16S-ITS-23S rRNA operon segment sequencing provides necessary and sufficient conditions for bacterial species-specific identification

Introduction. Sequencing of the 16S rRNA gene is the predominant method for assessing microbial communities and strain molecular identification. The short reads (2^nd generation sequencing)-based technology does not allow analysis beyond the 16S rRNA gene. The taxonomic verification level of samples usually remains at the genus or even family level. Currently, there have been proposed the latest versions of long-read technologies (Oxford Nanopore MinION, PacBio) for amplicon sequencing of near-complete ribosomal operon, including genes 16S, 23S, 5S, and internal transcribed spacer (ITS). At the moment, this approach has not been sufficiently studied, in addition, it involves PCR amplification of a very extended DNA region (more than 4000 bp-long). Materials and methods. The collection of non-tuberculous mycobacteria strains and their primary identification was carried out in the years 2019–2021. The strains were obtained by inoculation of positive cultures from the Bactec MGIT 960 bacteriological analyzer lacking MPT64 antigen in the MGIT TB Identification Test (Becton Dickinson, USA) on Lowenstein-Jensen medium. Preliminary species strain identification was carried out with the Speed-oligo Mycobacteria kit (Vircell, Spain) according to the manufacturer’s protocol. In this work, both known and newly developed universal bacterial primers flanking the near-complete 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene are used. In the present study, both known and newly developed universal bacterial primers are used to flank the near-complete 16S rRNA gene, ITS, and start of the 23S rRNA gene. Results and discussion. Sanger sequencing of the amplicons (about 2000 bp) obtained shows the taxonomic level sufficient to determine species up to 8 strains of non-tuberculous mycobacteria isolated from humans that caused clinically and bacteriologically confirmed diseases. The method proposed for PCR amplification of a bacterial operon a fragment containing most of the 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene is considered by us as an approbation of a methodological approach to study microbial communities in material with a high degree of degradation (necrotic foci, etc.). The results obtained indicate a significantly higher resolution of the approach used than the classical 16S rRNA gene sequencing.