SummaryIn order to elucidate the main predictors of V ibrio cholerae dynamics and to estimate the risk of V ibrio cholera‐related diseases, a recently developed direct detection approach based on fluorescence in situ hybridization and solid‐phase cytometry (CARD‐FISH/SPC) was applied in comparison to cultivation for water samples from the lake Neusiedler See, Austria and three shallow alkaline lakes over a period of 20 months. V ibrio cholerae attached to crustacean zooplankton was quantified via FISH and epifluorescence microscopy. Concentrations obtained by CARD‐FISH/SPC were significantly higher than those obtained by culture in 2011, but were mostly of similar magnitude in 2012. Maximum cell numbers were 1.26 × 106 V . cholerae per L in Neusiedler See and 7.59 × 107 V . cholerae per L in the shallow alkaline lakes. Only on a few occasions during summer was the crustacean zooplankton the preferred habitat for V . cholerae. In winter, V . cholerae was not culturable but could be quantified at all sites with CARD‐FISH/SPC. Beside temperature, suspended solids, zooplankton and ammonium were the main predictors of V . cholerae abundance in Neusiedler See, while in the shallow alkaline lakes it was organic carbon, conductivity and phosphorus. Based on the obtained concentrations a first estimation of the health risk for visitors of the lake could be performed.

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 ؋ 10 2 to 2.3 ؋ 10 4 cell equivalents liter ؊1 , whereas GR-corrected abundances ranged from 4.7 ؋ 10 3 to 1.6 ؋ 10 6 cell equivalents liter ؊1 . GR-corrected qPCR results were in good agreement with an independent cellbased direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. Vibrio cholerae is a waterborne bacterium found worldwide in brackish water, coastal areas, and estuarine environments (1). Although the species V. cholerae comprises more than 200 serotypes, only serotypes O1 and O139 are currently able to cause epidemic and pandemic cholera outbreaks, with more than 100,000 reported death cases per year (2). All other non-O1/non-O139 serotypes are usually associated with less-severe gastrointestinal, blood, wound, and ear infections (3). V. cholerae has been classified as a potential category B terrorism agent by the U.S. Centers for Disease Control and Prevention (4). Detection and quantification of V. cholerae, especially of serogroup O1/O139 strains in environmental samples, are still difficult tasks, and no international standard is available. Currently accepted cultivation-based methods take at least 48 h to obtain a final result (5) and severely underestimate the presence of O1/O139 serogroups, due to the fact that these strains predominantly enter a viable but nonculturable (VBNC) state once released from the human intestine into the environment (6, 7). Alternatively, fluorescence in situ hybridization (FISH) and the direct fluorescent-antibody assay (DFA) are direct cell-...

In recent years, global warming has led to a growing number of infections in bathing water users in regions formerly unaffected by this pathogen. It is therefore of high importance to monitor in aquatic environments and to elucidate the main factors governing its prevalence and abundance. For this purpose, rapid and standardizable methods that can be performed by routine water laboratories are prerequisite. In this study, we applied a recently developed multiplex quantitative PCR (qPCR) strategy (i) to monitor the spatiotemporal variability of abundance in two small soda pools and a large lake that is intensively used for recreation and (ii) to elucidate the main factors driving dynamics in these environments. was detected with qPCR at high concentrations of up to 970,000 genomic units 100 ml during the warm season, up to 2 orders of magnitude higher than values obtained by cultivation. An independent cytometric approach led to results comparable to qPCR data but with significantly more positive samples due to problems with DNA recovery for qPCR. Not a single sample was positive for toxigenic , indicating that only nontoxigenic (NTVC) was present. Temperature was the main predictor of NTVC abundance, but the quality and quantity of dissolved organic matter were also important environmental correlates. Based on this study, we recommend using the developed qPCR strategy for quantification of toxigenic and nontoxigenic in bathing waters with the need for improvements in DNA recovery. There is a definitive need for rapid and standardizable methods to quantify waterborne bacterial pathogens. Such methods have to be thoroughly tested for their applicability to environmental samples. In this study, we critically tested a recently developed multiplex qPCR strategy for its applicability to determine the spatiotemporal variability of abundance in lakes with a challenging water matrix. Several qPCR protocols for detection have been developed in the laboratory, but comprehensive studies on the application to environmental samples are extremely scarce. In our study, we demonstrate that our developed qPCR approach is a valuable tool but that there is a need for improvement in DNA recovery for complex water matrices. Furthermore, we found that nontoxigenic is present in very high numbers in the investigated ecosystems, while toxigenic is apparently absent. Such information is of importance for public health.