Intestinal microbiome in chronic intestinal failure and associations with intestinal failure associated liver disease

Background and Aims: Intestinal dysbiosis has been described in children with chronic intestinal failure (CIF) and in adults with short bowel syndrome (SBS), mostly with jejunocolic anastomosis (SBS-2) and jejuno-ileal anastomosis (SBS-3), linked to generic data with the pathogenesis of Intestinal Failure Associated Liver Disease (IFALD). Little is known about gut microbiome of adults with end-jejunostomy (SBS-1) and in CIF other than SBS and any specific associations with the onset of IFALD. We aimed to describe the fecal microbiome of adult patients with different mechanisms of CIF and any possible associations with the development of IFALD. Material and methods: Fecal samples from 61 patients with benign CIF. Phylogenetic characterization of the microbiome by amplification of the hypervariable regions V3 and V4 of the bacterial gene encoding 16S rRNA, and subsequent grouping of sequences in amplicon sequence variants (ASVs). Patient samples comparison to microbiome sequences from 61 healthy subjects, matched for sex and age, selected from the healthy subjects library of the Laboratory of the Microbial Ecology of Health Unit, Department of Pharmacy and Biotechnology, of the University of Bologna. IFALD was assessed by the diagnostic criteria of IFALD-cholestasis, IFALD-steatosis, IFALD-fibrosis. Results: Decreased bacterial α-diversity in CIF patients (increase of Proteobacteria and Actinobacteria and decrease in Bacteroidetes). Identification of microbial family-level signatures specific for CIF mechanisms (increase in Actinomycetaceae and Streptococcaceae in SBS-1, Bifidobacteriaceae and Lactobacillaceae in SBS-2, Bacteroidaceae and Porphyromonadaceae in dysmotility). Abundance of Lactobacillus and Lactobacillaceae strongly associated with IFALD-cholestasis and IFALD–fibrosis for SBS-1; Peptostreptococcus, Prevotellaceae (Prevotella) and Pasteurellaceae (Haemophilus) significantly increased in IFALD-fibrosis for other CIF mechanisms. Conclusions: CIF patients had a marked intestinal dysbiosis with microbial family-level signatures specific to the pathophysiological mechanism. Specific characteristics of microbiome may contribute to the pathogenesis of IFALD. Intestinal microbiome could become a therapeutic target in patients with CIF.

Molecular characterization of the human gut microbiota: the effect of aging

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

Genetic characterization, population history and evolutionary medicine perspective in two native south american populations: Yanesha and Wichi

Two Amerindian populations from the Peruvian Amazon (Yanesha) and from rural lowlands of the Argentinean Gran Chaco (Wichi) were analyzed. They represent two case study of the South American genetic variability. The Yanesha represent a model of population isolated for long-time in the Amazon rainforest, characterized by environmental and altitudinal stratifications. The Wichi represent a model of population living in an area recently colonized by European populations (the Criollos are the population of the admixed descendents), whose aim is to depict the native ancestral gene pool and the degree of admixture, in relation to the very high prevalence of Chagas disease. The methods used for the genotyping are common, concerning the Y chromosome markers (male lineage) and the mitochondrial markers (maternal lineage). The determination of the phylogeographic diagnostic polymorphisms was carried out by the classical techniques of PCR, restriction enzymes, sequencing and specific mini-sequencing. New method for the detection of the protozoa Trypanosoma cruzi was developed by means of the nested PCR. The main results show patterns of genetic stratification in Yanesha forest communities, referable to different migrations at different times, estimated by Bayesian analyses. In particular Yanesha were considered as a population of transition between the Amazon basin and the Andean Cordillera, evaluating the potential migration routes and the separation of clusters of community in relation to different genetic bio-ancestry. As the Wichi, the gene pool analyzed appears clearly differentiated by the admixed sympatric Criollos, due to strict social practices (deeply analyzed with the support of cultural anthropological tools) that have preserved the native identity at a diachronic level. A pattern of distribution of the seropositivity in relation to the different phylogenetic lineages (the adaptation in evolutionary terms) does not appear, neither Amerindian nor European, but in relation to environmental and living conditions of the two distinct subpopulations.

Fusarium head blight: characterization of secondary Fusarium species, evaluation of their pathogenetic role and ability to produce mycotoxins

Fusarium head blight (FHB) is a worldwide cereal disease caused by a complex of Fusarium species resulting in high yield losses, reduction in quality and mycotoxin contamination of grain. A shift in Fusarium head blight community has been observed worldwide. The present work aimed to analyze the evolution of Italian FHB community focusing the attention on species considered “secondary” in the past years such as members of Fusarium tricinctum species complex (FTSC) and F. proliferatum. The first goal of the study was to analyze the fungal community associated with Italian durum wheat in two different years. F. poae, F. avenaceum and F. proliferatum were the main species detected on Italian durum kernels. A variable mycotoxins contamination was observed in the analyzed samples. Considering, the increased incidence of F. avenaceum and other members of FTSC in Italian FHB, the second aim was to investigate genetic diversity among the FTSC and estimate the mycotoxin risk related to these species. Phylogenetic analyses revealed that F. avenaceum (FTSC 4) was the most common species in Italy, followed by an unnamed Fusarium sp., F. tricinctum and F. acuminatum. In addition to these four phylospecies, five other F. tricinctum clade species were sampled. These included strains of four newly discovered species (Fusarium spp. FTSC 11, 13, 14, 15) and F. iranicum (FTSC 6). Most isolates tested for mycotoxin production on rice cultures were able to produce quantitative levels of enniatins and moniliformin. In addition, a preliminary study was conducted to evaluate the ability of a selected F. proliferatum isolate to produce fumonisins on wheat in open field and under natural climatic conditions. The three analogues (FB1, FB2 and FB3) were quantified by HPLC-FLD analysis on kernels, chaff and rachis. Fumonisins were detected in all the three investigated fractions without significant differences.

Characterization and cloning of chemically-induced mutants in barley (Hordeum vulgare L.)

Induced mutagenesis has been exploited for crop improvement and for investigating gene function and regulation. To unravel molecular mechanisms of stress resilience, we applied state-of-the-art genomics-based gene cloning methods to barley mutant lines showing altered root and shoot architecture and disease lesion mimic phenotypes. With a novel method that we named complementation by sequencing, we cloned NEC3, the causal gene for an orange-spotted disease lesion mimic phenotype. NEC3 belongs to the CYP71P1 gene family and it is involved in serotonin biosynthesis. By comparative phylogenetic analysis we showed that CYP71P1 emerged early in angiosperm evolution but was lost in some lineages including Arabidopsis thaliana. By BSA-Seq, we cloned the gene whose mutation increased leaf width, and we showed that the gene corresponded to the previously cloned BROADLEAF1. By BSA coupled to WGS sequencing, we cloned EGT1 and EGT2, two genes that regulate root gravitropic set point angle. EGT1 encodes a Tubby-like F-box protein and EGT2 encodes a Sterile Alpha Motive protein; EGT2 is phylogenetically related to AtSAM5 in Arabidopsis and to WEEP in peach where it regulates branch angle. Both EGT1 and EGT2 are conserved in wheat. We hypothesized that both participate to an anti-gravitropic offset mechanism since their disruption causes mutant roots to grow along the gravity vector. By the MutMap+ method, we cloned the causal gene of a short and semi-rigid root mutant and found that it encodes for an endoglucanase and is the ortholog of OsGLU3 in rice whose mutant has the same phenotype, suggesting that the gene is conserved in barley and rice. The mutants and the corresponding genes which were cloned in this work are involved in the response to stress and can potentially contribute to crop adaptation.