Q-231. Novel Francisella Strains Isolated from Natural Water Sources in Utah

K. E. Kesterson1, C. A. Whitehouse2, M. Wolcott2;
1Dugway Proving Ground, Dugway, UT, 2USAMRIID, Fort Detrick, MD.

Utah is the source of the type species, Francisella novicida and Francisella philomiragia. In 2001, a F. tularensis novicida-like organism (UT 01-4992) was isolated from a patient with probable exposure being Wasatch Warm Springs just north of Salt Lake City. The objective of this study was to sample the probable exposure site and a similar site for the presence of F. novicida. Two collections, six months apart, were made from each spring. Sediment, water, pond scum, and vegetation were collected aseptically. Each sample was inoculated onto cystine heart blood agar containing antibiotics and chocolate agar, and then incubated at 37 oC for 48 h. Colonies with the gross appearance of Francisella sp. were selected for characterization and identification using microscopy, BIOLOGTM, 16S rRNA ribotyping, MIDI, PCR/ESI-MS, and VNTR. All 21 isolates were tiny gram-negative coccobacilli. BIOLOGTM identified ten as F. philomiragia; the identities of the remaining 11 isolates were inconclusive. Ribotyping of the isolates using EcoR1 and PvuII restriction enzymes did not yield conclusive identifications but cluster analysis of the patterns generally matched those seen with a reference set of Francisella strains including F. philomiragia. MIDI fatty acid analysis and PCR/ESI-MS data revealed that isolates were closely matched to F. tularensis, but results were unable to disclose information at a subspecies level. VNTR was performed on seven isolates which resulted in unique molecular fingerprints that did not match known strains. Phylogenetic analysis of 16S rRNA sequences was performed. These data revealed 13 isolates which clustered with F. philomiragia. Of considerable interest were the seven unique genotypes which were grouped into two distinct clusters. Based upon the use of microscopic, metabolic, and molecular assessment, these data suggest that we have successfully isolated several new strains of Francisella spp.