Biotyper software version 3

Inoculated blood agar (BA) plates were cultured aerobically at 37 °C for 20 h. The resulting bacterial growth was visually evaluated and if positive, bacteria were isolated, cultured on BA and checked for their identity with S. aureus CNCTC 5480. Species identification of isolates was based on whole-cell profiling by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS using the Microflex LT instrument and BioTyper software version 3.1 (Bruker Daltonics, Bremen, Germany). Isolates identified as S. aureus were then checked for their identity with CNCTC 5480 at the strain level using SmaI-based macrorestriction analysis based on a previously published protocol^{47 (link)}.

Staphylococcus aureus strains were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), which detects bacterial proteins in whole-cell extracts. Bacterial spectra were analyzed using the Biotyper® software version 3.1 (Bruker Daltonik GmbH, Bremen, Germany).

A total of 46 mcr-1-positive E. coli strains isolated from retail raw meat collected between March 2017 and January 2019 in the Czech Republic were analysed in this study. Altogether 45 strains were isolated from turkey meat and liver and 1 strain was obtained from chicken liver. Raw meat and liver samples (20 in total) were purchased from the Czech retail market and originated from the Czech Republic (3), Germany (3), Brazil (5), and Poland (9). More details about sample preparation and detection of colistin-resistant bacteria carrying mcr genes were described previously [14 (link)]. In brief, sample cultivation was performed in buffered peptone water under aerobic conditions at 37 °C overnight. After enrichment, the presence of mcr-1 gene was verified by PCR according to Liu et al., 2016 [15 (link)]. Positive samples were subsequently inoculated onto Brilliance UTI Clarity agar (Oxoid, Basingstoke, UK) supplemented with colistin sulphate (Discovery Fine Chemicals, Wimborne, UK) at a final concentration of 3.5 mg/L and incubated at 37 °C overnight. Presumptive colonies of E. coli (based on colour and colony morphology) were sub-cultured on Blood agar and were identified by MALDI-TOF MS with the use of Biotyper software (version 3.1, Bruker Daltonics GmbH, Bremen, Germany) with a score above 2.0. Up to 5 colonies from each sample were selected for further characterization.