Background: Several prominent bacterial species known to induce diarrhea in human hosts encompass Escherichia coli, Escherichia albertii, Escherichia fergusonii, and various Shigella spp. Given that these organisms contribute to the burden of food-borne illness, it is essential to rapidly and correctly identify them in a clinical laboratory or food microbiology units to prevent their transmission and spread. Because of the genetic and phenotypic similarities of these pathogens, they are often mistakenly identified. Phenotypic tests are not highly discriminatory and are time-consuming. Whole-genome sequencing (WGS) is expensive, and not available in most clinical laboratories. To simplify their rapid detection, we improved an available multiplex PCR assay targeting three species-specific primer including Eco (main target for E. coli identification), Ealb (specific for E. albertii), Efer (specific for E. fergusonii), by adding ipaH, and lacY to additionally discriminate the highly similar Shigella spp. and enteroinvasive Escherichia coli (EIEC) organisms. Primers were tested on 65 defined isolates including E. coli (n=29), Shigella spp (n=26), E. fergusonii (n=1), E. albertii (n=1), and other Enterobacterales (n=8). Results: All examined E. coli yielded two amplicons of the expected size (Eco and lacY) except for EIEC which had three bands (Eco, lacY , and ipaH); all Shigella spp. yielded two amplicons (Eco and ipaH); E. fergusonii had only one band (Efer); E. albertii also yielded one band (Ealb) ; and for other Enterobacterales that were tested for validation did not show a product except for Klebsiella pneumoniae and Klebsiella oxytoca (both lacY).

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