Antimicrobial resistance of Escherichia coli isolated from freshwaters and hospital effluents in Belgium.

The purpose of this work was to evaluate the level of antimicrobial resistant Escherichia coli isolates in freshwaters and hospital effluents in Belgium. The samples were collected from 24 locations along the Ourthe, Vesdre, Amblève and Meuse rivers and in the wastewater effluents of several hospitals. The sampling stations in rivers were classified according to the dominant land covers of the rivers (rural, urban and forest areas). Two sampling campaigns were organized in May and October 2019 to highlight a possible seasonal effect. A total of 938 E. coli strains were isolated on Chromogenic Selective Tryptone Bile X-glucuronide (TBX) and TBX supplemented with amoxicillin (TBX+AMX) media. Disk diffusion assays were performed following the EUCAST's recommendations to assess the antimicrobial resistance against 12 antibiotics. A total of 32·7% of strains were at least resistant to one antibiotic and 24·6% were multiple antimicrobial resistant strains on TBX. The highest resistance rates were found for ampicillin (AMP), amoxicillin coupled with clavulanic acid (AMC) and sulfamethoxazole/trimethoprim (SXT). The lowest resistance rates were observed for meropenem (MEM) and ertapenem (ETP), which are last resort antibiotics. No significant difference was observed between both campaigns for the resistance rate to antibiotics.

[Multiple myeloma in an elderly patient with history of breast cancer : the value of multidisciplinary oncogeriatric collaboration]. / Cas clinique. Prise en charge d'un myélome multiple chez une patiente âgée aux antécédents de néoplasie mammaire : intérêt d'une collaboration multidisciplinaire onco-gériatrique.

As the prevalence of cancers increases with age, some elderly patients are confronted with multiple tumoural pathologies. The clinical case of a 70-year-old patient with adenocarcinoma of the breast and multiple myeloma complicated by severe renal failure illustrates the complexity of oncogeriatric management. The geriatric assessment makes it possible to detect frailty and provides assistance in the development of a personalized care plan while respecting the quality of life.

Comme la prévalence des cancers augmente avec l'âge, certains patients âgés se trouvent confrontés à plusieurs pathologies tumorales. Ce cas clinique d'une patiente de 70 ans, avec un adénocarcinome du sein et un myélome multiple compliqué d'une insuffisance rénale sé-vère, illustre la complexité de la prise en charge oncogériatrique. En effet, l'évaluation gériatrique permet de dépister la fragilité et apporte une aide à l'élaboration d'un plan de soins personnalisé en respectant la qualité de vie.

Endocrine activity in an urban river system and the biodegradation of estrogen-like endocrine disrupting chemicals through a bio-analytical approach using DRE- and ERE-CALUX bioassays.

The Zenne River, crossing the Brussels region (Belgium) is an extremely urbanized river impacted by both domestic and industrial effluents. The objective of this study was to monitor the occurrence and activity of Endocrine Active Substances (EAS) in river water and sediments in the framework of the Environmental Quality Standards Directive (2008/105/EC and 2013/39/EU). Activities were determined using Estrogen and Dioxin Responsive Elements (ERE and DRE) Chemical Activated Luciferase Gene Expression (CALUX) bioassays. A potential contamination source of estrogen active compounds was identified in the river at an industrial area downstream from Brussels with a peak value of 938 pg E2 eq./L water (above the EQS of 0.4 ng/L) and 195 pg E2 eq./g sediment. Estrogens are more abundantly present in the sediments than in the dissolved phase. Principal Component Analysis (PCA) showed high correlations between Suspended Particulate Matter (SPM), Particulate (POC) and Dissolved Organic Carbon (DOC) and estrogenic EAS. The dioxin fractions comply with previous data and all were above the United States Environmental Protection Agency (US EPA) low-level risk, with one (42 pg TCDD eq./g sediment) exceeding the high-level risk value for mammals. The self-purifying ability of the Zenne River regarding estrogens was examined with an in vitro biodegradation experiment using the bacterial community naturally present in the river. Hill coefficient and EC50 values (Effective Concentration at 50%) revealed a process of biodegradation in particulate and dissolved phase. The estrogenic activity was decreased by 80%, demonstrating the ability of self-purification of estrogenic compounds in the Zenne River.

Gamma irradiation: a method to produce an abiotic control for biological activated carbon.

