Cefotaxime

Swabs from cloacal and fecal samples were incubated overnight in LB medium (Lennox) (Carl Roth, Karlsruhe, Germany) containing 2 µg/mL cefotaxime (VWR International GmbH, Darmstadt, Germany) at 37 °C while shaking at 200 rpm and subsequently cultivated on CHROMagar Orientation plates (MAST Diagnostica, Reinfeld, Germany) supplemented with 2 µg/mL cefotaxime at 37 °C. Putative cefotaxime-resistant E. coli colonies were identified based on colony morphology (pinkish colonies) and subjected to further cultivation until pure cultures were obtained. Pure cultures were used for further verification and characterization.

In the laboratory, 100 mL of water samples were first pre-filtered using a sterilized gauze (PZN: 04046708, FESMED Verbandmittel GmbH, Frankenberg/Sa., Germany) to remove the fine particles. The pre-filtered water was free from any macro-particles, sediment and most fine particles. Thereafter, each sample was filtered using the EZ-Fit filtration system with 0.45 µm pore size filter membranes (merckmillipore, Darmstadt, Germany). After filtration, filter membranes were transferred to 10 mL TSB (Carl Roth GmbH, Karlsruhe, Germany) containing 2 µg/mL cefotaxime (VWR International, Darmstadt, Germany) followed by overnight incubation at 37 °C and shaking at 200 rpm. Depending on the turbidity level, dilution of the overnight cultures followed (up to 10,000 fold dilution). For each sample, 100 µL of the dilutions were plated on chromogenic media CHROMID CARB/OXA, CHROMID ESBL, CHROMID Colistin, and CHROMID MRSA agar plates (bioMérieux, Nürtingen, Germany), and incubated overnight at 37 °C. Putative antibiotic-resistant colonies of E. coli (red-purple colonies) and KEC (Klebsiella spp., Enterobacter spp., Citrobacter spp. (blue colonies)) were subcultivated until pure cultures were achieved. Single pure colonies were picked for further verification and characterization.