High taxonomic level fingerprint of the human intestinal microbiota by Ligase Detection Reaction - Universal Array approach

<p>Abstract</p> <p>Background</p> <p>Affecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation.</p> <p>Results</p> <p>In order to specifically monitor microbiota unbalances that impact human physiology, here we develop and validate an original DNA-microarray (HTF-Microbi.Array) for the high taxonomic level fingerprint of the human intestinal microbiota. Based on the Ligase Detection Reaction-Universal Array (LDR-UA) approach, the HTF-Microbi.Array enables specific detection and approximate relative quantification of 16S rRNAs from 30 phylogenetically related groups of the human intestinal microbiota. The HTF-Microbi.Array was used in a pilot study of the faecal microbiota of eight young adults. Cluster analysis revealed the good reproducibility of the high level taxonomic microbiota fingerprint obtained for each of the subject.</p> <p>Conclusion</p> <p>The HTF-Microbi.Array is a fast and sensitive tool for the high taxonomic level fingerprint of the human intestinal microbiota in terms of presence/absence of the principal groups. Moreover, analysis of the relative fluorescence intensity for each probe pair of our LDR-UA platform can provide estimation of the relative abundance of the microbial target groups within each samples. Focusing the phylogenetic resolution at division, order and cluster levels, the HTF-Microbi.Array is blind with respect to the inter-individual variability at the species level.</p

Unbalance of intestinal microbiota in atopic children

BACKGROUND: Playing a strategic role in the host immune function, the intestinal microbiota has been recently hypothesized to be involved in the etiology of atopy. In order to investigate the gastrointestinal microbial ecology of atopic disease, here we performed a pilot comparative molecular analysis of the faecal microbiota in atopic children and healthy controls. RESULTS: Nineteen atopic children and 12 healthy controls aged 4–14 years were enrolled. Stools were collected and the faecal microbiota was characterized by means of the already developed phylogenetic microarray platform, HTF-Microbi.Array, and quantitative PCR. The intestinal microbiota of atopic children showed a significant depletion in members of the Clostridium cluster IV, Faecalibacterium prausnitzii, Akkermansia muciniphila and a corresponding increase of the relative abundance of Enterobacteriaceae. CONCLUSION: Depleted in key immunomodulatory symbionts, the atopy-associated microbiota can represent an inflammogenic microbial consortium which can contribute to the severity of the disease. Our data open the way to the therapeutic manipulation of the intestinal microbiota in the treatment of atopy by means of pharmaceutical probiotics

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

BACKGROUND: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. RESULTS: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. CONCLUSIONS: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria

Next-generation sequencing characterization of the gut bacterial community of gilthead sea bream (Sparus aurata, L.) fed low fishmeal based diets with increasing soybean meal levels

The present study was carried out to evaluate growth, gut histology and gut bacterial community of gilthead sea bream (Sparus aurata) fed with increasing dietary soybean meal (SBM) levels in a low fishmeal (FM) based diet, in comparison with a control diet. Five isoproteic and isolipidic experimental diets were formulated to contain increasing levels of SBM (0, 100, 200, and 300 g kg 121 named S0, S10, S20 and S30, respectively) with 150 g kg 121 of FM, and one control diet (C) without SBM and containing 350 g kg 121 of FM. Sixty sea bream (initial body weight 75.9 \ub1 1.9 g, n = 900) per tank were reared in a recirculation system at 23.0 \ub1 1.0 \ub0C and fed to satiation. The trial was run in triplicate and lasted 100 days. At the end of the trial fish fed the S30 diet showed a higher (P 64 0.05) specific growth rate (SGR) compared to S0 (SGR, 1.17 \ub1 0.03, 1.20 \ub1 0.01, 1.22 \ub1 0.01, 1.25 \ub1 0.01 and 1.21 \ub1 0.04 for S0, S10, S20, S30 and C, respectively), and a higher feed intake (FI) compared to S0, S10 and S20. Sea bream fed the C diet had a higher (P 64 0.05) FI compared to S0 (FI, 1.40 \ub1 0.01, 1.45 \ub1 0.01, 1.44 \ub1 0.03, 1.51 \ub1 0.03 and 1.46 \ub1 0.02 for S0, S10, S20, S30 and C, respectively). No significant differences in feed conversion rate, protein efficiency ratio, gross protein efficiency and gross lipid efficiency among the treatments were detected. No specific histopathological changes indicative of soy-induced enteritis were observed in the intestine of any fish examined. Gut bacterial community of the distal intestine content was analyzed by Next-Generation Sequencing. At the phylum level, the gut bacterial community was dominated by Firmicutes (relative abundance 71%), while the most represented family was Lactobacillaceae (26%). Even if no significant differences (P 64 0.05) in the gut bacterial community \u3b1 and \u3b2-diversity according to the different diets were detected, Cyanobacteria and Lactobacillaceae progressively increased from diet C to diet S30. In conclusion results of growth, nutrient utilization, gut histology and gut bacterial community indicate that SBM can be successfully incorporated up to a level of 300 g kg 121 with the inclusion of 150 g kg 121 of FM, without any deleterious effects on growth, protein utilization and gut health during the on-growing of sea bream

