Colistin Resistance Genes Detection

After BPW enrichment, 100 µL and 10 µL were spread on Tryptone Bile X-glucuronide agar plates (TBX) and Simmons citrate agar + inositol (SCAi) with and without colistin (3.5 mg/L) and incubated (TBX at 37 °C for 24 h; SCAi at 37 °C for 48 h) for E. coli and Klebsiella spp. detection, respectively. From each plate, between one and five colonies of each morphotype were spread on a CLED medium for further identification by matrix-assisted laser desorption-ionisation-time of flight mass spectrometry (MALDI-TOF VITEK MS, bioMérieux) and standard PCRs for E. coli and K. pneumoniae [20 (link)]. Colistin resistance genes (mcr-1 to mcr-5 and mcr-6 to mcr-9) were identified in E. coli, K. pneumoniae and S. enterica isolates using a multiplex PCR published previously [21 (link)]. Amplified simplex PCR products were purified using the NZYGelpure kit (NZYTech, Lisbon, Portugal) and sequenced at Eurofins Genomics (Konstanz, Germany).