Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease.

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease.

Long-term response of different Botulinum toxins in refractory neurogenic detrusor overactivity due to spinal cord injury.

PURPOSE: To assess the response in spinal cord injured patients alternatively treated with different types and dosages of Botulinum neurotoxin type A (BoNT/A) over 15 years. MATERIAL AND METHODS: Patients who underwent first BoNT/A from 1999-2001 and practiced intermittent catheterization were included. Baseline 3-day bladder diary (BD) and urodynamics were collected. BoNT/A failure was defined when patients asked for re-injection ≤ 3 months post-treatment. Criteria for re-injection was at least one daily episode of urinary incontinence at BD. Before re-injection, patients were asked if they had reached 6 months of dryness without antimuscarinics (YES response). RESULTS: Overall, 32/60 (53.4%) "No failure" (NF) group; 16 (26.6%) "occasional failure" (OF) and 12 (20%) "consecutive failure" (CF) were included. A total of 822 BoNT/A infiltrations were performed. The mean interval from previous injection to treatment re-scheduling was 8 months. No significant differences between treatments were found within the three groups (p>0.05). The percentage of YES responses increased from 19% (AboBoNT/A 500IU) to 29 % (OnaBoNT/A 300IU) in NF, and from 18% (AboBoNT/A 500IU) to 25% (OnaBoNT/A 300IU) for OF. Five NF cases (15.6%) maintained 6 months of dryness after each injection. Among the baseline variables, only low compliance (< 20mL/cmH2O) was found as predictor for failure (p=0.006). CONCLUSIONS: Long term BoNT/A for NDO did not increase failures, independent of the types of treatments and switching. Definition of failure and other criteria for continuing repetitive BoNT/A treatment is mandatory. CF was predictable for no response in earlier follow-up.

Evolution of Intra-specific Regulatory Networks in a Multipartite Bacterial Genome.

Reconstruction of the regulatory network is an important step in understanding how organisms control the expression of gene products and therefore phenotypes. Recent studies have pointed out the importance of regulatory network plasticity in bacterial adaptation and evolution. The evolution of such networks within and outside the species boundary is however still obscure. Sinorhizobium meliloti is an ideal species for such study, having three large replicons, many genomes available and a significant knowledge of its transcription factors (TF). Each replicon has a specific functional and evolutionary mark; which might also emerge from the analysis of their regulatory signatures. Here we have studied the plasticity of the regulatory network within and outside the S. meliloti species, looking for the presence of 41 TFs binding motifs in 51 strains and 5 related rhizobial species. We have detected a preference of several TFs for one of the three replicons, and the function of regulated genes was found to be in accordance with the overall replicon functional signature: house-keeping functions for the chromosome, metabolism for the chromid, symbiosis for the megaplasmid. This therefore suggests a replicon-specific wiring of the regulatory network in the S. meliloti species. At the same time a significant part of the predicted regulatory network is shared between the chromosome and the chromid, thus adding an additional layer by which the chromid integrates itself in the core genome. Furthermore, the regulatory network distance was found to be correlated with both promoter regions and accessory genome evolution inside the species, indicating that both pangenome compartments are involved in the regulatory network evolution. We also observed that genes which are not included in the species regulatory network are more likely to belong to the accessory genome, indicating that regulatory interactions should also be considered to predict gene conservation in bacterial pangenomes.

Exploring the dynamics of bacterial community composition in soil: the pan-bacteriome approach.

