Vania Patrone

Short-term modifications in the distal gut microbiota of weaning mice induced by a high-fat diet.

The gut microbiota has been shown to be involved in host energy homeostasis and diet-induced metabolic disorders. To gain insight into the relationships among diet, microbiota and the host, we evaluated the effects of a high-fat (HF) diet on the gut bacterial community in weaning mice. C57BL/6 mice were fed either a control diet or a diet enriched with soy oil for 1 and 2 weeks. Administration of the HF diet caused an increase in plasma total cholesterol levels, while no significant differences in body weight gain were observed between the two diets. Denaturing gradient gel electrophoresis (DGGE) profiles indicated considerable variations in the caecal microbial communities of mice on the HF diet, as compared with controls. Two DGGE bands with reduced intensities in HF-fed mice were identified as representing Lactobacillus gasseri and an uncultured Bacteroides species, whereas a band of increased intensity was identified as representing a Clostridium populeti-related species upon sequencing. Quantitative real-time PCR confirmed a statistically significant 1-log decrease in L. gasseri cell numbers after HF feeding, and revealed a significantly lower level of Bifidobacterium spp. in the control groups after 1 and 2 weeks compared with that in the HF groups. These alterations of intestinal microbiota were not associated with caecum inflammation, as assessed by histological analysis. The observed shifts of specific bacterial populations within the gut may represent an early consequence of increased dietary fat.

Effects of geographic area, feedstock, temperature, and operating time on microbial communities of six full-scale biogas plants

The objective of this study was to investigate the effect of different animal feedings operated in two distinct PDO (protected designation of origin) cheese production areas (Parmigiano Reggiano and Grana Padano) on the microbiome of six full-scale biogas plants, by means of Illumina sequencing and qPCR techniques. The effects of feedstock (cattle slurry manure, energy crops, agro-industrial by-products), temperature (mesophilic/thermophilic), and operating time were also examined, as were the relationships between the predominant bacterial and archaeal taxa and process parameters. The different feedstocks and temperatures strongly affected the microbiomes. A more biodiverse archaeal population was highlighted in Parmigiano Reggiano area plants, suggesting an influence of the different animal feedings. Methanosarcina and Methanosaeta showed an opposite distribution among anaerobic plants, with the former found to be related to ammonium concentration. The Methanoculleus genus was more abundant in the thermophilic digester whereas representation of the Thermotogales order correlated with hydraulic retention time.

Infant Early Gut Colonization by Lachnospiraceae: High Frequency of Ruminococcus gnavus

Lachnospiraceae is a bacterial family usually isolated from human and mammalian intestinal microbiota. However, its presence and role in the infant microbiota is not fully elucidated. This may be due to the strictly anaerobic behavior of its members that hampers the possibility of culture-dependent enumeration. Here, we report on the presence of this bacterial group, using biomolecular techniques, in stool samples from 25 babies aged between 1 and 24 months. Denaturing gradient gel electrophoresis (DGGE) was used as a first detection step, and data were confirmed by quantitative PCR (qPCR). The DGGE showed the presence of Lachnospiraceae in infant fecal specimens and indicated the prevalence of Ruminococcus gnavus (R. gnavus). The qPCR confirmed the presence of the Clostridium XVIa group, Blautia genus, and R. gnavus, which are the main members of this family. We detected R. gnavus in 22 of 25 (88%) samples with a qPCR probe assay. Despite the difficulties associated with their detection and enumeration, Lachnospiraceae, and in particular R. gnavus, should be included in future studies on the infant microbiota composition.

Gut microbiota profile in systemic sclerosis patients with and without clinical evidence of gastrointestinal involvement

Recent evidence suggests that there is a link between the gut microbial community and immune-mediated disorders. Systemic sclerosis (SSc) is an autoimmune disease characterized by immunonological abnormalities, vascular lesions, and extensive fibrosis. Since the gastrointestinal tract is one of the organs most involved, the goal of this study was to explore the composition of the intestinal microbiota in SSc patients with (SSc/GI+) and without gastrointestinal involvement (SSc/GI-) in comparison to healthy controls (HC). The fecal bacterial composition was investigated by Illumina sequencing of 16 S rRNA gene amplicons. The fecal microbiota of SSc/GI+ subjects was characterized by higher levels of Lactobacillus, Eubacterium and Acinetobacter compared with healthy controls, and lower proportions of Roseburia, Clostridium, and Ruminococcus. The gut microbiota of SSc/GI- subjects was more similar to the microbiota of HC than to that of SSc/GI+ subjects albeit Streptococcus salivarius was over-represented in SSc/GI- fecal samples compared with both SSc/GI+ subjects and controls. Our study reveals microbial signatures of dysbiosis in the gut microbiota of SSc patients that are associated with clinical evidence of gastrointestinal disease. Further studies are needed to elucidate the potential role of these perturbations in the onset and progression of systemic sclerosis, and gastrointestinal involvement in particular.

Detailed analyses of the bacterial populations in processed cocoa beans of different geographic origin, subject to varied fermentation conditions.

The quality of chocolate is influenced by several parameters, one of which is bacterial diversity during fermentation and drying; a crucial factor for the generation of the optimal cocoa flavor precursors. Our understanding of the bacterial populations involved in chocolate fermentation can be improved by the use of high-throughput sequencing technologies (HTS), combined with PCR amplification of the 16S rRNA subunit. Here, we have conducted a high-throughput assessment of bacterial diversity in four processed samples of cocoa beans from different geographic origins. As part of this study, we also assessed whether different DNA extraction methods could affect the quality of our data. The dynamics of microbial populations were analyzed postharvest (fermentation and sun drying) and shipment, before entry to the industrial process. A total of 691,867 high quality sequences were obtained by Illumina MiSeq sequencing of the two bacterial 16S rRNA hypervariable regions, V3 and V4, following paired-read assembly of the raw reads. Manual curation of the 16S database allowed us to assign the correct taxonomic classifications, at species level, for 83.8% of those reads. This approach revealed a limited biodiversity and population dynamics for both the lactic acid bacteria (LAB) and acetic acid bacteria (AAB), both of which are key players during the acetification and lactic acid fermentation phases. Among the LAB, the most abundant species were Lactobacillus fermentum, Enterococcus casseliflavus, Weissella paramesenteroides, and Lactobacillus plantarum/paraplantarum. Among the AAB, Acetobacter syzygii, was most abundant, then Acetobacter senegalensis and Acetobacter pasteriuanus. Our results indicate that HTS approach has the ability to provide a comprehensive view of the cocoa bean microbiota at the species level.

Abundance and Diversity of Hydrogenotrophic Microorganisms in the Infant Gut before the Weaning Period Assessed by Denaturing Gradient Gel Electrophoresis and Quantitative PCR.

Delivery mode (natural vs. cesarean) and feeding type (breast vs. formula feeding) are relevant factors for neonatal gut colonization. Biomolecular methods have shown that the ecological structure of infant microbiota is more complex than previously proposed, suggesting a relevant presence of unculturable bacteria. It has also been postulated that among unculturable bacteria, hydrogenotrophic populations might play a key role in infant health. Sulfate-reducing bacteria (SRB), acetogens, and methanogenic archaea use hydrogenotrophic pathways within the human colon. However, to date, few studies have reported detection of hydrogenotrophic microorganisms in newborns, possibly because of limitations on available group-specific, culture-independent quantification procedures. In the present work, we analyzed 16 fecal samples of healthy babies aged 1–6 months by means of quantitative PCR (qPCR) targeting the 16S rRNA or metabolic functional genes and by denaturing gradient gel electrophoresis (DGGE). qPCR data showed quantifiable levels of methanogens, SRB, and acetogens in all samples, indicating that the relative abundances of these microbial groups were not affected by delivery mode (natural vs. caesarian). DGGE revealed a high prevalence of the Blautia genus within the acetogenic bacteria despite strong interindividual variability. Our preliminary results suggest that hydrogenotrophic microorganisms, which have been a neglected group to date, should be included in future ecological and metabolic studies evaluating the infant intestinal microbiota.

Prebiotics, Probiotics, and Synbiotics: A Bifidobacterial View

Abstract The chapter begins with an overview of the definitions of the three terms presented in the title, from both a scientific and a regulatory point of view. Then the clinical evidences available on the beneficial effects obtained by dosing selected strains of bifidobacteria or feeding human subjects with prebiotic substances will be addressed, taking into consideration different stages in aging (neonatal life, adulthood, and elders in their “ golden years”). A section will be devoted to “therapeutic” use of bifidobacteria, that is, administration to subjects in pathological conditions. A final section will be devoted to an update on the mechanisms of action of probiotic bifidobacteria, as well as the impact of prebiotics studies by means of microbiome analysis.

Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan

Probiotics are actively used for treatment of diarrhoea, respiratory infections, and prevention of infectious gastrointestinal diseases. The efficacy of probiotics is due to strain-specific features and the number of viable cells; however, several reports of deviations from the label in the actual content of strains in probiotic products are a matter of concern. Most of the available data on quality focuses on probiotic products containing lactobacilli and/or bifidobacteria, while very few data are available on spore-forming probiotics. The present study evaluates the label claims for spore count and species identification in five commercial probiotic products marketed in India and Pakistan that claim to contain Bacillus clausii: Tufpro, Ecogro, Enterogermina, Entromax, and Ospor. Bacterial enumeration from three batches was done by microbiological plating methods by two independent operators. Species identification was done using PCR amplification and sequence analysis of the 16S rRNA gene, and determination of the total amount of species present in the products was done using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by DNA sequencing of the excised bands. Plate count methods demonstrated poor correlations between quantitative label indications and bacteria recovered from plates for Tufpro, Ecogro, and Ospor. The 16S rRNA analysis performed on bacteria isolated from plate counts showed that only Enterogermina and Ospor contained homogenous B. clausii. PCR-DGGE analysis revealed that only Enterogermina had a homogenous B. clausii population while other products had mixed bacterial populations. In conclusion, the current analysis clearly demonstrates that of the five analysed commercial probiotics, only Enterogermina followed the label claims.

Effect of bariatric surgery on serum levels of gastrointestinal hormones in obese patients

Free-fat yoghurt manufactured from skim milk treated with transglutaminase (TG; 1 Ugmilk protein) and buttermilk powder (BMP; 1% and 2%); either individually or in combination, was investigated. For comparison, the full-fat and free-fat variants without TG or BMP addition were also studied. The results reveal that TG did not interfere with the pH reduction during fermentation progress, while BMP addition had fairly accelerated the pH drop during incubation time. Although TG treatment or BMP addition has significantly (P<0.05) improved the yoghurt gel strength by means of improving the water holding capacity functionality, the free-fat yoghurts made from TG-treated milk in combination with BMP addition obtained the highest values which were similar to that of full-fat control yoghurt. Furthermore, addition of BMP enhanced the reactivity of TG as indicated by the extended and excessive appearance of high molecular weight protein polymers bands in electrophoreses patterns. This result was confirmed by scanning electron microscope (SEM) analysis as a more compact and dense structure accompanied by tortuous clusters of protein aggregates were observed within micrographs of free-fat yoghurt made with combination of TG and BMP. Addition of TG or BMP individually had a marked affirmative impact on free-fat gel network, represented in a denser and more homogeneous systematic protein aggregate network characterised by a finer-meshed network. It is worthwhile to state that this impact was more pronounced within BMP addition (either 1% or 2%) than for TG treatment. Free-fat yoghurts of individually BMP addition exhibited the most desirable organoleptic attributes as indicated by the assessors and were similar to the full-fat yoghurt perception, whereas, the combined TG-BMP treatments received fairly criticized scores due to its firmer and crumbly mouth-feel. Overall, addition of TG or BMP appears to be a valuable alternative in free-fat yoghurt production, and BMP can be worthy considered as a source of extra protein level, which in-turn offers a promising option to develop innovative functional free-fat yoghurt.A of the study is to evaluate dietary intake, nutritional assessment, plasma levels of gastrointestinal peptides that regulate food intake and fecal microbiota in severely obese patients before and after bariatric surgery. We studied 28 severely obese patients, 19 of which underwent biliointestinal bypass, and 28 healthy normal weight controls. In all subjects we evaluated food intake, body composition, plasma levels of peptide YY (PYY), glucagon-like peptide (GLP)-1, GLP-2, ghrelin (GHR), orexin (ORE) and cholecystokinin (CCK), and fecal microbiota. In severely obese patients all parameters were evaluated at 0 time and 6 months after bariatric surgery. In obese patients we found: 1) a higher intake of nutrients, both as calories and as macro and micronutrients in respect to controls (p<0.05); 2) a decrease of free fat mass (p<0.01) and an increase of BMI (p<0.01), fat mass (p<0.01) and trunk fat (p<0.01) in respect to controls; 3) a significant decrease of GLP-1 and an increase of GLP-2, GHR and PYY in respect to controls (p<0.05); 4) further increase in GLP-2, GHR and PYY, and increase over control values of GLP-1 after bariatric surgery (p<0.05 versus pre-surgery). Obese individuals were found to harbor a community dominated by members of the Clostridial clusters XIVa and IV, whereas prominent bands after surgery were identified as Lactobacillus crispatus and Megasphaera elsdenii-related phylotype. We postulate that the beneficial effects of bariatric surgery may at least in part be accounted for by changes in circulating GI peptides and fecal microbiota.

Probiotic Microorganisms for Shaping the Human Gut Microbiota - Mechanisms and Efficacy into the Future

The use of bacteria as beneficial biological agents being used as food ingredients or as active components of food supplements dates back to the early 1900s. These bacteria must reach the human gut in a viable form and in numbers sufficiently high to attain a substantial presence amongst the thousands of bacteria inhabiting the gut; they have also to exert some beneficial action. These beneficial actions have been supported by hundreds of papers, the vast majority of them dealing with subjects in which the ratio among the different bacterial groups of the gut were altered by pathological conditions; the administration of probiotic bacteria are able to restore the function of the gut microbiota. In healthy people the action of probiotics is possibly related to different mechanisms, linked to strain-specific action of probiotics and relying on the replacement of some autochthonous bacteria with specifically selected probiotic strains.