Using wasps as a tool to restore a functioning vine grape mycobiota and preserve the mycobial "terroir".

In the last one-hundred years, the exponential expansion of wine making has artificialized the agricultural landscape as well as its microbial diversity, spreading human selected Saccharomyces cerevisiae strains. Evidence showed that social wasps can harbor a significant fraction of the yeast phenotypic diversity of a given area of wine production, allowing different strains to overwinter and mate in their gut. The integrity of the wasp-yeast ecological interaction is of paramount importance to maintain the resilience of microbial populations associated to wine aromatic profiles. In a field experiment, we verified whether Polistes dominula wasps, reared in laboratory and fed with a traceable S. cerevisiae strain, could be a useful tool to drive the controlled yeast dispersion directly on grapes. The demonstration of the biotechnological potential of social insects in organic wine farming lays the foundations for multiple applications including maintenance of microbial biodiversity and rewilding vineyards through the introduction of wasp associated microbiomes.

Forests influence yeast populations vectored by insects into vineyards.

Introduction: In the vineyard, yeast communities impact the ripening and fermentation of grapes and are influenced by geographical location, climate, and soil characteristics. Despite the great advancement in our knowledge of the vineyard mycobiota, a key step of the process leading to the definition of the vineyard yeast community is still poorly understood: if geography, climate, and soil influence the mycobiota, potentially through selection, where do the yeast originate from, and how can they reach the vineyard? In this perspective, it is currently acknowledged that forests host several yeast species and that insects, particularly social wasps, can vector and maintain the yeasts known to populate the vineyard. Alas, the conveyance, fostered by insects, of yeasts from the forest to the vineyard has not been proven yet. In this study, we aimed to assess the existence of links between a potential natural source of yeasts (woods), the vectors (social wasps), and the composition of the vineyard mycobiota. Methods: For this purpose, the mycobiota of wasps caught in six Italian vineyards were analyzed over 2 years through culturomics approaches. Results: The results clearly indicate that the presence of wooded areas close to vineyards is associated with particular features of the mycobiota vectored by social wasps. Wasps caught in vineyards near wooded areas bear a higher number of yeast cells and higher biodiversity than insects caught in vineyards far from woods. Furthermore, insects caught in vineyards close to woods bear distinctive yeast populations, encompassing species such as Saccharomyces cerevisiae. Discussion: Overall, our work provides fundamental insights into the ecology of the vineyard mycobiota and highlights the need to maintain a vineyard-woodland mosaic landscape, thus preserving the suitable habitat for yeast species relevant to wine-making.

Genomic Assembly of Clinical Candida glabrata (Nakaseomyces glabrata) Isolates Reveals within-Species Structural Plasticity and Association with In Vitro Antifungal Susceptibility.

The opportunistic human pathogen Candida glabrata has become an increasingly important threat to human health, with infections globally characterized by high mortality rates and multidrug resistance. To face this threat, more efficient diagnostic and therapeutic approaches are required, underpinning research to help define the intraspecies epidemiology, genetic variability, and therefore, diagnostic and therapeutic target stability. Previous comparative genetics studies conducted on limited numbers of strains only revealed partial resolution of chromosomal settings. In this study, by combining short- and long-read genome sequencing, phenotypic characterization, and comparative genomics over a large set of strains, we detected strict relationships between large chromosomal rearrangements and phylogenetic clades, genes subjected to different selective pressures, and new sets of genes associated with resistance to antifungals. Overall, these results not only provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology but may also lay the foundations for the future development of tailored therapeutic approaches. IMPORTANCE The human pathogen Candida glabrata has become a global threat to human health, with infections characterized by high mortality and multidrug resistance. We have obtained nine fully assembled genomes from clinical isolates through a combination of short- and long-read sequencing approaches. The quality and completeness of such genomes and their subsequent comparison to the broadest set of genomes so far allowed us to pinpoint chromosomal rearrangements in several genomes and detect phylogenetic clades that were not associated with geographic location or isolation source. We identified a new set of genes associated with resistance to antifungals coding for adhesin or adhesin-like proteins, suggesting C. glabrata resists antifungals by forming aggregates or adhering to the host tissue. These results, which provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology, may initiate the development of precision medicine interventions for patients with suspected or proven invasive fungal infections.

Resistance to Arsenite and Arsenate in Saccharomyces cerevisiae Arises through the Subtelomeric Expansion of a Cluster of Yeast Genes.

