Soil salinity poses a major challenge to the legume-rhizobia symbiosis development, thereby affecting sustainable agriculture. Selecting NaCl-tolerant strains and enhancing the native strains' fitness under salt stress are essential steps for the restoration of marginal areas. In this work, 49 Sinorhizobium meliloti strains, the rhizobial species forming symbiotic nitrogen-fixing associations with alfalfa-including 21 de novo-sequenced field isolates-were subjected to a thorough in vitro screening for salt tolerance at progressively higher NaCl concentrations. Field isolates showed genome-based geographical clustering but contrasting salt tolerance abilities. Indeed, genome-wide association (GWA) analysis on the strains' whole-genome sequencing data indicated several loci associated with the variability in salt tolerance. Candidate genes were involved in various processes including cell wall organisation, LPS biosynthesis, quorum sensing, and carbohydrate transport and metabolism. The relationship with carbohydrate metabolism was further confirmed by Phenotype Microarray analysis which indicated salt-tolerant strains having enhanced capacity in carbon source usage. These findings reveal synergistic pathways underlying salt tolerance and suggest candidate traits (e.g., quorum sensing, carbohydrate synthesis and modification) for developing bioinoculants to enhance legume performance in saline soils.

The phenomenon of urbanization is associated with significant shifts in lifestyle and dietary habits, which can impact the composition of gut microbiota. While variations in gut bacterial communities between rural and urban residents are documented, changes in fungal communities (mycobiota) remain underexplored. This study investigates the impact of urbanization-related dietary shifts on the gut mycobiota in a sub-Saharan African context (Burkina Faso). We analyzed the gut mycobiota composition of individuals from households of rural and semi-urbanized areas, and that of wealthy families living in the capital city (Ouagadougou). We compared the gut mycobiota of three Burkinabè cohorts with that of a cohort of Italian families, as representative of a Western urban lifestyle. Using high-throughput sequencing, we characterized gut mycobiota composition and diversity, assessing changes in accordance with the different dietary patterns and lifestyle. Our findings revealed modifications in gut mycobiota composition along the rural-to-urban gradient. A significant reduction in the alpha-diversity of the gut mycobiota was observed in the cohorts residing in the urban setting compared to those living in rural and semi-urbanized areas. Members of rural households exhibited greater fungal richness and diversity compared to those in urban families, including affluent families in the capital city. Furthermore, we identified 33 fungal amplicon sequence variants (ASVs) significantly associated with the different lifestyle and dietary patterns related to the studied areas. The household-level survey of rural and urban settings in Burkina Faso highlighted the impact of urbanization-related dietary shifts on gut mycobiota diversity and composition. The observed loss of fungal diversity and the significant reduction of fungal taxa associated with a rural lifestyle are potential indicators of the shift from a rural to an urban context. In agreement with the hygiene hypothesis, these findings lay the foundation for further studies aiming at investigating the effect that these microbial losses will have on human health, similarly to those already observed for gut bacterial communities.

Resistance, tolerance, and persistence to antibiotics have mainly been studied at the level of a single microbial isolate. However, in recent years it has become evident that microbial interactions play a role in determining the success of antibiotic treatments, in particular by influencing the occurrence of persistence and tolerance within a population. Additionally, the challenge of resuscitation (the capability of a population to revive after antibiotic exposure) and pathogen clearance are strongly linked to the small size of the surviving population and to the presence of fluctuations in cell counts. Indeed, while large population dynamics can be considered deterministic, small populations are influenced by stochastic processes, making their behaviour less predictable. Our study argues that microbe-microbe interactions within a community affect the mode, tempo, and success of persister resuscitation and that these are further influenced by noise. To this aim, we developed a theoretical model of a three-member microbial community and analysed the role of cell-to-cell interactions on pathogen clearance, using both deterministic and stochastic simulations. Our findings highlight the importance of ecological interactions and population size fluctuations (and hence the underlying cellular mechanisms) in determining the resilience of microbial populations following antibiotic treatment.

