Genome-based re-evaluation of the ECA75F sequence and development of a dual-probe real-time PCR assay for differentiating Shigella species and enteroinvasive Escherichia coli

Shigella species and enteroinvasive Escherichia coli (EIEC) have highly similar biochemical, serological, and pathogenic profiles, making their differentiation challenging. Although the 25-base sequence ECA75F within the 16S rRNA gene has been used as a potential marker in PCR-based differentiation methods, some environmental Shigella spp. closely related to E. coli possess this sequence, while it remains absent in clinical Shigella spp. Hence, we aimed to further explore the relationships between E. coli and Shigella spp. employing multilocus sequence typing (MLST)-based phylogenetic analysis of 42 clinical Shigella spp. isolates and 32 E. coli isolates, among which 28 were classified as EIEC. Shigella isolates clustered within specific clades, whereas E. coli isolates, including EIEC, formed largely distinct clusters with limited overlap. Three O-untypable EIEC isolates clustered within the Shigella ST245 clonal complex (CC245), highlighting the complexity of classification within this group. Additionally, a dual-probe real-time PCR (DpRT-PCR) assay targeting both the ECA75F and the invasion plasmid antigen H gene ( ipaH ) was developed. This assay demonstrated sensitivities and specificities of 100% versus 96.7% for Shigella spp. and 88.3% and 100% for EIEC, respectively. Overall, these findings demonstrate that the DpRT-PCR assay provides a rapid and effective method for differentiating Shigella spp. from EIEC. Furthermore, for cases with ambiguous genetic profiles, such as EIEC isolates grouped with CC245, MLST-based phylogenetic analysis may provide additional resolution and clarity. IMPORTANCE Shigella and enteroinvasive Escherichia coli (EIEC) cause clinically indistinguishable diarrheal diseases, yet they differ in surveillance reporting, outbreak control measures, and public health responses. However, routine laboratory identification often fails to clearly differentiate these pathogens because of their close genetic relatedness and overlapping biochemical characteristics. In this study, we performed a genome-based reassessment of the ECA75F sequence and developed a rapid and robust dual-probe real-time PCR assay that accurately distinguishes Shigella spp. from EIEC. This method overcomes the limitations of conventional PCR assays and provides a reliable tool for frontline laboratories. The ability to differentiate these pathogens quickly and accurately will improve case classification, guide appropriate public health interventions, and strengthen epidemiological monitoring in both endemic settings and outbreak investigations.