Bacterial Identification via MALDI-TOF and 16S rRNA Sequencing

Bacterial isolates were identified by MALDI-TOF mass spectrometry (MALDI-TOF MS) with ethanol-formic acid extraction procedure with HCCA matrix solution according to the manufacturer’s instructions (Bruker Daltonik GmbH, Bremen, Germany) with the usage of an extended custom database in Biotyper software (Bruker) for identification of bifidobacteria [50 (link)].
Based on species variability, the selected isolates were then identified by 16S rRNA gene amplicon sequencing. DNA was isolated using PrepMan Ultra™ (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. For PCR amplifications of the 16S rRNA gene, the universal pair of primers fD1/rP2 [51 (link)] was used, except for pair 285F/261R [52 (link)] for the identification of bifidobacteria. PCR products were purified by the EZNA Cycle Pure Kit (Omega Bio-Tek, Norcross, GA, USA) and sequenced by Eurofins Genomics (Ebersberg, Germany). The obtained sequences were processed in Chromas Lite 2.5.1 (Technelysium Pty Ltd., Tewantin, Australia), BioEdit [53 ] with ClustalW algorithm [54 (link)], and compared with 16S rRNA gene sequences in BLAST rRNA/ITS (https://blast.ncbi.nlm.nih.gov/https://www.ezbiocloud.net/, accessed on 4 November 2022) and EZBioCloud databases (https://www.ezbiocloud.net/, accessed on 4 November 2022).