Cultivation of vancomycin-resistant enterococci and methicillin-resistant staphylococci from input and output samples of German biogas plants

Vancomycin-resistant enterococci (VRE) and methicillin-resistant staphylococci (MRS) were detected in two mesophilic German biogas plants (BGPs) using selective pre-enrichment methods combined with cultivation on CHROMagar media and antibiotic resistance gene screening. Genetic fingerprinting and 16S rRNA gene sequencing showed the presence of enterococci isolated by the VRE selective cultivation (67 isolates) in input and output samples of BGPs. In contrast, MRS (44 isolates) were detected in input, but in none of the output samples. Enterococcus isolates showed highest 16S rRNA gene sequence similarity (>99.8%) to E. lemanii, E. casseliflavus/E. gallinarium or E. devriesei/E. pseudoavium/E. viikkiensis and carried vanA, vanB and/or vanC1 genes. Enterococcus faecium and E. faecalis VRE were not detected, but isolates closely related to those species (>99.9% 16S rRNA gene sequence similarity) were detected by the MRS selective cultivation methods. Staphylococcus isolates shared highest 16S rRNA gene sequence similarity (>99.9%) with S. haemolyticus, S. lentus and S. sciuri and carried mecA genes. Methicillin-resistant S. aureus (MRSA) were not detected. In summary, manure from livestock husbandry contained both, VRE and MRS. VRE were also detected in output samples, indicating that enterococci with vancomycin resistance genes could be release into the environment by the application of BGP output material as biofertilizers.