| Summary: | Strains 335427<sup>T</sup> and 234509<sup>T</sup>, isolated from two 76-year-old patients with chronic pulmonary diseases, were the subject of polyphasic taxonomic studies and comparative genomic analyses for virulence factors. The 16 rRNA gene sequence similarity between strains 335427<sup>T</sup> and 234509<sup>T</sup> and their closest phylogenetic neighbors <i>Nocardia asiatica</i> NBRC 100129<sup>T</sup> and <i>Nocardia abscessus</i> NBRC 100374<sup>T</sup> were 99.5% and 100%, respectively. Digital DNA–DNA hybridization values between the aforementioned studied strains were well below the 70% threshold for assigning prokaryotic strains to a novel species. Strains 335427<sup>T</sup> and 234509<sup>T</sup> have genome sizes of 8.49 Mpb and 8.07 Mpb, respectively, with G + C content of 68.5%. Isolate 335427<sup>T</sup> has C<sub>16:0</sub>, C<sub>18:1</sub> ω<sub>9</sub>c, C<sub>18:0</sub> and C<sub>18:0</sub> 10 methyl as major fatty acids (>15%) and mycolic acids formed of 52–54 carbon atoms. However, only C<sub>18:1</sub> ω<sub>9</sub>c was detected for isolate 234509<sup>T</sup>, which had mycolic acids with 44–56 carbon. Based on phenotypic and genetic data, strains 335427<sup>T</sup> (DSM 109819<sup>T</sup> = CECT 9924<sup>T</sup>) and 234509<sup>T</sup> (DSM 111366<sup>T</sup> = CECT 30129<sup>T</sup>) merit recognition as novel species, which are named <i>Nocardia barduliensis</i> sp. nov. and <i>Nocardia gipuzkoensis</i> sp. nov., respectively. All the strains studied had homologous VF-associated genes to those described in <i>M. tuberculosis</i>, including experimentally verified virulence genes in humans related to tuberculosis. The <i>nar</i>GHIJ (nitrate reduction pathway) and <i>gvp</i>AFGOJLMK (gas vesicles) genetic maps of strains 335427<sup>T</sup>, 234509<sup>T</sup>, NBRC 100129<sup>T</sup> and NBRC 100374<sup>T</sup> showed the same syntenic block and raise the question of whether their functions are interlinked during the infection of the human host. However, further research is required to decipher the role of the gas vesicle in the pathogenicity mechanism of <i>Nocardia</i> spp.
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