The aim of this paper was to investigate the feasibility of using gamma irradiation to inhibit the microbial activity of biological powder activated carbon (PAC) without impacting its adsorptive properties. First of all, the range of dose of gamma rays required to produce abiotic PAC was selected on the basis of heterotrophic plate counts (HPC) inactivation and methylene blue (MB) adsorption kinetics. Doses inferior to 10 kGy were not sufficient to inhibit the culture of heterotrophic bacteria. On the other hand, doses superior to 15 kGy were demonstrated to affect the adsorption rate of MB. Consequently, a dose comprised between 10 and 15 kGy was selected for further investigation. In order to validate the adequacy of the range of dose (i.e. 10-15 kGy), adsorption characteristics were tested by monitoring the removal kinetics of refractory dissolved organic carbon (RDOC). No significant differences were observed between irradiated and non-irradiated biological PAC for the adsorption of RDOC. Irradiated, non-irradiated and virgin PAC were also evaluated in terms of abundance of viable (using the LIVE/DEAD BacLight method) bacteria and in terms of heterotrophic biomass activity. The results of the BacLight method demonstrated that attachment of the biofilm on the PAC was not impacted by the irradiation and heterotrophic activity measurements demonstrated that the latter could be radically reduced in the range of dose selected. In conclusion, when using a proper dose, the gamma irradiation of colonized activated carbon drastically reduced the heterotrophic activity on activated carbon without significantly impacting its adsorptive behaviour.

Rapid enumeration of Escherichia coli in marine bathing waters: potential interference of nontarget bacteria.

AIMS: To compare the Escherichia coli quantification given by the 'Coliplage' assay, based on the direct measurement of the beta-D-glucuronidase (GLUase) activity and the reference Most Probable Number (MPN) method from seawater sites and investigate the possible interference of non-E. coli strains in the GLUase activity measurement. METHODS AND RESULTS: Comparison performed from 69 French coastal bathing sites (1401 samples) showed nonconcordance between both methods, only for 8% of samples. Non-E. coli 4-methylumbelliferyl-beta-D-glucuronide (MUG+) were isolated from nonconcordant samples. Phylogenetic analysis showed that Gammaproteobacteria were dominants and mainly represented by Vibrio species, which displayed GLUase activities on the same order of magnitude and sometimes much higher as E. coli reference strains. CONCLUSIONS: The'Coliplage' assay is a rapid method for the quantification of E. coli showed few discordances with the standard MPN method. Some Vibrio species could interfere on the direct GLUase activity measurement of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Data present the first qualitative investigation on disagreement between Coliplage and the MPN results. If the interference of Vibrio species is confirmed in situ, appropriate treatments should be developed to remove the interfering signal.

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters.

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 procédé 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.

Respective contributions of point and non-point sources of E. coli and enterococci in a large urbanized watershed (the Seine river, France).

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.

Coliforms and other microbial indicators occurrence in water and biofilm in full-scale distribution systems.

Biofilm and microbial water quality were studied in four middle size full-scale distribution systems (DS) in France serving 5,000-30,000 inhabitants (maximum residence time 23-160h) through three sampling campaigns over 1 year. Three of these DSs were chosen because of a quite high occurrence of bacterial indicators (i.e. total coliforms), the last DS was considered as a reference. Biofilm was studied on cast iron coupons incubated for more than 1 month in devices continuously fed with water from the DS in conditions imitating those met in DS. The devices were located at different points (4-6) along each DS. The abundance of bacteria in biofilm was estimated by heterotrophic plate counts (HPC) after detachment of the biofilm from the support by sonication. Microbiological water quality was estimated in parallel; analysis of total coliforms, E. coli, enterococci and anaerobic sulphide-reducing bacteria spores (ASRB spores) was carried out in biofilm and water. Over the period of the study, 171 water samples and 57 biofilm samples were collected. Over these 171 waters, 19 (11%) were positive for at least one of the measured indicators while two biofilm samples were positive (3.5%). Significant differences were observed in the levels of contamination between the DSs. High residence time in the DS, low disinfectant residual and high temperature increased the risk of indicator occurrence in the water phase. Due to the low number of biofilm samples positive for bacterial indicators, the data collected in the present study did not allow observation of a direct association between biofilm and water contaminations, even if the occurrence of indicators in water appeared on DSs with the highest density of biofilm (HPC).

Modelling faecal coliforms dynamics in the Seine estuary, France.

