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BackgroundVibrio cholerae, the causative agent of cholera, is a human pathogen that thrives in estuarine environments. V. cholerae competes with neighboring microbes by the contact-dependent translocation of toxic effectors with the type VI secretion system (T6SS). Effector types are highly variable across V. cholerae strains, but all pandemic isolates encode the same set of distinct effectors. It is possible that acquisition of these effectors via horizontal gene transfer played a role in the development of pandemic V. cholerae.ResultsWe assessed the distribution of V. cholerae T6SS loci across multiple Vibrio species. We showed that the fish-pathogen V. anguillarum encodes all three V. cholerae core loci as well as two of the four additional auxiliary clusters. We further demonstrated that V. anguillarum shares T6SS effectors with V. cholerae, including every pandemic-associated V. cholerae effector. We identified a novel T6SS cluster (Accessory Aux1) that is widespread in V. anguillarum and encodes the pandemic V. cholerae effector TseL. We highlighted potential gene transfer events of Accessory Aux1 from V. anguillarum to V. cholerae. Finally, we showed that TseL from V. cholerae can be neutralized by the V. anguillarum Accessory Aux1 immunity protein and vice versa, indicating V. anguillarum as the donor of tseL to the V. cholerae species.ConclusionsV. anguillarum constitutes an environmental reservoir of pandemic-associated V. cholerae T6SS effectors. V. anguillarum and V. cholerae likely share an environmental niche, compete, and exchange T6SS effectors. Further, our findings highlight the fish as a potential reservoir of pandemic V. cholerae.

Most efforts to understand the biology ofVibrio choleraehave focused on a single group, the pandemic-generating lineage harbouring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in theV. choleraepopulations inhabiting in regions with varying history of cholera cases and how that might influence the abundance of pandemic strains, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka (cholera endemic) located in the Ganges delta, and of Falmouth (no known history of cholera), a small coastal town on the US East Coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny ofV. choleraespecies, sufficiently differentiated genetically and phenotypically to form a novel species. Strains from this species have been anecdotally isolated from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhoea. In 1935 Gardner and Venkatraman unofficially referred to a member of this group asVibrio paracholerae. In recognition of this earlier designation, we propose the nameVibrio paracholerae, sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species.ImportanceCholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution and environmental adaptation of the causative agentVibrio choleraeand tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics ofVibrio choleraein an inland locality which is known as endemic for cholera and compared with that of a cholera free coastal location. We found the consistent presence of the pandemic generatingV. choleraein cholera-endemic Dhaka and an exclusive presence of a lineage phylogenetically distinct from otherV. cholerae. Our study suggests that this lineage represents a novel species having pathogenic potential and a human link to its environmental abundance. The possible association with human population, co-existence and interaction with toxigenicV. choleraein the natural environment make this potential human pathogen an important subject for future studies.

Core genome multilocus sequence typing (cgMLST) has gained popularity in recent years in epidemiological research and subspecies level classification. cgMLST retains the intuitive nature of traditional MLST but offers much greater resolution by utilizing significantly larger portions of the genome. Here, we introduce a cgMLST scheme forVibrio cholerae, a bacterium abundant in marine and freshwater environments and the etiologic agent of cholera. A set of 2,443 core genes ubiquitous inV. choleraewere used to analyze a comprehensive dataset of 1,262 clinical and environmental strains collected from 52 countries, including 65 newly sequenced genomes in this study. We established a sublineage threshold based on 133 allelic differences that creates clusters nearly identical to traditional MLST types, providing backwards compatibility to new cgMLST classifications. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying strains from the same outbreak and closely related isolates which could give clues on outbreak origin. Using cgMLST, we confirmed the South Asian origin of modern epidemics and identified clustering affinity among sublineages of environmental isolates from the same geographic origin. Advantages of this method are highlighted by direct comparison with existing classification methods, such as MLST and single nucleotide polymorphism-based methods. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use. We anticipate this scheme will serve as a basis for a universally applicable and standardized classification system forV. choleraeresearch and epidemiological surveillance in the future. This cgMLST scheme is publicly available on PubMLST (https://pubmlst.org/vcholerae/).IMPORTANCEToxigenicVibrio choleraeof the O1 and O139 serogroups are the causative agent of cholera, an acute diarrheal disease that plagued the world for centuries, if not millennia. Here, we introduce a core genome multilocus sequence typing (cgMLST) scheme forV. cholerae. Using cgMLST, we established an outbreak threshold that can efficiently identify outbreak related strains and potential sources of introduction. We also defined a sublineage threshold that is similar to traditional MLST sequence type which will provide context to this new typing method by relating it to previous MLST results. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use, making this scheme the most suitable method forV. choleraetyping and surveillance worldwide.

Vibrio metoecusis a recently described and little studied causative agent of opportunistic infections in humans, often coexisting withV. choleraein aquatic environments. However, the relative abundance ofV. metoecuswithV. cholerae and their population dynamics in aquatic reservoirs is still unknown. We developed a multiplex qPCR assay with a limit of detection of three copies per reaction to simultaneously quantify totalV. metoecusandV. choleraeabundance, as well as the toxigenic and O1 serogroup subpopulations ofV. choleraefrom environmental samples. Four different genes were targeted as specific markers for individualVibriospecies or subpopulations;viuB, a gene encoding a vibriobactin utilization protein, was used to quantify the totalV. choleraepopulation. The cholera toxin genectxAprovided an estimation of toxigenicV. choleraeabundance, while therfbO1gene specifically detected and quantifiedV. choleraebelonging to the O1 serogroup, which includes almost all lineages of the species responsible for the majority of past and ongoing cholera pandemics. To measureV. metoecusabundance, the genemcp, encoding methyl accepting chemotaxis protein, was used. Marker specificity was confirmed by testing several isolates ofV. choleraeandV. metoecusalongside negative controls of isolates within and outside of theVibriogenus. Analysis of environmental water samples collected from four different geographic locations including cholera-endemic (Dhaka, Kuakata and Mathbaria in Bangladesh) and non-endemic (Oyster Pond in Falmouth, Massachusetts, USA) regions showed thatV. metoecuswas only present in the USA site, recurring seasonally. Within the coastal USA site, the non-toxigenic O1 serogroup represented up to ∼18% of the totalV. choleraepopulation.V. choleraetoxigenic O1 serogroup was absent or present in low abundance in coastal Bangladesh (Kuakata and Mathbaria) but constituted a relatively high proportion of the totalV. choleraepopulation sustained throughout the year in inland Bangladesh (Dhaka). A preference for host/particle attachment was observed, as the majority of cells from bothVibriospecies (>90%) were identified in the largest water size fraction sampled, composed of particles or organisms >63 μm and their attached bacteria. This is the first study to apply a culture-independent method to quantifyV. choleraeorV. metoecusdirectly in environmental reservoirs of areas endemic and non-endemic for cholera on significant temporal and spatial scales.SIGNIFICANCECholera is a life-threatening disease that requires immediate intervention; it is of prime importance to have fast, accurate and sensitive means to detectV. cholerae. Consistent environmental monitoring of the abundance ofV. choleraealong with its toxigenic and O1 serogroup subpopulations could facilitate the determination of the actual distribution of this organism in aquatic reservoirs and thus help to predict an outbreak before it strikes. The lack of substantial temporal and spatial environmental sampling, along with specific quantitative measures, has made this goal elusive so far. The same is true forV. metoecus, a close relative ofV. choleraewhich has been associated with several clinical infections and could likely pose an emerging threat, readily exchanging genetic material with its more famous relative.