Simultaneous quantification ofVibrio metoecusandVibrio choleraewith its O1 serogroup and toxigenic subpopulations in environmental reservoirs

Abstract

ABSTRACTVibrio 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.