Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

Abstract

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 ؋ 10 1 cells ml ؊1 in the large saline lake Neusiedler See to 5.6 ؋ 10 4 cells ml ؊1 in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.V ibrio cholerae and Vibrio mimicus are important human pathogens and two very closely related species. Only recently, their classification as two different species has been confirmed (35). V. cholerae comprises more than 200 serotypes, but only serotypes O1 and O139 cause the severe diarrheal disease cholera (9, 32). Interestingly, several V. mimicus isolates carrying cholera toxin genes (ctxAB) have also been identified. It was suggested that horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae have generated pathogenic V. mimicus strains carrying cholera toxin genes (35). All other V. cholerae serotypes, summarized as non-O1/non-O139, and V. mimicus strains are usually associated with less-severe gastrointestinal infections as well as blood, wound, and ear infections (7,16,28,35). Besides their role as important human pathogens, both organisms are found worldwide in brackish water, coastal areas, and estuarine environments (8,35) and are therefore considered to be natural components of the bacterial community in aquatic ecosystems.Rapid and precise methods for detection and quantification of pathogenic bacteria are fundamental for monitoring and risk assessment in regard to the use of water as well as for studying their ecology in the environment. For quantification of the V. cholerae/V. mimicus clade, various methods have been developed during the last decades. Culture-based techniques for detecting these pathogens, followed by biochemical confirmation via API 20E or PCR, are still regularly used (5, 8). These techniques often include an enrichment step with alkaline peptone water and can then be used quantitatively only as laborious most-probable-number (MPN) techniques. In addition, they cannot be applied when Vibrio cell...

Chlorination of Household Drinking Water Among Cholera Patients' Households to Prevent Transmission of Toxigenic Vibrio cholerae in Dhaka, Bangladesh: CHoBI7 Trial

“…Previous studies have found environmental water that contains 10 1 -10 4 CFU/mL V. cholerae cells. 18 Therefore, we decided to evaluate the efficacy of chlorine in deactivating V. cholerae one log higher at 10 5 CFU/mL. Each flask containing 500 mL water with different chlorine concentration was inoculated with 1 mL bacterial stock (10 7 CFU), which generated a 10 5 CFU/mL concentration of V. cholerae O1 (containers 1-11) or O139 (containers 12-22) in each flask.…”

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

“…Known numbers of V. cholerae O395 (source and growth conditions are listed in Table S1 in the supplemental material) were seeded into environmental water samples collected from a drinking water distribution system, the River Danube, and the Neusiedler See (an Austrian lake). The samples had been collected with sterile 2-liter glass bottles and transported to the laboratory at the in situ temperature (Ϯ2°C) in a cooling box within 3 h. The exact numbers of the added V. cholerae cell suspensions, prepared in 10% (final concentration) ethanol, were determined via epifluorescence microscopy and solidphase cytometry (SPC) as described elsewhere (9). Aliquots (2 ml; cell numbers of 5.4 ϫ 10 6 ml Ϫ1 ) were stored at Ϫ80°C to act as the standard for cell recovery estimations.…”

“…Permanent autochthonous occurrence of non-O1/non-O139 V. cholerae strains had been reported in these lakes, which are known for their high turbidity and high concentrations of DOC and humic substances (25,26). For solid-phase cytometry, 3 to 8 replicate subsamples of the appropriate volume (between 10 and 100 l, depending on the turbidity of the water) were taken and filled up with 1ϫ phosphatebuffered saline (PBS) to reach a final volume of 1 ml and processed as described by Schauer et al (9). Cultivation-based quantification of V. cholerae was done by membrane filtration as described by Schauer et al (9).…”

“…For solid-phase cytometry, 3 to 8 replicate subsamples of the appropriate volume (between 10 and 100 l, depending on the turbidity of the water) were taken and filled up with 1ϫ phosphatebuffered saline (PBS) to reach a final volume of 1 ml and processed as described by Schauer et al (9). Cultivation-based quantification of V. cholerae was done by membrane filtration as described by Schauer et al (9). Briefly, appropriate volumes (from 10 ml to 10 l, filled to reach 10 ml with sterile 1ϫ PBS as needed for an even distribution on the filter) were filtered through 0.45-m-pore-size cellulose nitrate filters, and the filter was directly placed on thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates (Merck, Darmstadt, Germany).…”

“…DFA is a monoclonal-antibody staining application that can be used only for serotype O1 (8). FISH based on 16S rRNAtargeted oligonucleotide probes cannot discriminate between toxigenic and nontoxigenic V. cholerae genotypes, and separation of V. mimicus and V. cholerae is not possible (9). New molecular biological methods based on real-time quantitative PCR (qPCR) technology have the ability to provide bacterial genome equivalent estimates in as little as 3 h (10,11).…”