Transcriptional Changes in Potato Sprouts upon Interaction with Rhizoctonia solani Indicate Pathogen-Induced Interference in the Defence Pathways of Potato.

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants' molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants' comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (-Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term "response to abiotic stimulus" was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

Nutritional Targeting of the Microbiome as Potential Therapy for Malnutrition and Chronic Inflammation.

Homeostatic interactions with the microbiome are central for a healthy human physiology and nutrition is the main driving force shaping the microbiome. In the past decade, a wealth of preclinical studies mainly using gnotobiotic animal models demonstrated that malnutrition and chronic inflammation stress these homeostatic interactions and various microbial species and their metabolites or metabolic activities have been associated with disease. For example, the dysregulation of the bacterial metabolism of dietary tryptophan promotes an inflammatory environment and susceptibility to pathogenic infection. Clinical studies have now begun to evaluate the therapeutic potential of nutritional and probiotic interventions in malnutrition and chronic inflammation to ameliorate disease symptoms or even prevent pathogenesis. Here, we therefore summarize the recent progress in this field and propose to move further towards the nutritional targeting of the microbiome for malnutrition and chronic inflammation.

Salmonella Establishment in Agricultural Soil and Colonization of Crop Plants Depend on Soil Type and Plant Species.

Human pathogenic bacteria, such as Salmonella enterica, are able to colonize crop plants. So far, not much is known about biotic and abiotic factors influencing this colonization in field soil. This understanding, however, is imperative for the provision of safe fresh produce to the consumer. In this study, we investigated the effects of soil type, organic fertilization, plant species and the way of Salmonella entry into the plant production system, on the survival of S. enterica in soil as well as the colonization of plants. The selected S. enterica serovar Typhimurium strain 14028s, S. Typhimurium strain LT2 and S. Senftenberg were able to persist in soil for several weeks. Salmonella's persistence in soil was prolonged in loamy, if compared to sandy soil, and when applied together with organic fertilizer. The leaves of lettuce and corn salad were colonized by S. enterica providing evidence for internalization from the soil via the root. Colonization rates were affected by soil type, plant species and S. enterica strain. Overall, S. enterica was detected in leaves of 0.5-0.9% of the plants, while lettuce was more frequently colonized than corn salad. Plants grown in sandy soil were more often colonized than plants grown in loamy soil. After spray inoculation, S. enterica could be detected on and in leaves for several weeks by cultivation-depending methods, confirmed by confocal microscopy using GFP-labeled S. Typhimurium 14028s. On the one hand, transcriptome data from S. Typhimurium 14028s assessed in response to lettuce medium or lettuce root exudates showed an upregulation of genes associated with biofilm formation and virulence. On the other hand, lettuce inoculated with S. Typhimurium 14028s showed a strong upregulation of genes associated with plant immune response and genes related to stress response. In summary, these results showed that organic fertilizers can increase the persistence of Salmonella in soil and that soil type and plant species play a crucial role in the interactions between human pathogens and crop plants. This understanding is therefore a starting point for new strategies to provide safe food for the consumer.

Changes in microbiota composition, bile and fatty acid metabolism, in successful faecal microbiota transplantation for Clostridioides difficile infection.

BACKGROUND: Alteration of the gut microbiota by repeated antibiotic treatment increases susceptibility to Clostridioides difficile infection. Faecal microbiota transplantation from donors with a normal microbiota effectively treats C. difficile infection. METHODS: The study involved 10 patients with recurrent C. difficile infection, nine of whom received transplants from individual donors and one who received a donor unit from a stool bank (OpenBiome). RESULTS: All individuals demonstrated enduring post-transplant resolution of C. difficile- associated diarrhoea. Faecal microbiota diversity of recipients significantly increased, and the composition of the microbiota resembled that of the donor. Patients with C. difficile infection exhibited significantly lower faecal levels of secondary/ bile acids and higher levels of primary bile acids. Levels of secondary bile acids were restored in all transplant recipients, but to a lower degree with the OpenBiome transplant. The abundance increased of bacterial genera known from previous studies to confer resistance to growth and germination of C. difficile. These were significantly negatively associated with primary bile acid levels and positively related with secondary bile acid levels. Although reduced levels of the short chain fatty acids, butyrate, propionate and acetate, have been previously reported, here we report elevations in SCFA, pyruvic and lactic fatty acids, saturated, ω-6, monounsaturated, ω-3 and ω-6 polyunsaturated fatty acids (PUFA) in C. difficile infection. This potentially indicates one or a combination of increased dietary FA intake, microbial modification of FAs or epithelial cell damage and inflammatory cell recruitment. No reversion to donor FA profile occurred post-FMT but ω-3 to ω-6 PUFA ratios were altered in the direction of the donor. Archaeal metabolism genes were found in some samples post FMT. CONCLUSION: A consistent metabolic signature was identified in the post-transplant microbiota, with reduced primary bile acids and substantial restoration of secondary bile acid production capacity. Total FA levels were unchanged but the ratio of inflammatory to non-inflammatory FAs decreased.

