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The absence of genetic tools for\n                      <jats:italic toggle=\"yes\">Orientia<\/jats:italic>\n                      has limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported\n                      <jats:italic toggle=\"yes\">Orientia<\/jats:italic>\n                      replication, and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi ank13<\/jats:italic>\n                      gene (OTT_RS04140) (CM\n                      <jats:italic toggle=\"yes\">ank13<\/jats:italic>\n                      ) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi tsa22<\/jats:italic>\n                      -up and\n                      <jats:italic toggle=\"yes\">tsa56<\/jats:italic>\n                      -down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      via electroporation or CaCl\n                      <jats:sub>2<\/jats:sub>\n                      , the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I-positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CM\n                      <jats:italic toggle=\"yes\">ank13<\/jats:italic>\n                      cassette and loss of wild-type\n                      <jats:italic toggle=\"yes\">ank13<\/jats:italic>\n                      were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      and building additional genetic tools to investigate this globally significant pathogen.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Orientia tsutsugamushi<\/jats:italic>\n                      causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease,\n                      <jats:italic toggle=\"yes\">Orientia<\/jats:italic>\n                      is the only one for which genetic tools have not been developed. This has limited understanding of\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      -host dynamics that drive the bacterium\u2019s pathobiology and hinder the development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      . Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for\n                      <jats:italic toggle=\"yes\">O. tsutsugamushi<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01284-26","type":"journal-article","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T13:23:27Z","timestamp":1783344207000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Transformation and allelic exchange in\n                    <i>Orientia tsutsugamushi<\/i>"],"prefix":"10.1128","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0299-7380","authenticated-orcid":false,"given":"Paige E.","family":"Allen","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02nkdxk79","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Microbiology and Immunology, School of Medicine, Virginia Commonwealth University 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