Cereal-legume intercropping and inoculation with Plant Growth-Promoting Rhizobacteria (PGPR) have been extensively documented to impact crop performance and the rhizosphere microbiome. However, information on their combined effects is limited, and the influence of PGPR-inoculation of legumes on the rhizosphere microbiome of neighboring cereals remains unknown. This study examines how legume intercropping with or without PGPR inoculation affects yield, grain quality, and rhizosphere bacterial diversity in durum wheat and barley. Field experiments were conducted using three cropping systems: cereal sole cropping (SC), intercropping with legumes (IC), and intercropping with PGPR-inoculated legumes (ICC), where legumes included fenugreek and clover inoculated with a PGPR consortium (Pseudomonas thivervalensis and Variovorax paradoxus). The IC system increased grain yield by 26

Ports are hot spots of pollution; they receive pollution from land-based sources, marine traffic and port infrastructures. Marine ecosystems of nearby areas can be strongly affected by pollution from port-related activities. Here, we investigated the microbiomes present in sea floor sediments along a transect from the harbour of Livorno (Central Italy) to a nearby marine protected area. Results of 16S rRNA amplicon sequencing and metagenome assembled genomes (MAGs) analyses indicated the presence of different trends of specific bacterial groups (e.g. phyla NB1-j, Acidobacteriota and Desulfobulbales) along the transect, correlating with the measured pollution levels. Human pathogenic bacteria and antibiotic resistance genes (ARGs) were also found. These results demonstrate a pervasive impact of human port activities and highlight the importance of microbiological surveillance of marine sediments, which may constitute a reservoir of ARGs and pathogenic bacteria.

Abstract Background How the evolution of dietary habits has impacted the yeasts associated with our gut is largely unknown. The impact of urbanization and globalization on human nutrition and the composition of gut microbial communities are considered driving forces behind the rise in non-communicable diseases. While previous studies in developing countries have investigated changes in the bacterial component of the gut microbiota during the transition from rural to urban areas, the modifications in the intestinal fungal communities are completely unexplored. In this study, we examined the impact of urbanization and dietary shifts on the composition of the gut mycobiota in families residing in rural, semi-urbanized, and urban areas in Burkina Faso. We compared these findings with families living in the urban area of Florence (Italy) as a reference for a globalized lifestyle. Results Our research revealed a significant reduction in the alpha diversity of the intestinal mycobiota as individuals transitioned from rural to urban areas. Members of rural households exhibited greater fungal richness and biodiversity compared to those in urban households, including affluent families in the capital city, Ouagadougou. We observed that the fungal diversity varied in households as a function of the rural-to-urban transition gradient, and we identified 33 fungal amplicon sequence variants (ASVs), including 12 fungal species, as associated with distinct areas with specific lifestyle and dietary patterns as indicators of the rural-to-urban transition. Conclusion The household-level survey of rural and urban communities in Burkina Faso highlighted the effect of urbanization on the lifestyle and subsequent composition of the participants' intestinal mycobiota. A greater diversity of fungal taxa emerged in the rural cohort, along with the presence of distinct species with potential pathogenic traits. This finding suggests that the continuous exposure to pathogenic fungi and the ensuing interaction with the immune system may contribute to the maintenance of lower incidence and severity of non-communicable diseases (NCDs) in non-globalized communities. In agreement with the “hygiene hypothesis”, the lack of yeast diversity could provide a potential explanation for the higher prevalence of inflammatory and immune-related disorders in urbanized regions across the world.

Extracellular vesicles (EVs) are lipid-bilayered particles, containing various biomolecules, including nucleic acids, lipids, and proteins, released by cells from all the domains of life and performing multiple communication functions. Evidence suggests that the interaction between host immune cells and fungal EVs induces modulation of the immune system. Most of the studies on fungal EVs have been conducted in the context of fungal infections; therefore, there is a knowledge gap in what concerns the production of EVs by yeasts in other contexts rather than infection and that may affect human health. In this work, we characterized EVs obtained by Saccharomyces cerevisiae and Pichia fermentans strains isolated from a fermented milk product with probiotic properties. The immunomodulation abilities of EVs produced by these strains have been studied in vitro through immune assays after internalization from human monocyte-derived dendritic cells. Results showed a significant reduction in antigen presentation activity of dendritic cells treated with the fermented milk EVs. The small RNA fraction of EVs contained mainly yeast mRNA sequences, with a few molecular functions enriched in strains of two different species isolated from the fermented milk. Our results suggest that one of the mechanisms behind the anti-inflammatory properties of probiotic foods could be mediated by the interactions of human immune cells with yeast EVs.