Tumour-associated and non-tumour-associated microbiota: Addendum.

In a recent study1 we reported that tissue-associated microbial Co-abundance Groups (CAGs) were differentially associated with colorectal cancer (CRC). Two of the CAGs, which we named Pathogen CAG and Prevotella CAG, were correlated with a gene expression signature indicative of a TH17 response. A TH17 response has been associated with decreased survival in patients with CRC2, and members of the Pathogen CAG such as Fusobacterium nucleatum, Escherichia coli and Bacteroides fragilis have been repeatedly reported to be associated with CRC-development. Thus we hypothesized that the abundance of these CAGs may be associated with poor survival. In this Addendum we extend our analysis of the at-surgery microbiota to microbiota profiles obtained after surgery for CRC which we analyzed in the context of survival data for patients with CRC. Surprisingly we found that high tissue-associated abundance of the previously defined Prevotella- and Pathogen-CAGs at surgery was associated with longer survival. Furthermore, we detected an association of the Bacteroidetes CAG in pre-surgery faecal microbiota with stability of the microbiota after surgery.

The oral microbiota in colorectal cancer is distinctive and predictive.

BACKGROUND AND AIMS: Microbiota alterations are linked with colorectal cancer (CRC) and notably higher abundance of putative oral bacteria on colonic tumours. However, it is not known if colonic mucosa-associated taxa are indeed orally derived, if such cases are a distinct subset of patients or if the oral microbiome is generally suitable for screening for CRC. METHODS: We profiled the microbiota in oral swabs, colonic mucosae and stool from individuals with CRC (99 subjects), colorectal polyps (32) or controls (103). RESULTS: Several oral taxa were differentially abundant in CRC compared with controls, for example, Streptococcus and Prevotellas pp. A classification model of oral swab microbiota distinguished individuals with CRC or polyps from controls (sensitivity: 53% (CRC)/67% (polyps); specificity: 96%). Combining the data from faecal microbiota and oral swab microbiota increased the sensitivity of this model to 76% (CRC)/88% (polyps). We detected similar bacterial networks in colonic microbiota and oral microbiota datasets comprising putative oral biofilm forming bacteria. While these taxa were more abundant in CRC, core networks between pathogenic, CRC-associated oral bacteria such as Peptostreptococcus, Parvimonas and Fusobacterium were also detected in healthy controls. High abundance of Lachnospiraceae was negatively associated with the colonisation of colonic tissue with oral-like bacterial networks suggesting a protective role for certain microbiota types against CRC, possibly by conferring colonisation resistance to CRC-associated oral taxa and possibly mediated through habitual diet. CONCLUSION: The heterogeneity of CRC may relate to microbiota types that either predispose or provide resistance to the disease, and profiling the oral microbiome may offer an alternative screen for detecting CRC.

Fecal microbiota variation across the lifespan of the healthy laboratory rat.

Laboratory rats are commonly used in life science research as a model for human biology and disease, but the composition and development of their gut microbiota during life is poorly understood. We determined the fecal microbiota composition of healthy Sprague Dawley laboratory rats from 3 weeks to 2 y of age, kept under controlled environmental and dietary conditions. Additionally, we determined fecal short-chain fatty acid profiles, and we compared the rat fecal microbiota with that of mice and humans. Gut microbiota and to a lesser extent SCFAs profiles separated rats into 3 different clusters according to age: before weaning, first year of life (12- to 26-week-old animals) and second year of life (52- to 104-week-old). A core of 46 bacterial species was present in all rats but its members' relative abundance progressively decreased with age. This was accompanied by an increase of microbiota α-diversity, likely due to the acquisition of environmental microorganisms during the lifespan. Contrastingly, the functional profile of the microbiota across animal species became more similar upon aging. Lastly, the microbiota of rats and mice were most similar to each other but at the same time the microbiota profile of rats was more similar to that of humans than was the microbiota profile of mice. These data offer an explanation as to why germ-free rats are more efficient recipients and retainers of human microbiota than mice. Furthermore, experimental design should take into account dynamic changes in the microbiota of model animals considering that their changing gut microbiota interacts with their physiology.

From Culture to High-Throughput Sequencing and Beyond: A Layperson's Guide to the "Omics" and Diagnostic Potential of the Microbiome.

