{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T03:48:20Z","timestamp":1792208900357,"version":"4.4.0"},"reference-count":23,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2024,4,3]],"date-time":"2024-04-03T00:00:00Z","timestamp":1712102400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003593","name":"CNPq","doi-asserted-by":"publisher","award":["402301\/2023-4"],"award-info":[{"award-number":["402301\/2023-4"]}],"id":[{"id":"10.13039\/501100003593","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003593","name":"CNPq","doi-asserted-by":"publisher","award":["169846\/2023-7"],"award-info":[{"award-number":["169846\/2023-7"]}],"id":[{"id":"10.13039\/501100003593","id-type":"DOI","asserted-by":"publisher"}]},{"name":"PIBIC\/Fiocruz Program","award":["402301\/2023-4"],"award-info":[{"award-number":["402301\/2023-4"]}]},{"name":"PIBIC\/Fiocruz Program","award":["169846\/2023-7"],"award-info":[{"award-number":["169846\/2023-7"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Bacillus and related genera are among the most important contaminants in the pharmaceutical production environment, and the identification of these microorganisms at the species level assists in the investigation of sources of contamination and in preventive and corrective decision making. The aim of this study was to evaluate three methodologies for the characterization of endospore-forming aerobic bacterial strains isolated from a pharmaceutical unit in Rio de Janeiro, Brazil. MALDI-TOF MS was performed using MALDI Biotyper\u00ae and VITEK\u00ae MS RUO systems, and complete 16S rRNA gene sequencing was performed using the Sanger methodology. The results showed the prevalence of the genera Bacillus (n = 9; 36.0%), Priestia (n = 5; 20.0%), and Paenibacillus (n = 4; 16.0%). Three (20.0%) strains showed &lt;98.7% of DNA sequencing similarity on the EzBioCloud Database, indicating possible new species. In addition, the reclassification of Bacillus pseudoflexus to the genus Priestia as Priestia pseudoflexus sp. nov. is proposed. In conclusion, 16S rRNA and MALDI TOF\/MS were not sufficient to identify all strains at the species level, and complementary analyses were necessary.<\/jats:p>","DOI":"10.3390\/microorganisms12040724","type":"journal-article","created":{"date-parts":[[2024,4,3]],"date-time":"2024-04-03T00:58:38Z","timestamp":1712105918000},"page":"724","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Characterization by MALDI-TOF MS and 16S rRNA Gene Sequencing of Aerobic Endospore-Forming Bacteria Isolated from Pharmaceutical Facility in Rio de Janeiro, Brazil"],"prefix":"10.3390","volume":"12","author":[{"given":"Nathalia Gon\u00e7alves Santos","family":"Caldeira","sequence":"first","affiliation":[{"name":"National Institute for Quality Control in Health, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"},{"name":"Integrated Laboratory\u2013Simulids and Onchocerciasis & Medical and Forensic Entomology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Luiza Soares","family":"de Souza","sequence":"additional","affiliation":[{"name":"Integrated Laboratory\u2013Simulids and Onchocerciasis & Medical and Forensic Entomology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebeca Vit\u00f3ria da Silva Lage","family":"de Miranda","sequence":"additional","affiliation":[{"name":"Institute of Technology in Immunobiologicals, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1873-4056","authenticated-orcid":false,"given":"Luciana Veloso","family":"da Costa","sequence":"additional","affiliation":[{"name":"Institute of Technology in Immunobiologicals, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6709-0712","authenticated-orcid":false,"given":"Stephen James","family":"Forsythe","sequence":"additional","affiliation":[{"name":"Foodmicrobe.com Ltd., Adams Hill, Keyworth, Nottingham NG12 5GY, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viviane","family":"Zahner","sequence":"additional","affiliation":[{"name":"Integrated Laboratory\u2013Simulids and Onchocerciasis & Medical and Forensic Entomology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1121-7312","authenticated-orcid":false,"given":"Marcelo Luiz Lima","family":"Brand\u00e3o","sequence":"additional","affiliation":[{"name":"Institute of Technology in Immunobiologicals, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,4,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"434","DOI":"10.5731\/pdajpst.2021.012636","article-title":"Microbial Contamination in Water Systems","volume":"76","author":"Sandle","year":"2022","journal-title":"PDA J. Pharm. Sci. Technol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"392","DOI":"10.5731\/pdajpst.2011.00765","article-title":"A review of cleanroom microflora: Types, trends, and patterns","volume":"65","author":"Sandle","year":"2011","journal-title":"PDA J. Pharm. Sci. Technol."},{"key":"ref_3","unstructured":"European Medicines Agency (2022). The Rules Governing Medicinal Products in the European Union, European Medicines Agency. Annex 1: Manufacture of Sterile Medicinal Products."},{"key":"ref_4","unstructured":"National Health Surveillance Agency (ANVISA) (2024, January 10). Resolution of the collegiate board\u2014RDC no 609, of 9 March 2022, Brazilian Pharmacopoeia, Available online: https:\/\/www.gov.br\/anvisa\/pt-br\/assuntos\/pharmacopoeia\/pharmacopoeia-brazilian."},{"key":"ref_5","unstructured":"United States Pharmacopeial Convention (USP) (2021). The United States Pharmacopeia, United States Pharmacopeial Convention Inc.. [43rd ed.]. Available online: http:\/\/www.uspnf.com\/uspnf\/login."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Costa, L.V.D., Miranda, R.V.D.S.L., Fonseca, E.L., Gon\u00e7alves, N.P., Reis, C.M.F., Fraz\u00e3o, A.M., Cruz, F.V., Brand\u00e3o, M.L.L., Ramos, J.N., and Vieira, V.V. (2022). Assessment of VITEK\u00ae 2, MALDI-TOF MS and full gene 16S rRNA sequencing for aerobic endospore-forming bacteria isolated from a pharmaceutical facility. J. Microbiol. Methods, 194.","DOI":"10.1016\/j.mimet.2022.106419"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Miranda, R.V.D.S.L., da Costa, L.V., Albuquerque, L.S., Dos Reis, C.M.F., Braga, L.M.P.D.S., de Andrade, J.M., Ramos, J.N., Mattoso, J.M.V., Forsythe, S.J., and Brand\u00e3o, M.L.L. (2023). Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility. Lett. Appl. Microbiol., 76.","DOI":"10.1093\/lambio\/ovad056"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Celandroni, F., Salvetti, S., Gueye, S.A., Mazzantini, D., Lupetti, A., Senesi, S., and Ghelardi, E. (2016). Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0152831"},{"key":"ref_9","first-page":"1","article-title":"Microbial Identification Methods in Pharmaceutical Analysis: Comparison and Evaluation","volume":"1","author":"Sakhno","year":"2016","journal-title":"Mathews J. Pharm. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1435","DOI":"10.2478\/s11696-014-0612-0","article-title":"Need for database extension for reliable identification of bacteria from extreme environments using MALDI TOF mass spectrometry","volume":"68","author":"Kopcakova","year":"2014","journal-title":"Chem. Pap."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Costa, L.V.D., Miranda, R.V.D.S.L., Reis, C.M.F., Andrade, J.M., Cruz, F.V., Fraz\u00e3o, A.M., Fonseca, E.L., Ramos, J.N., Brand\u00e3o, M.L.L., and Vieira, V.V. (2022). MALDI-TOF MS database expansion for identification of Bacillus and related genera isolated from a pharmaceutical facility. J. Microbiol. Methods, 203.","DOI":"10.1016\/j.mimet.2022.106625"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3022","DOI":"10.1093\/molbev\/msab120","article-title":"MEGA11: Molecular evolutionary genetics analysis version 11.0.13","volume":"38","author":"Tamura","year":"2021","journal-title":"Mol. Biol. Evol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1613","DOI":"10.1099\/ijsem.0.001755","article-title":"Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies","volume":"67","author":"Yoon","year":"2017","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_14","first-page":"95","article-title":"BioEdit: A User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95\/98\/NT","volume":"41","author":"Hall","year":"1999","journal-title":"Nucleic Acids Symp. Ser."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5753","DOI":"10.1099\/ijsem.0.004475","article-title":"Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: Description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species","volume":"70","author":"Gupta","year":"2020","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1099\/ijsem.0.003775","article-title":"A phylogenomic and comparative genomic framework for resolving the polyphyly of the genus Bacillus: Proposal for six new genera of Bacillus species, Peribacillus gen. nov., Cytobacillus gen. nov., Mesobacillus gen. nov., Neobacillus gen. nov., Metabacillus gen. nov. and Alkalihalobacillus gen. nov","volume":"70","author":"Patel","year":"2020","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2163","DOI":"10.1007\/s10096-014-2183-y","article-title":"Evaluation of the Bruker Biotyper and VITEK MS MALDI-TOF MS systems for the identification of unusual and\/or difficult-to-identify microorganisms isolated from clinical specimens","volume":"33","author":"TeKippe","year":"2014","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Mezian, L., Chincha, A.I.A., Vecchione, A., Ghelardi, E., Bonatto, J.M.C., Marsaioli, A.J., Campelo, P.H., Benamar, I., Moussaoui, A.A., and Sant\u2019Ana, A.S. (2022). Aerobic spore-forming bacteria in powdered infant formula: Enumeration, identification by MALDI-TOF mass spectrometry (MS), presence of toxin genes and rpoB gene typing. Int. J. Food Microbiol., 368.","DOI":"10.1016\/j.ijfoodmicro.2022.109613"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1007\/s10123-019-00080-6","article-title":"Phylogenetic diversity of aerobic spore-forming Bacillalles isolated from Brazilian soils","volume":"22","author":"Orem","year":"2019","journal-title":"Int. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4525","DOI":"10.1016\/j.ygeno.2020.08.008","article-title":"Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E","volume":"112","author":"Pereira","year":"2020","journal-title":"Genomics"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"895","DOI":"10.1007\/s13213-015-1174-2","article-title":"Bacillus pseudoflexus sp. Nov., a moderately halophilic bacterium isolated from compost","volume":"66","author":"Chandna","year":"2016","journal-title":"Ann. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5607","DOI":"10.1099\/ijsem.0.004332","article-title":"List of Prokaryotic names with Standing in Nomenclature (LPSN) moves to the DSMZ","volume":"70","author":"Parte","year":"2020","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_23","first-page":"1847","article-title":"A Numerical Classification of the Genus Bacillus","volume":"134","author":"Priest","year":"1988","journal-title":"J. Gen. Microbiol."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/12\/4\/724\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:22:44Z","timestamp":1760106164000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/12\/4\/724"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,4,3]]},"references-count":23,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["microorganisms12040724"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms12040724","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,4,3]]}}}