We performed a longitudinal study (repeated observations of the same sample over time) to investigate both the composition and structure of temporal changes of bacterial community composition in soil mesocosms, subjected to three different treatments (water and 5 or 25 mg kg(-1) of dried soil Cd(2+)). By analogy with the pan genome concept, we identified a core bacteriome and an accessory bacteriome. Resident taxa were assigned to the core bacteriome, while occasional taxa were assigned to the accessory bacteriome. Core and accessory bacteriome represented roughly 35 and 50 % of the taxa detected, respectively, and were characterized by different taxonomic signatures from phylum to genus level while 15 % of the taxa were found to be unique to a particular sample. In particular, the core bacteriome was characterized by higher abundance of members of Planctomycetes, Actinobacteria, Verrucomicrobia and Acidobacteria, while the accessory bacteriome included more members of Firmicutes, Clamydiae and Proteobacteria, suggesting potentially different responses to environmental changes of members from these phyla. We conclude that the pan-bacteriome model may be a useful approach to gain insight for modeling bacterial community structure and inferring different abilities of bacteria taxa.

Efficacy of the ND:YAG laser therapy on EBV and HSV1 contamination in periodontal pockets.

AIM: The aim of this retrospective multicenter study was to verify the efficacy of Nd:YAG laser in the treatment of periodontal pockets infected by Epstein-Barr Virus (EBV) and Herpes Simplex Virus 1 (HSV1). METHODS: Subgingival plaque samples of 291 Italian periodontal patients were analyzed by Real Time PCR to evaluate the frequency of both viruses before and after Nd:YAG laser-assisted periodontal treatment. RESULTS: Before treatment, EBV and HSV1 were observed in 29.9% and in 3.8% of periodontal patients respectively, while co-infection with both viruses was detected in 1.7% of cases. Periodontal Nd:YAG laser treatment ("Periodontal Biological Laser-Assisted Therapy", PERIOBLAST) produced statistical significant benefits, especially in EBV periodontal infection: 78.2% of EBV positive patients became EBV-negative following treatment. CONCLUSIONS: Results of this preliminary study highlight that EBV is found in periodontal pockets more frequently than HSV1, supporting the theory of the potential role of EBV in the onset and progression of periodontal disease. Moreover, our data showed that Nd:YAG laser-assisted periodontal treatment (Perioblast) is also effective in case of viral infection, validating evidences that it represents a successful alternative approach to traditional periodontal protocols.

Yeast metagenomics: analytical challenges in the analysis of the eukaryotic microbiome.

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.

Yeast strains isolated from fermented beverage produce extracellular vesicles with anti-inflammatory effects.

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.

Characterization of the Bacterial Communities Inhabiting Tropical Propolis of Puerto Rico.

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.

Conserved organ-specific microbial assemblages in different populations of a terrestrial crab.

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.

Species-specific gill's microbiome of eight crab species with different breathing adaptations.

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.

Novel Sources of Biodiversity and Biomolecules from Bacteria Isolated from a High Middle Ages Soil Sample in Palermo (Sicily, Italy).

The urban plan of Palermo (Sicily, Italy) has evolved throughout Punic, Roman, Byzantine, Arab, and Norman ages until it stabilized within the borders that correspond to the current historic center. During the 2012 to 2013 excavation campaign, new remains of the Arab settlement, directly implanted above the structures of the Roman age, were found. The materials investigated in this study derived from the so-called Survey No 3, which consists of a rock cavity of subcylindrical shape covered with calcarenite blocks: it was probably used to dispose of garbage during the Arabic age and its content, derived from daily activities, included grape seeds, scales and bones of fish, small animal bones, and charcoals. Radiocarbon dating confirmed the medieval origin of this site. The composition of the bacterial community was characterized through a culture-dependent and a culture-independent approach. Culturable bacteria were isolated under aerobic and anaerobic conditions and the total bacterial community was characterized through metagenomic sequencing. Bacterial isolates were tested for the production of compounds with antibiotic activity: a Streptomyces strain, whose genome was sequenced, was of particular interest because of its inhibitory activity, which was due to the Type I polyketide aureothin. Moreover, all strains were tested for the production of secreted proteases, with those belonging to the genus Nocardioides having the most active enzymes. Finally, protocols commonly used for ancient DNA studies were applied to evaluate the antiquity of isolated bacterial strains. Altogether these results show how paleomicrobiology might represent an innovative and unexplored source of novel biodiversity and new biotechnological tools. IMPORTANCE 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. However, in this work, investigations about the composition of the bacterial community of an ancient soil sample (harvested in Palermo, Italy) were carried out aiming to screen ancient culturable strains with biotechnological potential, such as the ability to produce bioactive molecules and secreted hydrolytic enzymes. Besides showing the biotechnological relevance of paleomicrobiology, this work reports a case of germination of putatively ancient bacterial spores recovered from soil rather than extreme environments. Moreover, in the case of spore-forming species, these results raise questions about the accuracy of techniques usually applied to estimate antiquity of DNA, as they could lead to its underestimation.

