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Occasionally, corynebacteria are isolated from clinical specimens associated with infection. In this study, 56 bacterial isolates were examined. These isolates were obtained from 52 patients with diverse infections such as keratitis, osteitis\/osteomyelitis, mastitis, (suspected) foreign body associated infections (spine, prosthetic joint), suspected meningitis, post-operative infections, among others. These isolates were identified as corynebacteria by MALDI-TOF mass spectrometry but could not be reliably assigned to a specific species. To resolve this issue, the isolates were genome-sequenced, and species identification was done with different approaches, including digital DNA\u2013DNA hybridization, phylogenomic tree placement and Average Nucleotide Identity (ANI) calculations. A subset of 34 strains was further investigated by biochemical characterization and antimicrobial susceptibility testing (AST).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The 56 isolates belonged to 28 distinct corynebacterial species. Species identification was particularly ambiguous for 13 isolates as the ANIs were below 95% to the closest identified reference genomes. Two isolates represented potentially novel species, since no close relative could be identified (ANI\u2009&lt;\u200990%). The majority of isolates belonged to the\n                      <jats:italic>Corynebacterium marquesiae\/tuberculostearicum<\/jats:italic>\n                      (\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u200910) and\n                      <jats:italic>Corynebacterium kroppenstedtii\/parakroppenstedtii<\/jats:italic>\n                      (\n                      <jats:italic>n<\/jats:italic>\n                      \u2009=\u20099) complexes. Biochemical tests and AST revealed species- and strain-level variability. AST demonstrated extensive antimicrobial resistance (AMR), particularly among\n                      <jats:italic>C. marquesiae<\/jats:italic>\n                      ,\n                      <jats:italic>C. tuberculostearicum<\/jats:italic>\n                      ,\n                      <jats:italic>C. lehmanniae<\/jats:italic>\n                      ,\n                      <jats:italic>C. hesseae<\/jats:italic>\n                      and\n                      <jats:italic>C. resistens<\/jats:italic>\n                      , with resistances observed against penicillin, clindamycin, ciprofloxacin, and rifampin. Resistance was frequently associated with acquired AMR genes, such as\n                      <jats:italic>erm(X)<\/jats:italic>\n                      ,\n                      <jats:italic>tet(W)<\/jats:italic>\n                      and genes encoding aminoglycoside-modifying enzymes. Among the tested antibiotics, clindamycin resistance was most common, detected in 23 of 34 tested strains (64.7%).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      This study expands our knowledge of\n                      <jats:italic>Corynebacterium<\/jats:italic>\n                      isolates derived from clinical specimens, particularly those differing from well-characterized species. It underscores the extensive geno- and phenotypic variability within most\n                      <jats:italic>Corynebacterium<\/jats:italic>\n                      species and challenges current species boundary definitions. The extensive level of detected AMR may complicate treatment of underlying infections. However, it remains uncertain whether these isolates represent true infectious agents or contaminants derived from the skin of the patients.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12866-025-04619-8","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T12:45:05Z","timestamp":1766407505000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Genomic and phenotypic diversity among taxonomically ambiguous clinical Corynebacterium isolates"],"prefix":"10.1186","volume":"26","author":[{"given":"Holger","family":"Br\u00fcggemann","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lise","family":"Hald Schultz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anja","family":"Poehlein","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bo","family":"S\u00f6derquist","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,12,22]]},"reference":[{"key":"4619_CR1","first-page":"1","volume-title":"Bergey\u2019s manual of systematics of archaea and bacteria","author":"KA Bernard","year":"2015","unstructured":"Bernard KA, Funke G. 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No human genetic material was present in the samples. According to national regulations (Sect.\u00a04\u00a7\u00a03 of the Swedish Act on Ethical Review 2003:460), ethical approval and informed consent are not required for research involving pure clinical bacterial isolates that cannot be linked back to patients.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"60"}}