Isolation and Characterization of ESBL-Producing E. coli from Aquatic Environments

ESBL-producing isolates were selected from an existing collection of isolates obtained from wastewater and surface water sampled between 2010 and 20125 (link)13 (link). Surface water samples (n = 20) were taken at ten different sites situated in four different regions in the Netherlands (including rivers, canals, lakes, North Sea), wastewater samples (n = 20) included influents and effluents from wastewater treatment plants (WWTP), an international airport WWTP, and from wastewater of health care institutions. Isolates were obtained by filtration of multiple volumes of water samples through 0.45 μm filters, followed by incubation of these filters on selective culture media for the isolation of ESB-producing E. coli: Tryptone Bile X-glucuronide medium supplemented with 1 μg/ml cefotaxime (TBX/CTX) or on ChromID^TM ESBL agar (Biomerieux). Incubation conditions were 18–24 hours at 36 ± 2 °C or 4 to 5 hours at 36 ± 2 °C followed by 18 to 19 hours at 44 ± 0.5 °C. Isolation procedures were based on standard isolation procedures for the selective isolation of E. coli from water and food using chromogenic media (NEN-EN-ISO 9308-1 and ISO 16649-2), adapted to enable selective growth of ESBL-producing variants. Variations in isolation procedures was related to isolates being obtained as part of different projects. Suspected ESBL-E. coli isolates (i.e. the ß-glucuronidase-positive colonies on TBX-CTX as well as on ChromID^TM ESBL agar) were further confirmed as E. coli by testing for indole-production using BBL Dry Slide^TM (BD), and subsequently tested for ESBL-production by disk diffusion following CLSI guidelines14 , using Sensi-Discs^TM (BD, Breda, the Netherlands). Zone diameters were determined for cefotaxime (30 μg) ± clavulanic acid (10 μg), ceftazidime (30 μg) ± clavulanic acid (10 μg). ESBL-producing isolates were defined as strains resistant to cefotaxime (zone diameter ≤22 mm) and/or ceftazidime (zone diameter ≤17 mm), and an increase in zone diameter of ≥5 mm with the disks containing clavulanic acid14 .
From this pre-existing collection of confirmed ESBL-producing E. coli isolates, 93 wastewater and 93 surface water isolates, from 20 samples each, were selected more or less randomly, but taking into account established ABR profiles (when available), to minimize the chance of including duplicate isolates from the same sample. After characterization of phylogenetic groups, ESBL-genes, virulence genes and antibiotic resistance profiles was completed, some isolates were retrospectively identified as duplicates and omitted, leaving 88 wastewater (63 from WWTP, 15 from the international airport and 10 from health care institutions) and 82 surface water isolates for analyses.