Propolis is a resinous material produced by honeybees from different plant sources and used in the hive as a building material and to protect the colony from parasites and pathogens. Despite its antimicrobial properties, recent studies showed that propolis hosts diverse microbial strains, some with great antimicrobial potential. In this study, the first description of the bacterial community of propolis produced by the gentle Africanized honeybee was reported. Propolis was sampled from hives of two different geographic areas of Puerto Rico (PR, USA), and the associated microbiota investigated by both cultivation and metataxonomic approaches. Metabarcoding analysis showed appreciable bacterial diversity in both areas and statistically significant dissimilarity in the taxa composition of the two areas, probably due to the different climatic conditions. Both metabarcoding and cultivation data revealed the presence of taxa already detected in other hive components and compatible with the bee's foraging environment. Isolated bacteria and propolis extracts showed antimicrobial activity against Gram-positive and Gram-negative bacterial tester strains. These results support the hypothesis that the propolis microbiota could contribute to propolis' antimicrobial properties.

Even if their impact is often underestimated, yeasts and yeast-like fungi represent the most prevalent eukaryotic members of microbial communities on Earth. They play numerous roles in natural ecosystems and in association with their hosts. They are involved in the food industry and pharmaceutical production, but they can also cause diseases in other organisms, making the understanding of their biology mandatory. The ongoing loss of biodiversity due to overexploitation of environmental resources is a growing concern in many countries. Therefore, it becomes crucial to understand the ecology and evolutionary history of these organisms to systematically classify them. To achieve this, it is essential that our knowledge of the mycobiota reaches a level similar to that of the bacterial communities. To overcome the existing challenges in the study of fungal communities, the first step should be the establishment of standardized techniques for the correct identification of species, even from complex matrices, both in wet lab practices and in bioinformatic tools.

Transitions to physically different environments, such as the water-to-land transition, proved to be the main drivers of relevant evolutionary events. Brachyuran crabs evolved remarkable morphological, behavioral, and physiological adaptations to terrestrial life. Terrestrial species evolved new respiratory structures devoted to replace or support the gills, a multifunctional organ devoted to gas exchanges, ion-regulation and nitrogen excretion. It was hypothesized that microorganisms associated with respiratory apparatus could have facilitated the processes of osmoregulation, respiration, and elimination of metabolites along this evolutionary transition. To test if crab species with different breathing adaptations may host similar microbial communities on their gills, we performed a comparative targeted-metagenomic analysis, selecting two marine and six terrestrial crabs belonging to different families and characterised by different breathing adaptations. We analysed anterior and posterior gills separately according to their different and specific roles. Regardless of their terrestrial or marine adaptations, microbial assemblages were strongly species-specific indicating a non-random association between the host and its microbiome. Significant differences were found in only two terrestrial species when considering posterior vs. anterior gills, without any association with species-specific respiratory adaptations. Our results suggest that all the selected species are strongly adapted to the ecological niche and specific micro-habitat they colonise.

Microorganisms are ubiquitous in the environment and provide genetic and physiological functions to multicellular organisms. Knowledge on the associated microbiota is becoming highly relevant to understand the host's ecology and biology. Among invertebrates, many examples of endosymbiosis have been described, such as those in corals, ants, and termites. At present, however, little is known on the presence, diversity, and putative roles of the microbiota associated to brachyuran crabs in relation to their environment. In this work we investigated the associated microbiota of three populations of the terrestrial brachyuran crab Chiromantes haematocheir to find evidence of a conserved organ-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. Despite the presence of truly marine larval stages and the absence of a gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated with the gut and the gills of crabs from the different populations (with more than 15% of the genera detected specifically enriched only in one organ). These findings suggest the presence of possible functional roles of the organ-specific microbiota.

One of the goals of paleomicrobiology is the characterization of the microbial community present in archaeological sites. These analyses can usually provide valuable information about past events, such as occurrence of human and animal infectious diseases, ancient human activities, and environmental changes.

Abstract Extracellular vesicles (EVs) are lipid-bilayered particles, containing various biomolecules, including nucleic acids, lipids, and proteins, released by cells from all the domains of life and performing multiple communication functions. Evidence suggests that the interaction between host immune cells and fungal EVs induces modulation of the immune system. Most of the studies on fungal EVs have been conducted in the context of fungal infections; therefore, there is still a knowledge gap regarding EVs produced by non-pathogenic yeasts. In this work, we characterized EVs obtained by Saccharomyces cerevisiae and Pichia fermentans strains isolated from a fermented milk product with probiotic properties. The immunomodulation abilities of EVs produced by these strains have been studied in vitro through immune assays after internalization from human monocyte-derived dendritic cells. Results showed a significant reduction in antigen presentation activity of dendritic cells treated with the fermented milk EVs. The small RNA fraction of EVs contained mainly yeast mRNA sequences, with a few molecular functions enriched in strains of two different species isolated from the fermented milk. Our results suggest that one of the mechanisms behind the anti-inflammatory properties of probiotic foods could be mediated by the interactions of human immune cells with yeast EVs.

