Search

Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

In 2000, the European Union (EU) launched the Water Framework Directive (WFD), calling for a good ecological status for water bodies by 2015 through integrated river basin management. Despite huge investments, the river Zenne (Belgium) still acquires high loads of pollutants. A multidisciplinary research project was therefore launched to evaluate the effects of river basin wastewater management plans on the river's ecological functioning. Different water quantity and quality processes were considered, and different models were used for each process. For the integration of these models – in view of a holistic analysis – we opted for the Open Modelling Interface (OpenMI). This paper discusses early results of integrated faecal bacteria modelling considering five models. The integrated model is shown to reproduce concentrations of Escherichia coli (used as indicator faecal bacteria) with ‘Very Good’, and ‘Satisfactory’ accuracy for dry weather, and long-term simulations respectively. For both cases, it is found that the E. coli concentrations in the river are well above EU level for bathing water, with occasional combined sewer overflows (CSOs) from the Brussels' sewer systems making the ecological status even worse. We also found OpenMI-based integration to be very useful. However, the calculation time overhead for such OpenMI integrated models remains significant.

Acetobacter pasteurianus, a Gram-negative bacterium belonging to the α-divison of Proteobacteria, produces acetic acid through ethanol oxidation. A genomic bank of A. pasteurianus 386B DNA was cloned in the low-copy cosmid pRG930Cm vector and the resulting clones were screened for the production of protease using the skimmed-milk agar assay whereby a clearing zone around the inoculated spots indicates casein degradation. Several positive clones were selected and restriction analysis revealed that many contained the same inserts. One clone was further analyzed and the cosmid DNA subjected to in vitro transposon insertion. After electroporation, several clones having lost the capacity to cause casein degradation were isolated and the sequence of the transposon-flanking regions analyzed. The majority of insertions mapped to one gene encoding an NAD(P)(+)-dependent aldehyde dehydrogenase (ALDH) of the PNTB superfamily, whereas one insert was found upstream in a gene encoding an ethanol dehydrogenase. Addition of phenol red to the medium confirmed the ethanol-dependent acidification around the inoculated spots of the clones without transposon insertion, suggesting that casein degradation is due to the production of acetic acid as a result of the combined activities of the alcohol dehydrogenase and ALDH. Quantitative data and pH measurements confirmed a significant acidification, and the presence of acetic acid.

Sewage-contaminated rivers are ecosystems deeply disturbed by human activity due to the release of heavy metals, organic pollutants and pharmaceuticals as well as faecal and pathogenic micro-organisms, which coexist with the autochthonous microbial population. In this study, we compared the percentage of resistance in faecal and heterotrophic bacteria in rivers with different degrees of sewage pollution. As a matter of fact, no correlation was found neither between the degree of sewage pollution and the percentage of antimicrobial resistant heterotrophic bacteria nor between the number of resistant faecal bacteria and that of resistant heterotrophic bacteria. Most of the resistant isolates from the Zenne river downstream Brussels were multi-resistant and the resistance patterns were similar among the strains of each phylogenetic group. The total microbial community in this polluted river (as evaluated through a 16S rRNA gene clone library analysis) appeared to be dominated by the phyla Proteobacteria and Bacteroidetes while the phylum TM7 was the third most represented.

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.

The Almendares River, located in Havana city, receives the wastewaters of more than 200,000 inhabitants. The high abundance of faecal bacterial indicators (FBIs) in the downstream stretch of the river reflects the very poor microbiological water quality. In this zone, the Almendares water is used for irrigation of urban agriculture and recreational activities although the microbiological standards for these uses are not met. Improvement of wastewater treatment is absolutely required to protect the population against health risk. This paper compares the removal of FBIs in three wastewater treatment plants (WWTPs) located in this watershed: a conventional facility using trickling filters, a constructed wetland (CW) and a solar aquatic system (SAS). The results indicate better removal efficiency in the two natural systems (CW and SAS) for all the measured parameters (suspended matters, biological oxygen demand, total coliforms, E. coli and enterococci). Removals of the FBIs were around two log units higher in both natural systems than in the conventional one. A longitudinal profile of the microbiological quality of the river illustrates the negative impact of the large conventional WWTP. This case study confirms the usefulness of small and natural WWTPs for tropical developing countries, even in urban and periurban areas.

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procede d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log-log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.

