Microbes growing onto solid surfaces form complex 3-D biofilm structures characterized by the production of extracellular polymeric compounds and an increased resistance to drugs. The quantification of biofilm relays currently on a number of different approaches and techniques, often leading to different evaluations of the ability to form biofilms of the studied microbial strains. Measures of biofilm biomass were carried out with crystal violet (CV) and a direct reading at 405 nm, whereas the activity was assessed with the XTT ((2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) method. The strains of four pathogenic species of the genus Candida (C. albicans, C. glabrata, C. parapsilosis and C. tropicalis) and of Staphylococcus aureus were employed to determine the effective relatedness among techniques and the specific activity of the biofilm, as a ratio between the XTT and the CV outcomes. Since the ability to form biomass and to be metabolically active are not highly related, their simultaneous use allowed for a categorization of the strains. This classification is putatively amenable of further study by comparing the biofilm type and the medical behavior of the strains.

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus Candida. Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.

During an investigation of yeast biota in the rhizosphere of lentisk in Sardinian semi-arid areas, a strain was isolated that could not be assigned to any known species. The sequence of the D1/D2 domain of the large subunit rDNA gene revealed that the strain belonged to the genus Kazachstania and was phylogenetically related to a clade including Kazachstania aerobia, Kazachstania servazzii, Kazachstania solicola and Kazachstania unispora. The novel isolate differed from members of this clade in its ability to assimilate d-glucono-1,5-lactone and its very weak fermentation of glucose and sucrose; its assimilation profile was unique within the genus Kazachstania. Monosporal colonies were able to sporulate, indicating that the species is homothallic. It is proposed that the isolate represents a novel species, Kazachstania ichnusensis sp. nov., with LCF 1675^T ( = CBS 11859^T) as type strain.

A yeast strain was isolated during a study on vineyard-associated yeast strains from Apulia in Southern Italy. ITS and LSU D1/D2 rDNA sequences showed this strain not to belong to any known species and was described as the type strain of Ogataea uvarum sp. nov., a close relative of O. philodendri. Several secondary peaks appeared in the sequences, suggesting internal heterogeneity among the copies of the rDNA. This hypothesis was tested by sequencing single clones of the marker region. The analyses showed different levels of variability throughout the operon with differences between the rRNA encoding genes and the internally transcribed regions. O. uvarum and O. philodendri share high frequency variants, i.e., variants frequently found in many clones, whereas there is a large variability of the low frequency polymorphisms, suggesting that the mechanism of homogenization is more active with the former than with the latter type of variation. These findings indicate that low frequency variants are detected in Sanger sequencing as secondary peaks whereas in Next Generation Sequencing (NGS) of metagenomics DNA would lead to an overestimate of the alpha diversity. For the first time in our knowledge, this investigation shed light on the variation of the copy number of the rDNA cistron during the yeast speciation process. These polymorphisms can be used to investigate on the processes occurring in these taxonomic markers during the separation of fungal species, it being a genetic process highly frequent in the complex microbial ecosystem existing in grape, must and wine.

Zosteric acid (ZA) is a secondary metabolite of the seagrass Zostera marina, with antibiofilm activity against fungi. Information concerning its mechanisms of action is lacking and this limits the development of more potent derivatives based on the same target and activity structure. The aim of this work was to investigate the ZA mode of action by analyzing the metabolic status of Candida albicans biofilm and its protein expression profile upon ZA treatment. Fourier-Transform Infrared Spectroscopy confirmed that ZA modified the metabolomic response of treated cells, showing changes in the spectral regions, mainly related to the protein compartment. Nano Liquid Chromatography–High-Resolution Mass Spectrometry highlighted that 10 proteins were differentially expressed in the C. albicans proteome upon ZA treatment. Proteins involved in the biogenesis, structure and integrity of cell walls as well as adhesion and stable attachment of hyphae were found downregulated, whereas some proteins involved in the stress response were found overexpressed. Additionally, ZA was involved in the modulation of non-DNA-based epigenetic regulatory mechanisms triggered by reactive oxygen species. These results partially clarified the ZA mechanism of action against fungi and provided insight into the major C. albicans pathways responsible for biofilm formation.