Gardnerella vaginalis clades in pregnancy: New insights into the interactions with the vaginal microbiome

Gardnerella vaginalis (GV) is an anaerobic bacterial species involved in the pathogenesis of bacterial vaginosis (BV), a condition of vaginal dysbiosis associated with adverse pregnancy outcomes. GV strains are categorized into four clades, characterized by a different ability to produce virulence factors, such as sialidase. We investigated the distribution of GV clades and sialidase genes in the vaginal ecosystem of a cohort of pregnant women, assessing the correlations between GV clades and the whole vaginal microbiome. A total of 61 Caucasian pregnant women were enrolled. Their vaginal swabs, collected both at the first and third trimester of pregnancy, were used for (i) evaluation of the vaginal status by Nugent score, (ii) vaginal microbiome profiling by 16S rRNA sequencing, (iii) detection and quantification of GV clades and sialidase A gene by qPCR assays. DNA of at least one GV clade was detected in most vaginal swabs, with clade 4 being the most common one. GV clade 2, together with the presence of multiple clades (&gt;2 simultaneously), were significantly associated with a BV condition. Significantly higher GV loads and sialidase gene levels were found in BV cases, compared to the healthy status. Clade 2 was related to the major shifts in the vaginal microbial composition, with a decrease in Lactobacillus and an increase in several BV-related taxa. As the number of GV clades detected simultaneously increased, a group of BV-associated bacteria tended to increase as well, while Bifidobacterium tended to decrease. A negative correlation between sialidase gene levels and Lactobacillus, and a positive correlation with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia were observed. Our results added knowledge about the interactions of GV clades with the inhabitants of the vaginal microbiome, possibly helping to predict the severity of BV and opening new perspectives for the prevention of pregnancy-related complications

A Deep Look at the Vaginal Environment During Pregnancy and Puerperium

A deep comprehension of the vaginal ecosystem may hold promise for unraveling the pathophysiology of pregnancy and may provide novel biomarkers to identify subjects at risk of maternal-fetal complications. In this prospective study, we assessed the characteristics of the vaginal environment in a cohort of pregnant women throughout their different gestational ages and puerperium. Both the vaginal bacterial composition and the vaginal metabolic profiles were analyzed. A total of 63 Caucasian women with a successful pregnancy and 9 subjects who had a first trimester miscarriage were enrolled. For the study, obstetric examinations were scheduled along the three trimester phases (9-13, 20-24, 32-34 gestation weeks) and puerperium (40-55 days after delivery). Two vaginal swabs were collected at each time point, to assess the vaginal microbiome profiling (by Nugent score and 16S rRNA gene sequencing) and the vaginal metabolic composition (1H-NMR spectroscopy). During pregnancy, the vaginal microbiome underwent marked changes, with a significant decrease in overall diversity, and increased stability. Over time, we found a significant increase of Lactobacillus and a decrease of several genera related to bacterial vaginosis (BV), such as Prevotella, Atopobium and Sneathia. It is worth noting that the levels of Bifidobacterium spp. tended to decrease at the end of pregnancy. At the puerperium, a significantly lower content of Lactobacillus and higher levels of Gardnerella, Prevotella, Atopobium, and Streptococcus were observed. Women receiving an intrapartum antibiotic prophylaxis for Group B Streptococcus (GBS) were characterized by a vaginal abundance of Prevotella compared to untreated women. Analysis of bacterial relative abundances highlighted an increased abundance of Fusobacterium in women suffering a first trimester abortion, at all taxonomic levels. Lactobacillus abundance was strongly correlated with higher levels of lactate, sarcosine, and many amino acids (i.e., isoleucine, leucine, phenylalanine, valine, threonine, tryptophan). Conversely, BV-associated genera, such as Gardnerella, Atopobium, and Sneathia, were related to amines (e.g., putrescine, methylamine), formate, acetate, alcohols, and short-chain fatty-acids (i.e., butyrate, propionate)

