Adriana Anzil

Using light scatter signal to estimate bacterial biovolume by flow cytometry

BACKGROUND In the past decade, flow cytometry has become a useful and precise alternative to microscopic bacterial cell counts in aquatic samples. However, little evidence of its usefulness for the evaluation of bacterial biovolumes has emerged in from the literature. METHODS The light scattering and cell volume of starved bacterial strains and natural bacterial communities from the Black Sea were measured by flow cytometry and epifluorescence microscopy, respectively, in order to establish a relationship between light scattering and cell volume. RESULTS With the arc-lamp flow cytometer, forward angle light scatter (FALS) was related to cell size in both the starved strains and natural communities, although regression parameters differed. We tested the predictive capacity of the FALS verous cell size relationship in a bacterial community from the North Sea. That analysis showed that a reliable bacterial biovolume prediction of a natural bacterial community can be obtained from FALS using a model generated from natural bacterial community data. CONCLUSIONS Bacterial biovolume is likely to be related to FALS measurements. It is possible to establish a generally applicable model derived from natural bacterial assemblages for flow cytometric estimation of bacterial biovolumes by light scatter.

Seasonal Variations and Resilience of Bacterial Communities in a Sewage Polluted Urban River

The Zenne River in Brussels (Belgium) and effluents of the two wastewater treatment plants (WWTPs) of Brussels were chosen to assess the impact of disturbance on bacterial community composition (BCC) of an urban river. Organic matters, nutrients load and oxygen concentration fluctuated highly along the river and over time because of WWTPs discharge. Tag pyrosequencing of bacterial 16S rRNA genes revealed the significant effect of seasonality on the richness, the bacterial diversity (Shannon index) and BCC. The major grouping: -winter/fall samples versus spring/summer samples- could be associated with fluctuations of in situ bacterial activities (dissolved and particulate organic carbon biodegradation associated with oxygen consumption and N transformation). BCC of the samples collected upstream from the WWTPs discharge were significantly different from BCC of downstream samples and WWTPs effluents, while no significant difference was found between BCC of WWTPs effluents and the downstream samples as revealed by ANOSIM. Analysis per season showed that allochthonous bacteria brought by WWTPs effluents triggered the changes in community composition, eventually followed by rapid post-disturbance return to the original composition as observed in April (resilience), whereas community composition remained altered after the perturbation by WWTPs effluents in the other seasons.

Bacterial Community Composition in Three Freshwater Reservoirs of Different Alkalinity and Trophic Status

In order to investigate the factors controlling the bacterial community composition (BCC) in reservoirs, we sampled three freshwater reservoirs with contrasted physical and chemical characteristics and trophic status. The BCC was analysed by 16S rRNA gene amplicon 454 pyrosequencing. In parallel, a complete dataset of environmental parameters and phytoplankton community composition was also collected. BCC in the analysed reservoirs resembled that of epilimnetic waters of natural freshwater lakes with presence of Actinobacteria, Alpha- and Betaproteobacteria, Cytophaga–Flavobacteria–Bacteroidetes (CFB) and Verrucomicrobia groups. Our results evidenced that the retrieved BCC in the analysed reservoirs was strongly influenced by pH, alkalinity and organic carbon content, whereas comparatively little change was observed among layers in stratified conditions.

Impact of Wastewater Release on the Faecal Contamination of a Small Urban River: The Zenne River in Brussels (Belgium)

In this study, we investigated the microbiological quality of the small urban Zenne River which flows through the city of Brussels (Belgium). The abundances of faecal indicator bacteria (FIB) (Escherichia coli and intestinal enterococci) revealed high levels of faecal contamination of the Zenne River, especially in Brussels area and downstream from this area where FIB were in the range of what is usually observed in secondary treated wastewaters. The origin of this faecal contamination is mainly attributed to the effluents of the two wastewater treatment plants (WWTPs) located along the river in Brussels. Comparison of the present situation with the 1990s situation (before the implementation of Brussels WWTPs) showed a sharp improvement of the river microbiological quality. This improvement should however be regarded with care as it was also observed that, during rain events, combined sewer overflows (CSO) outfalls were responsible of a strong increase of faecal contamination in the river downstream from Brussels, and such CSO occur frequently in Brussels. Altogether, these results document the variations of the microbiological quality of a sewage-polluted urban river in relation with long-term changes (implementation of WWTP) and the short-term disturbances (CSO).