A model describing the dynamics of faecal coliforms (FC) in the Seine estuary has been developed and coupled with a three-dimensional hydrodynamic model. As input to this model three types of FC sources were considered: (1) FC transported by the Seine river flow at the estuary entrance at Poses dam; (2) FC brought in through the tributaries of the Seine estuary; and (3) the outfalls of the treated effluents of the WWTPs located along the estuary. As previous studies in the Seine estuary showed that a large proportion of FC was attached to SM, two pools of FC were considered separately in the model: free FC and FC attached to SM. Two main processes controlling the fate of FC in the estuary were considered: mortality and settling. The model calculates for a given discharge of the Seine river at Poses the longitudinal distribution of FC along the estuary. The model was validated by comparison of model calculations with experimental data in various hydrological conditions. The model also correctly reproduced the impact of the main river flow rate on the level of estuarine water's contamination. Finally, the model was used to test different scenarios of sanitation, suggesting priorities for sanitation efforts.

An operational method for the real-time monitoring of E. coli numbers in bathing waters.

The aim of this study was to investigate the potential application of the beta-d-glucuronidase (GLUase) activity measurement for the routine detection and quantification of E. coli in marine bathing waters. GLUase activity was measured as the rate of hydrolysis of 4-methylumbelliferyl-beta-d-glucuronide. Culturable E. coli were quantified by the most probable number (MPN) microplate method. Both methods were applied to a large set of seawater samples. Significant correlation was found between the log of GLUase activity and the log of culturable E. coli. The mean coefficient of variation (CV) of the GLUase activity was less than 15% at concentrations around the current standards of International regulations whereas the CV of the microplate method was around 30%. When samples were stored at 4 degrees C and 20 degrees C, the mean CV of the GLUase activity remained below 15% up to 6 hours after sample collection whereas the range of variation of the microplate method varied between 10 and 50%. We concluded that the GLUase activity is an operational, reproducible, simple, very rapid and low cost method for the real-time enumeration of E. coli in bathing waters and should be preferred to the microplate method. The GLUase activity method should be routinely applied to the rapid enumeration of E. coli in recreational waters and recommendations for its application were suggested to water quality managers.

Beta-D-glucuronidase activity assay to assess viable Escherichia coli abundance in freshwaters.

AIMS: The relationships between the beta-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-beta-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.

Combining direct viable count (DVC) and fluorescent in situ hybridisation (FISH) to enumerate viable E. coil in rivers and wastewaters.

In this study, a combination of the direct viable count procedure (DVC) and the FISH method was used to monitor by epifluorescence microscopy the abundance of viable E. coli in river water and wastewater samples. The DVC procedure consisted of exposing bacterial cells to a resuscitation medium containing antibiotics preventing cellular division and, thus, inducing an elongation of the viable cells. The FISH was performed using the "Colinsitu" probe specific for E. coil 16S r-RNA. Accuracy and detection limit of the epifluorescence microscopic DVC-FISH procedure were investigated. The method was then applied to river-water and wastewater samples. A good correlation was found in a log-log plot between the abundance of E. coil enumerated by a classical culture-based method (MPN method) and the DVC-FISH procedure. However, the DVC-FISH procedure gave consistently higher numbers. The ratio between both enumerations (DVC-FISH/MPN), which also indicated the ratio between viable and culturable E. coli, ranged between 2 and >30. It increased with decreasing abundance of culturable E. coli.

Impact of backwashing on nitrification in the biological activated carbon filters used in drinking water treatment.

Nitrification during biological filtration is currently used in drinking water production to remove ammonia, which is the source of several water quality problems during treatment and distribution. We evaluated here the impact of backwashing on nitrification efficiency in filters used for drinking water treatment. Two different granular activated carbon (one open and one closed carbon superstructure) were tested. Ammonia removal and fixed nitrifying bacterial biomass before and after backwashing were compared in first-stage pilot filters and full-scale second-stage filters. Backwashing has a greater impact on nitrification on first-stage than an second-stage filters. Backwashing improved the ammonia removal in warm (> or = 18 degrees C) water in a first-stage filter containing an open-superstructure granular activated carbon, whereas a closed-superstructure support showed a removal capacity that is less after a regular backwashing than before, or similar to it. In cold water (< or = 4 degrees C), backwashing had a negative impact on nitrification capacity in an open-superstructure medium first-stage filter. In full-scale second-stage filters, backwashing had a slight negative impact on filter performances for both open- and closed-superstructure media at temperatures between 8 degrees C and 12 degrees C. In colder waters (< or = 3 degrees C), nitrification was very poor both before and after backwashing. Sampling of fixed, nitrifying biomass in the pilot filter columns showed that in all cases (low and high expansion backwash; both media tested) and at 20 +/- 3 degrees C the overall biomass levels remained unchanged before and after backwash. In the full-scale second-stage filters, nitrifying bionass was similar before and after backwashing for both types of media tested.

