{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T01:15:55Z","timestamp":1788225355930,"version":"build-2803163510"},"reference-count":38,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development","award":["24fk0108702j0001, 25fk0108702h0002, 26fk0108702h0003"],"award-info":[{"award-number":["24fk0108702j0001, 25fk0108702h0002, 26fk0108702h0003"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,7,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      species and enteroinvasive\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      (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\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      spp. closely related to\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      possess this sequence, while it remains absent in clinical\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      spp. Hence, we aimed to further explore the relationships between\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      spp. employing multilocus sequence typing (MLST)-based phylogenetic analysis of 42 clinical\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      spp. isolates and 32\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      isolates, among which 28 were classified as EIEC.\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      isolates clustered within specific clades, whereas\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      isolates, including EIEC, formed largely distinct clusters with limited overlap. Three O-untypable EIEC isolates clustered within the\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      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 (\n                      <jats:italic toggle=\"yes\">ipaH<\/jats:italic>\n                      ) was developed. This assay demonstrated sensitivities and specificities of 100% versus 96.7% for\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                        and enteroinvasive\n                        <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                        (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\n                        <jats:italic toggle=\"yes\">Shigella<\/jats:italic>\n                        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.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03988-25","type":"journal-article","created":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T12:01:02Z","timestamp":1781524862000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Genome-based re-evaluation of the ECA75F sequence and development of a dual-probe real-time PCR assay for differentiating\n                    <i>Shigella<\/i>\n                    species and enteroinvasive\n                    <i>Escherichia 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