Rapid Analysis of Toxin-Encoding ETEC Plasmids Using a Basic Optical DNA Mapping Setup with Combed DNA

Abstract

Plasmids are circular extrachromosomal DNA molecules and key vectors of virulence and antibiotic resistance in pathogenic bacteria. We have previously successfully used advanced nanochannel-based optical DNA mapping (ODM) for plasmid characterization. Here, we present a simplified ODM approach using DNA combing on functionalized glass slides and demonstrate the method by analysis of toxin-encoding plasmids from seven Enterotoxigenic Escherichia coli (ETEC) strains. We detect the toxin genes on the plasmids, identify the presence of single or multiple toxin genes, and determine whether these are located on the same or different plasmids. The method allows characterization of plasmids carrying any combination of genes, illustrated by analysis of an isolate carrying both toxin and antibiotic resistance genes. Moreover, we demonstrate that the method can be performed on a basic fluorescence microscope, thereby increasing its general usability in standard microbiology laboratories globally. Finally, we show that by comparing ODM data to a library of known plasmids encoding ETEC toxins, collected from public databases, plasmids with high sequence similarity can be identified. Thus, the ODM method can be used as a bioinformatics tool to find related plasmids and investigate sequence similarities, without sequencing the plasmid of interest.