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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.

SUBMITTER: Barns SM

PROVIDER: S-EPMC1214603 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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

Barns Susan M SM Grow Christy C CC Okinaka Richard T RT Keim Paul P Kuske Cheryl R CR

Applied and environmental microbiology 20050901 9

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 ...[more]

PMID: 16151142

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Project description:Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features-for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

Project description:Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective.

Project description:Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.

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

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

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