Microbiome mapping in beef processing reveals safety-relevant variations in microbial diversity and genomic features.

The microbiome of surfaces along the beef processing chain represents a critical nexus where microbial ecosystems play a pivotal role in meat quality and safety of end products. This study offers a comprehensive analysis of the microbiome along beef processing using whole metagenomics with a particular focus on antimicrobial resistance and virulence-associated genes distribution. Our findings highlighted that microbial communities change dynamically in the different steps along beef processing chain, influenced by the specific conditions of each micro-environment. Brochothrix thermosphacta, Carnobacterium maltaromaticum, Pseudomonas fragi, Psychrobacter cryohalolentis and Psychrobacter immobilis were identified as the key species that characterize beef processing environments. Carcass samples and slaughterhouse surfaces exhibited a high abundance of antibiotic resistance genes (ARGs), mainly belonging to aminoglycosides, ß-lactams, amphenicols, sulfonamides and tetracyclines antibiotic classes, also localized on mobile elements, suggesting the possibility to be transmitted to human pathogens. We also evaluated how the initial microbial contamination of raw beef changes in response to storage conditions, showing different species prevailing according to the type of packaging employed. We identified several genes leading to the production of spoilage-associated compounds, and highlighted the different genomic potential selected by the storage conditions. Our results suggested that surfaces in beef processing environments represent a hotspot for beef contamination and evidenced that mapping the resident microbiome in these environments may help in reducing meat microbial contamination, increasing shelf-life, and finally contributing to food waste restraint.

Exposure to environmental pollutants selects for xenobiotic-degrading functions in the human gut microbiome.

Environmental pollutants from different chemical families may reach the gut microbiome, where they can be metabolized and transformed. However, how our gut symbionts respond to the exposure to environmental pollution is still underexplored. In this observational, cohort study, we aim to investigate the influence of environmental pollution on the gut microbiome composition and potential activity by shotgun metagenomics. We select as a case study a population living in a highly polluted area in Campania region (Southern Italy), proposed as an ideal field for exposomic studies and we compare the fecal microbiome of 359 subjects living in areas with high, medium and low environmental pollution. We highlight changes in gut microbiome composition and functionality that were driven by pollution exposure. Subjects from highly polluted areas show higher blood concentrations of dioxin and heavy metals, as well as an increase in microbial genes related to degradation and/or resistance to these molecules. Here we demonstrate the dramatic effect that environmental xenobiotics have on gut microbial communities, shaping their composition and boosting the selection of strains with degrading capacity. The gut microbiome can be considered as a pivotal player in the environment-health interaction that may contribute to detoxifying toxic compounds and should be taken into account when developing risk assessment models. The study was registered at ClinicalTrials.gov with the identifier NCT05976126.

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.

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.

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.

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.