Negative plant-soil feedback in Arabidopsis thaliana: Disentangling the effects of soil chemistry, microbiome, and extracellular self-DNA.

Nutrient deficiency, natural enemies and litter autotoxicity have been proposed as possible mechanisms to explain species-specific negative plant-soil feedback (PSF). Another potential contributor to negative PSF is the plant released extracellular self-DNA during litter decay. In this study, we sought to comprehensively investigate these hypotheses by using Arabidopsis thaliana (L.) Heynh as a model plant in a feedback experiment. The experiment comprised a conditioning phase and a response phase in which the conditioned soils underwent four treatments: (i) addition of activated carbon, (ii) washing with tap water, (iii) sterilization by autoclaving, and (iv) control without any treatment. We evaluated soil chemical properties, microbiota by shotgun sequencing and the amount of A. thaliana extracellular DNA in the differently treated soils. Our results showed that washing and sterilization treatments mitigated the negative PSF effect. While shifts in soil chemical properties were not pronounced, significant changes in soil microbiota were observed, especially after sterilization. Notably, plant biomass was inversely associated with the content of plant self-DNA in the soil. Our results suggest that the negative PSF observed in the conditioned soil was associated to increased amounts of soilborne pathogens and plant self-DNA. However, fungal pathogens were not limited to negative conditions, butalso found in soils enhancing A.thaliana growth. In-depth multivariate analysis highlights that the hypothesis of negative PSF driven solely by pathogens lacks consistency. Instead, we propose a multifactorial explanation for the negative PSF buildup, in which the accumulation of self-DNA weakens the plant's root system, making it more susceptible to pathogens.

Fermented foods, their microbiome and its potential in boosting human health.

Fermented foods (FFs) are part of the cultural heritage of several populations, and their production dates back 8000 years. Over the last ~150 years, the microbial consortia of many of the most widespread FFs have been characterised, leading in some instances to the standardisation of their production. Nevertheless, limited knowledge exists about the microbial communities of local and traditional FFs and their possible effects on human health. Recent findings suggest they might be a valuable source of novel probiotic strains, enriched in nutrients and highly sustainable for the environment. Despite the increasing number of observational studies and randomised controlled trials, it still remains unclear whether and how regular FF consumption is linked with health outcomes and enrichment of the gut microbiome in health-associated species. This review aims to sum up the knowledge about traditional FFs and their associated microbiomes, outlining the role of fermentation with respect to boosting nutritional profiles and attempting to establish a link between FF consumption and health-beneficial outcomes.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Relationships between diet and gut microbiome in an Italian and Dutch cohort: does the dietary protein to fiber ratio play a role?

PURPOSE: To investigate the relationships between the habitual diet, the protein to fiber ratio (P/F), and the gut microbiome in one Italian and one Dutch cohort of healthy subjects consuming an omnivore diet. METHODS: The Italian cohort included 19 males (M_IT, BMI 25.2 ± 0.72 kg/m2, age 25.4 ± 0.96 years) and 20 females (F_IT, BMI 23.9 ± 0.81 kg/m2, age 23.8 ± 0.54 years); the Dutch cohort included 30 females (F_NL, BMI: 23.9 ± 0.81 kg/m2, age: 23.8 ± 0.54 years). Individual diets were recorded through Food Frequency Questionnaires and analyzed to assess the nutrient composition. Gut microbiome was assessed in fecal samples. RESULTS: M_IT consumed higher levels of proteins than F_NL and F_IT, whereas dietary fiber intake did not differ among groups. Data showed that consumption of plant protein to animal protein (PP/AP) and PP to total proteins ratio can determine a differentiation of F_NL more than the absolute amount of dietary fiber. Conversely, the protein to fiber (P/F) and AP to total proteins better characterized M_IT. M_IT harbored the highest abundance of proteolytic microorganisms and the lowest microbial gene richness. Conversely, F_NL had more fiber-degrading microorganisms like Bacteroides thetaiotaomicron, Bacteroides xylanisolvens, Roseburia sp., Coprococcus eutactus and Parabacteroides along with the highest number of genes encoding carbohydrate-active enzymes and gene richness. It was predicted that by each unit decrease in the P/F a 3% increase in gene richness occurred. CONCLUSION: Study findings suggested that dietary P/F, rather than the absolute amount of dietary fiber, could contribute to the shaping of the microbiome towards a more proteolytic or fiber-degrading gut ecosystem. CLINICALTRIALS: gov Identifier NCT04205045-01-10-2018, retrospectively registered. Dutch Trial Register NTR7531-05-10-2018.

