Fluorescence in situ Hybridization (FISH) is a versatile, widespread and widely- used technique in microbiology. The first step of FISH—fixation/permeabilization—is crucial to the outcome of the method. This work aimed to systematically evaluate fixation/permeabilization protocols employing ethanol, triton X-100 and lysozyme in conjugation with paraformaldehyde for Peptide Nucleic Acid (PNA)-FISH. Response surface methodology was used to optimize these protocols for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). In general, the optimal PNA-FISH fluorescent outcome in Gram-positive bacteria was obtained employing harsher permeabilization conditions when compared to Gram-negative optimal protocols. The observed differences arise from the intrinsic cell envelope properties of each species and the ability of the fixation/permeabilization compounds to effectively increase the permeability of these structures while maintaining structural integrity. Ultimately, the combination of paraformaldehyde and ethanol proved to have significantly superior performance for all tested bacteria, especially for Gram-positive species (p<0.05).

Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes’ milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3–V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01–1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

Serra da Estrela is the oldest and most recognizable traditional protected designation of origin (PDO) cheese from Portugal. It has been extensively studied over the years, but the latest microbial characterization is 20 years old. Hence, this work aimed to perform an updated characterization of Serra da Estrela PDO cheeses and raw materials. Our analysis showed that lactic acid bacteria content on Serra da Estrela cheeses exceeded 8.8 log CFUsg⁻¹, in all analyzed samples, with lactococci, lactobacilli and Leuconostoc spp. predominating over enterococci strains. Moreover, lactococci and lactobacilli abundance increased across the production season, while enterococci dropped considerably in late manufactures. Lastly, Leuconostoc spp. content remained unchanged in all analyzed periods. A correspondence analysis showed that L. paracasei, L. lactis, E. durans, E. faecium and L. mesenteroides are transversal in Serra da Estrela cheese manufacturing and were closely associated with milk, curd and cheese matrices. Additionally, L. casei, L. plantarum and L. curvatus were specifically associated with cheese matrices, possibly active during ripening and contributing for the development of these cheeses’ organoleptic characteristics.