Le nombre de coliformes fecaux (CF) a ete compare au nombre de Escherichia coli trouves dans des echantillons d'eau douce contamines de facon differentielle. La numeration des CF a ete realisee par comptage sur plaque sur du milieu chlorure triphenilique de 2,3,5-tetrazolium Tergitol. La numeration de E. coli a ete faite par la methode du nombre le plus probable (NPP) sur microplaque, basee sur la detection de l'activite glucuronidase. Les quantites de CF et de E. coli etaient en forte correlation; un ratio E. coli/FC moyen egal a 0,77 a ete trouve, indiquant qu'en moyenne E. coli constituait 77 % des CF. La connaissance du ratio E. coli/FC nous permet de convertir des donnees provenant de l'historique de la qualite microbiologique d'un site, exprimees en nombre de CF, en quantite de E. coli et ainsi les comparer avec les donnees de surveillance actuelles et futures qui sont (ou seront) basees sur la numeration de E. coli.

The Seine river watershed (France) is a deeply anthropogenically impacted area, due to the high population density, intense industrial activities and intensive agriculture. The water quality and ecological functioning of the different rivers of the Seine drainage network have been extensively studied during the last fifteen years within the framework of a large French multidisciplinary scientific program (PIREN Seine program). This paper presents a synthesis of the main data gained in the scope of this program concerning the microbiological water contamination of the rivers of the Seine drainage network. The more common indicator of fecal contamination (fecal coliforms) was mainly used; some complementary works used E. coli and intestinal enterococci as alternative fecal indicators. Point sources (outfall of wastewater treatment plants) and non point sources (surface runoff and soil leaching) of fecal pollution to the rivers of the watershed were quantified. Results showed that, at the scale of a large urbanised watershed as the Seine basin, the input of fecal micro-organisms by non-point sources is much lower than the inputs by point sources. However, the local impact of diffuse non-human sources (especially surface runoff of pastured fields) can be of major importance on the microbiological quality of small headwater rivers. Fecal contamination of the main rivers of the Seine watershed (Seine, Marne, Oise rivers) was studied showing high level of microbiological pollution when compared to European guidelines for bathing waters. The strong negative impact of treated wastewater effluents outfall on the microbiological quality of receiving rivers was observed in different areas of the watershed. Once released in rivers, culturable fecal bacteria disappeared relatively rapidly due to mortality (protozoan grazing, lysis) or loss of culturability induced by stress conditions (sunlight effect, nutrient concentration, temperature). Mortality rates of E. coli were studied in different types of rivers within the watershed showing, in summer conditions, no major difference in the mortality rates in small and large rivers. As a result of these studies, a module describing the dynamics of fecal bacteria has been developed and embedded within a hydro-ecological model describing the functioning of the rivers of the whole watershed (the SENEQUE model). Once validated, such a model can be used for testing predictive scenarios and thus can be a very useful tool for the management of microbiological water quality at the scale of the whole basin.

The waters of the Seine river estuary, located in a highly anthropogenicized area in the northern part of France, are of poor microbiological quality; the concentrations of faecal bacteria usually exceed the European Union bathing and recreational water directives. The aim of the present study was to identify the main sources of the faecal pollution of the Seine estuary in order to help define priorities for management and sanitation efforts. Budgets of faecal coliform (FC) inputs to the estuary were established for various hydrological conditions. Main sources of FC were the outfalls of the treated effluents of the wastewater treatment plants (WWTPs) located along the estuary, the faecal bacteria brought in through the tributaries of the Seine estuary, and the faecal bacteria transported by the Seine river flow at the estuary entrance at Poses dam. In order to quantify these inputs, FC were enumerated during sampling campaigns conducted for various hydrological conditions in the Seine at the entrance of the estuary, in the tributaries close to their confluence with the estuary, and in the effluents of some WWTPs located along the estuary. The importance of the flux of FC transported by the Seine river flow at the estuary entrance at Poses dam decreased from 92% of the total FC input when the flow rate was high (717 m3 s−1) to 5% when flow rate was low (143 m3 s−1). The release of the domestic wastewaters of the large city of Paris located 120 km upstream from the entrance of the estuary was mainly responsible for this microbiological pollution. At low flow rates, the tributaries represent the most important source of FC (64–76% for flow rates of the Seine at Poses at approximately 150 m3 s−1), mainly from the Robec and Eure rivers. The treated wastewater of the WWTPs located along the estuary was the second source of FC for low flow conditions (19–30%); it was less important for high to intermediate flow rate conditions.