Arsenic is one of the most prevalent toxic elements in the environment, and its toxicity affects every organism. Arsenic resistance has mainly been observed in microorganisms, and, in bacteria, it has been associated with the presence of the Ars operon. In Saccharomyces cerevisiae, three genes confer arsenic resistance: ARR1, ARR2, and ARR3. Unlike bacteria, in which the presence of the Ars genes confers per se resistance to arsenic, most of the S. cerevisiae isolates present the three ARR genes, regardless of whether the strain is resistant or sensitive to arsenic. To assess the genetic features that make natural S. cerevisiae strains resistant to arsenic, we used a combination of comparative genomic hybridization, whole-genome sequencing, and transcriptomics profiling with microarray analyses. We observed that both the presence and the genomic location of multiple copies of the whole cluster of ARR genes were central to the escape from subtelomeric silencing and the acquisition of resistance to arsenic. As a result of the repositioning, the ARR genes were expressed even in the absence of arsenic. In addition to their relevance in improving our understanding of the mechanism of arsenic resistance in yeast, these results provide evidence for a new cluster of functionally related genes that are independently duplicated and translocated.

Going wild: ecology and genomics are crucial to understand yeast evolution.

Improved and more accessible genome-sequencing approaches have allowed the analysis of large sets of natural yeast isolates. As a consequence, this unprecedented level of description of yeast-genome characteristics and variations in natural environments has provided crucial insights on yeast ecology and evolution. Here, we review some of the most relevant and intriguing aspects of yeast evolution pointed out, thanks to the combination of yeast ecology and genomics, and critically examine the resulting improvement of our knowledge on this field. Only integrated approaches, taking into consideration not only the characteristics of the microbe but also those of the hosting environment, will significantly move forward the exploration of yeast diversity, ecology, and evolution.

Profile of Bacterial Infections in COVID-19 Patients: Antimicrobial Resistance in the Time of SARS-CoV-2.

The global onset of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infections happened suddenly, hence imposing a rapid definition of effective therapeutic approaches. Antibiotics were included among the prophylactic agents because of both the similarity between SARS-CoV-2 and atypical pneumonia symptoms, and the immune-modulating and anti-inflammatory properties of such drugs. Although, this approach could exacerbate the emergence of antimicrobial resistance. To evaluate the impact of the COVID-19 pandemic on the spread and characteristics of bacterial infections, as well as on the frequency of antimicrobial resistance, we investigated and compared clinical bacterial strains isolated in an Italian hospital from COVID-19 patients and non-COVID-19 patients during and before the COVID-19 outbreak. Data clearly indicate the impact of the COVID-19 pandemic on bacterial infections: not only some bacterial species were found in either COVID-19 positive or in COVID-19 negative patients, but isolates from COVID-19 patients also showed higher levels of antimicrobial resistance. Nevertheless, despite some bacterial species were isolated only before or over the pandemic, no differences were observed among the antimicrobial resistance levels. Overall, these results recapitulate the current situation of microbial infections and could also provide an overview of the impact of COVID-19 on bacterial pathogens spread and resistance.

Antimicrobial Resistance, an Update from the Ward: Increased Incidence of New Potential Pathogens and Site of Infection-Specific Antibacterial Resistances.

In order to monitor the spread of antimicrobial resistance, the European Union requires hospitals to be equipped with infection control centers. With this aim, we analyzed 1583 bacterial strains isolated from samples of different origin from patients with community-onset and nosocomial infections in a public tertiary University Hospital on the outskirts of Turin, Italy. Statistical analyses of the isolates (source, type) and their antimicrobial resistance (AMR) were performed. The survey revealed infections associated with bacterial species considered as not-commensal and not-pathogenic, hence potentially emerging as new threats for human health. Conversely to the general observation of nosocomial strains being more resistant to antibiotics compared to community-acquired strains, nosocomial strains isolated in this study were more resistant only to 1/42 tested antibiotics (tetracycline). By adopting an ecological approach, we observed that blood infections are associated with the broadest range of species compared to infections affecting other areas and we obtained clear indications on the antibiotics that should be preferred in the treatment of infections at specific body sites. Future investigations carried out on a larger geographical scale will clarify whether these indications are limited to the geographical region investigated over this study, or whether the same trends are visible at national or international level.

Comparative immunophenotyping of Saccharomyces cerevisiae and Candida spp. strains from Crohn's disease patients and their interactions with the gut microbiome.

