Fate of two strains of extended-spectrum beta-lactamase producing Escherichia coli in constructed wetland microcosm sediments: survival and change in antibiotic resistance profiles.

Free water surface constructed wetlands (FWS CW) are efficient technologies to limit the transfer of antibiotic resistant bacteria (ARB) originating from urban effluents into the aquatic environment. However, the decrease in ARB from inflow to outflow through the FWS CW may be explained by their transfer from the water body to the sediment. To investigate the behavior of ARB in the sediment of a FWS CW, we inoculated three microcosms with two strains of extended-spectrum beta-lactamase producing Escherichia coli (ESBL E. coli) belonging to two genotypes. Microcosms were composed of two sediments collected at two locations of an FWS CW from which the strains were isolated. Phragmites were planted in one of the microcosms. The survival curves of the two strains were close regardless of the genotype and the type of sediment. After a rapid decline, both strains were able to survive at low level in the sediments for 50 days. Their fate was not affected by the presence of phragmites. Changes in the bla content and antibiotic resistance of the inoculated strains were observed after three weeks of incubation, indicating that FWS CW sediments are favorable environments for spread of antibiotic resistance genes and for the acquisition of new antibiotic resistance.

In situ continuous monitoring of nitrogen with ion-selective electrodes in a constructed wetland receiving treated wastewater: an operating protocol to obtain reliable data.

Constructed wetlands receiving treated wastewater (CWtw) are placed between wastewater treatment plants and receiving water bodies, under the perception that they increase water quality. A better understanding of the CWtw functioning is required to evaluate their real performance. To achieve this, in situ continuous monitoring of nitrate and ammonium concentrations with ion-selective electrodes (ISEs) can provide valuable information. However, this measurement needs precautions to be taken to produce good data quality, especially in areas with high effluent quality requirements. In order to study the functioning of a CWtw instrumented with six ISE probes, we have developed an appropriate methodology for probe management and data processing. It is based on an evaluation of performance in the laboratory and an adapted field protocol for calibration, data treatment and validation. The result is an operating protocol concerning an acceptable cleaning frequency of 2 weeks, a complementary calibration using CWtw water, a drift evaluation and the determination of limits of quantification (1 mgN/L for ammonium and 0.5 mgN/L for nitrate). An example of a 9-month validated dataset confirms that it is fundamental to include the technical limitations of the measuring equipment and set appropriate maintenance and calibration methodologies in order to ensure an accurate interpretation of data.

Free water surface constructed wetlands limit the dissemination of extended-spectrum beta-lactamase producing Escherichia coli in the natural environment.

The fates of Escherichia coli and extended-spectrum beta-lactamase-producing E. coli (ESBL E. coli) were studied over a period of one year in a free water surface constructed wetland (FWS CW) with a succession of open water zones and vegetation ponds (Typha or Phragmites), that received the effluent from a wastewater treatment plant. ESBL E. coli were detected and isolated from all sampling areas of the FWS CW throughout the study period. They represented 1‰ of the total E. coli population regardless of the origin of samples. Two main factors affected the log removal of E. coli and of ESBL E. coli: the season and the presence of vegetation. Between the inlet and the outlet of the FWS CW, the log removal of E. coli ranged from 1.5 in the warmer season (summer and fall) to 3.0 in the colder season (winter and spring). The concentrations of E. coli decreased significantly in the vegetated areas during the colder season, but increased in the warmer season, suggesting an effect of the plant growth stage on the survival of E. coli. Among the 369 ESBL E. coli isolates collected during our study, 84% harbored the CTX-M-ESBL type and 55.3% carried bla genes on plasmid DNA. Furthermore, 93% of the ESBL E. coli isolates were multidrug resistant but the proportion of resistant strains did not change significantly along the FWS CW. ESBL E. coli were characterized by MLST analysis using the 7 genes based Achtman Scheme. ESBL E. coli isolated from water, sediments, roots and feces of myocastors collected in the FWS CW and in the recipient river were genotypically related, suggesting persistence and circulation of the ESBL producing E. coli throughout the FWS CW and in the receiving river. Overall, these observations show that FWS CW could be an efficient treatment for ESBL E. coli disinfection of wastewater and could limit their dissemination in the aquatic environment.