Importance of aggR sequence variants detection for accurate molecular diagnosis of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is one of the main diarrhea causative agents. This E. coli 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 aggR and aatA detection to characterize EAEC. Recently, afpR 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 aatA+/aggR+ and aatA+/afpR+. Studies performed by our group, however, detected conflicting results using different primer pairs for aggR. Therefore, this study mainly aimed to analyze the variation of aggR sequences and its implication in EAEC characterization, defining consensus primers. As a consequence, a triplex-PCR targeting aggR, aatA, and afpR is proposed as a one-step molecular diagnosis for both typical and atypical EAEC. Previously reported primer sequences used for aggR, aatA, and afpR 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 aggR revealed numerous variant regions along its sequence, impairing the annealing of most reported primers, while significant variations in the annealing sites of aatA and afpR primers were not observed. Broad-range primers for aggR were determined and combined with aatA and afpR, resulting in a triplex-PCR. Our triplex-PCR targeting aggR, aatA, and afpR successfully detected only EAEC, covering all aggR variants, promoting a broad and specific detection of this pathotype. IMPORTANCE Phenotypical and genotypical characterization of enteroaggregative Escherichia coli (EAEC) is either time-consuming or of variable sensitivity. Therefore, developing an accurate molecular diagnostic method for this E. coli pathotype is relevant for public health. This study analyzed the primers currently used to detect typical EAEC-related genes (aatA and aggR), and afpR as a marker for atypical EAEC. After an extensive literature review, it was observed that the aatA primers are suitable for its broad detection. On the other hand, none of the aggR primer pairs described were able to detect all variants of the aggR gene. To address this issue, universal primers for aggR were proposed to improve the detection of all aggR 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 aggR primers in combination with primers for aatA and afpR, detecting both typical and atypical EAEC.