Microbiome dynamics, antibiotic resistance gene patterns and spoilage-associated genomic potential in fresh anchovies stored in different conditions.

Fresh fish is a highly perishable product and is easily spoiled by microbiological activity and chemical oxidation of lipids. However, microbial spoilage is the main factor linked with the rapid fish sensorial degradation due to the action of specific spoilage organisms (SSOs) that have the ability to dominate over other microorganisms and produce metabolites responsible for off-flavours. We explored the microbial dynamics in fresh anchovies stored in different packaging (air, modified atmosphere, under vacuum) and temperatures (0, 4 and 10 °C) using shotgun metagenomics, highlighting the selection of different microbial species according to the packaging type. Indeed, Pseudoalteromonas nigrifaciens, Psychrobacter cryohalolentis and Ps. immobilis, Pseudomonas deceptionensis and Vibrio splendidus have been identified as the main SSOs in aerobically stored anchovies, while Shewanella baltica, Photobacterium iliopiscarium, Ps. cryohalolentis and Ps. immobilis prevailed in VP and MAP. In addition, we identified the presence of spoilage-associated genes, leading to the potential production of biogenic amines and different off-flavors (H2S, TMA). In particular, the abundance of microbial genes leading to BA biosynthesis increased at higher storage temperature, while those related to H2S and TMA production were enriched in aerobically and VP packed anchovies, suggesting that MAP could be an effective strategy in delaying the production of these compounds. Finally, we provided evidence of the presence of a wide range of antibiotic resistance genes conferring resistance to different classes of antibiotic (ß-lactams, tetracyclines, polymyxins, trimethoprims and phenicols) and highlighted that storage at higher temperature (4 and 10 °C) boosted the abundance of ARG-carrying taxa, especially in aerobically and MAP packed fish.

Limosilactobacillus reuteri DSM 17938 reverses gut metabolic dysfunction induced by Western diet in adult rats.

Introduction: Microencapsulation of probiotic bacteria is an efficient and innovative new technique aimed at preserving bacterial survival in the hostile conditions of the gastrointestinal tract. However, understanding whether a microcapsule preserves the effectiveness of the bacterium contained within it is of fundamental importance. Methods: Male Wistar rats aged 90 days were fed a control diet or a Western diet for 8 weeks, with rats fed the Western diet divided into three groups: one receiving the diet only (W), the second group receiving the Western diet and free L. reuteri DSM 17938 (WR), and the third group receiving the Western diet and microencapsulated L. reuteri DSM 17938 (WRM). After 8 weeks of treatment, gut microbiota composition was evaluated, together with occludin, one of the tight junction proteins, in the ileum and the colon. Markers of inflammation were also quantified in the portal plasma, ileum, and colon, as well as markers for gut redox homeostasis. Results: The Western diet negatively influenced the intestinal microbiota, with no significant effect caused by supplementation with free and microencapsulated L. reuteri. However, L. reuteri, in both forms, effectively preserved the integrity of the intestinal barrier, thus protecting enterocytes from the development of inflammation and oxidative stress. Conclusion: From these whole data, it emerges that L. reuteri DSM 17938 can be an effective probiotic in preventing the unhealthy consequences of the Western diet, especially in the gut, and that microencapsulation preserves the probiotic effects, thus opening the formulation of new preparations to be able to improve gut function independent of dietary habits.

