decline, while host microorganism
populations consequently increase. Such cycling of microbial
populations in nature is common. Although it is somewhat difficult
to detect and study viruses that attack microorganisms other than
bacteria, those of skill in the art are aware that all populations
of microorganisms (e.g., algae, rickettsiae, fungi, mycoplasmas,
protozoas) are controlled and cycled in a similar manner by viruses
that are capable of infecting and destroying such
microorganisms.
[0004] Bacterial viruses, which are also referred to as
"bacteriophages" or "phages," are ubiquitous and can be isolated
from all bacterial populations where hosts can be cultivated and
used for isolation. Phages are naturally-occurring entities that
are found in or on animals (including humans), plants, soil, and
water. Viruses which infect algae, molds, mycoplasmas, protozoa,
rickettsiae, yeasts, and other microorganisms are also known.
[0005] Two methods are typically employed in order to determine the
concentration, which is also referred to as "quantification," of
viruses in natural environments. First, electron microscopy may be
used to visualize and count total viral particles in a sample of
known size. Second, viable viruses may be cultured, or grown, and
counted. An exemplary method of quantification by culturing and
counting includes a technique which is typically referred to as a
plaque assay. In plaque assays, the viruses that are to be
quantified are mixed with a predetermined concentration of host
cells and transferred to a liquid (e.g., buffer, mineral salts
diluent, or broth). The mixture is then transferred to a semisolid
growth medium. The concentration of host cells must be sufficiently
great to form a confluent layer, which is typically referred to as
a "lawn," in the semisolid growth medium as the cells grow. During
incubation of the phage-host mixture, many of the viable viruses
infect host cells. Subsequently, new viruses are produced within
infected host cells, which are eventually destroyed, or "lysed," so
that new viruses may be released therefrom. The new viruses then
attack and eventually lyse cells that are adjacent to host cells
from which the new viruses were released. This spread of infection,
which continues as long as host cells are metabolizing, results in
formation of clear areas, which are typically referred to as
"plaques," in the host cell lawn. The number of viruses that were
present in the original mixture is determined by counting the
number of plaques that are formed in the host cell lawn.
Accordingly, viruses that are quantified by this method are
referred to as plaque-forming units ("PFU").
[0006] In order to quantify all of the various types of viruses in
an environmental sample by culturing host cells and counting PFUs,
host cells for each of the different viruses in the sample must be
cultured. Many types of microorganisms in a given environmental
sample are not known. Some of the known microorganisms cannot be
cultivated. Therefore, the number of viruses that are present in a
given environment may be underestimated when quantified by
culturing and counting. Although it is estimated that one gram of
soil includes as many as 10.sup.8 to 10.sup.9 microorganisms,
quantification techniques such as direct plate counting, selective
isolation, microscopy, and reassociation kinetics of total DNA
isolated from soil suggest that only a very small percentage of
these microorganisms can be cultured. Thus, the development and
application of direct electron microscopic counting methods have
provided a better understanding of the number of viruses that are
present in various environments, as well as the impact that viruses
have in reducing microbial populations.
[0007] Phages have been quantified in water. Bergh et al. (1989),
High abundance of viruses found in aquatic environments, Nature
340:467, used electron microscopy to determine the total
concentration of bacterial viruses in a natural, unpolluted
Norwegian lake. Phage concentrations of up to about
2.5.times.10.sup.8 phages/ml were found in the water. Bacterial
counts were as high as about 1.5.times.10.sup.7 cells/ml. From
these relative concentrations of phage and bacteria, it was
estimated that as many as one-third of the bacterial population
experiences one or more phage attacks each day. Similarly, Demuth
et al. (1993) Direct electron microscopy study on the morphological
diversity of bacteriophage populations in Lake Plussee, Appl.
Environ. Microbiol. 59:3378, determined that phage levels in a
German lake without sewage influences were as high as about
10.sup.8 phages/ml of lake water. As many as eleven morphologically
different phages were identified in the water samples.
[0008] Phages have also been quantified in soil. Using the
culturing and counting method, with Bacillus stearothermophilus as
the host cell, Reanny, D. C. and Marsh S. C. N. (1973). The ecology
of viruses attacking Bacillus stearothermophilus in soil, Soil.
Biol. Biochem. 5:399, reported that, on average, about
4.0.times.10.sup.7 PFUs that would infect B. stearothermophilus
were present in a gram of soil. Only phages against a single host
were, however, quantified in the Reanny and Marsh study. Thus, had
other bacterial hosts been tested along with B. stearothermophilus,
or had electron microscopy quantification techniques been employed,
phage counts would probably have been much higher.
[0009] Phages are also present in foods. Kennedy et al. (1986)
Distribution of coliphages in various foods, J. Food Protect.
49:944, found Escherichia coli and phages that attack E. coli
("coliphages"), in 11 of 12 tested foods, each of which are
available in many retail markets. For example, all ten ground beef
samples tested by Kennedy et al. were contaminated with coliphages.
Coliphages were also present in samples of fresh chicken, fresh
pork, fresh oysters, fresh mushrooms, lettuce, chicken pot pie,
biscuit dough, deli loaf, deli roasted turkey and packaged roasted
chicken. Similarly, Gautier et al. (1995) Occurrence of
Propionibacterium freudenreichii bacteriophages in Swiss cheese,
Appl. Environ. Microbiol. 61:2572, detected Propionibacterium
freudenreichii phage concentrations of about 7.times.10.sup.5 PFU/g
in Swiss cheese.
[0010] Both undesirable and beneficial microorganisms are present
in the environment. Viruses infect and destroy both beneficial and
undesirable microorganisms. Soil microorganisms that enhance plant
growth and microorganisms that degrade toxic substances are
exemplary of beneficial microorganisms in the environment.
