Fig 1.
Phylogenetic identification of isolates representing most abundant colonies grown on CHROMagar ESBL after direct plating of input or output samples from German biogas plants.
A, Five German biogas plants investigated in 2012; B, Two biogas plants sampled in February, July and October 2013 and February 2014. For input samples 2012 26 isolates of input and 18 isolates of output samples were identified; 2013/14, 178 isolates of input (DP: 56; PE: 122) and 72 isolates of output samples (DP: 9; PE: 63). Identification based on partial 16S rRNA gene sequence analysis (approximately 1000 nt) with assignment at the genus level based on at least 98.7% 16S rRNA gene sequence similarities to type strains of respective genera determined by BLAST analysis using the EzTaxon database. DP: direct plating on CHROMAgar ESBL; PE: selective pre-enrichment and subsequent plating on CHROMAgar ESBL. Numbers (n =) above the bars represent the number of isolates investigated per sample. The isolates were representative for abundant colonies grown on CHROMagar ESBL plates.
Fig 2.
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.
Table 1.
Overview of the detected E. coli ST types and the affiliation to ST Complexes determined by MLST based on seven genes, adk, fumC, purA, recA, gyrB, icd, and mdh using the E. coli database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/).
Fig 3.
Differentiation of ESBL-producing E. coli isolates by molecular fingerprinting using BOX-PCR and detailed characterization of respective isolates.
Cluster analysis was performed in Gel Compare II (Applied Maths) with UPGMA clustering based on a similarity matric calculated using the Pearson correlation (1.0% optimization and 1.0% position tolerance). Characterization of the isolates included the determination of ST-types (MLST analysis), phylogenetic E. coli typing, ESBL-gene characterization. Growth on CHROMagar ESBL and STEC. CTX-M groups were assigned by sequencing CTX-M-type genes. E. coli phylogenetic groups were determined by multiplex PCR and a dichotomous decision tree. *: assigned in group A was but there was a yjaA gene present. C: CTX-M; T: TEM; +: positive;-: negative. BGA shows to which biogas plant isolates belong; I = input sample, O = output sample. Names of the strains include information of the origin of the isolates: pre-enrichment (ESBL before strain number) Bxx (Biogas plant shortcut)_12, 13 or 14 (year of cultivation/isolation: either 2012, 2013 or 2014)_input or output.