The chemistry of gut microbiome-derived lipopolysaccharides impacts on the occurrence of food allergy in the pediatric age.

Introduction: Food allergy (FA) in children is a major health concern. A better definition of the pathogenesis of the disease could facilitate effective preventive and therapeutic measures. Gut microbiome alterations could modulate the occurrence of FA, although the mechanisms involved in this phenomenon are poorly characterized. Gut bacteria release signaling byproducts from their cell wall, such as lipopolysaccharides (LPSs), which can act locally and systemically, modulating the immune system function. Methods: In the current study gut microbiome-derived LPS isolated from fecal samples of FA and healthy children was chemically characterized providing insights into the carbohydrate and lipid composition as well as into the LPS macromolecular nature. In addition, by means of a chemical/MALDI-TOF MS and MS/MS approach we elucidated the gut microbiome-derived lipid A mass spectral profile directly on fecal samples. Finally, we evaluated the pro-allergic and pro-tolerogenic potential of these fecal LPS and lipid A by harnessing peripheral blood mononuclear cells from healthy donors. Results: By analyzing fecal samples, we have identified different gut microbiome-derived LPS chemical features comparing FA children and healthy controls. We also have provided evidence on a different immunoregulatory action elicited by LPS on peripheral blood mononuclear cells collected from healthy donors suggesting that LPS from healthy individuals could be able to protect against the occurrence of FA, while LPS from children affected by FA could promote the allergic response. Discussion: Altogether these data highlight the relevance of gut microbiome-derived LPSs as potential biomarkers for FA and as a target of intervention to limit the disease burden.

Extension of the Segatella copri complex to 13 species with distinct large extrachromosomal elements and associations with host conditions.

The Segatella copri (formerly Prevotella copri) complex (ScC) comprises taxa that are key members of the human gut microbiome. It was previously described to contain four distinct phylogenetic clades. Combining targeted isolation with large-scale metagenomic analysis, we defined 13 distinct Segatella copri-related species, expanding the ScC complex beyond four clades. Complete genome reconstruction of thirteen strains from seven species unveiled the presence of genetically diverse large circular extrachromosomal elements. These elements are consistently present in most ScC species, contributing to intra- and inter-species diversities. The nine species-level clades present in humans display striking differences in prevalence and intra-species genetic makeup across human populations. Based on a meta-analysis, we found reproducible associations between members of ScC and the male sex and positive correlations with lower visceral fat and favorable markers of cardiometabolic health. Our work uncovers genomic diversity within ScC, facilitating a better characterization of the human microbiome.

Plastics shape the black soldier fly larvae gut microbiome and select for biodegrading functions.

BACKGROUND: In the last few years, considerable attention has been focused on the plastic-degrading capability of insects and their gut microbiota in order to develop novel, effective, and green strategies for plastic waste management. Although many analyses based on 16S rRNA gene sequencing are available, an in-depth analysis of the insect gut microbiome to identify genes with plastic-degrading potential is still lacking. RESULTS: In the present work, we aim to fill this gap using Black Soldier Fly (BSF) as insect model. BSF larvae have proven capability to efficiently bioconvert a wide variety of organic wastes but, surprisingly, have never been considered for plastic degradation. BSF larvae were reared on two widely used plastic polymers and shotgun metagenomics was exploited to evaluate if and how plastic-containing diets affect composition and functions of the gut microbial community. The high-definition picture of the BSF gut microbiome gave access for the first time to the genomes of culturable and unculturable microorganisms in the gut of insects reared on plastics and revealed that (i) plastics significantly shaped bacterial composition at species and strain level, and (ii) functions that trigger the degradation of the polymer chains, i.e., DyP-type peroxidases, multicopper oxidases, and alkane monooxygenases, were highly enriched in the metagenomes upon exposure to plastics, consistently with the evidences obtained by scanning electron microscopy and 1H nuclear magnetic resonance analyses on plastics. CONCLUSIONS: In addition to highlighting that the astonishing plasticity of the microbiota composition of BSF larvae is associated with functional shifts in the insect microbiome, the present work sets the stage for exploiting BSF larvae as "bioincubators" to isolate microbial strains and enzymes for the development of innovative plastic biodegradation strategies. However, most importantly, the larvae constitute a source of enzymes to be evolved and valorized by pioneering synthetic biology approaches. Video Abstract.

