MALDI-TOF MS database expansion for identification of Bacillus and related genera isolated from a pharmaceutical facility

• Published in: Journal of microbiological methods Vol. 203; p. 106625
• Main Authors: Costa, Luciana Veloso da, Miranda, Rebeca Vitoria da Silva Lage de, Reis, Cristhiane Moura Falavina dos, Andrade, Joyce Modesto de, Cruz, Fernanda Ventura, Frazão, Adriana Marques, Fonseca, Erica Louro da, Ramos, Juliana Nunes, Brandão, Marcelo Luiz Lima, Vieira, Verônica Viana
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-12-2022
• Subjects: 16S rRNA; Agar; Animals; Bacillus; Bacillus - genetics; Bacterial identification; Housekeeping genes; MALDI-TOF MS; Pharmaceutical Preparations; Phylogeny; Proteomics; RNA, Ribosomal, 16S - genetics; rpoB; Sheep; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Housekeeping genes; MALDI-TOF MS; 16S rRNA; rpoB; Bacillus; Bacterial identification
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2022.106625

Bacillus and related genera are among the main bacterial groups isolated from pharmaceutical production areas. The identification of Bacillus species and related genera by classical methods is particularly difficult, due to similarities between closely related species. The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is one of the most promising techniques for chemotaxonomic characterization of microorganisms, being an alternative to genotypic methods. This study aimed to identify Bacillus strains and related genera isolated from immunobiological production areas by phylogenetic analysis of housekeeping genes and expand the database associated with MALDI-TOF MS to improve their identification. In a previous study, 97 aerobic endospore-forming bacteria isolated from a pharmaceutical facility were analyzed by MALDI-TOF MS and 16S rRNA gene full-length sequencing. All strains were identified as Bacillus and related genera by the latest methodology. Among the 97 strains, 22 were unidentified and 2 strains were misidentified by MALDI-TOF MS. In the present study, these 24 strains were subjected to 16S rRNA gene phylogenetic analysis. Strains not identified at species level by this methodology were submitted to rpoB gene phylogenetic analysis. After identifying the strains, 19 of the 24 strains were incubated for 24, 48, and 72 h on Tryptic Soy Agar and Sheep Blood Agar and subjected to analysis by MALDI-TOF MS. A SuperSpectrum for each strain was created and entered into the equipment database. Finally, the 24 strains were again submitted to proteomic analysis by MALDI-TOF MS, and, at this time, all were correctly identified. The genotypic identification of in-house isolated strains and the introduction of these spectra in MALDI-TOF MS, in order to obtain a customized database, proved to be an extremely effective tool in the identification of Bacillus and related genera from pharmaceutical industry origin.
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•MALDI-TOF MS customized database was effective for spore-formers identification.•rpoB gene sequencing was not effective for all spore-formers species.•Species-level identification methodologies are necessary in pharmaceutical industry.•Nine isolates belonging to five possible new species were identified.•MALDI-TOF database must be improved to include pharmaceutical facilities species.