Detection of diverse new Francisella-like bacteria in environmental samples

Abstract

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

FIG. 1.

Phylogenetic tree showing relationships of small-subunit rRNA gene sequences obtained from environmental samples to those of Francisella and related species. The tree was rooted using sequences of Escherichia coli, Thiothrix ramosa, Caedibacter taenospiralis, Piscirikettsia salmonis, and Thiomicrospira thyasirae (not shown). The percentage of 100 bootstrap resamplings that support each topological element in maximum-likelihood analysis is indicated, for values of >70%. Phylogenetic groups of sequences obtained in this study are labeled I to V. Within these groups, sequences representative of the types obtained from Houston soil clone libraries (in red) are labeled with the sample name (soil samples 005 to 045 and one water sample) and by distinct sequence types within each sample (a to d). Numbers in parentheses indicate the percentage of clones with identical sequences obtained from that sample. Sequences in blue text are reference sequences obtained in this study. Sequences in black were obtained from GenBank, with accession numbers shown in parentheses. The scale bar corresponds to 0.05 substitution per nucleotide position.

FIG. 2.

Maximum-parsimony phylogenetic tree of sdhA sequences from Houston soil organisms and Francisella reference strains. Sequences representative of the types obtained from Houston soil clone libraries (in red) are labeled with the sample name (soil samples 015 to 045) and sequence type (0.1, 0.2, and 0.3). Numbers in parentheses indicate the percentage of clones with identical sequences obtained from that sample. Sequences in black text are reference sequences obtained in this study. F.t., F. tularensis. Numbers above branches indicate the number of single-nucleotide sequence polymorphisms that distinguish each group.