High Diversity and Abundance of Legionella spp. in a Pristine River and Impact of Seasonal and Anthropogenic Effects ▿ †

The diversity and dynamics of Legionella species along a French river watershed subject to different thermal and wastewater discharges during an annual cycle were assessed by 16S rRNA gene sequencing and by a fingerprint technique, single-strand conformation polymorphism. A high diversity of Legionella spp. was observed at all the sampling sites, and the dominant Legionella clusters identified were most closely related to uncultured bacteria. The monthly monitoring revealed that Legionella sp. diversity changes were linked only to season at the wastewater site whereas there was some evidence for anthropogenic effects on Legionella sp. diversity downstream of the thermal bath. Quantification of Legionella pneumophila and Legionella spp. by culture and quantitative PCR (qPCR) was performed. Whereas only L. pneumophila was quantified on culture media, the qPCR assay revealed that Legionella spp. were ubiquitous and abundant from the pristine source of the river to the downstream sampling sites. These results suggest that Legionella spp. may be present at significant concentrations in many more freshwater environments than previously thought, highlighting the need for further ecological studies and culturing efforts

Activity and diversity of bacterial cells with high and low nucleic acid content

In most aquatic environments, at least 2 subpopulations of bacterial cells can be discriminated by flow cytometry based on their nucleic acid content. Recent investigations have shown that the cells with a high nucleic acid (HNA) content have a higher cell-specific activity (CSA) cell than those with a low nucleic acid (LNA) content. In this study, the CSA and biomass-specific activities (BSA) of HNA and LNA cells from different aquatic ecosystems, including marine, brackish and freshwater, were investigated using radioactive leucine incorporation and cell sorting by flow cytometry. The genetic diversity of natural assemblages, HNA and LNA cells was investigated using the SSCP (PCR single-strand conformation polymorphism) method. Data showed that both CSA and BSA of HNA cells were always significantly higher than CSA and BSA of LNA cells. In addition, HNA cells had a dominant contribution to the production of the total community (77 to 98 %). For the different samples, the SSCP fingerprints from the natural assemblage and from the 2 sorted fractions were not significantly different. This clearly suggests that HNA and LNA subpopulations were composed by the same dominant species and, thus, confirms an important heterogeneity of physiological states within most natural populations.info:eu-repo/semantics/publishe

Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry

In most aquatic bacterial communities, it is possible to discriminate bacterial cells with a high nucleic acid content (HNA) from those with a low nucleic acid content (LNA) by flow cytometry. The distribution of leucine incorporation rate per cell (specific activity) within the fraction of bacteria with a high apparent nucleic acid content was investigated in coastal seawater using flow cytometry and cell sorting techniques. Bacterial cells from a natural seawater sample were first labeled with tritiated leucine, stained with a fluorescent nucleic acid stain and then sorted based on their fluorescence and scatter signals, assuming that fluorescence was proportional to the cellular nucleic acid content and scatter signals to biovolume. Our results clearly demonstrated that specific activity was heterogeneously distributed within the group of HNA cells and increased with both scatter and fluorescence signals. This shows that important differences in terms of cell-specific rates of activity exist within the HNA group and that specific activity is positively correlated with both the nucleic acid content of cells and their biovolume. We hypothesized that this heterogeneity may be partly due to the structure of bacterial communities. Therefore, further investigations were made by analyzing the nucleic acid content and scatter properties of a set of 10 species isolated from the same coastal area. Our results confirm that both fluorescence and scatter values vary greatly between species at a given growth stage and between growth stages for a given species. Variations reported at different growth stages suggest that both parameters are sensitive to the metabolic activity of individual cells and confirm the positive relationship reported in this study between scatter and fluorescence parameters and specific activity within the HNA cellular fraction of natural communities. Interestingly, it was shown that HNA and LNA cells were present in late stationary phase cultures. This suggests that LNA cells found in natural communities may be dead or dying cells.info:eu-repo/semantics/publishe

Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry ▿†

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter−1, and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 103 viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples

Legionella species diversity and dynamics from surface reservoir to tap water: from cold adaptation to thermophily

Water samples of the Drinking Water Supply System (DWSS) of the city of Braunschweig were analysed for its Legionella species composition using genus-specific PCR amplicons and single-strand conformation polymorphism (SSCP) fingerprint analyses based on 16S rRNA genes. These analyses comprised the whole supply chain including raw water, treatment process and large-scale storage, and a seasonal study of finished drinking water sampled monthly from cold and hot tap water. Treatment of raw water had a major impact on Legionella species by reducing their diversity and abundances. The Legionella species composition of the tap water was highly distinct from that of both source waters. In cold water, 8-14 different phylotypes of Legionella (PTLs) were observed per sample with relative abundances ranging from >1% to 53%. In hot water, L. pneumophila was present during all seasons at high relative abundances (8-40%) accompanied by 5-14 other PTLs of which 6 PTLs were in common with cold water. This thermophilic Legionella community, including L. pneumophila, was able to grow in the hot water above 50 °C. Such thermophilic Legionella populations are of general relevance for drinking water management and public health, but also for the ecology and evolution of the genus Legionella