Pathosphere.org: pathogen detection and characterization through a web-based, open source informatics platform.

BACKGROUND: The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. RESULTS: The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. CONCLUSIONS: By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available.

Pathoadaptive mutations in Salmonella enterica isolated after serial passage in mice.

How pathogenic bacteria adapt and evolve in the complex and variable environment of the host remains a largely unresolved question. Here we have used whole genome sequencing of Salmonella enterica serovar Typhimurium LT2 populations serially passaged in mice to identify mutations that adapt bacteria to systemic growth in mice. We found unique pathoadaptive mutations in two global regulators, phoQ and stpA, which increase the competitive indexes of the bacteria 3- to 5-fold. Also, all mouse-adapted lineages had changed the orientation of the hin invertable element, resulting in production of a FliC type of flagellum. Competition experiments in mice with locked flagellum mutants showed that strains expressing the FliC type of flagellum had a 5-fold increase in competitive index as compared to those expressing FljB type flagellum. Combination of the flagellum cassette inversion with the stpA mutation increased competitive indexes up to 20-fold. These experiments show that Salmonella can rapidly adapt to a mouse environment by acquiring a few mutations of moderate individual effect that when combined confer substantial increases in growth.

Comparative genomics of 2009 seasonal plague (Yersinia pestis) in New Mexico.

Plague disease caused by the gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19(th) century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen.

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

BACKGROUND: Despite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). RESULTS: Archival strains and two "present day" type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the "military" isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of "military" isolates on lactate-containing media, and showed that the "military" strains exhibited a hypersporulating phenotype. CONCLUSIONS: Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.