Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-08
2004-04-20
Souaya, Jehanne (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C536S024320
Reexamination Certificate
active
06723505
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable
BACKGROUND OF THE INVENTION
The present invention relates generally to methods for the identification of microorganisms in liquid or liquified samples, and more particularly, but not by way of limitation, to methods using DNA for the identification of microorganisms which are indicators of contamination in liquid or liquified samples.
BRIEF DESCRIPTION OF THE ART
Most water-borne human pathogens cause infections and human disease via ingestion of fecal contaminated water or food. Various human parasites and pathogens are transmitted via human fecal contamination of water used for drinking, bathing, recreation, or washing/preparation of foods. To ensure good public health, there is a need for readily available methods to detect and enumerate pathogens in water.
While the presence of pathogens in water presents a significant public health concern, recovery of pathogens from environmental samples is generally difficult. Many fecal pathogens are infective at densities so low that water sample collection and concentration is inconvenient. Also, unpredictable physiological and morphological changes are observed in these pathogens in response to nutrient limitations and environmental stressors, and these injuries cause the organisms to exhibit atypical reactions and require specialized handling for their resuscitation. In addition, viable but unculturable organisms may be present in the water sample.
The methods commonly used to detect these pathogens were initially designed for clinical, rather than environmental, samples. However, clinical isolates are usually provided an ideal environment in which needed nutrients as well as protection from harsh environmental conditions, such as cold, heat, damaging chemicals and radiation, are readily supplied to the isolate. In contrast, environmental isolates are exposed to harsh, environmental conditions and effectively compete with organisms naturally present and adapted to life in the environment.
Pathogenic organisms are rarely readily adaptable to prolonged survival in the environment. For this reason, fecal microbial water contamination often is assessed by testing for harder and more robust, but not necessarily pathogenic, microbes, referred to as indicator organisms, such as the coliforms, especially
Escherichia coli,
and Enterococcus species. Indicator organisms serve to indicate whether a given water supply may be generally contaminated with fecal material without actually testing for the presence of all enteric pathogens. This contamination is viewed as predictive of the potential presence of enteric pathogens (i.e., without the presence of fecal material, the chances of these indicator organisms being present is usually remote).
Criteria for the establishment of the “ideal” indicator include the following factors: (1) the indicator should always be present in the presence of pathogens; (2) the indicator should always be present in a predictable ratio with pathogenic organisms; (3) the indicator should be specific for fecal contamination; (4) the indicator should be able to resist water treatment and disinfection processes to the same or a slightly greater extent than the pathogens; and (5) the indicator should be detectable by simple and rapid methods.
Although coliforms have historically served as the indicator bacteria for fecal contamination in United States water supplies, the term “coliform” encompasses four genera (Escherichia, Citrobacter, Enterobacter and Klebsiella) which include many species that are commonly found in the environment in the absence of fecal contamination.
E. coli
is the only species of coliform bacteria which is consistently and exclusively found in feces, and therefore, coliform detection methods are not specific for detecting fecal contamination in a water supply. Nonetheless, regulations based on detection and enumeration of “total coliforms” are still in effect in the United States.
Tests have also been developed to detect “fecal coliforms”, a subgroup of total coliforms which are thermotolerant. However, this designation is also nonspecific, as it includes not only
E. coli,
but also various Klebsiella strains. Despite the fact that there are substantial environmental sources of Klebsiella, and this organism is infrequently found in human feces, the use of the designation “fecal coliform” as well as tests to identify these organisms remain routine.
Typically, fecal coliform identification has relied on methods for distinguishing phenotype aspects such as growth or motility characteristics, and for immunological and serological characteristics. Selective growth procedures and immunological methods are the traditional methods of choice for bacterial identification, and these can be effective for the presumptive detection of a large number of species within a particular genus. However, theses methods are time consuming and are subject to error.
Selective growth methods require culturing for one to several days in selective media, followed by subjective analysis, including various metabolic, biochemical and immunochemical tests to confirm the microbiological identities of the organisms. These methods are labor, time and supply intensive, and must be performed by an experienced investigator.
Immunological detection (e.g., ELISA) is more rapid and specific, however, it still requires growth of a significant population of organisms and isolation of the relevant antigens. The sensitivity levels of currently available ELISA tests are about 10
4
-10
5
organisms per milliliter, and organisms present at lower concentrations will not be detected; therefore, one or more culture steps are required in order to enrich the number of microorganisms present in the sample, and these culture steps are generally time-consuming. In addition, antigenic cross reactivity using these serological procedures can result in misidentification of organisms. Also, immunological detection typically does not distinguish living from dead cells.
Rapid processes for detecting fecal coliforms by growth in a medium containing a fluorogenic substrate (methylumbelliferyl galactoside) which is metabolized by the bacteria are described in U.S. Pat. No. 5,292,644 entitled “Rapid Process For Detecting Coliform Bacteria,” and U.S. Pat. No. 5,518,894 entitled “Rapid Coliform Detection System.” These processes are much less time intensive than the previously described methods, and the total amount of time required for a presence-absence result generally does not exceed seven hours.
Other methods rely on hydrolysis of methylumbelliferyl glucuronide by &bgr;-glucuronidase, such as the methods described in U.S. Pat. No. 5,429,933 entitled “Detection of First Generation Environmental Sourced Microbes in an Environmentally-Derived Sample.” However, recent findings have shown that many coliform bacteria obtained from fecal samples do not possess &bgr;-glucuronidase, which is required to metabolize this fluorogenic substrate. Further, although 97% of clinical isolates of
E. coli
were &bgr;-glucuronidase-positive, only about 66-70% of all
E. coli
obtained from fecal samples were &bgr;-glucuronidase-positive. Therefore, these processes would fail to detect a significant portion, about 30%, of
E. coli
present in the fecal samples.
Therefore, interest has turned to detection of bacterial pathogens on the basis of specific DNA sequences. Colony hybridization uses a radiolabelled unique DNA gene probe fragment isolated from chromosomal DNA of the coliform of interest to identify colonies of the organism isolated from a water sample; this technique has higher sensitivity than those described above. Methods for hybridization of oligonucleotide probes to samples for detection of an organism or a group of organisms are described in U.S. Pat. No. 5,693,469 entitled “Nucleic Acid Probes and Methods for Detecting
Escherichia coli,
” U.S. Pat. No. 5,792,854 entitled “Detection of Salmonella,” and U.S. Pat. No. 5,795,717 entitled “Oligonucleotides for Detecting Bacteria and Detect
Dunlap Codding & Rogers P.C.
Nye Colifast AS
Souaya Jehanne
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