Spatiotemporal Dynamics of Vibrio cholerae in Turbid Alkaline Lakes as Determined by Quantitative PCR

Abstract

In recent years, global warming has led to a growing number of Vibrio cholerae infections in bathing water users in regions formerly unaffected by this pathogen. It is therefore of high importance to monitor V. cholerae 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 V. cholerae abundance in two small soda pools and a large lake that is intensively used for recreation and (ii) to elucidate the main factors driving V. cholerae dynamics in these environments. V. cholerae was detected with qPCR at high concentrations of up to 970,000 genomic units 100 ml^-1 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 V. cholerae, indicating that only nontoxigenic V. cholerae (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 V. cholerae in bathing waters with the need for improvements in DNA recovery.IMPORTANCE 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 V. cholerae abundance in lakes with a challenging water matrix. Several qPCR protocols for V. cholerae 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 V. cholerae is present in very high numbers in the investigated ecosystems, while toxigenic V. cholerae is apparently absent. Such information is of importance for public health.

Keywords: Vibrio cholerae non-O1/non-O139; bathing water; detection; qPCR; quantification.

FIG 1

V. cholerae concentrations in 2011 as determined by qPCR, cultivation, and CARD-FISH/SPC. (A) Sampling site 5, Neusiedler See; (B) sampling site 36, Neusiedler See; (C) sampling site ZL (soda pool Zicklacke). qPCR-based concentrations are given without correction (blue bars) and with correction (corrected with global recovery rate; red bars). Cultivation-based (green bars) and CARD-FISH/SPC (purple bars) results are shown. A black dotted line indicates the water temperature. A missing bar means that an analysis with the respective method was not possible; bars between 0 and 1 indicate V. cholerae concentrations below the detection limit. Values and error bars represent the mean of three (cultivation, qPCR) and three to eight (SPC/CARD-FISH) replicates ± standard deviation. GU, genomic units.

FIG 2

V. cholerae concentrations in 2014 as determined by qPCR and cultivation. (A) Sampling site 5, Neusiedler See; (B) sampling site 24, Neusiedler See; (C) sampling site 29, Neusiedler See; (D) sampling site 36, Neusiedler See; (E) sampling site ZL (soda pool Zicklacke); (F) sampling site US (soda pool Unterstinker). qPCR-based concentrations are given without correction (blue bars) and with correction (corrected with global recovery rate; red bars). Cultivation-based results (green bars) are shown. A black line indicates the water temperature. A missing bar means that an analysis with the respective method was not possible; bars between 0 and 1 indicate V. cholerae concentrations below the detection limit. Values and error bars represent the mean of three (cultivation, qPCR) replicates ± standard deviation. GU, genomic units.