Identification of antimicrobial resistant bacteria in rivers: insights into the cultivation bias

In the present study, the antimicrobial resistant (AR) bacteria were quantified and identified in different river samples using in parallel a culture-based approach and a culture-independent one. The objective was to evaluate the importance of the cultivation bias when studying antimicrobial resistance among environmental bacteria. Three different river samples covering a gradient of anthropic influence were tested and three different antimicrobial compounds were used as selective agents: amoxicillin, tetracycline and sulfamethoxazole. From a quantitative point of view, our results highlight the importance of the culture media used, as for the same sample and the same selective agent significant differences were observed in the counts of culturable AR bacteria depending on the culture media used. The identification of AR bacteria through culture or culture-independent methods put on evidence AR bacterial communities that differ dramatically: γ-proteobacteria and more specifically Aeromonadaceae dominated among the isolates while β-proteobacteria (Comamonadaceae), dominated among the sequences obtained without culture. Altogether these results highlight the necessity to develop a methodological consensus preferably without culture, to approach this important topic in the coming years.

Antibiotic resistance along an urban river impacted by treated wastewaters

Urban rivers are impacted ecosystems which may play an important role as reservoirs for antibiotic-resistant (AR) bacteria. The main objective of this study was to describe the prevalence of antibiotic resistance along a sewage-polluted urban river. Seven sites along the Zenne River (Belgium) were selected to study the prevalence of AR Escherichia coli and freshwater bacteria over a 1-year period. Culture-dependent methods were used to estimate E. coli and heterotrophic bacteria resistant to amoxicillin, sulfamethoxazole, nalidixic acid and tetracycline. The concentrations of these four antibiotics have been quantified in the studied river. The antibiotic resistance genes (ARGs), sul1, sul2, tetW, tetO, blaTEM and qnrS were also quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed an effect of treated wastewaters release on the spread of antibiotic resistance along the river. Although an increase in the abundance of both AR E. coli and resistant heterotrophic bacteria was observed from upstream to downstream sites, the differences were only significant for AR E. coli. A significant positive regression was also found between AR E. coli and resistant heterotrophic bacteria. The concentration of ARGs increased from upstream to downstream sites for both particle-attached (PAB) and free-living bacteria (FLB). Particularly, a significant increase in the abundance of four among six ARGs analyzed was observed after crossing urban area. Although concentrations of tetracycline significantly correlated with tetracycline resistance genes, the antibiotic levels were likely too low to explain this correlation. The analysis of ARGs in different fractions revealed a significantly higher abundance in PAB compared to FLB for tetO and sul2 genes. This study demonstrated that urban activities may increase the spread of antibiotic resistance even in an already impacted river.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as “moderate” at the upstream station and as “major” at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

Occurrence and persistence of carbapenemases genes in hospital and wastewater treatment plants and propagation in the receiving river

This study aims to investigate the prevalence of clinically relevant carbapenemases genes (blaKPC, blaNDM and blaOXA-48) in water samples collected over one-year period from hospital (H), raw and treated wastewater of two wastewater treatment plants (WWTPs) as well as along the Zenne River (Belgium). The genes were quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed that absolute abundances were the highest in H waters. Although absolute abundances were significantly reduced in WWTP effluents, the relative abundance (normalized per 16S rRNA) was never lowered through wastewater treatment. Particularly, for the PAB the relative abundances were significantly higher in the effluents respect to the influents of both WWTPs for all the genes. The absolute abundances along the Zenne River increased from upstream to downstream, peaking after the release of WWTPs effluents, in both fractions. Our results demonstrated that blaKPC, blaNDM and blaOXA-48 are widely distributed in the Zenne as a consequence of chronic discharge from WWTPs. To conclude, the levels of carbapenemases genes are significantly lower than other genes conferring resistance to more widely used antibiotics (analyzed in previous studies carried out at the same sites), but could raise up to the levels of high prevalent resistance genes.