Investigation of the fungal communities in animal models of Inflammatory Bowel Diseases (IBD) showed a controversial role of Saccharomyces cerevisiae and Candida spp. In health and disease. These conflicting observations could be ascribed to immunogenic differences among co-specific strains. To assess the relevance of intra-strains differences on yeast immunogenicity and impact on the microbiota, we screened S. cerevisiae and Candida spp. Strains isolated from fecal samples of IBD patients. We compared the cytokine profiles, obtained upon stimulation of Peripheral Blood Mononuclear Cells (PBMCs) and Dendritic Cells with different yeast strains, and evaluated the relationship between strain's cell wall sugar amount and immune response. Moreover, the gut microbiota composition was explored in relation to fungal isolation from fecal samples by metabarcoding analysis. The comparison of cytokine profiles showed strain dependent rather than species-dependent differences in immune responses. Differences in immunogenicity correlated with the cell wall composition of S. cerevisiae intestinal strains. Stimulation of human healthy PBMCs with different strains showed a pro-inflammatory IL-6 response counterbalanced by IL-10 production. Interestingly, Crohn's (CD) patients responded differently to "self" and "non-self" strains, eliciting pure Th1 or Th17 cytokine patterns. The differences observed in vitro were recapitulated in vivo, where different strains contributed in dramatically different ways to local epithelial activity and to the inflammation of wild type and Interleukin-deficient mice. Furthermore, we observed that the gut microbiota profiles significantly differentiated according to the presence of Saccharomyces or Candida spp. or the absence of fungal isolates in fecal samples. Our results show the importance to deepen metagenomics and immunophenotyping analyses to the strain level, to elucidate the role of fungal and bacterial communities in health and disease.

Saccharomyces cerevisiae - Insects Association: Impacts, Biogeography, and Extent.

Over the last few years, an increasing number of studies have reported the existence of an association between the budding yeast Saccharomyces cerevisiae and insects. The discovery of this relationship has called into question the hypothesis that S. cerevisiae is unable to survive in nature and that the presence of S. cerevisiae strains in natural specimens is the result of contamination from human-related environments. S. cerevisiae cells benefit from this association as they find in the insect intestine a shelter, but also a place where they can reproduce themselves through mating, the latter being an event otherwise rarely observed in natural environments. On the other hand, insects also take advantage in hosting S. cerevisiae as they rely on yeasts as nutriment to properly develop, to localize suitable food, and to enhance their immune system. Despite the relevance of this relationship on both yeast and insect ecology, we are still far from completely appreciating its extent and effects. It has been shown that other yeasts are able to colonize only one or a few insect species. Is it the same for S. cerevisiae cells or is this yeast able to associate with any insect? Similarly, is this association geographically or topographically limited in areas characterized by specific physical features? With this review, we recapitulate the nature of the S. cerevisiae-insect association, disclose its extent in terms of geographical distribution and species involved, and present YeastFinder, a cured online database providing a collection of information on this topic.

Population genomics reveals evolution and variation of Saccharomyces cerevisiae in the human and insects gut.

The quest to discover the variety of ecological niches inhabited by Saccharomyces cerevisiae has led to research in areas as diverse as wineries, oak trees and insect guts. The discovery of fungal communities in the human gastrointestinal tract suggested the host's gut as a potential reservoir for yeast adaptation. Here, we report the existence of yeast populations associated with the human gut (HG) that differ from those isolated from other human body sites. Phylogenetic analysis on 12 microsatellite loci and 1715 combined CDSs from whole-genome sequencing revealed three subclusters of HG strains with further evidence of clonal colonization within the host's gut. The presence of such subclusters was supported by other genomic features, such as copy number variation, absence/introgressions of CDSs and relative polymorphism frequency. Functional analysis of CDSs specific of the different subclusters suggested possible alterations in cell wall composition and sporulation features. The phenotypic analysis combined with immunological profiling of these strains further showed that sporulation was related with strain-specific genomic characteristics in the immune recognition pattern. We conclude that both genetic and environmental factors involved in cell wall remodelling and sporulation are the main drivers of adaptation in S. cerevisiae populations in the human gut.

Metagenomic Approaches to Investigate the Contribution of the Vineyard Environment to the Quality of Wine Fermentation: Potentials and Difficulties.

