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.

Upward movement of Verticillium dahliae from soil to olive plants detected by qPCR.

Olive trees play an important role in cultural, ecological, environmental and social fields, constituting in large part the Mediterranean landscape. In Tuscany, an important economic activity is based on olive. Unfortunately, the Verticillium wilt affects this species and causes vascular disease. In the present study, a real-time quantitative PCR approach has been used to detect and quantify Verticillium dahliae in soil and in olive tree tissues both in micropropagated and in seedling olives. The minimum amounts of V. dahliae DNA sequences detected in soil were 11.4 fg which is equivalent to less than one fungal haploid genome. In micropropagated olive the pathogen was detected in the leaves after 43 days, showing a vertical upward movement of the fungus from the culture medium to stem and leaves. A similar fungal behaviour was observed in inoculated olive stem where after 15 days the fungal DNA was detected from symptomless stem tissue above 8 cm the inoculation site. The described molecular approach is expected to provide a more sensitive and less time-consuming alternative detection method for V. dahliae than plating assay procedures, which were traditionally proposed as an early diagnosis method for Verticillium wilt to farmers and tree nursery growers.

The extracellular DNA can baffle the assessment of soil bacterial community, but the effect varies with microscale spatial distribution.

Environmental DNA is made-up of intracellular (iDNA) and extracellular (eDNA) pools. In soils, eDNA can be present up to 40% and could distort the assessment of living microorganisms. Distribution of microbial community is inconsistent among different size-aggregates, and the persistence and turnover of eDNA are thus uneven. Uneven persistence and distribution of eDNA could lead to heterogeneity in community analysis biases that arise due to eDNA sequences at micro-scale distribution. Here, we investigated the diversity and structure of eDNA and iDNA bacterial communities in bulk soil and different size-aggregates. Significant differences were observed between eDNA and iDNA bacterial diversity and composition. Changes in community composition are more important than the amount of eDNA to assess the biases caused by eDNA in community analysis. Furthermore, variations were also observed in aggregates-levels for eDNA and iDNA community which indicates that colonization pattern of iDNA community and protection of eDNA through absorbance on particle surface within soil-matrix is heterogeneous. Our work provides empirical evidence that eDNA presence could mask the detection of aggregates-level spatial dynamics in soil microbial community and have potential to qualitatively baffle observed live effects of given treatment by adequately muting the actual response dynamics of the soil microbiome.

Oral Lactobacillus Species in Systemic Sclerosis.

In systemic sclerosis (SSc), the gastrointestinal tract (GIT) plays a central role in the patient's quality of life. The microbiome populates the GIT, where a relationship between the Lactobacillus and gastrointestinal motility has been suggested. In this study, the analysis of oral Lactobacillus species in SSc patients and healthy subjects using culture-independent molecular techniques, together with a review of the literature on microbiota and lactobacilli in SSc, has been carried out. Twenty-nine SSc female patients (mean age 62) and twenty-three female healthy subjects (HS, mean age 57.6) were enrolled and underwent tongue and gum swab sampling. Quantitative PCR was conducted in triplicate using Lactobacillus specific primers rpoB1, rpoB1o and rpoB2 for the RNA-polymerase ß subunit gene. Our data show significantly (p = 0.0211) lower LactobacillusspprpoB sequences on the tongue of patients with SSc compared to HS. The mean value of the amount of Lactobacillus ssprpoB gene on the gumsofSSc patients was minor compared to HS. A significant difference between tongue and gums (p = 0.0421) was found in HS but not in SSc patients. In conclusion, our results show a lower presence of Lactobacillus in the oral cavity of SSc patients. This strengthens the hypothesis that Lactobacillus may have both a protective and therapeutic role in SSc patients.

Preliminary evidences of the presence of extracellular DNA single stranded forms in soil.

The relevance of extracellular DNA (eDNA) in the soil ecosystem is becoming more and more evident to the scientific community by the progressive discovery of functions accompanying to natural gene transformation. However, despite the increased number of published articles dedicated to eDNA in soil, so far only few are focused on its single stranded form (eDNAss). The present paper is the first to investigate the quantitative relevance of eDNAss in the total soil eDNA pool, discriminating between its linear (eDNAssl) and circular (eDNAssc) forms and the respective weakly (wa) and tightly (ta) adsorbed fractions. The results showed the prevalence of eDNAss and its linear form in both the total soil eDNA pool and its wa and ta fractions. Both of the eDNAss fractions (linear and circular) were characterized by small fragments.

Experimental discrimination and molecular characterization of the extracellular soil DNA fraction.

We experimentally discriminated and qualitatively-quantitatively characterized the extracellular fraction of a forest soil DNA pool. We sequentially extracted and classified the components of extracellular DNA by its strength of interaction with soil colloids as: (1) extractable in water, free in the extracellular soil environment or adsorbed on soil colloids; and as (2) extractable in alkaline buffer after previous extraction in water, bound on soil colloids. The comparative molecular analysis (fluorometer, gel electrophoresis, genetic fingerprinting) of directly and sequentially extracted extracellular DNA revealed quantitative and qualitative differences, also in terms of genetic information about microbial communities. The sequential extraction of extracellular DNA revealed differences in molecular weight, indicating a relationship between DNA fragment length and strength of interaction with soil colloids. The sequential extraction was also suitable to assess the presence of tightly bound DNA, providing information about the DNA-colloid interactions naturally occurring in the soil environment.

