Evaluation of gene targets for identification of tsukamurella species

Abstract

Using DNA-DNA hybridization as the reference method, the usefulness of six gene targets, secA, ssrA, rpoB, 16S rRNA, groEL and gyrB sequencing for identification of 28 strains isolated from human and snake, and five control strains of Tsukamurella species were evaluated. Phenotypic identification methods, using API 20C AUX and API 50 CH identification kits were also performed and analyzed. Among the 28 test strains, DNA-DNA hybridization confirmed that there were 13 strains of T. tyrosinosolvens, 15 strains of T. paurometabola. Phenotypic identification failed to differentiate the control strains T. inchonensis and T. tyrosinosolvens, indicating phenotypic characteristics are not useful to identify Tsukamurella to species levels. As for genotypic identification, 16S rRNA gene sequencing showed that there was >97% sequence similarity among the 28 test strains and the five control strains, revealing that 16S rRNA gene sequencing is not useful to identify Tsukamurella to species levels. The other five gene loci, secA, ssrA, gyrB, groEL and rpoB were found to be having more sequence variation than 16S rRNA gene, and the discriminatory power of secA gene was found to be the highest among the other four housekeeping genes. The present study also showed that combination of secA and ssrA genes analysis can achieve the best discriminatory power for Tsukamurella identification and the results are concordant with DDH result. More test strains from other clinical important Tsukamurella species such as T. paurometabola, T. strandjordae and T. inchonensis could further verify the usefulness of these gene targets for speciation of Tsukamurella species in future studies.