Fingerprinting 2 v4

Repetitive extragenic palindromic PCR (REP-PCR) typing was performed on all isolates as described elsewhere^{39 (link)}. Amplicons were run in a 1.5% agarose gel for 100 min, stained with ethidium bromide, and photographed. When at least two different bands were observed among isolates clonal relationship was also determined by PFGE. For this purpose, bacterial DNA embedded in agarose plugs was digested with the restriction enzyme XbaI and DNA separation was then performed in a CHEF-DRIII variable angle system (Bio-Rad, California, USA). Finally, the PFGE patterns were analysed with Fingerprinting II v4.5 software (Bio-Rad) and interpreted according to the criteria established by Tenover et al.^{40 (link)}.

We obtained XbaI macro-restriction patterns of the 63 C. striatum with a published protocol^{48 (link)} and a CHEF-DRIII variable angle system (Bio-Rad, Hercules, California, USA). The PFGE patterns were analysed with Fingerprinting II v4.5 software (Bio-Rad). Each isolate was compared with all other isolates using the Dice similarity coefficient and the unweighted pair Group method with arithmetic means (UPGMA), with 1% of optimization and tolerance. Isolates were classified as indistinguishable if they showed 100% similarity, as closely related subtypes if they showed 95–99% similarity, and as different strains if they showed <95% similarity.

Genetic relatedness was first studied by repetitive-extragenic-palindromic PCR (REP-PCR)^{29 (link)} in all 541 MDR-E. Subsequently, the clonal relationship was also determined by pulsed-field gel electrophoresis (PFGE) in at least one strain per patient or strains from the same patient if they had two or more differing bands on REP-PCR.
Bacterial DNA was embedded in agarose plugs and digested with 20 U of XbaI at 37ºC overnight. The fragments were separated in 1% agarose gel at 6 V/cm and 14 °C with 0.5xTBE buffer using the CHEF-DRII-system (BIO-RAD, California, USA). Pulse times ranged from 1-5 s for 7 h and 15-35 s for 16 h.
PFGE-patterns were analysed with Fingerprinting II v4.5 software (BIO-RAD). Isolates were classified as indistinguishable if they showed > 95% similarity, as closely related subtypes with 85–95% similarity, and as different strains with similarity < 85%.