Psychrotrophic Bacteria Equipped with Virulence and Colonization Traits Populate the Ice Cream Manufacturing Environment.

Several microbial taxa have been associated with food processing facilities, and they might resist by attaching on tools and equipment even after sanitation procedures, producing biofilms that adhere to the surfaces and might embed other microorganisms, including spoilers and pathogens. There is increasing evidence that these communities can be transferred to the final product. To explore the microbial contamination routes in a facility producing ice creams, we collected foods and environmental swabs from industrial surfaces of equipment and tools and performed taxonomic and functional analyses of the microbial DNA extracted from the environmental samples. Our results suggest that complex communities dominated by psychrotrophic bacteria (e.g., Pseudomonas and Acinetobacter spp.) inhabit the food processing environment, and we demonstrate that these communities might be transferred from the surfaces to the products. Functional analysis performed on environmental samples highlighted the presence of several genes linked to antimicrobial resistance and adherence on abiotic surfaces; such genes were more abundant on food contact (FC) than on other surfaces. Metagenome-assembled genomes (MAGs) of Pseudomonas stutzeri showed genes linked with biofilm formation and motility, which are surely linked to colonizing capabilities in the processing lines. The study highlights clear potential advantages of applying microbiome mapping in the food industry for source tracking of microbial contamination and for planning appropriate ad hoc sanitization strategies. IMPORTANCE Several microbial species might permanently establish in food processing facilities, thus contributing to food loss. In fact, food contact surfaces might transfer microorganisms to intermediates and products, potentially representing a hazard to human health. In this work, we provide evidence of the existence of complex microbial communities overcoming sanitation in an ice cream-producing facility. These communities harbored several genes that could potentially lead to attachment to surfaces and antimicrobial resistance. Also, prediction of routes of contamination showed that several potential spoilage taxa might end up in the final product. Importantly, in this work, we show that mapping the environmental microbiome is a high-resolution technique that might help food business operators ensure food quality and safety through detection of potentially hazardous microorganisms.

Milk protein digestion and the gut microbiome influence gastrointestinal discomfort after cow milk consumption in healthy subjects.

Many healthy people suffer from milk-related gastrointestinal discomfort (GID) despite not being lactose intolerant; the mechanisms underpinning such condition are unknown. This study aimed to explore milk protein digestion and related physiological responses (primary outcome), gut microbiome and gut permeability in 19 lactose-tolerant healthy nonhabitual milk consumers [NHMCs] reporting GID after consuming cow milk compared to 20 habitual milk consumers [HMCs] without GID. NHMCs and HMCs participated in a milk-load (250 mL) test, underwent blood sample collection at 6 time points over 6 h after milk consumption and collected urine samples and GID self-reports over 24 h. We measured the concentrations of 31 milk-derived bioactive peptides (BAPs), 20 amino acids, 4 hormones, 5 endocannabinoid system mediators, glucose and the dipeptidyl peptidase-IV (DPPIV) activity in blood and indoxyl sulfate in urine samples. Subjects also participated in a gut permeability test and delivered feces sample for gut microbiome analysis. Results showed that, compared to HMCs, milk consumption in NHMCs, along with GID, elicited a slower and lower increase in circulating BAPs, lower responses of ghrelin, insulin, and anandamide, a higher glucose response and serum DPPIV activity. The gut permeability of the two groups was similar, while the habitual diet, which was lower in dairy products and higher in the dietary-fibre-to-protein ratio in NHMCs, possibly shaped the gut microbiome; NHMCs exhibited lower abundance of Bifidobacteria, higher abundance of Prevotella and lower abundance of protease-coding genes, which may have reduced protein digestion, as evidenced by lower urinary excretion of indoxyl sulfate. In conclusion, the findings showed that a less efficient digestion of milk proteins, supported by a lower proteolytic capability of the gut microbiome, may explain GID in healthy people after milk consumption.

