Molecular identification and characterization of filamentous fungi and yeasts isolated in a pharmaceutical industry environment

Microbiological monitoring of pharmaceutical industry aseptic areas is an important procedure to assess the efficiencyof contamination control measures in these areas. Once the permitted microbiological level has been exceeded, themicrobial contaminant should be identified, and the actions to eliminate this agent should be adopted. The objective ofthis study was to identify the filamentous fungi and yeasts isolated from the pharmaceutical industry environment by thematrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) proteomic approachas well as to analyze fungal universal region polymerase chain reaction- restriction fragment length polymorphism(PCR-RFLP) capacity in discriminating inter- and intraspecies differences in these isolates. The MALDI-TOF methodwas able to identify 100% of the samples to the genus level; however, only 68.42% of the samples were identifiedto the species level. Candida guilliermondii, Penicillium spp., Rhodosporidium toruloides, and Aspergillus specieswere the most prevalent microorganisms among the samples. The PCR-RFLP analysis of the fungal universal regionrevealed a heterogeneous profile in the analyzed samples. The isolates of the same species, collected from differentsampling points, presented the same restriction profile. MALDI-TOF and PCR-RFLP methods of the fungal universalregion were able to, respectively, identify and characterize the genetic diversity and differentiate interspecifically theyeast and fungi samples analyzed in this study. The genetic profiles established by the PCR-RFLP methods of thisstudy can be used to create a database for in-house identification of yeasts and fungi in the laboratory, where this studywas carried out.

Genomic and proteomic approach as a tool to discrimination of Escherichia coli strains of biotechnological interest

The Methods based on genomic and proteomic approaches are described as effective tools for the identificationof microorganisms. The development of methodologies capable of differentiating, interspecifically, pathogenic,wild and genetically modified Escherichia coli strains is desirable for the fields of healthcare and Biotechnology.The purpose of this study was to verify the viability of ERIC-PCR and MALDI TOF methods in differentiatinglineages of Escherichia strains. For this purpose, laboratory Escherichia coli ATCC8739, Escherichia coliW3110, Escherichia coli BL21DE3+, Escherichia coli JM109, Escherichia coli MC 1061 and Escherichiacoli DH5α were subjected to ERIC-PCR and MALDI TOF mass spectrometry analyzes. Genomic (ERIC-PCR)and proteomic (MALDI-TOF) methods were able to discriminate between different lineages of Escherichiacoli strains including lineages of Escherichia coli K-12. However, the MALDI TOF proteomic approachrevealed being able to differentiate interspecifically lineages of Escherichia coli strains. The determination ofthe most frequent masses found in the studied Escherichia coli strains in addition to future experiments ofpeptide sequencing profile and SDS-PAGE can be used as a guideline for validating a method for proteomicidentification of these strains.