Fast, Ungapped Reads Mapping Using Squid.

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.

Host Trait Prediction from High-Resolution Microbial Features.

Predicting host traits from metagenomes presents new challenges that can be difficult to overcome for researchers without a strong background in bioinformatics and/or statistics. Profiling bacterial communities using shotgun metagenomics often leads to the generation of a large amount of data that cannot be used directly for training a model. In this chapter we provide a detailed description of how to build a working machine learning model based on taxonomic and functional features of bacterial communities inhabiting the lungs of cystic fibrosis patients. Models are built in the R environment by using different freely available machine learning algorithms.

A Protocol for Teaching Basic Next Generation Sequencing (NGS) Analysis Skills to Undergraduate Students Using Bash and R.

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).

Nonadditive Transcriptomic Signatures of Genotype-by-Genotype Interactions during the Initiation of Plant-Rhizobium Symbiosis.

Rhizobia are ecologically important, facultative plant-symbiotic microbes. In nature, there is a large variability in the association of rhizobial strains and host plants of the same species. Here, we evaluated whether plant and rhizobial genotypes influence the initial transcriptional response of rhizobium following perception of a host plant. RNA sequencing of the model rhizobium Sinorhizobium meliloti exposed to root exudates or luteolin (an inducer of nod genes, involved in the early steps of symbiotic interaction) was performed on a combination of three S. meliloti strains and three alfalfa varieties as host plants. The response to root exudates involved hundreds of changes in the rhizobium transcriptome. Of the differentially expressed genes, 35% were influenced by the strain genotype, 16% were influenced by the plant genotype, and 29% were influenced by strain-by-host plant genotype interactions. We also examined the response of a hybrid S. meliloti strain in which the symbiotic megaplasmid (∼20% of the genome) was mobilized between two of the above-mentioned strains. Dozens of genes were upregulated in the hybrid strain, indicative of nonadditive variation in the transcriptome. In conclusion, this study demonstrated that transcriptional responses of rhizobia upon perception of legumes are influenced by the genotypes of both symbiotic partners and their interaction, suggesting a wide spectrum of genetic determinants involved in the phenotypic variation of plant-rhizobium symbiosis.IMPORTANCE A sustainable way for meeting the need of an increased global food demand should be based on a holobiont perspective, viewing crop plants as intimately associated with their microbiome, which helps improve plant nutrition, tolerance to pests, and adverse climate conditions. However, the genetic repertoire needed for efficient association with plants by the microbial symbionts is still poorly understood. The rhizobia are an exemplary model of facultative plant symbiotic microbes. Here, we evaluated whether genotype-by-genotype interactions could be identified in the initial transcriptional response of rhizobium perception of a host plant. We performed an RNA sequencing study to analyze the transcriptomes of different rhizobial strains elicited by root exudates of three alfalfa varieties as a proxy of an early step of the symbiotic interaction. The results indicated strain- and plant variety-dependent variability in the observed transcriptional changes, providing fundamentally novel insights into the genetic basis of rhizobium-plant interactions. Our results provide genetic insights and perspective to aid in the exploitation of natural rhizobium variation for improvement of legume growth in agricultural ecosystems.

Melanoma diagnosis: traumatic impact of the event on the patient.

