Identification of nontuberculous mycobacteria using multilocous sequence analysis of 16S rRNA, hsp65, and rpoB

Abstract

Background The isolation of nontuberculous mycobacteria (NTM) from clinical specimens has increased, and they now are considered significant opportunistic pathogens. The aims of this study were to develop a database and interpretive criteria for identifying individual species. In addition, using clinical isolates, we evaluated the clinical usefulness of 16S rRNA, hsp65, and rpoB as target genes for this method. Methods The sequences of NTM for 16S rRNA, hsp65, and rpoB were collected from GenBank and checked by manual inspection. Clinical isolates collected between 2005 and 2010 were used for DNA extraction, polymerase chain reaction, and sequencing of these three genes. We constructed a database for the genes and evaluated the clinical utility of multilocus sequence analysis (MLSA) using 109 clinical isolates. Results A total 131, 130, and 122 sequences were collected from GenBank for 16S rRNA, hsp65, and rpoB, respectively. The percent similarities of the three genes ranged from 96.57% to 100% for the 16S rRNA gene, 89.27% to 100% for hsp65, and 92.71% to 100% for rpoB. When we compared the sequences of 109 clinical strains with those of the database, the rates of species‐level identification were 71.3%, 86.79%, and 81.55% with 16S rRNA, hsp65, and rpoB, respectively. We could identify 97.25% of the isolates to the species level when we used MLSA. Conclusion There were significant differences among the utilities of the three genes for species identification. The MLSA technique would be helpful for identification of NTM.

Isolation, identification and associated risk factors of non‐tuberculous mycobacteria infection in humans and dromedary camels in Samburu County, Kenya

“…This was consistent with the findings by Bolanos et al. (2018) and Kim and Shin (2018). Due to the limited availability of sequencing facilities in many settings, most studies may be missing valuable information on the identity of NTM for the management of human and animal health.…”

Phylogenomics and Comparative Genomic Studies Robustly Support Division of the Genus Mycobacterium into an Emended Genus Mycobacterium and Four Novel Genera

“…A number of studies have also been performed on a limited number of mycobacterial species using multilocus sequence analysis based on concatenated sequences of nucleotides or amino acid fragments from several gene sequences viz. 16S rRNA, rpoB , and hsp65 (Kim and Shin, 2017 ); 16S rRNA, hsp65, sodA, recA, rpoB (Adékambi and Drancourt, 2004 ) and hsp65, tuf , rpoB, smpB , 16S rRNA, sodA , tmRNA (Mignard and Flandrois, 2008 ). The results of these studies have provided useful insights into the relationships between members of the genus Mycobacterium .…”

Non-Tuberculous Mycobacteria: Molecular and Physiological Bases of Virulence and Adaptation to Ecological Niches

“…Conserved or variable regions of the 16S rDNA amplicon are amplified depending on the goal of detection or identification. Still, due to the high similarity found across mycobacterial sequences, this analysis is frequently coupled with rpoB , associated with rifampicin resistance, and also by analysis of hsp65 that together enhance discriminatory power [ 138 , 337 ]. The single-use of the 16S rRNA gene may lead to misidentification of species, contrary to rpoB sequencing which seems to achieve better resolution at the species level [ 338 ].…”