| Summary: | The genus <i>Corynebacterium</i> is frequently found in the female urinary microbiome (FUM). In-depth characterization of <i>Corynebacterium</i> at the species level has been barely exploited. During ongoing FUM research studies, eight strains (c8Ua_144<sup>T</sup>, c8Ua_172<sup>T</sup>, c8Ua_174<sup>T</sup>, c8Ua_181<sup>T</sup>, c9Ua_112<sup>T</sup>, c19Ua_109<sup>T</sup>, c19Ua_121<sup>T</sup>, and c21Ua_68<sup>T</sup>) isolated from urine samples of healthy women or diagnosed with overactive bladder could not be allocated to any valid <i>Corynebacterium</i> species. In this work, we aimed to characterize these strains based on a polyphasic approach. The strains were Gram stain positive, rod to coccoid shaped, nonmotile, catalase positive, and oxidase negative. Phylogenetic analysis based on 16S rRNA and <i>rpoB</i> gene sequences indicated that all strains belonged to the genus <i>Corynebacterium</i>. The average nucleotide identity and digital DNA–DNA hybridization values among the genomes of the above eight strains and closely related type strains of the <i>Corynebacterium</i> genus were <95 (74.1%–93.9%) and <70% (22.2%–56.5%), respectively. Mycolic acids were identified in all strains. MK-8(H2) and/or MK-9(H2) were identified as the major menaquinones. The polar lipids’ pattern mostly consisted of diphosphatidylglycerol, phosphatidylglycerol, and glycophospholipids. The major fatty acid was C<sub>18:1</sub><i>ω</i>9<i>c</i>. <i>Corynebacterium lehmanniae</i> (c8Ua_144<sup>T</sup> = DSM 113405<sup>T</sup> = CCP 74<sup>T</sup>), <i>Corynebacterium meitnerae</i> (c8Ua_172<sup>T</sup> = DSM 113406<sup>T</sup> = CCP 75<sup>T</sup>), <i>Corynebacterium evansiae</i> (c8Ua_174<sup>T</sup> = DSM 113407<sup>T</sup> = CCP 76<sup>T</sup>), <i>Corynebacterium curieae</i> (c8Ua_181<sup>T</sup> = DSM 113408<sup>T</sup> = CCP 77<sup>T</sup>), <i>Corynebacterium macclintockiae</i> (c9Ua_112<sup>T</sup> = DSM 113409<sup>T</sup> = CCP 78<sup>T</sup>), <i>Corynebacterium hesseae</i> (c19Ua_109<sup>T</sup> = DSM 113410<sup>T</sup>= CCP 79<sup>T</sup>), <i>Corynebacterium marquesiae</i> (c19Ua_121<sup>T</sup> = DSM 113411<sup>T</sup> = CCP 80<sup>T</sup>), and <i>Corynebacterium yonathiae</i> (c21Ua_68<sup>T</sup> = DSM 113412<sup>T</sup> = CCP 81<sup>T</sup>) are proposed. This study evidenced that commonly used methodologies on FUM research presented limited resolution for discriminating <i>Corynebacterium</i> at the species level. Future research studying the biological mechanisms of the new <i>Corynebacterium</i> species here described may shed light on their possible beneficial role for healthy FUM.
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