Undesirable microorganisms include pathogenic microorganisms and
algae that cause algal blooms and fish kills.
[0011] In addition to naturally-occurring microbial populations, in
recent decades disease-causing microorganisms resistant to
antibiotics have become epidemic in many hospitals, and have been
notoriously difficult to control. During the past fifty or more
years, the widespread use of antibiotics has resulted in the
selection of antibiotic-resistant bacterial strains. Staphylococcus
aureus, Streptococcus pneumoniae, Streptococcus pyogenes,
Enterococcus faecalis, Salmonella typhi, Hemophilus ducreyi,
Hemophilus influenzae, Mycobacterium tuberculosis, Neisseria
gonorrhoeae, Pseudomonas aeruginosa, various Shigella species,
members of the Enterobacteriaceae and Pseudomonas families, and
other bacterial species are resistant to many of the
conventionally-employed antibiotics. Infections that are acquired
during hospitalization, which are typically referred to as
nosocomial infections, cause an estimated 60,000 deaths per year,
and require treatment, which has been estimated to cost about $4.5
billion per year recently.
[0012] Statistics from the Centers for Disease Control and
Prevention (CDC) indicate that the majority of nosocomial
infections are caused by E. coli, S. aureus, coagulase-negative
staphylococci, enterococci, pneumococci, and pseudomonads. In
addition, according to the 1996 World Health Organization (WHO)
annual report, "drug-resistant strains of microbes have evaded
common treatments for tuberculosis, cholera, and pneumonia."
[0013] Consequently, the occurrence of infections that are caused
by antibiotic-resistant bacteria has steadily increased in
hospitals, localized communities, and at-risk populations worldwide
since the 1940s, shortly after antibiotics were first used for
treating bacterial infections. For example, in 1941 practically all
strains of S. aureus throughout the world were susceptible to
penicillin G. By 1944 however, some strains of S. aureus were
capable of making penicillinase, which is also typically referred
to as .beta.-lactamase, which degrades penicillin. In 1996, some
strains of S. aureus were not only resistant to various forms of
penicillin, but also to six of the seven other antibiotics that are
conventionally used to treat S. aureus ("staph") infections. [0014]
1) Since 1988, the potential for selection of vancomycin-resistant
mutants was a concern in that such resistance had been identified
in Gram-positive bacteria, such as vancomycin-resistant E.
faecalis, or faecium ("VREF"); VREF are also of great concern to
health care professionals due to their deadly combination of
antibiotic resistance, rapid spread, and high mortality rates in
patients with VREF-associated infections. [0015] 2) Infections by
methicillin-resistant S. aureus ("MRSA") pose an especially serious
public health threat. MRSA typically display various patterns of
multiple-drug resistance (i.e., are resistant to multiple types of
antibiotics). Many strains of MRSA are susceptible only to the
antibiotic vancomycin. [0016] 3) Although new and alternative drugs
for treating infections of antibiotic-resistant strains of bacteria
have been developed and discovered, many bacteria also develop
resistance to such new and alternative drugs. For example, certain
MRSA strains quickly developed resistance to the antibiotic
ciprofloxacin. Moreover, in 1997, a strain of S. aureus was
isolated from an infection that resisted 29 days of vancomycin
treatment. To put the threat posed by this S. aureus strain in
perspective, this S. aureus strain was categorized by the CDC as
having intermediate resistance somewhat short of full resistance,
and was labeled a medical red alert. It was reported that if MRSA
strains which have resistance to vancomycin develop, death rates
for all surgeries, including elective surgeries, may increase.
[0017] 4) In 2001 the isolation of MRSA from three heart patients
at McKay-Dee Hospital in Ogden, Utah, resulted in closure of its
cardiac surgical units to all but emergency surgeries.
Subsequently, vancomycin-resistant S. aureus (VRSA) have been
isolated from clinical patients in Michigan (2002), Pennsylvania
(2003) and New York (2004).
[0018] Similarly, about half of the known strains of S. pneumoniae
are resistant to penicillins, which have conventionally been
employed as the initial and primary treatment for S. pneumoniae
infections. Some S. pneumoniae strains are resistant to
cephalosporin antibiotics, which have conventionally been employed
as a secondary treatment for S. pneumoniae infections. Penicillin
and cephalosporin-resistant S. pneumoniae strains may be treated
with vancomycin. The use of vancomycin, however, is undesirable
because of severe side effects that vancomycin has on many patients
and the possibility that vancomycin-resistant strains of S.
pneumoniae may emerge.
[0019] The problem of antibiotic resistance is further compounded
by the fact that microorganisms may transfer genetic information,
which is referred to as "genes," or "DNA" for simplicity. Methods
by which microorganisms, such as bacteria, can transfer DNA, and
even entire genes, include conjugation, transformation, and
transduction. Various genes, including genes that impart bacteria
with resistance to antibiotic drugs, may be transferred from a
first, or donor, microorganism to a second, or recipient,
microorganism. In addition to transferring genes for antibiotic
resistance, microorganisms may transfer genes that enable a
microorganism to produce toxins, which are typically harmful to an
infected host. Virulence factors, which determine the types of
hosts and host cells that a microorganism can infect may also be
transferred from one microorganism to another.
[0020] In conjugation, plasmid or chromosomal DNA is transferred
directly from a donor microorganism to a recipient microorganism by
means of specialized pili or "sex pili," which are small, hollow,
filamentous appendages, which bind to and penetrate the cell
membrane of recipient microorganisms. Conjugation is a process by
which genes that code for antibiotic resistance in the "donor"
microorganism pass to a recipient microorganism, transforming the
recipient into an antibiotic-resistant microorganism.
