Abstract
The presence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli 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 Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU) per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%), few SHV-type beta lactamase (6%). Sixty-four bla CTX-M 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 E. coli 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 E. coli 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 E. coli in output samples of German biogas plants.
Highlights
Intensive application of antibiotics in livestock husbandry increases the abundance of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in animals and in consequence in their manure [1]
The concentration of bacteria cultured on CHROMagar ESBL was in the range of 103 to 106 colony forming units (CFU) (g dry weight)-1 for biogas plant input and 101 to 103 CFUs(g dry weight)-1 for biogas plant output samples
ESBL-producing E. coli were determined in four input samples of the five 2012 investigated biogas plants with CFUs per g dry weight in the range of 101 (BGA 002 and 005) to 103 (BGA 001 and 006)
Summary
Intensive application of antibiotics in livestock husbandry increases the abundance of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in animals and in consequence in their manure [1]. Recent studies showed that ESBL-producing E. coli strains isolated from humans and livestock husbandry represented the same sequence types (ST types) and/ or harbored identical ESBL gene carrying plasmids. Organic material is degraded to smaller compounds as amino acids, sugars and fatty acids by secretion of extracellular enzymes Most of these bacteria are strict anaerobes such as Bacterioides and Clostridium species. As reported by Phillipp and Hölzle [8] E. coli were not detected in output samples of thermophilic biogas plants. Detailed studies about the detection of ESBL-producing E. coli in output samples of biogas plants in general were not published so far. We compared the abundance of ESBL-producing E. coli in input and output material of six mesophilic and one thermophilic German biogas plants compared to the occurrence in respective input material (liquid and solid manure mixtures from livestock husbandry). Isolated ESBL-producing E. coli were characterized in detail by genomic fingerprinting, phylogenetic assignment, multilocus sequence typing (MLST), ESBL gene characterization and antimicrobial susceptibility testing
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