| Summary: | Vancomycin-resistant (VR) <i>Enterococcus</i> spp. can be detected in high concentrations in wastewaters and pose a risk to public health. During a one-year study (September 2022–August 2023), 24 h composite raw wastewater samples (<i>n</i> = 192) of a municipal wastewater treatment plant were investigated for cultivable enterococci. After growth on Slanetz–Bartley agar (SBA), a mean concentration of 29,736 ± 9919 cfu/mL was calculated. Using MALDI-TOF MS to characterize randomly picked colonies (<i>n</i> = 576), the most common species were found to be <i>Enterococcus faecium</i> (72.6%), <i>E. hirae</i> (13.7%), and <i>E. faecalis</i> (8.0%). Parallel incubation of wastewater samples on SBA and VRESelect agar resulted in a mean rate of VR enterococci of 2.0 ± 1.5%. All the tested strains grown on the VRESelect agar (<i>n</i> = 172) were <i>E. faecium</i> and carried the <i>vanA</i> (54.6%) or <i>vanB</i> gene (45.4%) with limited sequence differences. In susceptibility experiments, these isolates showed a high-level resistance to vancomycin (>256 µg/mL). Concentration of vancomycin was determined in 93.7% of 112 wastewater samples (mean: 123.1 ± 64.0 ng/L) and varied between below 100 ng/L (the detection limit) and 246.6 ng/L. A correlation between the concentration of vancomycin and the rate of VR strains among the total enterococci could not be found. The combination of incubation of samples on SBA and a commercial vancomycin-containing agar applied in clinical microbiology with a multiplex PCR for detection of van genes is an easy-to-use tool to quantify and characterize VR <i>Enterococcus</i> spp. in water samples.
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