Rapid Bacterial Testing for Spacecraft Water

New methods are urgently needed to rapidly enumerate specific viable bacteria in water and other systems. This is a particular concern in relation to water which will be reclaimed for potable use on the Space Station. Our principal objective is to further develop our methodology which permits the detection of specific marker bacteria used to monitor the performance of the water reclamation and storage systems. In addition, the techniques will be applicable to the detection of particular pathogenic bacteria. Our novel approach (U.S. Pat. No. 5,821,066), which utilizes membrane filtration and combines fluorochromes for assessment of physiological activity with specific fluorescent antibody detection of waterborne bacteria, will be evaluated in comparison with molecular methods which will be developed in this project. These include fluorescent in situ hybridization following membrane filtration and microcolony formation, to permit rapid quantitation of specific, viable bacteria. Fluorescent in situ PCR and PNA probes will also be investigated for sensitive detection of specific bacteria. Results will be applicable not only to spacecraft systems but will also have applications for earth-based situations. Similar methodologies would be of great value for the examination of clinical and fecal specimens, potable waters, natural waters, foods, and soils for more timely and reliable detection of specific microbial contamination. Other applications include the examination of purified waters used in the pharmaceutical industry, laboratories, and the electronic industry. We will develop analytical procedures to identify and quantify bacteria in wastewater and product water on spacecraft, permitting more timely measurement and control of bacterial contaminants and facilitating development of standards and countermeasures to optimize crew health, safety, and productivity.

A technique for staining bacteria in suspension and after capture on membrane filters for enumeration by a solid phase laser cytometer (ChemScan, Chemunex) has been developed and is undergoing further evaluation. It is anticipated that Chemunex will market this procedure and materials for use with their ChemScan or Scan RDI systems. Research into the use of nucleotide probes to identify bacteria and assess their metabolic status showed that the signals were too weak for detection by laser cytometry. In situ hybridization techniques did however permit detection of mRNA in whole cells using epifluorescence microscopy with a sensitive CCD camera.

The assays being developed are applicable to Earth-based systems, including the rapid examination of clinical and fecal specimens, potable and natural waters, foods, soils, and ultrapure water systems used in the pharmaceutical and electronics industries. Our experiments with disinfection and starvation, using these new methods to detect bacteria, have generated more reliable data on bacterial injury, lethality, and survival. The combination of methodological approaches for bacterial concentration, detection and viability assessment being investigated has significant benefits both immediately and in the long-term. For example, one target bacterium (E. coli O157:H7) is currently a persistent and significant food borne health threat in the U.S. Other aspects of developing this technology have been funded by the U.S. Department of Defense (Army) and the National Institutes of Health (Environmental Health Institute). U.S. Patent No. 5,821,066 was awarded in October 1998, and we have partnered with LigoCyte Pharmaceuticals, Inc., a local biotechnology development company, to bring this technique to the U.S. and international market place. Ultimately, the development of novel antibodies for bacterial detection may lead to therapeutics and possibly vaccines for the treatment of a range of enteric diseases which are caused by bacteria. It is anticipated that our new methodological approach will be applied in studies by others to determine the ecology of E. coli O157 and related bacteria in the food source animal population and their environment and, as a consequence, assist in its reduction or elimination. This technology is also applicable to the rapid detection of other target bacteria in a range of contexts including industrial, clinical, and environmental settings. We are also working with Chemunex, Inc. (New Jersey and Paris) to expand the applications of their solid phase laser scanner (Scan RDI or ChemScan). Collaboration is now being established to focus on adapting this system for spaceflight and alternative earth-based applications.