In the past decade, the number of publicly available bacterial genomes has increased dramatically. These genomes have been generated for impactful initiatives, especially in the field of genomic epidemiology (Brown, Dessai, McGarry, & Gerner-Smidt, 2019; Timme et al., 2017). Genomes are sequenced, shared publicly, and subsequently analyzed for phylogenetic relatedness. If two genomes of epidemiological interest are found to be related, further investigation might be prompted. However, comparing the multitudes of genomes for phylogenetic relatedness is computationally expensive and, with large numbers, laborious. Consequently, there are many strategies to reduce the complexity of the data for downstream analysis, especially using nucleotide stretches of length k (kmers).

Vibrio vulnificus (Vv) is a multi-host pathogenic species currently subdivided into three biotypes (Bts). The three Bts are human-pathogens, but only Bt2 is also a fish-pathogen, an ability that is conferred by a transferable virulence-plasmid (pVvbt2). Here we present a phylogenomic analysis from the core genome of 80 Vv strains belonging to the three Bts recovered from a wide range of geographical and ecological sources. We have identified five well-supported phylogenetic groups or lineages (L). L1 comprises a mixture of clinical and environmental Bt1 strains, most of them involved in human clinical cases related to raw seafood ingestion. L2 is formed by a mixture of Bt1 and Bt2 strains from various sources, including diseased fish, and is related to the aquaculture industry. L3 is also linked to the aquaculture industry and includes Bt3 strains exclusively, mostly related to wound infections or secondary septicemia after farmed-fish handling. Lastly, L4 and L5 include a few strains of Bt1 associated with specific geographical areas. The phylogenetic trees for ChrI and II are not congruent to one another, which suggests that inter- and/or intra-chromosomal rearrangements have been produced along Vv evolution. Further, the phylogenetic trees for each chromosome and the virulence plasmid were also not congruent, which also suggests that pVvbt2 has been acquired independently by different clones, probably in fish farms. From all these clones, the one with zoonotic capabilities (Bt2-Serovar E) has successfully spread worldwide. Based on these results, we propose a new updated classification of the species based on phylogenetic lineages rather than on Bts, as well as the inclusion of all Bt2 strains in a pathovar with the particular ability to cause fish vibriosis, for which we suggest the name “piscis.”

Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The Gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence.

Vibrio vulnificus is a natural inhabitant of estuarine waters worldwide and is of medical relevance due to its ability to cause grievous wound infections and/or fatal septicemia. Genetic polymorphisms within the virulence-correlated gene (vcg) serve as a primary feature to distinguish clinical (C-) genotypes from environmental (E-) genotypes. C-genotypes demonstrate superior survival in human serum relative to E-genotypes, and genome comparisons have allowed for the identification of several putative virulence factors that could potentially aid C-genotypes in disease progression. We used RNA sequencing to analyze the transcriptome of C-genotypes exposed to human serum relative to seawater, which revealed two divergent genetic programs under these two conditions. In human serum, cells displayed a distinct “virulence profile” in which a number of putative virulence factors were upregulated, including genes involved in intracellular signaling, substrate binding and transport, toxin and exoenzyme production, and the heat shock response. Conversely, the “environmental profile” exhibited by cells in seawater revealed upregulation of transcription factors such as rpoS, rpoN, and iscR, as well as genes involved in intracellular signaling, chemotaxis, adherence, and biofilm formation. This dichotomous genetic switch appears to be largely governed by cyclic-di-GMP signaling, and remarkably resembles the dual life-style of V. cholerae as it transitions from host to environment. Furthermore, we found a “general stress response” module, known as the stressosome, to be upregulated in seawater. This signaling system has been well characterized in Gram-positive bacteria, however its role in V. vulnificus is not clear. We examined temporal gene expression patterns of the stressosome and found it to be upregulated in natural estuarine waters indicating that this system plays a role in sensing and responding to the environment. This study advances our understanding of gene regulation in V. vulnificus, and brings to the forefront a number of previously overlooked genetic networks.

A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations ("outbreaks") that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila. This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. IMPORTANCEWe demonstrate that whole-genome sequencing helps to improve resolution of Legionella pneumophila isolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources of legionellosis outbreaks. Moreover, we evaluated various methods to analyze genome sequence data to help resolve outbreak-related isolates.