[0021] Transformation is the transfer of DNA that has been released
into the environment by a donor microorganism and incorporated by a
recipient microorganism. Transformation experiments have been
conducted in sterile soil that was inoculated with two parental
strains of Bacilus subtilis with differentially marked, or tagged,
DNA. Bacteria were isolated which carried the markers of both
parental strains. Even under the best laboratory conditions,
however, transformation is relatively inefficient and requires high
densities of donor DNA and recipient cells. Conditions that would
permit transformation in many microorganisms are typically not
present in a natural, or uncontrolled, environment. Consequently,
transformation is typically perceived as a laboratory
phenomenon.
[0022] Transduction is the transfer of host genes to recipient
microorganisms by viruses, such as phages. There are two kinds of
phages, virulent, or lytic, and temperate. When a host cell is
infected with a virulent phage, new phages, which are typically
referred to as progeny, are grown in the host cell, and the host
cell is subsequently lysed, or destroyed, so that the progeny may
be released. In contrast, temperate phages typically infect host
cells without destroying their host. Following infection of a host
cell, temperate phages typically incorporate their genetic
information into the DNA of the host cell. Many temperate
phage-infected host cells can be subsequently induced, by
ultraviolet light, mutagens, or otherwise, to enter a lytic cycle,
wherein genetic information of the temperate phage produces progeny
which then lyse the host cell.
[0023] Transduction of host DNA may be either "specialized" or
"generalized." In specialized transduction, a temperate phage's
genome is integrated into the chromosome of a host donor
microorganism without lysing the host. The phage genome that was
inserted into the host chromosome, is referred to as a "provirus,"
or "prophage," and is passively replicated as the host cell and its
chromosome replicate. Bacteria that carry proviruses are said to be
lysogenic. Certain events, such as exposing the host microorganism
and the provirus to ultraviolet light, may cause the provirus to
act as a virulent phage, whereby the provirus is excised from the
bacterial chromosome. Such excised proviruses may carry bacterial
genes, or "donor" genes, with them. Upon infecting a new host, or
recipient microorganism, these "donor" genes may be expressed,
which may alter the phenotype, or physical gene expression, of the
recipient microorganism.
[0024] Temperate and, possibly, some virulent phages may effect
generalized transduction. During viral replication, a section of
DNA of the donor microorganism, which is referred to as a "donor"
gene, rather than the phage genome, may be enclosed inside a phage
head. Phages that include only DNA of a host microorganism are
referred to as transducing particles. A typical phage is only
capable, however, of containing about one percent of the chromosome
of a host, or "donor," microorganism. Thus, the simultaneous
transfer of more than one gene by a single transducing particle is
unlikely. Since transducing particles do not include a phage
genome, transducing particles cannot produce progeny upon infecting
a recipient microorganism. Instead, the donor gene has to be
incorporated into the chromosome of the recipient microorganism. If
the recipient microorganism is infected with only one transducing
particle, it will survive and its phenotype may be altered by the
integrated donor gene. It is very important to remember if the
multiplicity of infection ("MOI") of transducing particles per
recipient microorganism is high, the cell will probably be
destroyed, which is typically referred to as "lysis from
without."
[0025] The transfer of genetic information from one microorganism
to another may have beneficial or undesirable effects. For example,
a beneficial transfer of genetic information was disclosed by
Chakrabarty, A. M. (1996) Microbial degradation of toxic chemicals:
Evolutionary insights and practical considerations, ASM News
62:130. Microorganism-rich soil was introduced into a chemostat
which contained a single industrial pollutant as a nutrient. In
less than a year, pseudomonads which had acquired all of the
enzymes needed to degrade the pollutant were isolated from the
soil.
[0026] Similarly, genes that exhibit undesirable traits may also be
transferred. Examples of such detrimental gene transfer include
transfer of genes carrying resistance to antibiotics, and genes
that code for production of toxins, such as shiga, diphtheria, and
botulism toxins. Outbreaks of toxin-related diseases, such as toxic
shock syndrome in 1980, the "flesh-eating streptococci" of 1994,
and illnesses caused by E. coli 0157:H7 in undercooked hamburger,
have been traced to the transfer of toxin genes by temperate
phages. Genes that code for cholera toxin are also reported to have
been transmitted by a temperate phage, which created yet another
epidemic strain, Vibrio cholerae 0139.
[0027] Viruses have been isolated and employed in treating various
types of bacterial infections. U.S. Pat. No. 4,375,734, which
issued to Kozloff et al. on Mar. 8, 1983 ("Kozloff"), discloses use
of a wild-type phage, Erh1, for protecting plants against frost
injury caused by an ice nucleation-promoting bacterium, Erwinia
herbicola. The treatment of corn plants with Erh 1 reduced the
incidence of ice nucleation damage by about 20% to 25%. Kozloff et
al. also discloses that Erh1 killed only about 90% of cultured E.
herbicola, which suggests that some of the remaining 10% were
resistant to wild-type Erh1.
[0028] U.S. Pat. No. 4,828,999, which issued to Jackson, one of the
present inventors, on May 9, 1989 ("Jackson"), discloses host
range, or "h-mutant," phages which attack phage-resistant strains
of various plant bacteria, and methods of treating bacterially
infected plants. The h-mutant phages, compositions containing such
phages, and methods of treatment that are disclosed in Jackson are,
however, limited to phages for plant bacteria and the treatment of
plants infected with such bacteria.
[0029] Similarly, some measures have been taken to address the
problem of bacterial diseases in humans, and to otherwise control
and prevent bacterial growth. Patent application Ser. No.
08/222,956 (the "'956 application"), which was published on Oct.
12, 1995 as WO 95/27043, discloses a type of phage therapy whereby
mutant phage strains are introduced into a bacterially infected
host. The mutant phages, which are thought to be resistant to
degradation by the bacterially infected host's defense systems,
particularly organs of the reticulo endothelial system, are
believed to attack the harmful bacteria with which the host is
infected. Thus, phages of the '956 application are believed to act
as an in vivo antibacterial agent, and may be used either alone or
as an adjunct to antibiotic therapy.
