In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C–3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL–2050 and 2009EL–2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL–2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

An optical detection method, Raman chemical imaging spectroscopy (RCIS), is reported, which combines Raman spectroscopy, fluorescence spectroscopy, and digital imaging. Using this method, trace levels of biothreat organisms are detected in the presence of complex environmental backgrounds without the use of amplification or enhancement techniques. RCIS is reliant upon the use of Raman signatures and automated recognition algorithms to perform species-level identification. The rationale and steps for constructing a pathogen Raman signature library are described, as well as the first reported Raman spectra from live, priority pathogens, including Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Francisella tularensis, Brucella abortus, and ricin. Results from a government-managed blind trial evaluation of the signature library demonstrated excellent specificity under controlled laboratory conditions.

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Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.