Determination of the effect of the bacteriocin enterocin AS-48 on the microbial loads and bacterial diversity of blueberries.

Fresh fruits have been involved in transmission of foodborne pathogens. In the present work, five different batches of blueberries were used. One aliquot from each batch was washed with sterile saline solution (SSS) and the other one with a solution of the circular bacteriocin enterocin AS-48 in SSS. Then, the surface microbiota of controls and bacteriocin-treated samples was recovered and used for microbiota analyses, both using viable counts and high-throughput amplicon sequencing. Total aerobic mesophilic loads ranged from 2.70 to 4.09 log CFU/g in most of the samples. Only two samples yielded detectable viable counts on selective media (Enterobacteriaceae, presumptive Salmonella and coliforms), with values ranging from 2.84 to 3.81 log CFU/g. The bacteriocin treatment reduced viable cell counts of total aerobic mesophiles to a range of 1.40-1.88 log CFU/g. No viable cells were detected on selective media. Amplicon sequencing indicated large batch-to-batch variations in the surface microbiota of blueberries and also an effect of the bacteriocin treatment on microbiota composition.

Impact of oral antiviral therapy against HCV on gut microbiota. A prospective study.

The intestinal microbiota plays a fundamental role in physiological homeostasis as well as in pathologic conditions. Hepatitis C virus is the leading cause of chronic liver diseases worldwide. The treatment of this infection has been revolutionized by the availability of direct-acting antiviral agents which guarantee a high rate (about 95%) of viral clearance. Few studies have assessed the change in the gut microbiota of patients treated with direct-acting antiviral agents against HCV, and many aspects still need to be clarified. The aim of the study was to evaluate the effects of antiviral therapy on gut microbiota. We enrolled patients with HCV-related chronic liver disease attending the Infectious Diseases Unit of the A.O.U. Federico II of Naples from January 2017 to March 2018 and treated with DAAs. For each patient, a fecal sample was collected and analyzed for the assessment of microbial diversity before the start of therapy and by SVR12 time. We excluded patients who had received antibiotics in the previous 6 months. Twelve patients were enrolled (6 male, 8 genotype 1 (1 subtype 1a), 4 genotype 2). Fibrosis scores were F0 in 1 patient, F2 in 1 patient, F3 in 4 patients and cirrhosis in the remaining 6 (all in Child-Pugh class A). All were treated with DAAs for 12 weeks (5 with Paritaprevir-Ombitasvir-Ritonavir-Dasabuvir, 3 with Sofosbuvir-Ledipasvir, 1 with Sofosbuvir-Ribavirin, 1 with Sofosbuvir-Daclatasvir, 1 with Sofosbuvir-Velpatasvir) and 100% achieved SVR12. In all patients, we observed a trend in reduction of potentially pathogenic microorganisms (i.e., Enterobacteriaceae). Furthermore, a trend of increase in α-diversity was observed in patients by SVR12 compared to baseline. This trend was markedly more evident in patients without liver cirrhosis than in those with cirrhosis. Our study shows that viral eradication obtained with DAA is associated with a trend in restoring the heterogeneity of α-diversity and in reducing the percentage of potentially pathogenic microbial species, although this benefit is less evident in patients with cirrhosis. Further studies with larger sample size are needed to confirm these data.

Therapeutic Effects of Butyrate on Pediatric Obesity: A Randomized Clinical Trial.