[0030] Although phages disclosed in the '956 application are
introduced into bacterially infected hosts for the purpose of
attacking undesirable bacteria, these phages included not only
lytic, but also temperate viruses which are able to transfer pieces
of donor bacterial DNA to recipient bacteria. Further, the '956
application lacks any disclosure that phages disclosed therein are
able to attack, and thereby prevent or otherwise control the
further growth of, phage-resistant bacterial strains.
[0031] Shortly after the discovery of phages as lytic agents of
bacteria by Twort in 1915 and by d'Herelle in 1917, the
investigation of their use for treating bacterial infections, which
is typically referred to as phage therapy, began. Various phages
are active against bacteria of many diseases in plants and animals,
such as mammals. Phages that are active against bacteria which
cause human diseases, such as anthrax, bronchitis, diarrhea,
scarlet fever, typhus, cholera, diphtheria, gonorrhea, paratyphus,
bubonic plague, osteomyelitis, and other bacterially induced
diseases, are known. While many in the art were initially convinced
of the efficacy of phage therapy, particularly in controlling
cholera, many phages were ineffective for in vivo treatment. It was
believed that such ineffectiveness was due to the inactivation of
phage by the host's immune system when administered parenterally,
denaturation by gastric juices when taken orally, and the rapid
emergence of phage-resistant bacterial mutants.
[0032] With the introduction and use of antibiotics, and their
initial effectiveness in controlling bacterial diseases, much of
the research for using phages as therapeutic agents ceased.
Recently, phage therapy was successfully employed to treat
nosocomial infections caused by antibiotic-resistant bacteria and
certain opportunistic pathogens, namely, pyogenic infections and
septicemias, especially staphylococcal, but also pseudomonads,
enterobacteria (E. coli, Klebsiella, Proteus, Providencia,
Serratia), injuries (infected wounds and burns, postoperative
infections, osteomyelitis), diseases of the skin and subcutaneous
tissue (furunculosis, abscesses, acute lymphangitis, decubitus
ulcers), urinary infections (chronic cystitis and pyelonephritis),
respiratory diseases (sinusitis, mucopurulent bronchitis,
pleuritis) and other diseases, for example, infantile diarrhea
caused by enteropathogenic E. coli (7,8). In treating bacterial
infections, phages may be administered orally in liquids, tablets
and capsules, topically by aerosols and direct application, and
intravenously. Phage therapy was conducted alone and in combination
with antibiotics. Phages were also used as antiseptics, including
uses such as disinfecting operating rooms, surgical instruments and
lesions on patients, and medical care professionals.
[0033] Microorganisms such as bacteria can develop phage-resistant
strains, however. Thus, phage therapy (or virus therapy for
non-bacterial microorganisms) is somewhat undesirable from the
standpoint that virus-resistant strains of a target strain of
microorganism may persist in an infected host that is being
treated, or in any other treated environment.
[0034] Conversely, many beneficial microorganism populations are
threatened by viruses that will interfere with the beneficial
properties of such microorganisms. Exemplary beneficial processes
that are facilitated by microorganisms include industrial
fermentation (e.g. in making food products), bioremediation of
toxic chemicals, pollutants, and other undesirable substances,
leaching of metals from low grade ores, extraction of petroleum and
related products from shale, and drug manufacture. The efficiency
of many beneficial processes is degraded by the ubiquitous nature
of many viruses that will attack the microorganisms that facilitate
these processes.
[0035] Thus, a need exists for an alternative method of
controlling, reducing, or eliminating microorganism populations,
which method addresses the ever-increasing emergence of
antimicrobial resistance and the virus-resistance of
microorganisms. A need also exists for a treatment which selects
and destroys undesirable microorganisms while permitting beneficial
microorganisms to survive. A need also exists for providing
virus-resistant beneficial microorganisms.
BRIEF SUMMARY OF THE INVENTION
[0036] Although viruses have been used to control populations of
microorganisms as previously described, many microorganisms can
readily develop resistance to infection by viruses. Moreover, the
use of temperate viruses in controlling populations of
microorganisms is often ineffective since temperate viruses do not
always proliferate in and lyse the infected host
microorganisms.
[0037] "Wild-type" is defined herein as those viruses isolated from
the wild or nature which display the most frequently observed
phenotype, or physical characteristic, and is typically referred to
as "normal," in contrast to "mutant." "Wild-type viruses" exhibit
normal host-range virulence. "Wild-type microorganisms" do not
resist infection by wild-type viruses specific for the particular
target strain of microorganism.
[0038] "Host-range mutant viruses," which are also referred to as
"h-mutant viruses," are defined herein as viruses which exhibit
broader than normal host-range virulence. H-mutant viruses infect
both wild-type microorganisms and virus-resistant variants of the
target strain of microorganisms.
[0039] The invention thus includes one or more viruses which do not
carry unwanted genes and are specific for one or more target
strains of microorganisms. The viruses are lytic viruses which may
be employed in processes including the control, reduction, or
elimination of populations of a target strain of a microorganism.
Preferably, a virus or virus mixture according to the present
invention includes one or more h-mutant viruses. A mixture of one
or more h-mutant viruses and one or more wild-type viruses is also
within the scope of the present invention. Wild-type and h-mutant
viruses "recognize" receptors on the surfaces of target strains,
including one or more virus-resistant variant thereof, and infect
these virus-resistant variants. Since the viruses of the present
invention comprise lytic viruses, infected host cells will be lysed
by the viruses. Viruses which are able to infect the wild-type of
the target strain as well as a variety of virus-resistant variants
of the target strain are preferred.
