Bbl dryslide

Suspected ESBL-producing E. coli isolates (n = 1,140) were confirmed to be indole-positive using BBL Dry Slide (BD), and subsequently tested for ESBL-production by disk diffusion following CLSI guidelines [36 ], using Sensi-Discs (BD, Breda, the Netherlands). Zone diameters were determined for cefotaxime (30μg) ± clavulanic acid (10μg), ceftazidime (30μg) ± clavulanic acid (10μg), and cefoxitin (30 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 acid [36 ]. ESBL-producing E. coli concentrations were calculated from the numbers of β-glucuronidase-positive colonies, and the fraction of isolates confirmed to be indole-positive and ESBL-producing. Some isolates (n = 22) did not appear to be resistant to either cefotaxime or ceftazidime, and then ESBL-production was confirmed using an alternative AmpC and ESBL detection test, which is based on cefpodoxime (Mastgroup Ltd., Bootle, UK). Third-generation cephalosporin-resistant isolates with no significant inhibitory effect of clavulanic acid (as defined by CLSI) were defined as non-ESBL-producers and excluded from further analyses (n = 32).

From each sample, multiple volumes were filtered through 0.45 μm pore size membrane filters (Millipore, Amsterdam, the Netherlands). Because samples were analysed as part of different projects, the method used for isolation and enumeration of E. coli varied and entailed either the use of tryptone soya agar (TSA) and tryptone bile agar (TBA), as described in to ISO 9308–1 ‘Rapid test’ [31 ], or alternatively, tryptone bile x-glucuronide agar (TBX) in accordance with ISO 16649–2 [32 ]. In short, filters were incubated on TSA or TBX for 4–5 hours at 36±2°C, and subsequently transferred to TBA or maintained on TBX and incubated for 19–20 hours at 44±0.5°C. Presumptive E. coli identified using the TSA/TBA method (i.e. indole-positive), were additionally confirmed as E. coli by testing for ß-glucuronidase-activity on Brilliance E. coli/coliform agar (BECSA; Oxoid, Badhoevedorp, the Netherlands) or using API20E (Biomerieux). Beta-glucuronidase-positive colonies identified using TBX were additionally confirmed by testing for indole-activity using BBL Dry Slide (BD, Breda, The Netherlands). E. coli concentrations were based on the number of indol-positive (TSA/TBA) or ß-glucuronidase-positive (TBX) colonies and the fraction of these colonies that was confirmed to be E. coli. The concentrations were calculated using Mathematica software 9.0.1 (WolframResearch, Champaign, IL, USA).