Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.

The key genes required forBacillus anthracisto cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution ofB. anthracisvirulence, we obtained high-redundancy genome sequences of 45 strains of theBacillus cereussensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for theB. cereuss.l. group was ∼1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss inB. anthracisor for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, severalB. cereusgenomes isolated from soil and not previously associated with human disease were degraded to various degrees. AlthoughB. anthracishas undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.

The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown.

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.

The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especial...

Sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. Hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing. This study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the U.S. Department of Defense and various international public health agencies. The newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. Target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with Ct values suggesting that amplicon sequencing would be likely to fail. Increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage. Hybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. As the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations.