Although rare in the U.S., outbreaks due to Neisseria meningitidis do occur. Rapid, early outbreak detection is important for timely public health response. In this study, we characterized U.S. meningococcal isolates (N = 201) from 15 epidemiologically defined outbreaks (2009–2015) along with temporally and geographically matched sporadic isolates using multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), and six whole genome sequencing (WGS) based methods. Recombination-corrected maximum likelihood (ML) and Bayesian phylogenies were reconstructed to identify genetically related outbreak isolates. All WGS analysis methods showed high degree of agreement and distinguished isolates with similar or indistinguishable PFGE patterns, or the same strain genotype. Ten outbreaks were caused by a single strain; 5 were due to multiple strains. Five sporadic isolates were phylogenetically related to 2 outbreaks. Analysis of 9 outbreaks using timed phylogenies identified the possible origin and estimated the approximate time that the most recent common ancestor emerged for outbreaks analyzed. U.S. meningococcal outbreaks were caused by single- or multiple-strain introduction, with organizational outbreaks mainly caused by a clonal strain and community outbreaks by divergent strains. WGS can infer linkage of meningococcal cases when epidemiological links are uncertain. Accurate identification of outbreak-associated cases requires both WGS typing and epidemiological data.

USA500 isolates are clonal complex 8 (CC8) Staphylococcus aureus strains closely related to the prominent community- and hospital-associated USA300 group. Despite being relatively understudied, USA500 strains cause a significant burden of disease and are the third most common methicillin-resistant S. aureus (MRSA) strains identified in the U.S. Emerging Infections Program (EIP) invasive S. aureus surveillance. To better understand the genetic relationships of the strains, we sequenced the genomes of 539 USA500 MRSA isolates from sterile site infections collected through the EIP between 2005 and 2013 in the United States. USA500 isolates fell into three major clades principally separated by their distribution across different U.S. regions. Clade C1 strains, found principally in the Northeast, were associated with multiple IS256 insertion elements in their genomes and higher levels of antibiotic resistance. C2 was associated with Southern states, and E1 was associated with Western states. C1 and C2 strains all shared a frameshift in the gene encoding AdsA surface-attached surface protein. We propose that the term "USA500" should be used for CC8 strains sharing a recent common ancestor with the C1, C2, and E1 strains but not in the USA300 group.IMPORTANCE In this work, we have removed some of the confusion surrounding the use of the name "USA500," placed USA500 strains in the context of the CC8 group, and developed a strategy for assignment to subclades based on genome sequence. Our new phylogeny of USA300/USA500 will be a reference point for understanding the genetic adaptations that have allowed multiple highly virulent clonal strains to emerge from within CC8 over the past 50 years.

Genome sequencing is an increasingly common component of infectious disease outbreak investigations. However, the relationship between pathogen transmission and observed genetic data is complex, and dependent on several uncertain factors. As such, simulation of pathogen dynamics is an important tool for interpreting observed genomic data in an infectious disease outbreak setting, in order to test hypotheses and to explore the range of outcomes consistent with a given set of parameters. We introduce 'seedy', an R package for the simulation of evolutionary and epidemiological dynamics (http://cran.r-project.org/web/packages/seedy/). Our software implements stochastic models for the accumulation of mutations within hosts, as well as individual-level disease transmission. By allowing variables such as the transmission bottleneck size, within-host effective population size and population mixing rates to be specified by the user, our package offers a flexible framework to investigate evolutionary dynamics during disease outbreaks. Furthermore, our software provides theoretical pairwise genetic distance distributions to provide a likelihood of personto-person transmission based on genomic observations, and using this framework, implements transmission route assessment for genomic data collected during an outbreak. Our open source software provides an accessible platform for users to explore pathogen evolution and outbreak dynamics via simulation, and offers tools to assess observed genomic data in this context.

Legionella species inhabit freshwater and soil ecosystems where they parasitize protozoa. L. pneumonphila (LP) serogroup-1 (Lp1) is the major cause of Legionnaires' Disease (LD), a life-threatening pulmonary infection that can spread systemically. The increased global frequency of LD caused by Lp and non-Lp species underscores the need to expand our knowledge of evolutionary forces underlying disease pathogenesis. Whole genome analyses of 43 strains, including all known Lp serogroups 1-17 and 17 emergent LD-causing Legionella species (of which 33 were sequenced in this study) in addition to 10 publicly available genomes, resolved the strains into four phylogenetic clades along host virulence demarcations. Clade-specific genes were distinct for genetic exchange and signal-transduction, indicating adaptation to specific cellular and/or environmental niches. CRISPR spacer comparisons hinted at larger pools of accessory DNA sequences in Lp than predicted by the pan-genome analyses. While recombination within Lp was frequent and has been reported previously, population structure analysis identified surprisingly few DNA admixture events between species. In summary, diverse Legionella LD-causing species share a conserved core-genome, are genetically isolated from each other, and selectively acquire genes with potential for enhanced virulence.

Background: The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes - the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans - its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature's Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. Results: We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. Conclusions: This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of Chlamydia trachomatis, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the C. trachomatis genome with 500 1.1–1.3 kb overlapping amplicons (5-fold amplicon redundancy). We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called binstrain. We used binstrain to estimate the proportion of SNPs originating from 14 C. trachomatis reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of C. trachomatis mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.

Background Multiple locus sequence typing (MLST) has become a central genotyping strategy for analysis of bacterial populations. The scheme involves de novo sequencing of 6–8 housekeeping loci to assign unique sequence types. In this work we adapted MLST to a rapid microfluidics platform in order to enhance speed and reduce laboratory labor time. Methodology/Principal Findings Using two integrated microfluidic devices, DNA was purified from 100 Bacillus cereus soil isolates, used as a template for multiplex amplification of 7 loci and sequenced on forward and reverse strands. The time on instrument from loading genomic DNA to generation of electropherograms was only 1.5 hours. We obtained full-length sequence of all seven MLST alleles from 84 representing 46 different Sequence Types. At least one allele could be sequenced from a further 15 strains. The nucleotide diversity of B. cereus isolated in this study from one location in Rockville, Maryland (0.04 substitutions per site) was found to be as great as the global collection of isolates. Conclusions/Significance Biogeographical investigation of pathogens is only one of a panoply of possible applications of microfluidics based MLST; others include microbiologic forensics, biothreat identification, and rapid characterization of human clinical samples.

Background Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated. Results In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF. Conclusions To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.

Background Chlamydia trachomatis is an obligate intracellular bacterial parasite, which causes several severe and debilitating diseases in humans. This study uses comparative genomic analyses of 12 complete published C. trachomatis genomes to assess the contribution of recombination and selection in this pathogen and to understand the major evolutionary forces acting on the genome of this bacterium. Results The conserved core genes of C. trachomatis are a large proportion of the pan-genome: we identified 836 core genes in C. trachomatis out of a range of 874-927 total genes in each genome. The ratio of recombination events compared to mutation (ρ/θ) was 0.07 based on ancestral reconstructions using the ClonalFrame tool, but recombination had a significant effect on genetic diversification (r/m = 0.71). The distance-dependent decay of linkage disequilibrium also indicated that C. trachomatis populations behaved intermediately between sexual and clonal extremes. Fifty-five genes were identified as having a history of recombination and 92 were under positive selection based on statistical tests. Twenty-three genes showed evidence of being under both positive selection and recombination, which included genes with a known role in virulence and pathogencity (e.g., ompA, pmps, tarp). Analysis of inter-clade recombination flux indicated non-uniform currents of recombination between clades, which suggests the possibility of spatial population structure in C. trachomatis infections. Conclusions C. trachomatis is the archetype of a bacterial species where recombination is relatively frequent yet gene gains by horizontal gene transfer (HGT) and losses (by deletion) are rare. Gene conversion occurs at sites across the whole C. trachomatis genome but may be more often fixed in genes that are under diversifying selection. Furthermore, genome sequencing will reveal patterns of serotype specific gene exchange and selection that will generate important research questions for understanding C. trachomatis pathogenesis.

Background: Spontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix. Methods: Here we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection. Results: Using whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains. Conclusions: Taken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).