Fpquest software v4

Genetic relatedness of the isolates was examined by PFGE in a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, and CA) following digestion of genomic DNA with XbaI enzyme (New England Biolabs, Massachusetts) according to Tenover et al. [20 (link)]. The PFGE images were processed and the dendrogram was calculated by FPQuest software v4.5 (Biorad laboratories inc, Hercules, California, USA.) using Dice coefficient and UPGMA (unweighted pair group method using arithmetic averages). Isolates having more than 95% similarity were considered identical.

PFGE was employed to determine clonal relatedness using the PulseNet protocol (http://www.cdc.gov/pulsenet/protocols.htm) in a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules and CA). DNA digestion was carried out for 12 h at 37°C using XbaI enzyme. Digested DNA fragments were electrophoresed for 20 h at 14°C at a 120° included angle, linear ramp factor with switch times of 2.2 and 54.2 s at 6 V/cm. Salmonella serotype Braenderup H9812 was used as the size determining marker. The PFGE results were interpreted according to Tenover criteria (44 (link)). The Dice coefficient (1% tolerance and 1% optimization) and the unweighted pair-group method with the arithmetic mean (UPGMA) were used for the cluster analysis, using the FPQuest Software v4.5 (Bio-Rad Laboratories).
For sequence typing analyses, sequences of 7 conserved housekeeping genes of K. pneumoniae (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were submitted to the multilocus sequence typing (MLST) database (https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?db=pubmlst__klebsiella_seqdef).

More than one colony of same colour with mcr gene was stocked from each sample and were subjected to repetitive extragenic palindromic elements-PCR (rep-PCR) [20 (link)]. Additionally, all mcr-negative isolates (of same colour as the mcr-positive ones) were subjected to rep-PCR to check for similar clones. Clonality among mcr-positive isolates were determined by pulsed-field gel electrophoresis (PFGE) in a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, and CA) using XbaI macro digestion and visually interpreted as per Tenovar criteria [21 (link)]. With FP Quest software v4.5 (Biorad Laboratories Inc, Hercules, California, USA), a dendrogram was prepared using Dice coefficient and UPGMA (unweighted pair group method using arithmetic averages). Tolerance and optimization were set at 1.5% and isolates with ≥90% similarity were considered identical. The phylogenetic classification of the isolates was performed via phylogroup multiplex PCR as described previously [22 (link)].