Use of beta-D-galactosidase and beta-D-glucuronidase activities for quantitative detection of total and fecal coliforms in wastewater.

Two enzymatic methods based on the measurement of the beta-D-galactosidase activity of total coliforms and the beta-D-glucuronidase activity of Escherichia coli were used to assess coliform levels in wastewater alongside traditional culture-based techniques, which can be biased by the aggregation of bacteria or their attachment to particles. Enzymatic methods were precise (i.e., coefficients of variation were 9%-15%), rapid (response in 20 min), and correlated well (in log units) with traditional techniques for raw and treated sewage (r2 > 0.75). They were used for rapid assessment of coliform removal efficiency in two different wastewater treatments. These methods could be useful for the estimation of the abundance of coliforms in domestic sewage and their removal by wastewater treatment processes.

Impact of temperature on nitrification in biological activated carbon (BAC) filters used for drinking water treatment.

The impact of temperature on nitrification in biological granular activated carbon (GAC) filters was evaluated in order to improve the understanding of the nitrification process in drinking water treatment. The study was conducted in a northern climate where very cold water temperatures (below 2 degrees C) prevail for extended periods and rapid shifts of temperature are frequent in the spring and fall. Ammonia removals were monitored and the fixed nitrifying biomass was measured using a method of potential nitrifying activity. The impact of temperature was evaluated on two different filter media: an opened superstructure wood-based activated carbon and a closed superstructure activated carbon-based on bituminous coal. The study was conducted at two levels: pilot scale (first-stage filters) and full-scale (second-stage filters) and the results indicate a strong temperature impact on nitrification activity. Ammonia removal capacities ranged from 40 to 90% in pilot filters, at temperatures above 10 degrees C, while more than 90% ammonia was removed in the full-scale filters for the same temperature range. At moderate temperatures (4-10 degrees C), the first stage pilot filters removed 10-40% of incoming ammonia for both media (opened and closed superstructure). In the full-scale filters, a difference between the two media in nitrification performances was observed at moderate temperatures: the ammonia removal rate in the opened superstructure support (more than 90%) was higher than in the closed superstructure support (45%). At low temperatures (below 4 degrees C) both media performed poorly. Ammonia removal capacities were below 30% in both pilot- and full-scale filters.

Use of rapid enzymatic assays to study the distribution of faecal coliforms in the Seine river (France).

In 1997 and 1998 faecal contamination of the Seine river and its estuary was studied for the first time by rapid enzymatic methods, based on the presence of the beta-D-glucuronidase enzyme in E. coli, in parallel with traditional plate counts of faecal coliforms on specific culture medium. Our study focused on a 450 km stretch of the river, including the Parisian area, and presenting highly variable levels of faecal pollution. Both methods showed that wastewater outfalls of the Parisian area and the presence of a maximum turbidity zone (at the mouth of the estuary) had a strong impact on the abundance of faecal coliforms in the river. Downstream from the Parisian outfalls, beta-D-glucuronidase activity measurements decreased 5-6x less rapidly than plate counts suggesting that rapid enzymatic assays could detect enzymatically-active but non-culturable bacteria.

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.

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.

Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments?

The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another. Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using (3)H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production.

Bacterial dynamics in the drinking water distribution system of Brussels.

Water samples and pipe coupons were collected from the Brussel's drinking water distribution system (DS). A treated surface water and various groundwaters feed this DS. Parameters related to bacterial regrowth have been measured on these samples: temperature, concentrations of free residual chlorine, concentration of biodegradable dissolved organic carbon (BDOC), abundance of suspended bacteria, densities of fixed bacteria and levels of bacterial activity. Results showed that groundwaters were less susceptible to favor bacterial regrowth in the DS pipes. Treated surface water and mixed waters had the highest potential of bacterial regrowth in the DS dead ends. Results also showed that the potential regrowth induced by the distribution of a treated surface water could be reduced if: (1) the BDOC levels were below 0.25 mg C/l at the outlet of the surface water treatment plant; (2) a significant free chlorine residual was present within the whole DS. Second-stage biological filtration using granular activated carbon is now under construction at the surface water treatment plant feeding a part of this DS. This treatment implementation should reduce BDOC levels and chlorine demand of the treated surface water and will further reduce the slight regrowth phenomena observed in this DS.