Importance: The pediatric obesity disease burden imposes the necessity of new effective strategies. Objective: To determine whether oral butyrate supplementation as an adjunct to standard care is effective in the treatment of pediatric obesity. Design, Setting, and Participants: A randomized, quadruple-blind, placebo-controlled trial was performed from November 1, 2020, to December 31, 2021, at the Tertiary Center for Pediatric Nutrition, Department of Translational Medical Science, University of Naples Federico II, Naples, Italy. Participants included children aged 5 to 17 years with body mass index (BMI) greater than the 95th percentile. Interventions: Standard care for pediatric obesity supplemented with oral sodium butyrate, 20 mg/kg body weight per day, or placebo for 6 months was administered. Main Outcomes and Measures: The main outcome was the decrease of at least 0.25 BMI SD scores at 6 months. The secondary outcomes were changes in waist circumference; fasting glucose, insulin, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglyceride, ghrelin, microRNA-221, and interleukin-6 levels; homeostatic model assessment of insulin resistance (HOMA-IR); dietary and lifestyle habits; and gut microbiome structure. Intention-to-treat analysis was conducted. Results: Fifty-four children with obesity (31 girls [57%], mean [SD] age, 11 [2.91] years) were randomized into the butyrate and placebo groups; 4 were lost to follow-up after receiving the intervention in the butyrate group and 2 in the placebo group. At intention-to-treat analysis (n = 54), children treated with butyrate had a higher rate of BMI decrease greater than or equal to 0.25 SD scores at 6 months (96% vs 56%, absolute benefit increase, 40%; 95% CI, 21% to 61%; P < .01). At per-protocol analysis (n = 48), the butyrate group showed the following changes as compared with the placebo group: waist circumference, -5.07 cm (95% CI, -7.68 to -2.46 cm; P < .001); insulin level, -5.41 µU/mL (95% CI, -10.49 to -0.34 µU/mL; P = .03); HOMA-IR, -1.14 (95% CI, -2.13 to -0.15; P = .02); ghrelin level, -47.89 µg/mL (95% CI, -91.80 to -3.98 µg/mL; P < .001); microRNA221 relative expression, -2.17 (95% CI, -3.35 to -0.99; P < .001); and IL-6 level, -4.81 pg/mL (95% CI, -7.74 to -1.88 pg/mL; P < .001). Similar patterns of adherence to standard care were observed in the 2 groups. Baseline gut microbiome signatures predictable of the therapeutic response were identified. Adverse effects included transient mild nausea and headache reported by 2 patients during the first month of butyrate intervention. Conclusions and Relevance: Oral butyrate supplementation may be effective in the treatment of pediatric obesity. Trial Registration: ClinicalTrials.gov Identifier: NCT04620057.

Evidence of virulence and antibiotic resistance genes from the microbiome mapping in minimally processed vegetables producing facilities.

Daily consumption of fresh vegetables is highly recommended by international health organizations, because of their high content of nutrients. However, fresh vegetables might harbour several pathogenic microorganisms or contribute to spread antibiotic resistance, thus representing a hazard for consumers. In addition, little is known about the transmission routes of the residential microbiome from the food handling environment to vegetables. Therefore, we collected environmental and food samples from three manufactures producing fresh vegetables to estimate the relevance of the built environment microbiome on that of the finished products. Our results show that food contact surfaces sampled after routine cleaning and disinfection procedures host a highly diverse microbiome, including pathogens such as the enterotoxigenic Bacillus cereus sensu stricto. In addition, we provide evidence of the presence of a wide range of antibiotic resistance and virulence genes on food contact surfaces associated with multiple taxa, thus supporting the hypothesis that selection of resistant and pathogenic taxa might occur on sanitized surfaces. This study also highlights the potential of microbiome mapping routinely applied in food industries monitoring programs to ensure food safety.

Effects of the Mediterranean Diet during pregnancy on the onset of allergy in at risk children: A study protocol of a multi-center, randomized- controlled, parallel groups, prospective trial (the PREMEDI study).