Advances in Next Generation Sequencing technologies allow us to inspect and unlock the genome to a level of detail that was unimaginable only a few decades ago. Omics-based studies are casting a light on the patterns and determinants of disease conditions in populations, as well as on the influence of microbial communities on human health, just to name a few. Through increasing volumes of sequencing information, for example, it is possible to compare genomic features and analyze the modulation of the transcriptome under different environmental stimuli. Although protocols for NGS preparation are intended to leave little to no space for contamination of any kind, a noticeable fraction of sequencing reads still may not uniquely represent what was intended to be sequenced in the first place. If a natural consequence of a sequencing sample is to assess the presence of features of interest by mapping the obtained reads to a genome of reference, sometimes it is useful to determine the fraction of those that do not map, or that map discordantly, and store this information to a new file for subsequent analyses. Here we propose a new mapper, which we called Squid, that among other accessory functionalities finds and returns sequencing reads that match or do not match to a reference sequence database in any orientation. We encourage the use of Squid prior to any quantification pipeline to assess, for instance, the presence of contaminants, especially in RNA-Seq experiments.

Aims Seed endophytic bacteria (SEB) are able to improve plant growth and to protect them against abiotic or biotic stresses. This work aimed to characterize the seed endophytic bacterial communities associated with different species of the nickel hyperaccumulator Odontarrhena , which is adapted to extreme environments such as serpentine soils. Moreover, this work also aimed to study any potential congruency between SEB community diversity and plant phylogeny. Methods Endophytic bacterial communities were characterized for seeds from 9 Odontarrhena populations, using high throughput sequencing. The plant genomes and environmental properties of the sites had previously been described. Results and discussion All Odontarrhena populations shared more than 95% of their OTUs and metabarcoding revealed a large SEB core microbiome. The plant species was more determinant than the site in explaining the dissimilarities between SEB communities. Nonetheless, both site and Odontarrhena species factors were significant diversity drivers of the SEB communities and the best explanatory factor was the interaction between them. When focusing only on plant populations, some OTUs were over- or under-represented in the O . chalcidica SEB communities in comparison with the SEB communities of the 4 other Odontarrhena species. With the current genetic markers, the cophylogenetic analysis revealed a non-significant coherence of phylogenies between seed microbiota and corresponding host plants. The OTUs based prediction of metabolic functions, is a first step that would potentially allow the power of the microbiome to be harnessed, thereby improving hyperaccumulator production in an agromining context.

Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land.

Next generation sequencing (NGS) is routinely used to study crucial aspects of biological systems, including differentially expressed genes identification, microbiome taxonomic composition and structure, enrichment of specific cellular functions in a given environment, and so on. Current research laboratories are facing a serious lack in the availability of properly trained researchers capable of carrying out basic NGS analysis computational pipelines. This reflects a gap in most academic curricula concerning the basics of NGS data management, analysis, and interpretation. Indeed, most of the times, the knowledge necessary to undertake these tasks is acquired through the use of one-shot tutorial, without a thorough explanation of the concepts behind the practical steps. With this protocol we aim to fill this gap by providing teachers with a hands-on protocol to guide bachelor and master students in a more focused analysis of NGS data, from basic and standard operations on sequencing reads (e.g., quality check and trimming) to more advanced analysis techniques (e.g., data normalization).

BackgroundThe human microbiota plays several roles in health and disease but is often difficult to determine which part is in intimate relationships with the host vs. the occasional presence. During the Mars500 mission, six crewmembers lived completely isolated from the outer world for 520 days following standardized diet regimes. The mission constitutes the first spaceflight simulation to Mars and was a unique experiment to determine, in a longitudinal study design, the composition and importance of the resident vs. a more variable microbiota—the fraction of the human microbiota that changes in time and according to environmental conditions—in humans. MethodsHere we report the characterization of the salivary microbiota from 88 samples taken during and after Mars500 mission for a total of 720 days. Amplicon sequencing of the V3-V4 region of 16S rRNA gene was performed and results were analyzed monitoring the diversity of the microbiota while evaluating the effect of the three main variables present in the experimental system: time, diet, and individuality of each subject. ResultsResults, showed statistically significant effects for either time, diet, and individuality of each subject. The main contribution came from the individuality of each subject, emphasizing salivary microbiota personalized features and an individual-based resilience of the microbiota. ConclusionsThe uniqueness of Mars500 mission, allowed to clearly dissect the environmental variables of salivary microbiota, highlighting its pronounced personalization even after sharing the same physical space for more than a year.