Bacterial Identification via 16S rRNA Sequencing

Isolates were cultured in Tryptic soy broth (Oxoid, Hants, UK) at 22 °C for 12–24 h and centrifuged at 9000 g for 3 min using an Eppendorf 5415D microcentrifuge to obtain a pellet. DNA extraction was carried out using the Wizard Genomic DNA Purification commercial kit (Promega, Madison, WI, USA) following the supplier’s instructions, and the obtained DNA samples were stored at −20 °C until analysis. The amplification of the 16S ribosomal genes of the isolates was carried out by PCR, following the methodology described by Opazo et al. [32 (link)]. The resulting amplified PCR products were sequenced by Macrogen (Rockville, MD, USA) using the ABI PRISM 373 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequences were edited and matched to the Ribosomal Database Project [33 ] to identify the bacterial isolates. Isolates exhibiting in their 16S rRNA gene sequence a similarity score of ≤99.4% with a nearest neighbor were not identified to the species level.