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.

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial Saccharomyces cerevisiae 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains’ tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome.

Fungal species delimitation was traditionally carried out with multicopy ribosomal RNA (rRNA) genes, principally for their ease of amplification. Since the efficacy of these markers has been questioned, single-copy protein-encoding genes have been proposed alone or in combination for Multi-Locus Sequence Typing (MLST). In this context, the role of the many sequences obtained with Next-Generation Sequencing (NGS) techniques, in both genomics and metagenomics, further pushes toward an analysis of the efficacy of NGS-derived markers and of the metrics to evaluate the marker efficacy in discriminating fungal species. This paper aims at proposing MeTRe (Mean Taxonomic Resolution), a novel index that could be used both for measuring marker efficacy and for assessing the actual resolution (i.e., the level of separation) between species obtained with different markers or their combinations. In this paper, we described and then employed this index to compare the efficacy of two rRNAs and four single-copy markers obtained from public databases as both an amplicon-based approach and genome-derived sequences. Two different groups of species were used, one with a pathogenic species of Candida that was characterized by relatively well-separated taxa, whereas the other, comprising some relevant species of the sensu stricto group of the genus Saccharomyces, included close species and interspecific hybrids. The results showed the ability of MeTRe to evaluate marker efficacy in general and genome-derived markers specifically.

Yeast taxonomy was introduced based on the idea that physiological properties would help discriminate species, thus assuming a strong link between physiology and taxonomy. However, the instability of physiological characteristics within species configured them as not ideal markers for species delimitation, shading the importance of physiology and paving the way to the DNA-based taxonomy. The hypothesis of reconnecting taxonomy with specific traits from phylogenies has been successfully explored for Bacteria and Archaea, suggesting that a similar route can be traveled for yeasts. In this framework, thirteen single copy loci were used to investigate the predictability of complex Fourier Transform InfaRed spectroscopy (FTIR) and High-performance Liquid Chromatography–Mass Spectrometry (LC-MS) profiles of the four historical species of the Saccharomyces sensu stricto group, both on resting cells and under short-term ethanol stress. Our data show a significant connection between the taxonomy and physiology of these strains. Eight markers out of the thirteen tested displayed high correlation values with LC-MS profiles of cells in resting condition, confirming the low efficacy of FTIR in the identification of strains of closely related species. Conversely, most genetic markers displayed increasing trends of correlation with FTIR profiles as the ethanol concentration increased, according to their role in the cellular response to different type of stress.

Homoplasy is a sort of noise in phylogenetic reconstructions, due to the accumulation of backmutations, convergent evolution and horizontal gene transfer (HGT), which is considered the major trigger of homoplasy in microorganism for its massive presence. It is also known that homoplasy increases with the complexity of the tree with both real and simulated data. In this paper, we analyzed the variation of homoplasy with the two widely used taxonomic markers ITS and LSU in four taxonomic models characterized by differences in the intra-specific distances. An algorithm (HomoDist) was developed to analyze the homoplasy index (HI) variation upon addition of a single element (strain or species) in increasing distance from a starting element. This algorithm allows to follow changes of the consistency index (CI), complementary to the HI, with the increase of the number of taxa and with the increase of the distance among elements. Results show that homoplasy increases—as expected—with the number of taxa, but also as a function of the overall distance among species, often with an almost linear relationship between distance and HI. No HI change was observed in trees with few taxa spanning through short distances, indicating that this noise is not prohibitive in this context, although the analysis of the ratio between HI and distance can be recommended as a criterion for tree acceptance. The absence of large changes of the HI within the species, and its increase when new species are added by HomoDist, suggest that homoplasy variation can be used as an auxiliary test in distance-based species delimitation with any type of marker.