Genomic Profiling of E. coli Isolates

Genomic DNA was extracted from E. coli isolates using the Maxwell 16-cell DNA purification kit (Promega, Madison, WI, USA). STs of each strain were determined using multilocus sequence typing (MLST), with the Achtman scheme (24 (link)), by allele numbering the seven housekeeping genes, namely, adk, fumC, gyrB, icd, mdh, purA, and recA. A minimum spanning tree was constructed using PHYLOViZ 2.0 (25 (link)) with the allelic profile of each ST. Subgroups of the ST131 isolates were identified by (i) fimH typing, using the database for the single nucleotide polymorphism (SNP)-based numbering system (26 (link)), (ii) ciprofloxacin resistance, and (iii) SNP-based x typing (27 (link)). For resistance genotyping, the ESBL bla_CTX-M gene and the plasmid-mediated ampC gene primer pairs were used (21 (link)). To genotype the virulence, eight pairs of primers each for adhesin genes, i.e., afaA, papC, papG, and focG, and for toxin genes, i.e., cnf1, hlyF, sat, and cdtB (2 (link)), were designed and used to screen for the presence of the gene.