Bacterial strains, transformants and plasmids

A total of 129 non duplicate bla_SHV-12 encoding E. coli, consecutively recovered during national antimicrobial resistance monitoring programmes or national projects between 2009 and 2014 were included in the study⁶⁰. Identification of the isolates was performed by MALDI-TOF Mass spectrometry (Brucker, Coventry, UK). Genes conferring an ESC^R phenotype were sought by microarray analysis followed by PCR amplification and sequencing⁶¹. Plasmid location of bla_SHV-12 was determined using a transformation-based approach. Briefly, plasmids encoding bla_SHV-12 were extracted from the parental strain using a miniprep method and transformed into E. coli DH10b cells (Invitrogen, Van Allen Way, CA USA) by electroporation under the following conditions: 1.25 kV/cm, 200 Ω, 25 μFar, as previously described⁶¹. Transformants were selected on Luria-Bertani (LB) agar plates supplemented with cefotaxime (1 mg/L) and confirmed for the presence of bla_SHV-12 gene. The presence of a single plasmid in the transformants was confirmed by S1-PFGE on the transformants, followed by Southern blot hybridization using DIG-labelled probe (DIG DNA Labeling and Detection Kit, Roche, Mannheim, Germany) targeting the bla_SHV-12 gene, as previously described⁶². Plasmid typing was confirmed by PCR-based replicon typing (PBRT KIT, DIATHEVA, Fano, Italy) on the transformants. Host E. coli sequence type were assigned by MLST based on the allelic profiles of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA)⁶³.