[0040] The h-mutant viruses of the present invention may be
generated by isolating a wild-type of a target strain of a
microorganism and growing this wild-type in the presence of a
wild-type virus which is specific for the target strain.
Virus-resistant variants of the target strain will grow in the
presence of the wild-type virus. The virus-resistant variants of
the target strain are isolated and then grown in the presence of
wild-type virus in order to generate h-mutant viruses. H-mutant
virus-resistant variants of the target strain may then be obtained
in a similar manner to the generation of virus-resistant variants
of the target strain. These h-mutant virus-resistant variants may
then be grown in the presence of h-mutant viruses in order to
generate secondary h-mutants which will infect one or more
virus-resistant variants of the target strain, imparting these
h-mutants with a broader host range than their predecessors.
[0041] The invention also includes the virus-resistant and h-mutant
virus-resistant variants of the microorganism, which are generated
as described previously, and as hereinafter further described.
[0042] The viruses of the present invention may then be employed in
a method of controlling, reducing, or eliminating populations of
target strain microorganisms. The method includes introducing lytic
viruses that are substantially devoid of undesirable genes into an
environment where an undesirable target strain microorganism is
present. As the target strain microorganism is exposed to the
viruses, it is infected and eventually lysed. Since h-mutant
viruses preferably infect wild-type and virus-resistant variants of
the target strain, depending upon the concentration of h-mutant
viruses, the preferred use of h-mutant viruses in the inventive
method may effectively control, reduce, or eliminate the target
strain microorganisms from the environment into which viruses are
introduced.
[0043] In another aspect of the method of controlling populations
of microorganisms includes introducing virus-resistant or h-mutant
virus-resistant variants of a microorganism into an environment
where the presence of the microorganism is desired. The
introduction of such virus-resistant and h-mutant virus-resistant
microorganisms is desirable in situations where the microorganism
facilitates a beneficial process.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention preferably includes host range-mutant
lytic viruses, which are also referred to as h-mutant virulent
viruses, or simply as h-mutant viruses, that infect and destroy
virus-resistant strains of microorganisms. The present invention
may also include wild-type lytic, or virulent, viruses, which are
collectively referred to as "viruses" for simplicity. The viruses
are preferably substantially free of undesirable genes. The present
invention also includes a process for generating h-mutant viruses
or mixtures of h-mutant and wild-type viruses that lack undesirable
genes, such as genes that impart the virus with the ability to
infect multicellular organisms, the ability to transfer undesirable
genes to infected host microorganisms, and the ability to convert
from a lytic state to a temperate state; a process for reducing,
eliminating or otherwise controlling the growth of microorganism
populations with h-mutant viruses or mixtures of h-mutant and
wild-type viruses; and a process that utilizes h-mutant viruses or
mixtures of h-mutant and wild-type viruses to generate
virus-resistant and h-mutant virus-resistant strains of
microorganisms. The virus-resistant and h-mutant virus-resistant
strains of microorganisms that are generated by the inventive
process are also within the scope of the present invention.
H-Mutant Viruses
[0045] The h-mutant viruses of the present invention are lytic, or
virulent, viruses, which infect host microorganisms, utilize the
various components of the host microorganisms to replicate and
assemble progeny, and destroy the host, target strain
microorganisms. Preferably, the h-mutant viruses of the present
invention lack undesirable characteristics, including, without
limitation, the ability to infect multicellular organisms, the
ability to transfer undesirable genes to infected host
microorganisms, and the ability to convert from a lytic state to a
temperate state.
[0046] The viruses include an outer protein coat, or "capsid,"
which is capable of "recognizing" a receptor, or receptor site, on
the outer surface of a target strain microorganism, including some
receptors which have been altered, or "mutated," to impart the
target strain microorganism with resistance to wild-type viruses or
resistance to one or more h-mutant viruses. The ability of h-mutant
viruses to recognize mutated receptors of the target strain
microorganism enables h-mutant viruses to infect virus-resistant
variations of the target strain microorganism.
[0047] Due to their ability to "recognize" receptors on the target
strain microorganism, viruses of the present invention specifically
infect the target strain, and do not infect other, non-targeted
strains of a same species of microorganism, other non-targeted
microorganisms, or other non-targeted cells. Thus, the inventive
viruses are not as likely to inhibit the activity of beneficial
microorganisms as antimicrobial drugs, which lack the specificity
of viruses for a target microorganism.
[0048] When employed in a treatment method according to the present
invention, as more fully described below, the inventive viruses
proliferate as they destroy target strain microorganisms, reducing
the need for repeated dosing in treatment which include the
administration of viruses. In contrast, antimicrobial therapies
require repeated doses since antimicrobial concentrations decrease
during treatment.
[0049] After target strain microorganism populations are reduced or
eliminated such that target strains are no longer present for the
viruses to infect, the viruses become inactive, and will eventually
be degraded. Following their degradation, the various components of
the viruses may be utilized by other organisms as nutrients.
[0050] The process of generating h-mutant viruses of the present
invention includes isolating virus-resistant microorganisms, and
growing the virus-resistant microorganisms in the presence of
wild-type viruses in order to generate and isolate h-mutant
viruses.
[0051] A target strain of a microorganism is isolated by techniques
which are known in the art. The target strain may then be
identified or otherwise analyzed by known processes.
Virus-resistant members of the target strain are then isolated by
culturing the target strain in a medium that facilitates growth, or
proliferation, of the target strain. Preferably, target strain
microorganisms are grown on a sterilized, semi-solid medium, such
as an agar. The target strain is grown in the presence of a
wild-type virus that is capable of infecting the former. The
concentration of the wild-type virus depends upon the desired MOI.
