Evaluation of MALDI-TOF MS, sequencing of D2 LSU rRNA and internal transcribed spacer regions (ITS) for the identification of filamentous fungi isolated from a pharmaceutical facility.

The identification of filamentous fungi through culture characterization may be hampered by phenotypic variability. Information obtained from the identification of microorganisms are important for investigation of sources of contamination of a product or process. The aim of this study was to identify filamentous fungal strains (n = 50) isolated from a pharmaceutical facility by using Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as well as D2 domain of the large-subunit (LSU) ribosomal RNA gene and internal transcribed spacer regions (ITS) sequencing. MALDI-TOF MS system only identified five strains at the species level, while 45 were not identified. The analysis through GenBank allowed the identification of up to 19 strains at the species level, while MycoBank allowed the identification of up to nine strains at the species level. The databases identified up to 11 genera: Penicillium, Aspergillus, Cladosporium, Chaetomium, Coniochaeta, Curvularia, Diaporthe, Fusarium, Trichoderma, Rhizopus and Microdochium. MALDI-TOF MS showed an insufficient database to identify the species of fungi. DNA sequencing was the best methodology to identify to the genus level but was unable to differentiate between closely related species. Therefore further methods for the identification of filamentous fungi from pharmaceutical areas at species level need to be developed.

Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility.

The pharmaceutical industry must comply with the requirements for good manufacturing practices to reduce inherent contamination risks in the production process. Bacillus and related genera are among the main bacterial isolated from clean areas, raw material, and products in the pharmaceutical industries, but the correct identification of these species is still a challenge. The aim of this study was to characterize by phenotyping, protein profiling, and 16S rRNA gene sequencing Sutcliffiellahorikoshii strains (n = 6) isolated from an immunobiological pharmaceutical facility, and to propose the reclassification of Bacillus tianshenii to the genus Sutcliffiella, and Sutcliffiella tianshenii sp. nov. The strains were characterized by VITEK®2, matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) using VITEK®MS, and 16S rRNA gene sequencing analysis. MALDI-TOF/MS did not identify any strains that were identified by 16S rRNA as S. horikoshii. VITEK®2 showed false-positive results, with misidentification as B. sporothermodurans (reclassified as Heyndrickxia sporothermodurans) and Geobacillus thermoleovorans. After MALDI-TOF/MS database expansion, with the creation of SuperSpectrum, the strains were correctly identified as S. horikoshii. This study is the first report of isolation of S. horikoshii strains from a pharmaceutical industry. More studies are necessary to better understand the ability of S. horikoshii to contaminate the environment and products.