THE DISEASE-CAUSING ORGANISMS BURKHOLDERIA MULTIVORANS, B. CEPACIA, GENOMOVARS OF THE B. CEPACIA COMPLEX (BCC), AS WELL AS AN UNCLASSIFIED SPECIES OF BURKHOLDERIA, AND ITS RELATIVES RALSTONIA PICKETTII AND R. INSIDIOSA ACCOUNT FOR 60% OF THE BACTERIAL ISOLATES OBTAINED FROM THE FILTRATED WATER SYSTEM ON BOARD THE INTERNATIONAL SPACE STATION (ISS). MEMBERS OF THESE GENERA CAN SURVIVE LONG PERIODS IN A PLANKTONIC STATE IN DISTILLED WATER OR AS BIOFILMS WITHIN AN INFECTED INDIVIDUAL. THEY ARE ALSO NATIVELY RESISTANT TO SEVERAL ANTIBIOTICS LEAVING TREATMENT OPTIONS COMPLEX. FOR BCC ORGANISMS, THIS COMPLEXITY IS INCREASED BY THE PHENOMENON OF BACTERIAL PERSISTENCE WHERE A GENETICALLY IDENTICAL SUBPOPULATION OF THE BACTERIA SUSTAINS UNDER HIGH ANTIBIOTIC CHALLENGE BY A REDUCTION IN METABOLISM AND OTHER UNKNOWN MECHANISMS. A HEALTHY ASTRONAUT COULD EASILY CLEAR A BCC OR RALSTONIA SP. INFECTION; HOWEVER, AFTER PROLONGED SPACEFLIGHT, A POTENTIALLY IMMUNOCOMPROMISED ASTRONAUT WITH REPEATED EXPOSURE TO THESE ORGANISMS COULD BE AT RISK FOR DISEASE. IN OUR WORK WITH BURKHOLDERIA PSEUDOMALLEI -THE CAUSATIVE AGENT OF MELIOIDOSIS- WE HAVE OBSERVED BY MEANS OF TRANSPOSON SEQUENCING (TNSEQ), THAT A COMMON SET OF GENES HELP THE WILDTYPE BACTERIA PERSIST REGARDLESS OF THE MODE OF ACTION OF THE ANTIBIOTIC USED FOR CHALLENGE. WE HAVE FOUND A CYSTEINE SYNTHASE GENE, WHOSE INHIBITION COULD ENHANCE EXISTING ANTIBIOTICS BY INTERFERING WITH THE BACTERIASPECIFIC SULFUR ASSIMILATION AND CYSTEINE SYNTHESIS PATHWAYS. IT IS LIKELY THAT METABOLIC REGULATION PLAYS A ROLE IN THE ABILITY FOR BCC AND RALSTONIA STRAINS TO PERSIST IN THE ISS POTABLE WATER IN A PLANKTONIC STATE. IT IS ALSO PROBABLE THAT THE SAME MECHANISMS WHICH INCREASE SURVIVORSHIP CAN IN TURN ENHANCE VIRULENCE AND LEND TO PERSISTENT BIOFILMS IN THE GASTROINTESTINAL OR RESPIRATORY TRACT OF THE INFECTED INDIVIDUAL. TO UNDERSTAND THE MECHANISMS THAT WOULD CONTRIBUTE TO ALTERED VIRULENCE IN ISS BCC AND RALSTONIA SPECIES, THE GENOMES OF 67 ISOLATES FROM 22 MISSIONS WILL BE SEQUENCED AND COMPARED TO THEIR RESPECTIVE ATCC BCC OR RALSTONIA STRAIN. THE POTENTIAL FOR INCREASED VIRULENCE WILL BE DETERMINED BY THE PRESENCE OF GENES CLUSTERED AS PATHOGENICITY ISLANDS IN THE RESPECTIVE GENOME. TO ESTABLISH IF THE STRAINS ARE BECOMING MORE RESISTANT TO ANTIBIOTICS, A MINIMUM INHIBITORY CONCENTRATION (MIC) WILL BE DETERMINED FOR EACH ISOLATE USING SIX ANTIBIOTICS CURRENTLY USED TO TREAT THE INFECTIONS AND THEN COMPARED TO THE RESPECTIVE ATCC CONTROL STRAIN. FINALLY, A TRANSPOSON LIBRARY WILL BE GENERATED FOR THE MOST VIRULENT, RESISTANT, TOLERANT OR PERSISTENT STRAIN, AND THIS LIBRARY WILL BE CULTURED IN DISTILLED WATER TO SIMULATE CONDITIONS ON THE ISS. HERE WE WILL IDENTIFY GENES WHICH AID IN THE SURVIVORSHIP OF BCC AND RALSTONIA STRAINS IN THE WATER SYSTEM OF THE ISS. THE KNOWLEDGE GAINED FROM THIS GENOMIC SEQUENCING AND TNSEQ STUDY WILL ALLOW US TO ASSESS ANY INCREASED VIRULENCE OR INFECTION POTENTIAL, AS WELL AS HELP TO MAKE ANTIBIOTIC RECOMMENDATIONS, PREDICT TARGET GENES WHERE INHIBITION BY ADJUVANTS WILL ENHANCE ANTIBIOTIC THERAPY, OR PRESENT NOVEL WATER TREATMENT OPTIONS TO ERADICATE THE BCC/RALSTONIA STRAINS FROM THE POTABLE WATER OF THE ISS.