Preferably, the relative concentrations of target strain
microorganisms to wild-type viruses are about one-to-one, for an
MOI of about one. Due to their ability to resist infection by the
wild-type virus or otherwise survive a virus infection, some of the
target strain microorganisms will grow in the presence of the
wild-type virus. Such microorganisms are referred to as wild-type
virus-resistant microorganisms, and will grow on the agar as
"colonies." Thus, wild-type virus-resistant microorganisms may be
isolated in the form of colonies by culturing target strain
microorganisms in the presence of a wild-type virus that will
infect, or is specific for, the target strain.
[0052] H-mutant viruses may then be generated and isolated by
transferring a sample of the wild-type virus-resistant
microorganism from a "colony" on agar, to a liquid or semi-solid
growth medium that includes a high concentration of wild-type
viruses. Thus, the MOI is preferably greater than one. The
concentration of wild-type virus-resistant microorganisms will
preferably facilitate growth of a confluent layer, which is also
typically referred to as a "lawn," in a semi-solid growth medium.
Although many of the viruses will have no effect on wild-type
virus-resistant microorganisms, some mutants will infect and lyse
the virus resistant microorganisms. These viruses are the
h-mutants, and are isolated within substantially transparent areas
of the lawn, which are typically referred to as "plaques."
[0053] The processes of isolating virus-resistant target strain
microorganisms and generating, selecting, and isolating h-mutant
viruses may be repeated in order to increase the range of
virus-resistant microorganisms of a target strain that the h-mutant
viruses will infect. Such a process may be performed by growing
virus-resistant microorganisms in the presence of h-mutant viruses
rather than wild-type viruses. Alternatively, various h-mutants
with different host ranges may be generated and isolated by
conducting these processes several different times.
Screening for Undesirable Genes
[0054] After the viruses have been isolated, the presence or
absence of undesirable genes (e.g., genes for virulence factors,
toxins, and antibiotic resistance) may be determined by comparison
techniques that are known to those in the art, such as conventional
agarose gel electrophoresis, pulsed-field gel electrophoresis, or
use of nucleic acid hybridization probes. Such techniques include
hybridization of any undesirable genes with complementary
polymerase chain reaction (PCR)-amplified strands of DNA which
include known undesirable genes (e.g., genes that impart the virus
with the ability to infect multicellular organisms, the ability to
transfer undesirable genes to infected host microorganisms, and the
ability to convert from a lytic state to a temperate state).
Hybrids may then be detected by known techniques, such as
radio-assays.
[0055] As an example of such comparative screening, since the
viruses of the present invention include only lytic viruses,
temperate viruses will be screened by comparing the genes of these
viruses to known genes that impart viruses with temperate
characteristics. Temperate h-mutant viruses may then be excluded
from virus mixtures of the present invention and from use in
treatment methods of the present invention.
[0056] Temperate wild-type viruses may be screened and excluded in
similar fashion from viruses and virus mixtures of the present
invention. As previously identified, temperate viruses may transfer
undesirable characteristics to a host target strain of a
microorganism. Moreover, temperate viruses do not readily destroy
the target strain microorganism. Thus, the use of temperate viruses
in controlling, reducing or eliminating microorganism populations
is not as desirable as the use of lytic viruses for these
purposes.
[0057] The virus-resistant microorganisms that are generated in the
foregoing process may be screened for other undesirable
characteristics, such as antibiotic resistance, in a similar
fashion.
[0058] Following screening for undesirable genes, viruses of the
present invention which lack undesirable characteristics may then
be proliferated and utilized in virus mixtures of the present
invention, and in accordance with methods of the present
invention.
Proliferating H-Mutant and Wild-Type Viruses
[0059] A process for proliferating the viruses of the present
invention includes growing large quantities of the target strain
microorganisms, including one or more virus-resistant variations
thereof. The desired virus or viruses, such as one or more h-mutant
variations or one or more wild-type variations of each desired
virus, are then introduced into the presence of the target strain
of microorganism at a desired MOI. An exemplary growth chamber
comprises a bioreactor, into which nutrients may be continually
introduced and from which microorganisms and/or viruses may be
continually removed. The virus or viruses may also be proliferated
in sterilized liquid growth medium in large flasks, or otherwise as
known in the art.
Concentrating and Storage of H-Mutant Viruses, Wild-Type Viruses
and Virus-Resistant Microorganisms
[0060] The viruses or virus mixtures may be concentrated by methods
that are known in the art, such as chemical precipitation and
ultrafiltration. Another method of concentrating h-mutant viruses
includes isolating and concentrating infected, non-lysed target
strain host microorganisms, which are referred to as "carriers."
The use of carriers is desirable because a single carrier will
eventually be lysed by viruses growing therein, and during lysis
release a large number of viruses. In addition, it is easier to
concentrate carrier microorganisms by conventional methods, such as
centrifugation, than it is to concentrate viruses by many
conventional methods. Preferably, carriers are avirulent variations
of the target strain microorganism, so that little or no risk
exists of introducing a virulent target strain into a virus
treatment site.
[0061] Viruses and carrier microorganisms of viruses may be stored
as known in the art (e.g., by refrigeration at about 4.degree. C.,
freezing or lyophilization processes) prior to use in the process
of the invention. Alternatively, the viruses and carriers including
the viruses of the present invention may be employed in accordance
with a process of the present invention and/or concentration
thereof.
[0062] The virus-resistant microorganisms of the present invention
may be concentrated and/or stored in a manner that is similar to
the processes for concentrating and storing the viruses.
[0063] Stored or unstored viruses and virus mixtures, and
virus-resistant microorganisms may then be utilized in accordance
with the microorganism population control processes of the present
invention, examples of which are set forth in detail below.
