AimsTo utilize comparative accessory gene fingerprinting to discriminate between naturalized and faecal Escherichia coli, with particular emphasis on strains from phylogroup B1. Methods and ResultsFourteen accessory genes that were potentially ecotype-specific were selected on the basis of comparative genomic DNA sequence analysis between faecal and environmental strains and also using a literature-based strategy. PCR assays were designed for each gene, and used to screen 107 faecal strains from various hosts and 106 environmental strains from surface water and sediment. While none of the 14 accessory genes were ecotype-specific, six of the genes were ecotype-enriched. Specifically, toxin-antitoxin system genes were more abundant among faecal strains, whereas genes involved in iron acquisition, complement resistance/surface exclusion, and biofilm formation were more abundant among environmental strains. These six genes were used to form composite fingerprints which revealed the presence of several ecotype-specific and -enriched fingerprints. Notably, some of the environmental strain-specific or -enriched fingerprints consisted of strains putatively belonging to clade ET-1, which has been previously recognized as a naturalized subpopulation. ConclusionsUnlike single genes which did not reliably distinguish between faecal and naturalized phylogroup B1 E.coli strains, composite fingerprints of ecotype-enriched accessory genes may offer a novel method for distinguishing between these two populations. Significance and Impact of the StudyAccessory gene fingerprinting may have important practical implications for improving the specificity of methods that are widely used for quantifying and identifying the sources of faecal contamination in surface water.