Filter holder manifold

A standard plate count method on CHROMagar ESBL (CHROMagar, Paris, France) plates was used for the enumeration of ESBL E. coli in all water samples. Depending on the expected bacterial concentration, serial decimal dilutions were prepared in sterile 0.2% buffered peptone water (BPW, Scharlab, Barcelona, Spain) and, subsequently, spread plating (0.1 mL), pour plating (1 mL) or membrane filtration (10 and 100 mL) methods were used. Samples were filtered through 0.45 μm cellulose nitrate membrane filters (Sartorius, Madrid, Spain) using a filter holder manifold (Millipore, Madrid, Spain). Plates were then incubated for 24 h at 37 °C, and dark pink-reddish colonies were counted. The analysis was performed in duplicate, and the results were expressed as cfu/100 mL. The detection limit (LOD) for ESBL E. coli counts in the raw water samples was 3.0 log cfu/100 mL (100 cfu/100 mL), while in the tertiary effluents, the LOD was 0 log cfu/100 mL (1 cfu/100 mL). When possible, five dark pink-reddish colonies on E. coli ESBL agar were picked from each positive sample and sub-cultured in brain infusion (BHI) at 37 °C for 24 h. After incubation, 1 mL of each culture was supplemented with 30% glycerol and kept at −20 °C until further analysis.