Introduction: Maternal diet during pregnancy has been linked to offspring allergy risk and it could represent a potential target for allergy prevention. The Mediterranean Diet (MD) is considered one of the healthiest dietary models. Randomized-controlled trials on the effect of MD in preventing pediatric allergic diseases are still needed. Methods and analysis: The Mediterranean Diet during Pregnancy study (PREMEDI) will be a 9-month multi-center, randomized-controlled, parallel groups, prospective trial. Healthy women (20-35 years) at their first trimester of pregnancy at risk for atopy baby, will be randomly allocated to Group 1 (standard obstetrical and gynecological follow-up and nutritional counseling to promote MD) or Group 2 (standard obstetrical and gynecological follow-up alone). 138 mother-child pair per group will be needed to detect a reduction in cumulative incidence of ≥1 allergic disease at 24 months of age. The primary study aim will be the evaluation of the occurrence of allergic disorders in the first 24 months of life. The secondary aims will be the evaluation of maternal weight gain, pregnancy/perinatal complications, growth indices and occurrence of other chronic disorders, mother-child pair adherence to MD and gut microbiome features, breastfeeding duration and breast milk composition, epigenetic modulation of genes involved in immune system, and metabolic pathways in the offspring. Ethics and dissemination: The study protocol has been approved by the Ethics Committee of the University of Naples Federico II (number 283/21) and it will be conducted in accordance with the Helsinki Declaration (Fortaleza revision, 2013), the Good Clinical Practice Standards (CPMP/ICH/135/95), the Italian Decree-Law 196/2003 regarding personal data and the European regulations on this subject. The study has been registered in the Clinical Trials Protocol Registration System. Clinical trial registration: [http://clinicaltrials.gov], identifier [NCT05119868].

Stuck or sluggish fermentations in home-made beers: Beyond the surface.

In the last several years, the popularity of homebrewed beers has skyrocketed. However, this type of product is extremely vulnerable to microbial deterioration. Twelve homemade beers, some characterized by defects or stuck fermentation, were analysed by using a polyphasic approach encompassing culturomics and culture-independent techniques to better understand mechanisms that drive microbiota evolution throughout production and to highlight determinants responsible for crowning with success. Two sour beers, one apple-flavoured ale, two Italian grape ales, and seven standard ales were sampled. Microbiological characterization was obtained by plating on nine different media coupled with High-throughput sequencing analysis of fungal and bacterial communities by targeting ITS1-2 and the V3-V4 regions of the 16S rRNA, respectively. Total microflora on PCA largely varied among samples, ranging from <102 CFU/mL up to around 107 CFU/mL often reflecting yeast counts on WL and LM. LAB population's levels on MRS and SDBm did not overlap, with the counts on the latter being even 5 Log CFU/mL greater. Acetic Acid bacteria were retrieved in Sour beers, as well as in one IGA, even though acetic acid was not detectable by HPLC in this last sample. Brettanomyces spp. were only found in sour beers, as expected, whereas Enterobacteriaceae were never counted. A total of 63 yeasts were randomly isolated from countable plates. Saccharomyces cerevisiae and Wickerhamomyces anomalus were the most frequently isolated species. In many cases, Interdelta analysis biotyping of S. cerevisiae isolates consistently allowed the detection of the starter strain. By HST S. cerevisiae dominated the mycobiota in four samples, even if in one of them residual maltose and ethanol contents suggested a stuck fermentation. W. anomalus was found to be the dominant species in two beers. Fifty-five LAB cultures were isolated and identified. Pediococcus damnosus was the only species retrieved in sour beers and two Ales, while Levilactobacillus brevis was found in two Ale samples. HTS did not confirm this result in one Ale sample since the genus Panotea spp. accounted for over 90 % of the microbiota. Enterobacteriaceae which were never counted dominated the microbiome of two Ale beers. Biogenic amines content largely varied with three Ale samples greatly contaminated. Based on chemical and microbiological outcomes only one beer ASAle out of 12 could be considered acceptable. Furthermore, the widespread presence of LAB by culturomics and Enterobacteriaceae by HTS raises concerns about the final products' safety.