Methods of Microorganism Population Control
A. Use of Virulent Viruses to Control Microorganism Populations
[0064] A first embodiment of the inventive method includes
employing viruses or virus mixtures of the present invention to
control populations of target strain microorganisms. This first
embodiment includes introducing the inventive viruses into a
treatment site in order to lyse target strain microorganisms.
[0065] Foods or food products, such as raw meat and poultry, are
exemplary treatment sites. The undesirable microorganisms that are
typically present in raw meat and poultry treatment sites include,
without limitation, the genera Salmonella, Campylobacter, and
Escherichia (e.g., E. coli.) An exemplary target strain of E. coli
is the infamous strain designated O157:H7. Introducing viruses that
will infect and lyse undesirable microorganisms into raw meat and
poultry treatment sites includes, but is not limited to,
introducing the viruses into food and water of live animals,
applying viruses to the living spaces of such animals, applying and
otherwise introducing viruses to animal carcasses, meat, and
surfaces in meat packing plants, storage and transportation
containers, markets, and homes. Applying viruses to meat and
poultry reduces or eliminates populations of undesirable
microorganisms, which are thought to reduce or eliminate the
incidences of disease and food spoilage caused by such
microorganisms. Similarly, vegetation and other food products may
be treated with the inventive viruses to control an increase in
populations of undesirable microorganisms thereon.
[0066] Another exemplary treatment site into which the viruses may
be introduced includes living animals (such as mammals, e.g.,
humans), or "subjects." The inventive viruses may be employed in
the prevention (i.e., prophylaxis) or treatment (i.e., therapy) of
diseases that are caused by a target strain of microorganism.
Treatment and prophylaxis both include introducing the viruses into
the subject by a known method. The viruses are preferably orally
administered by a known enteral dosage form. The viruses may be
topically administered in various known forms, such as aerosols,
liquids, creams, lotions, soaps, powders, and salves. The viruses
of the present invention may also be administered in accordance
with processes that are known in the art, such as those disclosed
in WO95/27043, the disclosure of which is hereby incorporated by
reference in its entirety.
[0067] While being used in therapy of microbial infections, the
viruses of the present invention may be introduced alone or in
combination with one or more antibiotics, which are also referred
to herein as "antimicrobial agents" or "bacteriocins." The term
"bacteriocin" was coined for antibacterial agents that are
synthesized by bacteria and require specific receptors on the
target microorganism. Various antibiotics and other antimicrobial
agents are known in the art (see, e.g., Handbook of Antimicrobial
Therapy, The Medical Letter (1984), the disclosure of which is
hereby incorporated by reference in its entirety). The viruses of
the present invention are especially useful for preventing
infection by and treating antibiotic-resistant strains of
bacteria.
[0068] The inventive viruses may also be employed to disinfect a
target strain of a microorganism from an object. In disinfection, a
composition including the viruses is applied to the object and the
viruses lytically infect the target strain. Exemplary objects which
may be disinfected in this manner include, but are not limited to,
infected areas of healthcare facilities, operating rooms and
treatment rooms in healthcare facilities, and equipment that is
used by healthcare professionals.
[0069] Plant diseases that are caused by microorganisms may also be
treated in accordance with this first embodiment of the method. The
viruses that will infect target strains of plant disease-causing or
harmful, e.g., ice-nucleation, microorganisms may be applied to
infected or contaminated plants, seedlings, seeds, or soil or other
matter which supports the foregoing by spraying or introduction
into the plant's water supply. As an example of the treatment of
plants, legume seed may be treated with a virus or virus mixture
that will infect and lyse undesirable strains of rhizobia that are
present in soil, and that will not infect beneficial strains of
rhizobia. The virus, which preferably includes an h-mutant virus,
will reduce or eliminate undesirable rhizobia strains, while the
desirable rhizobia strains will benefit the plant as the plant
grows.
[0070] The first embodiment of the method of the present invention
may also be employed to control microorganism populations that are
detrimental to the environment. As an example, the inventive
viruses may be employed to reduce populations of microorganisms
which deplete oxygen from bodies of water, and permit an increase
of oxygen levels in these waters. Nutrients from sewage and
fertilizer that are introduced into pond water, river water, or sea
water can create algal blooms. The algae eventually die and are
then decomposed by various microorganisms, which proliferate and
continue decomposing the dead algae. During proliferation of such
microorganisms, oxygen is depleted from the water, which inhibits
growth of most other organisms therein. The introduction of viruses
that infect and lyse specific algae species which may form blooms
at a particular site would control the populations of such algae
microorganisms and, therefore, the formation of algal blooms,
thereby permitting oxygen levels in the water to increase, and
facilitating reintroduction of other types of life into these
previously oxygen-depleted treatment sites.
[0071] As another example of this method, viruses of the present
invention may be employed to reduce the occurrence of "acid mine
drainage," which is an environmental problem associated with coal
mining. Thiobacillus ferrooxidans, a bacterial species that
oxidizes iron sulfide, is a major cause of acid mine drainage. As
acidic mine drainage pollutes the water in nearby lakes, rivers and
streams, the quality of these waters deteriorates. Acid and metals
that are dissolved in acid mine drainages are toxic to aquatic life
and render the water unsafe for consumption and human activity. The
introduction of viruses that will infect and lyse T. ferrooxidans
would therefore be useful in reducing or eliminating this type of
bacteria from coal mines, and reduce the occurrence of acid mine
drainage.
[0072] The control, reduction, and elimination of pathogenic agents
is another example of this method of the invention. Exemplary
pathogenic agents include, but are not limited to, various types of
bacterial (e.g., Bacillus anthracis, Salmonella typhi, Vibrio
cholerae, Yersina pestis, Xanthomonas albilineans, A. campestris
pv. citri, and X. campestris pv. oryzae) rickettsial (e.g.,
Coxiella burnetii and Rickettsia prowazeki), and fungal organisms.