The microbial community composition in meromictic Lake Kivu, with one of the largest CH4 reservoirs, was studied using 16S rDNA and ribosomal RNA (rRNA) pyrosequencing during the dry and rainy seasons. Highly abundant taxa were shared in a high percentage between bulk (DNA-based) and active (RNA-based) bacterial communities, whereas a high proportion of rare species was detected only in either an active or bulk community, indicating the existence of a potentially active rare biosphere and the possible underestimation of diversity detected when using only one nucleic acid pool. Most taxa identified as generalists were abundant, and those identified as specialists were more likely to be rare in the bulk community. The overall number of environmental parameters that could explain the variation was higher for abundant taxa in comparison to rare taxa. Clustering analysis based on operational taxonomic units (OTUs at 0.03 cutoff) level revealed significant and systematic microbial community composition shifts with depth. In the oxic zone, Actinobacteria were found highly dominant in the bulk community but not in the metabolically active community. In the oxic-anoxic transition zone, highly abundant potentially active Nitrospira and Methylococcales were observed. The co-occurrence of potentially active sulfur-oxidizing and sulfate-reducing bacteria in the anoxic zone may suggest the presence of an active yet cryptic sulfur cycle. ispartof: Microbial Ecology vol:3 issue:3 pages:596-611 ispartof: location:United States status: published

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).

We review available data on archaea, bacteria and small eukaryotes in an attempt to provide a general picture of microbial diversity, abundances and microbe-driven processes in Lake Kivu surface and intermediate waters (ca. 0–100 m). The various water layers present contrasting physical and chemical properties and harbour very different microbial communities supported by the vertical redox structure. For instance, we found a clear vertical segregation of archaeal and bacterial assemblages between the oxic and the anoxic zone of the surface waters. The presence of specific bacterial (e.g. Green Sulfur Bacteria) and archaeal (e.g. ammonia-oxidising archaea) communities and the prevailing physico-chemical conditions point towards the redoxcline as the most active and metabolically diverse water layer. The archaeal assemblage in the surface and intermediate water column layers was mainly composed by the phylum Crenarchaeota, by the recently defined phylum Thaumarchaeota and by the phylum Euryarchaeota. In turn, the bacterial assemblage comprised mainly ubiquitous members of planktonic assemblages of freshwater environments (Actinobacteria, Bacteroidetes and Betaproteobacteria among others) and other less commonly retrieved phyla (e.g. Chlorobi, Clostridium and Deltaproteobacteria). The community of small eukaryotes (

Approved methods traditionally used for Escherichia coli enumeration in waters are culture-based. However, these methods can underestimate the E. coli abundance in aquatic systems because they do not take into account cells that remain viable but have lost the ability to grow in or on culture media. We investigated, in freshwater samples, the abundance of (i) culturable E. coli, enumerated by the most probable number microplate method and (ii) viable E. coli, estimated using a procedure called DVC–FISH, which couples fluorescent in situ hybridization (FISH) and a viability testing technique (direct viable count (DVC)). The ratio of culturable to viable E. coli was close to 1 in highly contaminated waters (samples with a high concentration of culturable E. coli), but decreased drastically for weakly contaminated samples. This indicates a large fraction of viable but nonculturable (VBNC) E. coli in the latter samples. Microcosm experiments showed that some environmental factors, such as nutrient scarcity and solar irradiation, could lead to the presence of a high proportion of VBNC E. coli.

Attachment of fecal bacteria to suspended matter in the water column has important implications for its fate in rivers. We examined the part of Escherichia coli (E. coli) associated with suspended matter in natural river water samples, using a combination of 5-microm filtration and beta-D-glucuronidase (GLUase) assay to estimate the E. coli abundance and attachment. We observed that the fraction of particle-associated E. coli was positively correlated with suspended matter concentration. The settling rate of particle-associated E. coli was found to be positively correlated with suspended matter concentration for samples with suspended matter content lower than 50 mg/L. For samples with higher suspended matter concentration, the settling rate was quite constant (0.066 m/h, on average). In batch experiments using river waters, we observed that free E. coli had a decay rate approximately 2 times higher than particle-associated E. coli. This information can be used to improve the models on the fate of E. coli in rivers.

