Crew health is a dominant issue in manned space flight. Microbiological issues in particular have repeatedly emerged as determinants of flight readiness. For example, in at least one case, suspected contamination of the potable water supply nearly forced a launch delay. In another instance a crew member's urinary track infection nearly led to early termination of the mission, in part due to the difficulty of accurately diagnosing the nature of the infection in-flight. In both cases traditional microbiological screening would not be sufficient to address the problem. In the first case the need for information arose just hours before scheduled launch. Hence, time to conduct traditional tests was not available. With respect to in-flight studies it is of course obvious that it is not be possible to operate a traditional microbiology laboratory in space. We propose to address these problems by using novel versions of the rRNA-targeted DNA probe diagnostics which are revolutionizing clinical identification of difficult bacterial pathogens in terrestrial medicine.

Ribosomal RNA sequences have been experimentally determined in several thousand bacterial species. Each of these sequences contains short sub-sequences that are totally preserved throughout the data set as well as other sub-sequences which are totally unique to, and hence characteristic of, a particular species. This pattern of sequence conservation makes it possible to design oligonucleotide hybridization probes that can distinguish individual organisms or groupings of historically related organisms. Practical diagnostic kits, based on this technology e.g. for Legionella and Chlamydia, have been successfully introduced into the market place. These single organism diagnostics rely on the natural amplification associated with ribosomal RNA (as many as 10,000 copies per cell), to provide a probe target and use manual detection procedures based on either chemiluminescence or fluorescence. In order for this technology to be of value during space flight applications it must simultaneously detect many organisms of interest, be subject to miniaturization and be highly automated. An especially promising format for doing this is being investigated by the UH PI's in conjunction with Genometrix Inc. The essence of the idea is to immobilize an array of unique oligonucleotide probes for each organism of interest on a solid support. Each probe in the array will be designed to be specific for a particular organism or organism group of interest. All probes are simultaneously exposed to the same sample. Since the location of the probes on the grid is known, the location of the hybridization signal on the grid will serve to identify which probes are responsible for the signal This information will be obtained by straightforward computer imaging and processing.

The design of individual hybridization chips is governed by the choice of organisms that need to be distinguished. It is anticipated that an initial in-flight system will monitor compliance with standards established by NASA microbiologists. Currently, microbiological data collected from recent missions flown by the United States and Russia are being analyzed. Recommendations currently exist for allowable microbial load in air, water, and surfaces and these are subject to refinement as more knowledge is gained. For example, the water standard currently specifies no more than 100 colony forming units per 100 ml of water with no coliform bacteria present. In general the level of certain organisms that have caused problems in prior space missions might be monitored as well. Thus, in addition to a total bacteria probe and a coliform specific probe one would likely want specific probes for Legionella, Streptococcus pyrogenes, Staphylococcus aureus, and Enterooccus. A small number of eukaryotic fungi will also be of interest. Candida albicans has frequently been problematic and an extended system would likely include Aspergillus fumigatus and Crptococcus neoformans as well. In designing probes for these particular organisms or genera a conservative strategy will be employed. Rather than attempting to distinguish the known pathogen from all very closely related species and strains, an indicator approach will be taken. A well resolved cluster of organisms will be identified in the existing rRNA sequence data which contains the organism or species of special interest. Potential probes are then identified that distinguish that small cluster of organisms from all others.

Before incorporation into chips, each probe is being separately evaluated in collaboration with a group at the Baylor College of Medicine under the direction of Dr. Michael Hogan to verify that it performs the desired discrimination. Cells from a representative member of each target group are being grown and ribosomes are being prepared from which pure preparations of the target rRNA can be obtained. The probes are then assayed using a sandwich assay system in which biotinilated capture probes are immobilized onto a streptavidin plate. In this manner, it is possible to determine which probes are most promising for use with a particular organism group. In the final design, it will be essential that the various probes do not cross hybridize with each other or with the capture probes. A computer comparison of the various probes will be undertaken in order to determine which combination of probes is most likely to work in a mixed probe system. These will again be tested in the streptavidin system.

Another major project component is sample preparation. Laboratory experiments are being done on highly purified RNA samples or mixed RNA samples. Although the instrumentation to be used with the chip based assay is likely to be compatible with in-flight needs it is clear that the standard RNA preparation procedures will not be. The restrictions on in-flight experimental work are severe, as one is greatly restricted in terms of power, auxiliary equipment availability, expertise of the astronauts, and mission time. Existing procedures can be used to collect samples from the air and water supply. However, in order to utilize an automated hybridization system in a space craft environment a very simple procedure for RNA extraction from the cell material will need to be devised. In the low gravity space environment, liquid handling capability is largely restricted to what can be accomplished with syringe based systems. Thus, a promising approach which is being explored is one in which the collected cells are chemically lysed and the ribosomal RNA is concentrated from the cell extract by passage through a packed column. Boronate chemistry is being explored for this purpose.

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