Improved Detection of Extended Spectrum Beta-Lactamase (ESBL)-Producing <i>Escherichia coli</i> in Input and Output Samples of German Biogas Plants by a Selective Pre-Enrichment Procedure

<div><p>The presence of extended-spectrum beta-lactamase (ESBL)-producing <i>Escherichia coli</i> was investigated in input (manure from livestock husbandry) and output samples of six German biogas plants in 2012 (one sampling per biogas plant) and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing <i>Escherichia coli</i> were cultured by direct plating on CHROMagar ESBL from input samples in the range of 10<sup>0</sup> to 10<sup>4</sup> colony forming units (CFU) per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing <i>E</i>. <i>coli</i> by the biogas plant process. Detected non target bacteria were assigned to the genera <i>Acinetobacter</i>, <i>Pseudomonas</i>, <i>Bordetella</i>, <i>Achromobacter</i>, <i>Castellaniella</i>, and <i>Ochrobactrum</i>. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing <i>E</i>. <i>coli</i> in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing <i>E</i>. <i>coli</i> were isolated from input and 46 from output samples. Most of the <i>E</i>. <i>coli</i> isolates carried CTX-M-type and/or TEM-type beta lactamases (94%), few SHV-type beta lactamase (6%). Sixty-four <i>bla</i><sub>CTX-M</sub> genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85%) and 9 (13%), and one to group 2. Phylogenetic grouping of 80 <i>E</i>. <i>coli</i> isolates showed that most were assigned to group A (71%) and B1 (27%), only one to group D (2%). Genomic fingerprinting and multilocus sequence typing (MLST) showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing <i>E</i>. <i>coli</i> revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT) genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing <i>E</i>. <i>coli</i> in output samples of German biogas plants.</p></div>