The Seine river watershed is characterized by a high population density and intense agricultural activities. Data show low microbiological water quality in the main rivers (Seine, Marne, Oise) of the watershed. Today, there is an increasing pressure from different social groups to restore microbiological water quality in order to both increase the safety of drinking water production and to restore the possible use of these rivers for bathing and rowing activities, as they were in the past. A model, appended to the hydro-ecological SENEQUE/Riverstrahler model describing the functioning of large river systems, was developed to describe the dynamics of faecal coliforms (FC), the most usual faecal contamination indicator. The model is able to calculate the distribution of FC concentrations in the whole drainage network resulting from land use and wastewater management in the watershed. The model was validated by comparing calculated FC concentrations with available field data for some well-documented situations in different river stretches of the Seine drainage network. Once validated, the model was used to test various predictive scenarios, as, for example, the impact of the modifications in wastewater treatment planned at the 2012 horizon in the Seine watershed in the scope of the implementation of the european water framework directive. The model was also used to investigate past situations. In particular, the variations of the microbiological water quality in the Parisian area due to population increase and modifications in wastewater management were estimated over the last century. It was shown that the present standards for bathing and other aquatic recreational activities are not met in the large tributaries upstream from Paris since the middle of the 1950's, and at least since the middle of the XIXth century in the main branch of the Seine river downstream from Paris. Efforts carried out for improving urban wastewater treatment in terms or organic matter and nutrient loading resulted in a sensible reduction of microbiological contamination, but were not specific enough toward bacteriological contamination for achieving the objective of restoring levels compatible with bathing activities in the Parisian area.

Because the large rivers of the Seine watershed have a low microbiological water quality, the main sources of fecal contamination were investigated in the present study. The inputs of the point (wastewater treatment plants (WWTPs) effluents) and non-point sources (surface runoff and soil leaching) of fecal bacteria were quantified for Escherichia coli and intestinal enteroccoci used as bacterial indicators. In order to assess the contamination through non-point sources, fecal indicators abundance was estimated in samples collected in small streams located in rural areas upstream from all point sources; these small rivers were characterized by the land use of their watershed. Bacterial indicator numbers were also measured in effluents of WWTPs, some using classical treatment (settling followed by activated sludge process) and some using an additional disinfection stage (UV irradiation). These data were used to estimate the respective importance of each type of source at the scale of the whole Seine river watershed taking into account the land use and the population density. It shows the predominant importance of the point sources of fecal indicator bacteria at the scale of the whole watershed. In a scenario in which activated sludge treatment would be complemented with UV in all WWTPs located in this watershed, the non-point sources of fecal indicator bacteria would be dominant.

Aims: The relationships between the β-d-glucuronidase (GLUase) activity, the abundance of culturable Escherichia coli and the number of viable E. coli were investigated in river and wastewater samples. Methods and Results: GLUase activity was measured as the rate of hydrolysis of 4-methylumbelliferyl-β-d-glucuronide. Culturable E. coli were enumerated by the most probale number (MPN) microplate method. Viable E. coli were estimated by fluorescent in situ hybridization (FISH) coupled with a procedure of viability testing (DVC-FISH procedure). Significant correlations were found between the log of GLUase activity and both, the log culturable E. coli and the log of viable E. coli. Conclusions: GLUase activity per viable E. coli gave a broadly constant value from low to highly contaminated waters while GLUase activity per culturable E. coli strongly increased at low contaminated waters because of an underestimation of the number of active E. coli by the culture-based method. Significance and Impact of the Study: GLUase activity is a reliable parameter for the rapid quantification of viable E. coli in waters.

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culture-based method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water.

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species., SCOPUS: ar.j, info:eu-repo/semantics/published

In 2000, the European Union (EU) launched the Water Framework Directive (WFD), calling for a good ecological status for water bodies by 2015 through integrated river basin management. Despite huge investments, the river Zenne (Belgium) still acquires high loads of pollutants. A multidisciplinary research project was therefore launched to evaluate the effects of river basin wastewater management plans on the river's ecological functioning. Different water quantity and quality processes were considered, and different models were used for each process. For the integration of these models – in view of a holistic analysis – we opted for the Open Modelling Interface (OpenMI). This paper discusses early results of integrated faecal bacteria modelling considering five models. The integrated model is shown to reproduce concentrations of E. coli (used as indicator faecal bacteria) with ‘Very Good’, and ‘Satisfactory’ accuracy for dry weather, and long-term simulations respectively. For both cases, it is found that the E. coli concentrations in the river are well above EU level for bathing water, with occasional Combined Sewer Overflows (CSOs) from the Brussels' sewer systems making the ecological status even worse. We also found OpenMI-based integration to be very useful. However, the calculation time overhead for such OpenMI integrated models remains significant.