The dissemination of inventive viruses that infect and lyse such
pathogenic agents into treatment sites where such pathogenic agents
are present would control, reduce, and potentially eliminate
populations of such pathogenic agents.
[0073] The first embodiment of the process of the present invention
may also be employed to selectively control, reduce or eliminate
populations of undesirable microorganisms that inhibit the ability
of beneficial microorganisms to perform beneficial processes. As
those of skill in the art are aware, several types of
microorganisms, which are referred to as beneficial microorganisms
or beneficial agents, benefit their hosts. The ability of a
beneficial microorganism to benefit its host may, however, be
interfered with by an undesirable microorganism. A preferred virus
or virus mixture that would be useful in treating a target strain
of undesirable microorganisms in accordance with the first
embodiment of the process would infect and lyse the target strain,
and would not infect or lyse any of the beneficial
microorganisms.
[0074] Similarly, the bioremediation of toxic chemicals by
beneficial microorganisms may be interfered with by undesirable
target strains of microorganisms. For example, pseudomonads, which
produce a variety of antimicrobial substances, may be present in a
mixture of bioremediating microorganisms. The presence of
antimicrobial substances in such mixtures, however, is undesirable
since it may destroy the ability of many of the microorganisms to
bioremediate toxic chemicals. Accordingly, the viruses of the
present invention would be useful in the present method for
controlling the number of undesirable antimicrobial-producing
microorganisms in such a mixture.
[0075] Other microorganisms are beneficial for some purposes, but
may be detrimental in other regards. One such microorganism, P.
aeruginosa occurs naturally in soil, and is useful in the
bioremediation of many environmental pollutants. P. aeruginosa,
however, also causes various diseases in plants and animals. Thus,
this method of the invention would be useful for controlling,
reducing, or eliminating the population of P. aeruginosa after that
microorganism has performed its beneficial task.
[0076] Populations of genetically engineered microorganisms may be
controlled, reduced, or eliminated in a similar manner. Since many
people fear that the use of genetically engineered microorganisms
for beneficial purposes may also have adverse effects, the
elimination of such genetically engineered microorganisms may be
desirable. Thus, viruses that infect and lyse a target strain of
genetically engineered microorganism may be utilized in accordance
with this embodiment of the invention to control, reduce, or
eliminate populations of genetically engineered target strain
microorganisms from a treatment site following their use for
beneficial purposes.
B. Methods of Using Virus-Resistant Microorganisms to Control
Microorganism Populations
[0077] As described previously, many beneficial microorganisms, or
"beneficial agents," perform beneficial processes. Such
microorganisms, however, are susceptible to being infected and
lysed by viruses. Accordingly, a second embodiment of the method of
the present invention includes the use of virus-resistant strains
of beneficial microorganisms in beneficial processes.
[0078] Virus-resistant microorganisms are generated, as previous
described, by growing a target strain of microorganism in the
presence of wild-type and/or h-mutant viruses that will infect and
lyse the target strain microorganism. Virus-resistant
microorganisms may then be isolated as discussed previously, and
proliferated in a growth medium under otherwise substantially
sterile and preferably controlled conditions.
[0079] As an example of the use of the second embodiment of the
process, virus-resistant Pantoea ananus, which is parasitic for the
rust fungi, Puccinia spp., is useful for controlling the growth of
rust fungus on wheat. Phages which attack P. ananus are, however,
also present in proximity to the rust fungus, and have a
detrimental effect on the ability of P. ananus to control rust
fungus. Accordingly, the application of virus-resistant strains of
P. ananus to wheat would be useful in controlling the growth of
rust fungus on the wheat. Similarly, the application of
virus-resistant P. ananus to rust fungus-infected wheat would be
useful for preventing the spread of rust fungus to other wheat
plants, and for treating the rust fungus-infected wheat plants.
[0080] Similarly, some bacteria, such as Serratia entomophilia,
control the proliferation and spread of insect populations, such as
New Zealand grass grub, or Costelytra zealandica, and may be
applied to treat plants or to prevent the spread of such insects to
other plants. Other virus-resistant bacteria used for biological
pest control, such as Enterobacter aerogenes for locusts, are also
useful in the second embodiment of the process of the present
invention.
[0081] Other beneficial microorganisms are useful for performing
processes which include, without limitation, leaching in order to
oxidize the sulfide of sulfide-rich minerals to sulfuric acid so as
to liberate and concentrate valuable minerals, such as copper and
uranium, from low-grade ores (i.e., T. ferrooxidans and
Acidiphilium spp.); releasing petroleum and related substances from
bituminous shale (e.g., Rhodococcus spp., Sulfobus spp., and/or
Thiobacillus spp.); acidifying sulfur or other matter to acidify
alkaline soils that have been selected for agricultural uses (e.g.,
Rhodococcus spp., Suljobus spp., and/or Thiobacillus spp.);
decomposing tires and other rubber products for recycling (e.g.,
Rhodococcus spp., Sulfobus spp., and/or Thiobacillus spp.);
bioremediation of hazardous chemicals and pollutants (e.g.,
Rhodococcus spp., Sulfobus spp., and/or Thiobacillus spp.);
treatment of wastewater discharges and sludges in which other
microorganisms produce foul-smelling odors (e.g., from dairy and
hog farms, kennels, farms, etc.); and in industrial fermentation
processes (e.g., lactic acid bacteria for the production of
cheese). The use of virus-resistant microorganisms in such
processes in accordance with the second embodiment of the process
of the present invention reduces the likelihood that the beneficial
microorganisms will be infected or lysed by viruses specific
therefor.
[0082] Although the invention has been described with regard to
certain preferred embodiments, the scope of the invention is to be
defined by the appended claims and their legal equivalents.
* * * * *