Therapeutic effects elicited by the probiotic Lacticaseibacillus rhamnosus GG in children with atopic dermatitis. The results of the ProPAD trial.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 20% of the pediatric population associated with alteration of skin and gut microbiome. Probiotics have been proposed for AD treatment. The ProPAD study aimed to investigate the therapeutic effects of the probiotic Lacticaseibacillus rhamnosus GG (LGG) in children with AD. METHODS: In total, 100 AD patients aged 6-36 months were enrolled in a randomized, double-blind, controlled trial to receive placebo (Group A) or LGG (1 x 1010 CFU/daily) (Group B) for 12 weeks. The primary outcome was the evaluation of the efficacy of LGG supplementation on AD severity comparing the Scoring Atopic Dermatitis (SCORAD) index at baseline (T0) and at 12-week (T12). A reduction of ≥8.7 points on the SCORAD index was considered as minimum clinically important difference (MCID). The secondary outcomes were the SCORAD index evaluation at 4-week (T16) after the end of LGG treatment, number of days without rescue medications, changes in Infant Dermatitis Quality Of Life questionnaire (IDQOL), gut microbiome structure and function, and skin microbiome structure. RESULTS: The rate of subjects achieving MCID at T12 and at T16 was higher in Group B (p < .05), and remained higher at T16 (p < .05)The number of days without rescue medications was higher in Group B. IDQOL improved at T12 in the Group B (p < .05). A beneficial modulation of gut and skin microbiome was observed only in Group B patients. CONCLUSIONS: The probiotic LGG could be useful as adjunctive therapy in pediatric AD. The beneficial effects on disease severity and quality of life paralleled with a beneficial modulation of gut and skin microbiome.

Specific Microbial Communities Are Selected in Minimally-Processed Fruit and Vegetables according to the Type of Product.

Fruits and vegetables (F&V) products are recommended for the daily diet due to their low caloric content, high amount of vitamins, minerals and fiber. Furthermore, these foods are a source of various phytochemical compounds, such as polyphenols, flavonoids and sterols, exerting antioxidant activity. Despite the benefits derived from eating raw F&V, the quality and safety of these products may represent a source of concern, since they can be quickly spoiled and have a very short shelf-life. Moreover, they may be a vehicle of pathogenic microorganisms. This study aims to evaluate the bacterial and fungal populations in F&V products (i.e., iceberg lettuces, arugula, spinaches, fennels, tomatoes and pears) by using culture-dependent microbiological analysis and high-throughput sequencing (HTS), in order to decipher the microbial populations that characterize minimally-processed F&V. Our results show that F&V harbor diverse and product-specific bacterial and fungal communities, with vegetables leaf morphology and type of edible fraction of fruits exerting the highest influence. In addition, we observed that several alterative (e.g., Pseudomonas and Aspergillus) and potentially pathogenic taxa (such as Staphylococcus and Cladosporium) are present, thus emphasizing the need for novel product-specific strategies to control the microbial composition of F&V and extend their shelf-life.

Omics-based monitoring of microbial dynamics across the food chain for the improvement of food safety and quality.

The diffusion of high-throughput sequencing has dramatically changed the study of food microbial ecology. Amplicon-based description of the microbial community may be routinary implemented in the food industry to understand how the processing parameters and the raw material quality may affect the microbial community of the final product, as well as how the community changes during the shelf-life. In addition, application of shotgun metagenomics may represent an invaluable resource to understand the functional potential of the microbial community, identifying the presence of spoilage-associated activities or genes related to pathogenesis. Finally, retrieving Metagenome-Assembled Genomes (MAGs) of relevant species may be useful for strain-tracking along the food chain and in case of food poisoning outbreaks. This review gives an overview of the possible applications of sequencing-based approaches in the study of food microbial ecology, highlighting limitations that still prevent the spreading of these techniques to the food industry.