The winemaking is a complex process that begins in the vineyard and ends at consumption moment. Recent reports have shown the relevance of microbial populations in the definition of the regional organoleptic and sensory characteristics of a wine. Metagenomic approaches, allowing the exhaustive identification of microorganisms present in complex samples, have recently played a fundamental role in the dissection of the contribution of the vineyard environment to wine fermentation. Systematic approaches have explored the impact of agronomical techniques, vineyard topologies, and climatic changes on bacterial and fungal populations found in the vineyard and in fermentations, also trying to predict or extrapolate the effects on the sensorial characteristics of the resulting wine. This review is aimed at highlighting the major technical and experimental challenges in dissecting the contribution of the vineyard and native environments microbiota to the wine fermentation process, and how metagenomic approaches can help in understanding microbial fluxes and selections across the environments and specimens related to wine fermentation.

Deciphering the mechanism of action of 089, a compound impairing the fungal cell cycle.

Fungal infections represent an increasingly relevant clinical problem, primarily because of the increased survival of severely immune-compromised patients. Despite the availability of active and selective drugs and of well-established prophylaxis, classical antifungals are often ineffective as resistance is frequently observed. The quest for anti-fungal drugs with novel mechanisms of action is thus important. Here we show that a new compound, 089, acts by arresting fungal cells in the G2 phase of the cell cycle through targeting of SWE1, a mechanism of action unexploited by current anti-fungal drugs. The cell cycle impairment also induces a modification of fungal cell morphology which makes fungal cells recognizable by immune cells. This new class of molecules holds promise to be a valuable source of novel antifungals, allowing the clearance of pathogenic fungi by both direct killing of the fungus and enhancing the recognition of the pathogen by the host immune system.

Yeast-insect associations: It takes guts.

Insects interact with microorganisms in several situations, ranging from the accidental interaction to locate attractive food or the acquisition of essential nutrients missing in the main food source. Despite a wealth of studies recently focused on bacteria, the interactions between insects and yeasts have relevant implications for both of the parties involved. The insect intestine shows several structural and physiological differences among species, but it is generally a hostile environment for many microorganisms, selecting against the most sensitive and at the same time guaranteeing a less competitive environment to resistant ones. An intensive characterization of the interactions between yeasts and insects has highlighted their relevance not only for attraction to food but also for the insect's development and behaviour. Conversely, some yeasts have been shown to benefit from interactions with insects, in some cases by being carried among different environments. In addition, the insect intestine may provide a place to reside for prolonged periods and possibly mate or generate sexual forms able to mate once back in the external environments. YEA-May-17-0084.R3.

SaccharomycesIDentifier, SID: strain-level analysis of Saccharomyces cerevisiae populations by using microsatellite meta-patterns.

Saccharomyces cerevisiae is a common yeast with several applications, among which the most ancient is winemaking. Because individuals belonging to this species show a wide genetic and phenotypic variability, the possibility to identify the strains driving fermentation is pivotal when aiming at stable and palatable products. Metagenomic sequencing is increasingly used to decipher the fungal populations present in complex samples such as musts. However, it does not provide information at the strain level. Microsatellites are commonly used to describe the genotype of single strains. Here we developed a population-level microsatellite profiling approach, SID (Saccharomyces cerevisiae IDentifier), to identify the strains present in complex environmental samples. We optimized and assessed the performances of the analytical procedure on patterns generated in silico by computationally pooling Saccharomyces cerevisiae microsatellite profiles, and on samples obtained by pooling DNA of different strains, proving its ability to characterize real samples of grape wine fermentations. SID showed clear differences among S. cerevisiae populations in grape fermentation samples, identifying strains that are likely composing the populations and highlighting the impact of the inoculation of selected exogenous strains on natural strains. This tool can be successfully exploited to identify S. cerevisiae strains in any kind of complex samples.

Survival of metastatic melanoma patients after dendritic cell vaccination correlates with expression of leukocyte phosphatidylethanolamine-binding protein 1/Raf kinase inhibitory protein.

Immunotherapy for metastatic melanoma offers great promise but, to date, only a subset of patients have responded. There is an urgent need to identify ways of allocating patients to the most beneficial therapy, to increase survival and decrease therapy-associated morbidity and costs. Blood-based biomarkers are of particular interest because of their straightforward implementation in routine clinical care. We sought to identify markers for dendritic cell (DC) vaccine-based immunotherapy against metastatic melanoma through gene expression analysis of peripheral blood mononuclear cells. A large-scale microarray analysis of 74 samples from two treatment centers, taken directly after the first round of DC vaccination, was performed. We found that phosphatidylethanolamine binding protein 1 (PEBP1)/Raf Kinase inhibitory protein (RKIP) expression can be used to identify a significant proportion of patients who performed poorly after DC vaccination. This result was validated by q-PCR analysis on blood samples from a second cohort of 95 patients treated with DC vaccination in four different centers. We conclude that low PEBP1 expression correlates with poor overall survival after DC vaccination. Intriguingly, this was only the case for expression of PEBP1 after, but not prior to, DC vaccination. Moreover, the change in PEBP1 expression upon vaccination correlated well with survival. Further analyses revealed that PEBP1 expression positively correlated with genes involved in T cell responses but inversely correlated with genes associated with myeloid cells and aberrant inflammation including STAT3, NOTCH1, and MAPK1. Concordantly, PEBP1 inversely correlated with the myeloid/lymphoid-ratio and was suppressed in patients suffering from chronic inflammatory disease.