BACKGROUND: The aim of our study was to consider the distressing impact of the diagnosis in a group of patients with metastatic melanoma, and the effects it could have on the quality of life of the patients. METHODS: We proposed an Impact Event Scale (IES-R) to a group of 31 patients. The patients were positive to the distress thermometer (DS) and accepted the psychological support. After six months from the start of the treatment we made a semi-structured interview of 10 multiple choice questions. RESULTS: Sixty-five per cent of women and 50% of men report that all the event related to the disease, cause emotions that recall the disease. Eighty-two per cent of women compared to 50% of men, report that the thought of their medical condition tends to affect their quality of sleep; the patients report feelings of anger and irritation (41% of the women and 78% of the men). CONCLUSIONS: The traumatic aspects following the diagnosis of melanoma burst powerfully into the life of these patients, who show different reactions, also according to gender.

Defining the resilience of the human salivary microbiota by a 520-day longitudinal study in a confined environment: the Mars500 mission.

BACKGROUND: The 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. METHODS: Here, 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 regions 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. RESULTS: Results 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. CONCLUSIONS: The uniqueness of Mars500 mission, allowed to dampen the effect of environmental variables on salivary microbiota, highlighting its pronounced personalization even after sharing the same physical space for more than a year. Video abstract.

Competitiveness for Nodule Colonization in Sinorhizobium meliloti: Combined In Vitro-Tagged Strain Competition and Genome-Wide Association Analysis.

Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species specific, different rhizobial strains may colonize the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial genome-wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. For this, 13 tester strains were green fluorescent protein (GFP) tagged and assayed versus 3 red fluorescent protein (RFP)-tagged reference competitor strains (Rm1021, AK83, and BL225C) in a Medicago sativa nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on k-mers. Among the k-mers with the highest scores, 51 k-mers mapped on the genomes of four strains showing the highest competition phenotypes (>60% single strain nodule occupancy; GR4, KH35c, KH46, and SM11) versus BL225C. These k-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors, and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays versus BL225C, retrieving k-mers mapped on the genes previously found and on vir genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components. IMPORTANCE Decoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, the single-gene approach has mainly been used to uncover the bases of nodulation, but there is still a knowledge gap regarding the main features that a priori characterize rhizobial strains able to outcompete indigenous rhizobia. Therefore, tracking down which traits make different rhizobial strains able to win the competition for plant infection over other indigenous rhizobia will improve the strain selection process and, consequently, plant yield in sustainable agricultural production systems. We proved that a k-mer-based GWAS approach can efficiently identify the competition determinants of a panel of strains previously analyzed for their plant tissue occupancy using double fluorescent labeling. The reported strategy will be useful for detailed studies on the genomic aspects of the evolution of bacterial symbiosis and for an extensive evaluation of rhizobial inoculants.

Exploring the Gut Microbiome Alteration of the European Hare (Lepus europaeus) after Short-Term Diet Modifications.

This study aimed to characterise the gut microbiome composition of European hares (Lepus europaeus) and its potential changes after a short-term diet modification. The high sensitivity of European hare to habitat changes makes this species a good model to analyse possible alterations in gut microbiome after the introduction of additional nourishment into the diet. In total, 20 pairs were chosen for the experiments; 10 pairs formed the control group and were fed with standard fodder. The other 10 pairs represented the experimental group, whose diet was integrated with apples and carrots. The DNA from fresh faecal pellets collected after 4 days from the start of the experiment was extracted and the V3-V4 hypervariable regions were amplified and sequenced using the Illumina MiSeq® platform. The obtained amplicon sequence variants were classified into 735 bacterial genera belonging to 285 families and 36 phyla. The control and the experimental groups appeared to have a homogenous dispersion for the two taxonomic levels analysed with the most abundant phyla represented by Bacteroidetes and Firmicutes. No difference between control and experimental samples was detected, suggesting that the short-term variation in food availability did not alter the hares' gut microbiome. Further research is needed to estimate significant time threshold.