The increasing problem of antimicrobial resistances is recognized all over the world and in all compartments of the One Health principle. Especially resistances to carbapenems, which are categorized as Critically Important Antimicrobials, are of particular importance. For years, carbapenemase-producing (CP) Enterobacteriaceae (CPE) were almost exclusively reported from clinical samples. Nowadays, CPE from wild animals, domestic animals, livestock and food are reported worldwide. These resistances are mostly caused by the production of carbapenemhydrolyzing enzymes, so called carbapenemases. The genetic information for their synthesis is often located on plasmids or other mobile genetic elements, which are transferable between different strains and even bacterial species. To detect the dynamics and occurrence of CPE, the EU has established a monitoring for CP Escherichia (E.) coli from food and livestock. In the past, some observations indicated a lack of sensitivity of the monitoring method. One example is an isolate from 2017 that was not detected in the German CP E. coli monitoring, but in monitoring for ESBL/AmpC β-lactamases. Therefore, one goal of this thesis was to optimize the microbiological isolation methods for CPE from meat and caecal samples. Next to this method optimization, the detailed characterization and comparison of recently isolated German CPE between 2017 and 2020 in frame of the EU wide CP monitoring programme was a second aim of this thesis. The phenotypic and genotypic characterization of such isolates included i.e. the assessment of MIC values and typing by pulsed-field gel electrophoresis and whole genome sequencing. To improve the sensitivity of the isolation method, different approaches were tested to stimulate the growth of Enterobacteriaceae while reducing the interfering accompanying flora. These approaches included i.e. a second enrichment step (i.e. different media, different supplements in different concentrations and some incubation conditions), the choice of selective agar (in-house produced MacConkey agar supplemented with 1 mg/L cefotaxim and 0.125 mg/L meropenem (McC+CTX+MEM) and MacConkey agar supplemented with 0.125 mg/L meropenem (McC+MEM) or commercial chromID® CARBA SMART (bioMérieux, Nürtingen, Germany)) and the influence of pre-additions to the samples. These approaches demonstrated a significant improvement in detectability when the recommended in-house selective agar was used instead of commercial agars. The original EURLprotocol in combination with in-house produced McC+MEM and McC+CTX+MEM agar plates led to a sensitivity and specificity of 100 % (by screening 54 samples, spiked with 100 cfu/ml of CPE), whereas the sensitivity decreased to ~75 % by using the same procedure in combination with the tested commercial agar. If this recommended method fails to isolate a putative colony due to the accompanying flora, an alternative method (including a second anaerobic selective enrichment followed by real-time PCR) should be used. Furthermore, a higher sensitivity of the isolation method can be assumed if the samples are prepared within a few hours. This is because a stepwise reduction of 2 log units within 24 hours after CPE could be demonstrated in faecal samples. The detailed characterization of recently isolated CPE revealed several observations, which could support two conclusions. On one hand, CPE with high similarity regarding the sequence type, phylogenetic group and the plasmids have been repeatedly isolated in the German food chain. Repeated isolation of strains with such high similarity raises concerns about stability and a possible adaption of CPE to a new reservoir, i.e. pigs. On the other hand, the variance of detected CPE increased. While initially only VIM-1 producing E. coli and Salmonella enterica were detected, in 2019, OXA-48 and GES-5 producing E. coli from pig production were detected. In addition, certain similarities to human CPE were described for all isolates. These observations indicate a repeated, independent and possibly human entry, which underlines the One Health aspect. In conclusion, the sensitivity of the isolation method for CPE from meat and ceacal samples could be increased. By using selective and sensitive cultivation methods, the number of isolated CPE may be more in line with the actual prevalence. The comparison of isolated CPE from animal origin could provide insights into possible adaptation, plasmid evolution, and other questions. The variance of isolates increased, whereas some persistence of VIM-1 producing E. coli isolates was observed at the same time.