Non-invasive real time monitoring of yeast volatilome by PTR-ToF-MS.

INTRODUCTION: Producing a wide range of volatile secondary metabolites Saccharomyces cerevisiae influences wine, beer, and bread sensory quality and hence selection of strains based on their volatilome becomes pivotal. A rapid on-line method for volatilome assessing of strains growing on standard solid media is still missing. OBJECTIVES: Methodologically, the aim of this study was to demonstrate the automatic, real-time, direct, and non-invasive monitoring of yeast volatilome in order to rapidly produce a robust large data set encompassing measurements relative to many strains, replicates and time points. The fundamental scope was to differentiate volatilomes of genetically similar strains of oenological relevance during the whole growing process. METHOD: Six different S. cerevisiae strains (four meiotic segregants of a natural strain and two laboratory strains) inoculated onto a solid medium have been monitored on-line by Proton Transfer Reaction-Time-of-Flight-Mass Spectrometry for 11 days every 4 h (3540 time points). FastGC PTR-ToF-MS was performed during the stationary phase on the 5th day. RESULTS: More than 300 peaks have been extracted from the average spectra associated to each time point, 70 have been tentatively identified. Univariate and multivariate analyses have been performed on the data matrix (3640 measurements × 70 peaks) highlighting the volatilome evolution and strain-specific features. Laboratory strains with opposite mating type, and meiotic segregants of the same natural strain showed significantly different profiles. CONCLUSIONS: The described set-up allows the on-line high-throughput screening of yeast volatilome of S. cerevisiae strains and the identification of strain specific features and new metabolic pathways, discriminating also genetically similar strains, thus revealing a novel method for strain phenotyping, identification, and quality control.

Relations between Effects and Structure of Small Bicyclic Molecules on the Complex Model System Saccharomyces cerevisiae.

The development of compounds able to modify biological functions largely took advantage of parallel synthesis to generate a broad chemical variance of compounds to be tested for the desired effect(s). The budding yeast Saccharomyces cerevisiae is a model for pharmacological studies since a long time as it represents a relatively simple system to explore the relations among chemical variance and bioactivity. To identify relations between the chemical features of the molecules and their activity, we delved into the effects of a library of small compounds on the viability of a set of S. cerevisiae strains. Thanks to the high degree of chemical diversity of the tested compounds and to the measured effect on the yeast growth rate, we were able to scale-down the chemical library and to gain information on the most effective structures at the substituent level. Our results represent a valuable source for the selection, rational design, and optimization of bioactive compounds.

Core Microbiota and Metabolome of Vitis vinifera L. cv. Corvina Grapes and Musts.

The composition and changes of the fungal population and of the metabolites present in grapes and in ferments of Vitis vinifera L. cv. Corvina, one of the major components of the Amarone musts, were dissected aiming at the identification of constant characteristics possibly influenced by the productive process. The fungal populations and metabolomic profiles were analyzed in three different vintages. 454-pyrosequencing on the ribosomal ITS1 region has been used to identify the fungal population present in Corvina grapes and fresh must. Samples were also subjected to metabolomics analysis measuring both free volatile compounds and glycosylated aroma precursors through an untargeted approach with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. Albeit strongly dependent on the climate, both the mycobiota and metabolome of Corvina grapes and fresh musts show some characteristics recursive in different vintages. Such persistent characteristics are likely determined by the method adopted to produce Amarone or other dry wines made from partially dried grapes. In particular, the harsh conditions imposed by the prolonged withering appear to contribute to the shaping of the fungal populations. The fungal genera and metabolites present in different vintages in V. vinifera L. cv. Corvina grapes and fresh musts represent core components of the peculiar technique of production of Amarone. Their identification allows the in-depth understanding and improved control of the process of production of this economically and culturally relevant wine.

Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan.

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley.