Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-07-20
2003-04-08
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S004000, C435S006120, C435S007200, C435S007320, C435S008000, C422S082080
Reexamination Certificate
active
06544729
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of The Invention
The invention pertains to methods and devices for detecting targeted microorganisms such as bacteria by inducing bioluminescence in bioreporter cells. Genetically engineered bacteriophage are employed to infect target bacteria in the presence of genetically engineered bioreporter cells. The bioreporter cells respond by producing light upon stimulation by an inducer. The inducer is produced as a result of infection of the target bacteria by the bacteriophage.
1.2 Description of the Related Art
Current technology focusing on the development of biologically-based detection systems has prompted efforts to address the need for methods for detecting specific microbial pathogens. Numerous methods for determining the presence of microbial contaminants have been used over the years; typically, culture methods were employed in the past but these methods were slow and inefficient. Recent developments in bioreporter technology have prompted use of genetically engineered bacteria or bacteriophage to identify toxic chemical compounds, and, in some cases, to identify particular species of bacteria.
Bioreporters are genetically engineered organisms designed to detect specific compounds by incorporating a gene responsive to a selected external compound, for example by using a heterologous promoter responsive to a target compound where the promoter then induces expression of a detectable gene product in the bioreporter cell. Bioluminescent bioreporters, as used in the present context, are genetically engineered bacteria incorporating genes that when expressed result in bioluminescence. Upon detection of a specific compound, the bioreporter cell responds by producing light. A popular gene for this purpose is the lux gene. Under proper conditions, the lux genes are expressed and the subsequent bioluminescence is detectable by any of a variety of optical methods. Many of the constructs incorporated in bioluminescent bioreporter organisms derive from the bioluminescent marine bacterium
Vibrio fischeri
(King et al., 1990).
Sayler et al. (1998) have described bioluminescent bacterial-based bioreporters that respond to specific compounds via the production of visible light. A variety of lux-based bacterial bioreporters has been used to detect and monitor naphthalene (Heitzer et al., 1994), BTEX (benzene, toluene, ethylbenzene, and xylene) (Applegate et al., 1998), polychlorinated biphenyls (PCBs) (Layton et al., 1998), 2,4-dichlorophenoxyacetic acid (2,4-D) (Hay et al,. 2000), ammonia (Simpson et al., 2001), and the food spoilage indicator chemical &bgr;-phenylethylamine (Ripp et al., 2000a).
Genetic constructs for imparting bioluminescence to bacterial bioreporter cells have generally employed a lux gene cassette derived from the marine bacterium
Vibrio fischeri
(Engebrecht, et al., 1983). As used herein, “cassette” refers to a recombinant DNA construct made from a vector and inserted DNA sequences. The complete lux cassette consists of five genes, i.e. luxA, B, C, D and E. LuxA and luxB encode the proteins that are responsible for generating bioluminescence while luxC and D encode an aldehyde required for the bioluminescence reaction.
The light response generated by bioluminescent bioreporters is typically measured with optical transducers such as photomultiplier tubes, photodiodes, microchannel plates, or charge-coupled devices. Some means of transferring the bioluminescent signal to the transducer is required, which necessitates the need for fiber optic cables, lenses or liquid light guides. Such instruments are generally unsuitable for field use. What typically results is a large, bulky instrument anchored to power and optic cables. For example, in field release experiments described by Ripp et al. (2000b), a bioluminescent bioreporter for the detection of naphthalene was used for monitoring of polyaromatic hydrocarbon degradation in soil. Bioluminescent signals were detected using a multiplexed photomultiplier tube linked to a network of fiber optic cables that proved to be expensive, fragile, and cumbersome to work with.
Battery-operated, hand-held photomultiplier units that may be interfaced with a laptop computer have been described and used in conjunction with bioreporters for field analysis of hydrocarbon contamination in groundwater (Ripp, et al., 1999a). Special bioluminescent bioreporter integrated circuits (BBICs) have been reported (Simpson, et al., 2001) and these self contained units have been shown to detect environmental contaminants such as naphthalene and BTEX by simply exposing the BBIC device to samples containing these compounds (Ripp et al, 1999b). The bioluminescent bioreporters utilized in these devices are genetically modified bacterial bioreporters that respond to specific chemicals in the environment via production of visible light.
Detection of pathogenic organisms, as opposed to chemical agents, is another area of current interest. Pathogens such as those causing human and animal diseases, foodbome pathogens and those used in biological warfare are of great significance for the safety of human populations. Furthermore, the continual appearance of new strains of bacteria underscores the need for sophisticated detection systems.
In the food industry as an example, microbial contamination of fresh fruits and vegetables has become a mounting concern during the last decade due to an increased emphasis of these products in a healthy diet and the recognition of new foodborne pathogens such as
Campylobacter jejuni, Escherychia coli
O157:H7, and
Listeria monocytogenes
(Tauxe, 1992). Federal agencies have published recommended safe food handling practices for minimizing risk; however rapid, real-time methods for detection of pathogens in the production, processing, and distribution systems are not yet available. Of particular concern in monitoring food safety is the need to identify the bacteria that cause the majority of food-related deaths in the United States, including Salmonella,
Listeria monocytogenes, Escherychia coli
O157:H7 and Campylobacter.
Bioluminescent methods to determine bacterial contamination are currently in use in the food industry. One technology, based on detection of ATP, relies on the biochemical requirement of all bacteria to utilize ATP for the energy production that is necessary for survival and growth. Unfortunately the ATP detection method is non-specific in nature; thus it does not differentiate among bacterial species nor does it distinguish non-pathogenic bacteria from pathogens that pose significant health risks (Vanne, et al., 1996).
Several reports have documented bioluminescent detection of a target bacterium using bacteriophage infection. Table 1 summarizes select pathogens that have been detected by these procedures.
TABLE 1
Bioluminescence detection of bacterial pathogens by
bacteriophage containing a luxAB insert.
Pathogen
Bacteriophage
Detection Limit
Test Source
Reference
Enterobacteriaceae
Unspecified
10 cells/g/cm
2
Surface and meat
Kodikara
carcass swabs
et al.,
1991
Escherichia coli
&lgr; Charon
100 cells/ml
Milk
Ulitzer and
species
Kuhn,
1987
Escherichia coli
&phgr; V10
Not determined
Pure culture
Waddell
O157:H7
and
Poppe,
1999
Listeria
A511
10 cells/g
Cheese, pudding,
Loessner
monocytogenes
cabbage
et al.,
1996
Salmonella
P22
10 cfu/ml
Eggs
Chen and
species
Griffiths,
1996
Salmonella
P22
100 cells/ml
Pure culture
Stewart et
typhimurium
al., 1989
Staphylococcus
Unspecified
1000 cfu/ml
Pure culture
Pagotto et
aureus
al., 1996
In all of these cases, the bacteriophage contained only an incomplete lux gene, i.e. luxAB. While useful in detection of some pathogenic species, the technique suffers from several disadvantages. When only the luxAB genes are employed, an exogenous source of the aldehyde substrate for the luciferase reaction is required for detection of the bioluminescent response. This can raise problems with detection. Moreover, there are further difficulties because conditions such as the amount of added inducer may have to be adjusted. This is particularl
Applegate Bruce
Ripp Steven A.
Sayler Gary S.
Akerman & Senterfitt
Housel James
Lucas Zachariah
University of Tennessee
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