Populations of the bacterium Vibrio cholerae consist of dozens of distinct lineages, with primarily (but not exclusively) members of the pandemic generating lineage capable of causing the diarrhoeal disease cholera. Assessing the composition and temporal dynamics of such populations requires extensive isolation efforts and thus only rarely covers large geographic areas or timeframes exhaustively. We developed a culture-independent amplicon sequencing strategy based on the protein-coding gene viuB (vibriobactin utilization) to study the structure of a V. cholerae population over the course of a summer. We show that the 26 co-occurring V. cholerae lineages continuously compete for limited space on nutrient-rich particles where only a few of them can grow to large numbers. Differential abundance of lineages between locations and size-fractions associated with a particle-attached or free-swimming lifestyle could reflect adaptation to various environmental niches. In particular, a major V. cholerae lineage occasionally grows to large numbers on particles but remain undetectable using isolation-based methods, indicating selective culturability for some members of the species. We thus demonstrate that isolation-based studies may not accurately reflect the structure and complex dynamics of V. cholerae populations and provide a scalable highthroughput method for both epidemiological and ecological approaches to studying this species.
Freshly squeezed or freshly prepared fruit juices sold by local market vendors in Dhaka city were analyzed for the microbiological quality.
Vibrio metoecus is a recently described aquatic bacterium and opportunistic pathogen, closely related to and often coexisting with Vibrio cholerae. To study the relative abundance and population dynamics of both species in aquatic environments of cholera-endemic and cholera-free regions, we developed a multiplex qPCR assay allowing simultaneous quantification of total V. metoecus and V. cholerae (including toxigenic and O1 serogroup) cells. The presence of V. metoecus was restricted to samples from regions that are not endemic for cholera, where it was found at 20% of the abundance of V. cholerae. In this environment, non-toxigenic O1 serogroup V. cholerae represents almost one-fifth of the total V. cholerae population. In contrast, toxigenic O1 serogroup V. cholerae was also present in low abundance on the coast of cholera-endemic regions, but sustained in relatively high proportions throughout the year in inland waters. The majority of cells from both Vibrio species were recovered from particles rather than free-living, indicating a potential preference for attached versus planktonic lifestyles. This research further elucidates the population dynamics underpinning V. cholerae and its closest relative in cholera-endemic and non-endemic regions through culture-independent quantification from environmental samples.
Most efforts to understand the biology of Vibrio cholerae have 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 the V. cholerae populations inhabiting regions with a history of cholera cases and those lacking such a history, 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 United States 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 for the V. cholerae species, sufficiently differentiated genetically and phenotypically to form a novel species. A retrospective search revealed that strains from this species have been anecdotally found 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 as Vibrio paracholerae . In recognition of this earlier designation, we propose the name Vibrio 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. Importance Cholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution, and environmental adaptation of the causative agent Vibrio cholerae and tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics of Vibrio cholerae in 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 generating V. cholerae in cholera-endemic Dhaka and an exclusive presence of a lineage phylogenetically distinct from other V. 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 toxigenic V. cholerae in 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 for Vibrio cholerae, a bacterium abundant in marine and freshwater environments and the etiologic agent of cholera. A set of 2,443 core genes ubiquitous in V. cholerae were 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 for V. cholerae research and epidemiological surveillance in the future. This cgMLST scheme is publicly available on PubMLST (https://pubmlst.org/vcholerae/). IMPORTANCE Toxigenic Vibrio cholerae of the O1 and O139 serogroups are the causative agents of cholera, an acute diarrheal disease that plagued the world for centuries, if not millennia. Here, we introduce a core genome multilocus sequence typing scheme for V. cholerae. Using this scheme, we have standardized the definition for subspecies-level classification, facilitating global collaboration in the surveillance of V. cholerae. In addition, this typing scheme allows for quick identification of outbreak-related isolates that can guide subsequent analyses, serving as an important first step in epidemiological research. This scheme is also easily scalable to analyze thousands of isolates at various levels of resolution making it an invaluable tool for large-scale ecological and evolutionary analyses.