Detailed knowledge of the community of organisms in the gut has become possible in recent years because of the development of culture-independent methods. Largely based on latest DNA sequencing platforms, it is now possible to establish the composition of the microbiota and the repertoire of biochemical functions it encodes. Variations in either or both of these parameters have been linked to intestinal and extraintestinal disease. This article summarizes how these methods are applied, with special reference to gastroenterology, and describes the achievements and future potential of microbiota analysis as a diagnostic tool.

Tumour-associated and non-tumour-associated microbiota in colorectal cancer.

OBJECTIVE: A signature that unifies the colorectal cancer (CRC) microbiota across multiple studies has not been identified. In addition to methodological variance, heterogeneity may be caused by both microbial and host response differences, which was addressed in this study. DESIGN: We prospectively studied the colonic microbiota and the expression of specific host response genes using faecal and mucosal samples ('ON' and 'OFF' the tumour, proximal and distal) from 59 patients undergoing surgery for CRC, 21 individuals with polyps and 56 healthy controls. Microbiota composition was determined by 16S rRNA amplicon sequencing; expression of host genes involved in CRC progression and immune response was quantified by real-time quantitative PCR. RESULTS: The microbiota of patients with CRC differed from that of controls, but alterations were not restricted to the cancerous tissue. Differences between distal and proximal cancers were detected and faecal microbiota only partially reflected mucosal microbiota in CRC. Patients with CRC can be stratified based on higher level structures of mucosal-associated bacterial co-abundance groups (CAGs) that resemble the previously formulated concept of enterotypes. Of these, Bacteroidetes Cluster 1 and Firmicutes Cluster 1 were in decreased abundance in CRC mucosa, whereas Bacteroidetes Cluster 2, Firmicutes Cluster 2, Pathogen Cluster and Prevotella Cluster showed increased abundance in CRC mucosa. CRC-associated CAGs were differentially correlated with the expression of host immunoinflammatory response genes. CONCLUSIONS: CRC-associated microbiota profiles differ from those in healthy subjects and are linked with distinct mucosal gene-expression profiles. Compositional alterations in the microbiota are not restricted to cancerous tissue and differ between distal and proximal cancers.

Studying the Microbiome: "Omics" Made Accessible.

The term microbiome refers to the collection of microbes or microbial genes in a specified location or clinical sample. Identifying micro-organisms has historically relied upon bacteriological culture, which is time consuming and difficult to effectively implement. The recent adaptation of culture-independent techniques for profiling microbial communities, allied with next-generation massively parallel DNA sequencing, allows clinician scientists to determine the entire microbial content of a specimen to a forensic level of detail within 48 hours. The technology is still young, and the main thrust of current efforts is to identify how changes in the microbiome covary with a variety of syndromes and diseases, and to determine if these changes are causative or consequential. Regardless of the outcome of these investigations, it is already apparent that the gut microbiome is a useful biomarker for intestinal and extraintestinal disease. In this review, the authors summarize the main concepts in microbiome analysis, and prospects for the microbiome's clinical deployment.

Evidence of a putative deep sea specific microbiome in marine sponges.

The microbiota of four individual deep water sponges, Lissodendoryx diversichela, Poecillastra compressa, Inflatella pellicula, and Stelletta normani, together with surrounding seawater were analysed by pyrosequencing of a region of the 16S rRNA gene common to Bacteria and Archaea. Due to sampling constraints at depths below 700 m duplicate samples were not collected. The microbial communities of L. diversichela, P. compressa and I. pellicula were typical of low microbial abundance (LMA) sponges while S. normani had a community more typical of high microbial abundance (HMA) sponges. Analysis of the deep sea sponge microbiota revealed that the three LMA-like sponges shared a set of abundant OTUs that were distinct from those associated with sponges from shallow waters. Comparison of the pyrosequencing data with that from shallow water sponges revealed that the microbial communities of all sponges analysed have similar archaeal populations but that the bacterial populations of the deep sea sponges were distinct. Further analysis of the common and abundant OTUs from the three LMA-like sponges placed them within the groups of ammonia oxidising Archaea (Thaumarchaeota) and sulphur oxidising γ-Proteobacteria (Chromatiales). Reads from these two groups made up over 70% of all 16S rRNA genes detected from the three LMA-like sponge samples, providing evidence of a putative common microbial assemblage associated with deep sea LMA sponges.

Archaea appear to dominate the microbiome of Inflatella pellicula deep sea sponges.

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ~60% and ~72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (~11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ~88% and ~89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (~0.2% and ~0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.

Marine metagenomics: new tools for the study and exploitation of marine microbial metabolism.

The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome.