Importance of <i>aggR</i> sequence variants detection for accurate molecular diagnosis of enteroaggregative <i>Escherichia coli</i>

Enteroaggregative <i>Escherichia coli</i> (EAEC) is one of the main diarrhea causative agents. This <i>E. coli</i> pathotype is defined by an aggregative adherence pattern observed in HEp-2 cells. Molecular diagnosis is still a challenge due to EAEC genetic heterogeneity. Different publications described primers for <i>aggR</i> and <i>aatA</i> detection to characterize EAEC. Recently, <i>afpR</i> has been suggested as EAEC molecular marker of some strains, demonstrating the existence of two subpopulations within this pathotype: typical and atypical EAEC, respectively, comprising <i>aatA</i>+/<i>aggR</i>+ and <i>aatA</i>+/<i>afpR</i>+. Studies performed by our group, however, detected conflicting results using different primer pairs for <i>aggR</i>. Therefore, this study mainly aimed to analyze the variation of <i>aggR</i> sequences and its implication in EAEC characterization, defining consensus primers. As a consequence, a triplex-PCR targeting <i>aggR</i>, <i>aatA,</i> and <i>afpR</i> is proposed as a one-step molecular diagnosis for both typical and atypical EAEC. Previously reported primer sequences used for <i>aggR, aatA</i>, and <i>afpR</i> detection, and their annealing sites were analyzed using sequence databases. The best primers for each target were selected, and a triplex-PCR was standardized and validated. Database alignment of <i>aggR</i> revealed numerous variant regions along its sequence, impairing the annealing of most reported primers, while significant variations in the annealing sites of <i>aatA</i> and <i>afpR</i> primers were not observed. Broad-range primers for <i>aggR</i> were determined and combined with <i>aatA</i> and <i>afpR</i>, resulting in a triplex-PCR. Our triplex-PCR targeting <i>aggR, aatA</i>, and <i>afpR</i> successfully detected only EAEC, covering all <i>aggR</i> variants, promoting a broad and specific detection of this pathotype. IMPORTANCE Phenotypical and genotypical characterization of enteroaggregative <i>Escherichia coli</i> (EAEC) is either time-consuming or of variable sensitivity. Therefore, developing an accurate molecular diagnostic method for this <i>E. coli</i> pathotype is relevant for public health. This study analyzed the primers currently used to detect typical EAEC-related genes (<i>aatA</i> and <i>aggR</i>), and <i>afpR</i> as a marker for atypical EAEC. After an extensive literature review, it was observed that the <i>aatA</i> primers are suitable for its broad detection. On the other hand, none of the <i>aggR</i> primer pairs described were able to detect all variants of the <i>aggR</i> gene. To address this issue, universal primers for <i>aggR</i> were proposed to improve the detection of all <i>aggR</i> variants. Considering the demand for a fast and accurate molecular approach for EAEC diagnosis, a triplex-PCR assay was developed, validated, and optimized using the universal <i>aggR</i> primers in combination with primers for <i>aatA</i> and <i>afpR</i>, detecting both typical and atypical EAEC.