ICT for pneumococcal antigen in CSF is 100% sensitive and specific in diagnosing pyogenic pneumococcal meningitis and can detect approximately 30% more pneumococcal meningitis cases than with culture alone. The simplicity of the test procedure and the longevity of CSF antigen detection suggest the potential utility of ICT to estimate the true burden of pneumococcal disease, as for Haemophilus influenzae type b using data from meningitis, and to guide selection of appropriate antibiotic treatment, especially in resource-poor countries with widespread prehospital antimicrobial use.
A total of 109 water samples were collected from around Dhaka city and examined for microbial contamination. Samples were collected in sterilized screw capped glass bottles, transported to the laboratory in cold and processed within 6 hours of their collection. All river water, pond water and household water were found heavily contaminated with coliform, faecal coliform, E. Coli and Salmonella whereas tube well water and bottled water was devoid of faecal coliform, E. Coli and Salmonella. A significant correlation between total number of total coliform and percentage of Salmonella was also investigated. It is suggested that water must be boiled before use.
24Vibrio metoecus is a recently described and little studied causative agent of opportunistic 25 infections in humans, often coexisting with V. cholerae in aquatic environments. However, the 26 relative abundance of V. metoecus with V. cholerae and their population dynamics in aquatic 27 reservoirs is still unknown. We developed a multiplex qPCR assay with a limit of detection of 28 three copies per reaction to simultaneously quantify total V. metoecus and V. cholerae 29 abundance, as well as the toxigenic and O1 serogroup subpopulations of V. cholerae from 30 environmental samples. Four different genes were targeted as specific markers for individual 31 Vibrio species or subpopulations; viuB, a gene encoding a vibriobactin utilization protein, was 32 used to quantify the total V. cholerae population. The cholera toxin gene ctxA provided an 33 estimation of toxigenic V. cholerae abundance, while the rfbO1 gene specifically detected and 34 quantified V. cholerae belonging to the O1 serogroup, which includes almost all lineages of the 35 species responsible for the majority of past and ongoing cholera pandemics. To measure V. 36 metoecus abundance, the gene mcp, encoding methyl accepting chemotaxis protein, was used. 37 Marker specificity was confirmed by testing several isolates of V. cholerae and V. metoecus 38 alongside negative controls of isolates within and outside of the Vibrio genus. Analysis of 39 environmental water samples collected from four different geographic locations including 40 cholera-endemic (Dhaka, Kuakata and Mathbaria in Bangladesh) and non-endemic (Oyster Pond 41 in Falmouth, Massachusetts, USA) regions showed that V. metoecus was only present in the 42 USA site, recurring seasonally. Within the coastal USA site, the non-toxigenic O1 serogroup 43 represented up to ~18% of the total V. cholerae population. V. cholerae toxigenic O1 serogroup 44 was absent or present in low abundance in coastal Bangladesh (Kuakata and Mathbaria) but 45 constituted a relatively high proportion of the total V. cholerae population sustained throughout 48 fraction sampled, composed of particles or organisms >63 μ m and their attached bacteria. This is 49the first study to apply a culture-independent method to quantify V. cholerae or V. metoecus 50 directly in environmental reservoirs of areas endemic and non-endemic for cholera on significant 51 temporal and spatial scales. 52 53 SIGNIFICANCE 54Cholera is a life-threatening disease that requires immediate intervention; it is of prime 55 importance to have fast, accurate and sensitive means to detect V. cholerae. Consistent 56 environmental monitoring of the abundance of V. cholerae along with its toxigenic and O1 57 serogroup subpopulations could facilitate the determination of the actual distribution of this 58 organism in aquatic reservoirs and thus help to predict an outbreak before it strikes. The lack of 59 substantial temporal and spatial environmental sampling, along with specific quantitative 60 measures, has made this goal elusive so far. The same is true...
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