Torquetenovirus in pregnancy: Correlation with vaginal microbiome, metabolome and pro-inflammatory cytokines

Torquetenovirus (TTV) is a negative sense, single-stranded DNA virus present in many body fluids of apparently healthy individuals. At present, it is considered a non-pathogenic endogenous virus. TTV can be detected in the vagina of pregnant women, its abundance being modulated with the extent of immune system activation. Until now, there is only scarce information regarding the association between TTV and the composition of the vaginal environment. Therefore, this study aimed to assess the presence of TTV in the vaginal ecosystem of a cohort of white women with a normal pregnancy (n = 60) at different gestational stages (first, second and third trimester) and in 9 subjects suffering a first trimester miscarriage. For each woman, we determined (i) the presence and titer of TTV, (ii) the vaginal bacterial composition by means of Nugent score and 16S rRNA gene sequencing, (iii) the vaginal metabolic profiles through H-1-NMR spectroscopy, and (iv) the vaginal concentration of two pro-inflammatory cytokines (IL-6 and IL-8). More than one third of women were found negative for TTV at all gestational stages. Although not statistically significant, the positivity for TTV dropped from 53.3% in the first to 36.6% in the third trimester. TTV loads varied greatly among vaginal samples, ranging between 2 x 10(1) and 2 x 10(5) copies/reaction. No difference in TTV prevalence and loads was observed between women with normal pregnancies and miscarriages. The presence of TTV was more common in women with a higher vaginal leucocyte count (p = 0.02). The levels of IL-6 (p = 0.02), IL-8 (p = 0.03), propionate (p = 0.001) and cadaverine (p = 0.006) were significantly higher in TTV-positive samples. TTV titer was positively correlated with the concentrations of 4-hydroxyphenyllactate (p &lt; 0.0001), isoleucine (p = 0.01) and phenylalanine (p = 0.04). TTV-positive samples were characterized by a higher relative abundance of Sneathia (p = 0.04) and Shuttleworthia (p = 0.0009). In addition, a trend toward a decrease of Lactobacillus crispatus and Lactobacillus jensenii, and an increase of Lactobacillus iners was observed for TTV-positive samples. In conclusion, we found that TTV is quite common in women with normal pregnancy outcomes, representing a possible predictor of local immune status

ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design

16S rRNA gene is one of the preferred targets for resolving species phylogenesis issues in microbiological-related contexts. However, the identification of single-nucleotide variations capable of distinguishing a sequence among a set of homologous ones can be problematic. Here we present ORMA (Oligonucleotide Retrieving for Molecular Applications), a set of scripts for discriminating positions search and for performing the selection of high-quality oligonucleotide probes to be used in molecular applications. Two assays based on Ligase Detection Reaction (LDR) are presented. First, a new set of probe pairs on cyanobacteria 16S rRNA sequences of 18 different species was compared to that of a previous study. Then, a set of LDR probe pairs for the discrimination of 13 pathogens contaminating bovine milk was evaluated. The software determined more than 100 candidate probe pairs per dataset, from more than 300 16S rRNA sequences, in less than 5 min. Results demonstrated how ORMA improved the performance of the LDR assay on cyanobacteria, correctly identifying 12 out of 14 samples, and allowed the perfect discrimination among the 13 milk pathogenic-related species. ORMA represents a significant improvement from other contexts where enzyme-based techniques have been employed on already known mutations of a single base or on entire subsequences

Early gut microbiota signature of aGvHD in children given allogeneic hematopoietic cell transplantation for hematological disorders

The onset of acute Graft-versus-Host Disease (aGvHD) has been correlated with the gut microbiota (GM) composition, but experimental observations are still few, mainly involving cohorts of adult patients. In the current scenario where fecal microbiota transplantation has been used as a pioneer therapeutic approach to treat steroid-refractory aGvHD, there is an urgent need to expand existing observational studies of the GM dynamics in Hematopoietic Stem Cell Transplantation (HSCT). Aim of the present study is to explore the GM trajectory in 36 pediatric HSCT recipients in relation to aGvHD onset

Modulation of gut microbiota dysbioses in type 2 diabetic patients by macrobiotic Ma-Pi 2 diet

The study was carried out with internal funding