Salmonella colistin resistance screening

PCR screening was performed on 407 initially phenotypically colistin-resistant (MIC >2 mg/L) S. enterica isolates (with exception of serogroup D) obtained from livestock, animals, food, feed and the environment, collected in the frame of routine diagnostics as well as national monitoring and surveillance programs in the years 2011 to 2018 in Germany at the National Reference Laboratory for the Analysis and Testing of Zoonoses (NRL Salmonella). All strains selected have no epidemiological link (neither isolated at the same place, time, animal or food). All isolates were routinely subjected to serotyping by slide agglutination with antisera raised against O-, H1- and H2- antigens and MIC testing as described before (Borowiak et al., 2017a (link)). For PCR screening, isolates were cultivated from stock cultures by inoculating liquid LB medium and subsequent incubation under shaking conditions (250 × rpm) for 16 h at 37°C. Thermal cell lysis preparations were produced as previously described (Borowiak et al., 2017a (link)). DNA suitability for PCR amplification was confirmed by a standard PCR protocol for amplification of the housekeeping gene hemD using the primers recommended on Enterobase¹. The hemD PCR was prepared in 25 μL including 12.5 μL 2x DreamTaq Green PCR Master Mix (Thermo Scientific, Vilnius, Lithuania), 0.5 μL of each 10 μM primer dilution, 9.5 μL PCR grade water and 2 μL of thermal cell lysis preparation as template DNA and carried out as follows: initial denaturation for 3 min at 95°C, 35 cycles denaturation for 30 s at 95°C, primer annealing for 30 s at 55°C and elongation for 30 s at 72°C followed by a final elongation step for 10 min at 72°C.