Pulse field certified agarose gel

All S. aureus isolates were genotyped by pulsed-field gel electrophoresis (PFGE) using the SmaI digestion enzyme (Sigma, Poole, Dorset, UK) as described before [29 (link)]. PFGE of the H. influenzae isolates was performed similarly, with some supplementation taken from the protocol by Saito et al [30 (link)]. Briefly, after solidification, the plugs were incubated first in 100 mM EDTA containing lysozyme for three hours at 37°C, then in 100 mM EDTA containing proteinase K overnight at 54°C. The purified chromosomal DNA was digested by SmaI enzyme. DNA fragments were separated in 1% pulse-field certified agarose gel (Bio-Rad) in 0.5x TBE (Tris-boric acid-EDTA) buffer in a CHEF-DR® II apparatus, at 14°C, 6 V/cm, for 10 h with pulse times of 5 s to 15 s, and for 10.5 h with pulse times of 15 s to 60 s. S. pneumoniae isolates were also compared based on their SmaI restriction patterns as described before [31 (link)]. PFGE profiles were analysed with the BioNumerics software version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium), applying unweighted pair group method using arithmetic averages (UPGMA) and the different bands similarity coefficient, with a band position tolerance of 2.0%. During interpretation, the Tenover’s criteria [32 (link)] and the suggested designation by van Belkum et al. [33 ] were applied.

A total of 21 randomly selected V. cholerae O1 strains isolated from 2007 to 2019 were pulsotyped by PFGE technique in a CHEF Mapper system (Bio-Rad, USA), according to the procedures described elsewhere [22 (link)]. The genomic DNA of V. cholerae in agarose plugs were restricted with 50U of Not I (New England Biolabs, USA). The Not I digested restriction DNA fragments were separated through 1% pulse-field certified agarose gel (Bio-Rad, USA) in 0.5X TBE buffer. The DNA was visualized after staining and destaining of the gel and the images were digitized by gel documentation system (Bio-Rad). The fingerprint patterns in gel were analyzed using BioNumerics software V7.6.