Rapid growth of planktonic Vibrio cholerae non-O1/non-O139 strains in a large alkaline lake in Austria: dependence on temperature and dissolved organic carbon quality

Abstract

Vibrio cholerae non-O1/non-O139 strains have caused several cases of ear, wound, and blood infections, including one lethal case of septicemia in Austria, during recent years. All of these cases had a history of local recreational activities in the large eastern Austrian lake Neusiedler See. Thus, a monitoring program was started to investigate the prevalence of V. cholerae strains in the lake over several years. Genetic analyses of isolated strains revealed the presence of a variety of pathogenic genes, but in no case did we detect the cholera toxin gene or the toxin-coregulated pilus gene, both of which are prerequisites for the pathogen to be able to cause cholera. In addition, experiments were performed to elucidate the preferred ecological niche of this pathogen. As size filtration experiments indicated and laboratory microcosms showed, endemic V. cholerae could rapidly grow in a free-living state in natural lake water at growth rates similar to those of the bulk natural bacterial population. Temperature and the quality of dissolved organic carbon had a highly significant influence on V. cholerae growth. Specific growth rates, growth yield, and enzyme activity decreased markedly with increasing concentrations of high-molecular-weight substances, indicating that the humic substances originating from the extensive reed belt in the lake can inhibit V. cholerae growth.

FIG. 1.

View of Neusiedler See, shared by Austria (A) and Hungary (H), with the five routine sampling points (1 to 5) and the extra sampling point (Ruster Poschen [RP]) for the laboratory batch culture experiments indicated. The darkly shaded area indicates the Phragmites australis reed belt; the lightly shaded area indicates the open-water area.

FIG. 2.

Seasonal pattern of V. cholerae prevalence, crustacean zooplankton biomass, and water temperature in Neusiedler See during the period from 2001 to 2004. V. cholerae prevalence data indicate the percentages of samples that tested positive for V. cholerae that were taken from five sampling points. DW, dry weight; WIN, winter; SPR, spring; SUM, summer; AUT, autumn.

FIG. 3.

Growth of V. cholerae in sterile lake water at 15°C (top), 20°C (middle), and 37°C (bottom) with different ratios of HMW-humic-organic-matter concentrations (expressed in ratios relative to the in situ concentrations). Results of experiments performed at 25°C and 30°C are not shown.

FIG. 4.

Exponential dependence of the growth rate of V. cholerae (μ) on temperature in the batch culture experiments.

FIG. 5.

Linear dependence of the growth rate of V. cholerae on the concentration of HMW humic organic matter in the batch culture experiments. To enable a comparison of the results of the different experiments run at different temperatures, the growth rates are expressed in relative units. (Top) HMW substances expressed as a ratio relative to the actual concentration in the lake; (bottom) HMW substances expressed in absolute concentrations (mg liter⁻¹).

FIG. 6.

Decreasing trend of chitinase (A) and beta-glucosidase (B) activities with increasing HMW-substance concentrations, expressed as a ratio relative to the actual concentration in the lake. Activity values were expressed in relative units to make experiments comparable, and box whisker plots represent pooled data from four experiments. For both enzymes, results from two representative experiments are shown in the small insets. Data in the insets show rates measured at the beginning (left y axis) and at the end (right y axis) of the logarithmic growth phase.