title: A nitrocefin-based amperometric assay for the rapid quantification of extended-spectrum β-lactamase-producing Escherichia coli in wastewaters.
abstract: A sensitive and inexpensive amperometric assay based on the electrochemical detection of the β-lactamase activity using the nitrocefin as substrate was developed for the rapid and quantitative detection of extended spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) in urban wastewaters. The specific detection of ESBL-EC was achieved by culturing the filtered sample in a medium containing the cefotaxime supplemented or not with the potassium clavulanate inhibitor. This step was followed by the incubation of each subculture filtrate with the nitrocefin substrate which hydrolysis was monitored by amperometry using disposable carbon screen-printed sensors. Current intensities iCefand iClavcorrespond to the intensity of the anodic current measured (∼+ 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The intensity value i = iCef- iClavwas chosen as the analytical response. ESBL-EC calibration plots were established with artificially contaminated wastewater samples. This assay allowed the detection of ESBL-EC amounts as low as 10 cfu in treated effluents and 100 cfu in raw wastewaters with short time analysis of 5.5 h and 4.5 h, respectively. The amperometric method was applied to the analysis of 38 wastewater samples and the results were in good agreement with CFU counts on a selective chromogenic medium for 24 h. Owing to its rapidity, convenience, low-cost and portability, this assay is a promising tool to obtain quantitative data on antimicrobial-resistant E. coli in wastewater effluents. Furthermore, this assay might be used to improve wastewater treatment plant processes in order to minimize the release of antibiotic resistant bacteria into the aquatic environment.
authors.affiliation: Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: Chantemesse
authors.affiliation: Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Betelli
authors.affiliation: Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Solanas
authors.affiliation: Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Vienney
authors.affiliation: Laboratoire Chrono-environnement UMR 6249, Univ. Bourgogne Franche-Comté, 25000 Besançon, France.
authors.forename: Loïc
authors.initials: L
authors.lastname: Bollache
authors.affiliation: Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France. Electronic address: Murielle.dequaire@u-bourgogne.fr.
authors.forename: Murielle
authors.initials: M
authors.lastname: Rochelet
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects*; D004927:Escherichia coli Infections; D062065:Wastewater / Q000382:microbiology; D001618:beta-Lactamases
chemical_list: D000900:Anti-Bacterial Agents; D062065:Wastewater; D001618:beta-Lactamases
keywords: Carbon sensors; Electrochemistry; Extended-spectrum β-lactamase; Nitrocefin; Wastewaters