Effect of Mediterranean Diet Enriched in High Quality Extra Virgin Olive Oil on Oxidative Stress, Inflammation and Gut Microbiota in Obese and Normal Weight Adult Subjects.

Introduction: The Mediterranean Diet (MD) is useful in the prevention of overweight, obesity and metabolic disease. High Quality-Extra Virgin Olive Oil (HQ-EVOO), an essential component of this diet, exerts protective effects against chronic diseases. Gut Microbiota (GM), recognized as a key factor in driving metabolic activities, is involved in the regulation of host immunity. Lactic Acid Bacteria (LAB) and their probio-active cellular substances produce beneficial effects in the gastrointestinal tract. Materials and Methods: Eighteen overweight/obese subjects (cases, BMI ≥25 kg/m2) and 18 normal weight controls (BMI 18.5-24.9 kg/m2) were fed with MD enriched with 40 g/die HQ-EVOO for three months. Feces and blood samples were collected at time 0 (T0) and after three months (T1) for LAB composition, oxidative stress, metabolic and inflammation parameter determinations. Results: Myeloperoxidase and 8-hydroxy-2-deoxyguanosine, markers of inflammation and oxidative stress, were significantly decreased after MD rich in HQ-EVOO both in controls and in cases. Proinflammatory cytokines levels were significantly decreased in cases in comparison to controls, while IL-10 and adiponectin were significantly increased in cases. LAB's rpoB copies/ng of DNA increased 55.6 folds in cases compared to their baseline after MD rich in HQ-EVOO. MD rich in HQ-EVOO increased adiponectin and IL-10 concentration in overweight/obese subjects and decreased oxidative stress and inflammation parameters and at the same time, increased LAB number in GM. Discussion: Our results indicate that MD rich in HQ-EVOO induces an increase of LAB in GM and could have a potential role in the prevention of inflammation. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03441802.

Long-term effects of aided phytostabilisation of trace elements on microbial biomass and activity, enzyme activities, and composition of microbial community in the Jales contaminated mine spoils.

We studied the effectiveness of remediation on microbial endpoints, namely microbial biomass and activity, microbial and plant species richness, of an As-contaminated mine spoil, amended with compost (C) alone and in combination with beringite (B) or zerovalent iron grit (Z), to increase organic matter content and reduce trace elements mobility, and to allow Holcus lanatus and Pinus pinaster growth. Untreated spoil showed the lowest microbial biomass and activity and hydrolase activities, and H. lanatus as sole plant species, whereas the presented aided phytostabilisation option, especially CBZ treatment, significantly increased microbial biomass and activity and allowed colonisation by several plant species, comparable to those of an uncontaminated sandy soil. Microbial species richness was only increased in spoils amended with C alone. No clear correlation occurred between trace element mobility and microbial parameters and plant species richness. Our results indicate that the choice of indicators of soil remediation practices is a bottleneck.

Persistence of transgenic and not transgenic extracellular DNA in soil and bacterial transformation.

The study of the fate of transgenic and not transgenic extracellular DNA in soil is of extreme relevance because the soil extracellular DNA pool represents a genetic reservoir that could be utilized as a source of food by any heterotrophic microorganism or genetic information by recipient eukaryotic and prokaryotic cells. Several data have clearly evidenced that extracellular DNA could persist in soil for long time maintaining a sufficient integrity of the molecule. Recent microcosm studies under laboratory conditions have evidenced that extracellular DNA molecule could be leached or raised up by capillarity. The persistence and movement of extracellular DNA molecule in soil suggest that the genetic information of extracellular DNA could be taken up by microorganisms temporarily and spatially separated. Several authors have studied the persistence and transformation efficiency of the extracellular DNA in soil demonstrating that there is a sharp discrepancy between its biological efficiency and its persistence; fragments of target DNA were detected after a long time in soil but no transformations were determined probably because the genetic information originally present in the complete DNA molecule could be lost by degradation. It is also important to underline that the frequency of gene transfer in soil is markedly limited by the few number of bacteria able to develop competence and that this physiological state is reached only under certain conditions. Furthermore the dilution of the transgene in the soil extracellular DNA pool drastically decreases chances for the uptake of the transgene. Anyway the importance of transformation in evolutionary terms, represents a valid reason to continue the investigation on the fate of extracellular DNA in soil.

Evaluation of the Performances of Ribosomal Database Project (RDP) Classifier for Taxonomic Assignment of 16S rRNA Metabarcoding Sequences Generated from Illumina-Solexa NGS.

Here we report a benchmark of the effect of bootstrap cut-off values of the RDP Classifier tool in terms of data retention along the different taxonomic ranks by using Illumina reads. Results provide guidelines for planning sequencing depths and selection of bootstrap cut-off in taxonomic assignments.

Long-term persistence and bacterial transformation potential of transplastomic plant DNA in soil.

The long-term physical persistence and biological activity of transplastomic plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic plant DNA in soil.