Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes.

Bacterial species with high DNA sequence similarity to pathogens could affect the specificity of assays designed to detect biological threat agents in environmental samples. The natural presence of four pathogenic bacteria, Bacillus anthracis, Clostridium perfringens, Francisella tularensis, and Yersinia pestis and their closely related species, was determined for a large collection of soil and aerosol samples. Polymerase chain reaction (PCR) and gene sequencing were used using group-specific 16S rRNA primers to identify pathogens and related species, and pathogen-specific virulence genes. Close relatives of B. anthracis (B. cereus group species) were detected in 37% of the soils and 25% of the aerosol samples. The B. anthracis protective antigen (pag) gene or a close homolog was detected in 16 of these samples. For the other three pathogen groups, the frequency of detection was much lower, and none of the samples were positive with both the phylogenetic and virulence gene primer sets.

Detection of diverse new Francisella-like bacteria in environmental samples.

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.

Changes in nitrogen-fixing and ammonia-oxidizing bacterial communities in soil of a mixed conifer forest after wildfire.

This study was undertaken to examine the effects of forest fire on two important groups of N-cycling bacteria in soil, the nitrogen-fixing and ammonia-oxidizing bacteria. Sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis of nifH and amoA PCR amplicons was performed on DNA samples from unburned, moderately burned, and severely burned soils of a mixed conifer forest. PCR results indicated that the soil biomass and proportion of nitrogen-fixing and ammonia-oxidizing species was less in soil from the fire-impacted sites than from the unburned sites. The number of dominant nifH sequence types was greater in fire-impacted soils, and nifH sequences that were most closely related to those from the spore-forming taxa Clostridium and Paenibacillus were more abundant in the burned soils. In T-RFLP patterns of the ammonia-oxidizing community, terminal restriction fragments (TRFs) representing amoA cluster 1, 2, or 4 Nitrosospira spp. were dominant (80 to 90%) in unburned soils, while TRFs representing amoA cluster 3A Nitrosospira spp. dominated (65 to 95%) in fire-impacted soils. The dominance of amoA cluster 3A Nitrosospira spp. sequence types was positively correlated with soil pH (5.6 to 7.5) and NH(3)-N levels (0.002 to 0.976 ppm), both of which were higher in burned soils. The decreased microbial biomass and shift in nitrogen-fixing and ammonia-oxidizing communities were still evident in fire-impacted soils collected 14 months after the fire.