Flowsheet of the cultivation-dependent detection of ESBL-producing Enterobacteriaceae in input and output samples of biogas plants including a specifically established pre-enrichment method compared to direct plating (DP) on CHROMagar ESBL.

For direct plating (DP), bacteria were detached from 10 g fresh samples by shaking in sterile 0.02% tetrasodium-pyrophophate buffer (TSPP) and horizontal shaken at room temperature for 5 min. After 30 min sedimentation the supernatant (10⁰) was serially diluted (up to 10^-3) and 100 μL of each dilution step were plated in triplicates on CHROMagar ESBL. For pre-enrichment (PE), 0.1, 1, or 10 g fresh samples were pre-incubated (in triplicates) in LB broth containing 0.5 mg L^-1 cefotaxim (CEF) and 0.5 mg L^-1 ceftazidim (CAZ) for 24 h at 37°C. Thereafter 10 μL of the pre-enrichments were streaked on CHROMagar ESBL. After 24 h incubation at 37°C for both methods, cream, pink and blue coloured colonies were screened from the presence of CTX-M, TEM and SHV-type ESBL genes using a multiplex PCR.