Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-05-12
2003-07-29
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S242000
Reexamination Certificate
active
06599715
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to viability detection of bacterial spores and more particularly to real time detection of viable bacterial spores. This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
Some medically important bacteria such as
Bacillus anthracis, Bacillus cereus, Clostridium botulinum,
and
Clostridium perfringens
form endospores. Currently, detection of these and other bacterial spores are typically accomplished through antibody or DNA bases systems. One shortfall of these techniques is their inability to determine whether or not the detected spores are viable. As an example, naked DNA or spore coats (which do not constitute pathogenicity) may be erroneously perceived as infectious agents.
The need for rapid microbiological viability assays is well recognized in virtually all industries. Currently, culturing is the accepted method for viability detection, yet, viability testing for bacterial spores is typically a time consuming process that requires aseptic culture techniques and extended incubation periods upwards of 24 hours. Currently, there is no real time, positive viability detection available.
Recently, U.S. Pat. No. 5,795,730 described a rapid read-out biological indicator to assess or determine the effectiveness of a sterilization process including contacting an indicator containing microbial spores with a sterilant to give exposed spores, contacting the exposed spores with a medium selected to germinate the spores and calculating a rate of germination of the exposed spores to determine the effectiveness of the sterilization. The germination rate was determined using spectrophotometric or light scattering techniques related to measuring the light absorbance or scattering of ungerminated spores. The germination medium preferably included L-asparagine or L-glutamine.
Nevertheless, a need remains for a rapid, sensitive method for determining viability of bacteria spores in real time, e.g., for determining the effectiveness of sterilization procedures. Further, such a rapid, sensitive method would enhance the safety and protection of a civilian population from a terrorist-induced panic.
Some of the most potent biological agents are spore-forming bacteria. These bacterial can be produced and maintained in a dormant state and upon entering the human body, convert into their pathogenic form. The best known bacterial warfare agent is
Bacillus anthracis,
which causes the disease anthrax. This endospore-forming microbe can survive long periods of time in a dormant (spore) form. When these spores enter the lungs through airborne dispersal, they are converted from their dormant state to a vegetative state. In the vegetative state, these microbes produce toxins that enter cells and cause them to lyse. Cell lysis leads to extensive oedema of the tissues and is fatal.
The entire process by which dormant bacterial spores become vegetative cells is called germination. The length of time required to complete such a process has been documented as taking from a matter of minutes up to 40 minutes or more. Differences in bacterial strains can affect the duration of this process, but the reasons for these differences are unknown. However, early events in germination, generally referred to as initiation, can occur within minutes when the spores come in contact with the proper environment. Events in initiation of spore germination are commitment, in which some change in the spore begins an irreversible process not fully understood but including loss of heat resistance, release of cations, dipicolinic acid (DPA) and sulfolactic acid, and loss of spore cortex, refractivity and UV resistance. Initiation may only take 5 minutes for a spore population, so detection of these events can serve as an ideal target for a real time viability monitor.
Terbium chelation and other lanthanide chelation have been used to detect free DPA in solutions, and to detect chemical germination of
B. megaterium.
U.S. Pat. No. 5,876,960 by Rosen describes the use of this assay to detect the presence of bacterial spores, but there is no suggestion regarding the viability of the spores.
It is an object of this invention to provide a method for rapid viability detection of bacterial spores.
It is another object of this invention to provide a system for such a rapid viability detection of bacterial spores.
It is still another object of the present invention to provide a germination medium capable of promoting high germination levels of bacterial spores.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, provides a process for detecting the presence of viable bacterial spores in a sample including placing a sample in a germination medium for a period of time sufficient for commitment of any present viable bacterial spores to occur, mixing said sample with a solution of a lanthanide capable of forming a fluorescent complex with dipicolinic acid, and, measuring said sample for the presence of dipicolinic acid.
The present invention further provides germination reaction mixture useful for promoting commitment of any viable bacterial spores including a combination of L-alanine, L-asparagine and D-glucose.
The present invention still further provides a system including a germination chamber having inlets from a sample chamber, a germinant chamber and a bleach chamber, said germination chamber further including an outlet through a filtering means, said outlet connected to a detection chamber, said detection chamber having an inlet from a fluorescence promoting metal chamber and said detection chamber including a spectral excitation source and a means of measuring emission spectra from a sample, said detection chamber further connected to a waste chamber.
REFERENCES:
patent: 3687815 (1972-08-01), Scharpf
patent: 5536645 (1996-07-01), Jay
patent: 5795730 (1998-08-01), Tautvydas
patent: 5800821 (1998-09-01), Acheson et al.
patent: 5876960 (1999-03-01), Rosen
CAPLUS abstract (Acc No. 1960:98190). Demain et al. (1960). Dissociation of spore germination from outgrowth by use of auxotrophic mutants ofBacillus subtilis. J. Bacteriol. 79: 783-788.*
Obiso et al, “Characterization of Bacillus Globigii Endospore Germination: A Real-Time Method of Detecting Endospore Viability,” equivalent to Abstracts of the General Meeting of the American Society for Microbiology, May 1998, vol. 98, p. 308.
Thacker et al., “A Method for the Rapid Detection and Enumeration of Viable Bacteria,” American Laboratory, pp. 20-23 (May 1998).
Rosen, “Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence,” Analytical Chemistry, vol. 69, No. 6, pp. 1082-1085 (Mar. 15, 1997).
Sacks, “Chemical Germination of Native and Cation-Exchanged Bacterial Spores with Trifluoperazine,” Applied and Environmental Microbiology, vol. 56, No. 4, pp. 1185-1187 (Apr. 1990).
Lamture et al., “Intensely Luminescent Immunoreactive Conjugates of Proteins and Dipicolinate-Based Polymeric Tb(III) Chelates,” Bioconjugate Chem. No. 6, pp. 88-92 (1995).
Herdendorf Timothy J.
Obiso Richard J.
Vanderberg Laura A.
Cottrell Bruce H.
Gitomer Ralph
The Regents of the University of California
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