16S rRNA Gene Sequencing and Identification

The complete sequencing of the 16S rRNA gene was performed using the MicroSEQ™ Full Gene 16S rDNA kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The sequences were obtained using the 3500 Series Genetic Analyzer (Applied Biosystems, Waltham, MA, USA) and were processed using DNA Star LaserGene SeqMan software, version 7.0.0. The identification results were obtained from the website https://www.ezbiocloud.net/ (database update: 23 August 2023; last access: 23 November 2023) [12 (link)]. All sequences were deposited at https://www.ncbi.nlm.nih.gov/ (database update: 29 December 2023), and accession numbers are provided in Table 2. Complete 16S rRNA gene sequencing results were considered valid when the identification percentage was ≥96%. When the identification was ≥98.7%, the strain was considered identified at the species level [13 (link)].
Phylogenetic analysis was conducted through the alignment of the sequences obtained by sequencing the 16S rRNA gene using BioEdit Sequence Alignment Editor software, version 7.0.5.3 [14 ]. MEGA 11, software version 11.0.13 [12 (link)], was used to construct maximum likelihood trees, employing the Kimura-2 parameter model with branching support based on 1000 bootstrap replicates.