Effective cleaning and sanitation are essential in cheese manufacturing to control microbial contamination and prevent biofilm formation. A multi-step approach was undertaken to assess microbial persistence and cleaning-in-place (CIP) strategies at an industrial Cheddar cheese facility with a production capacity of similar to 40,000 tonnes. Pre- and post-CIP environmental swabs were analysed using plate counts and high-throughput (16S rRNA amplicon and shotgun) sequencing to identify microbial hotspots. Traditional CIP was broadly effective, however residual contamination persisted in critical zones such as the Alfomatic weir, curd distribution tank (CDT), and block formers. Industrial-scale trials comparing caustic and acid-based CIP with an enzymatic alternative revealed complete microbial elimination post traditional CIP, while enzymatic CIP left DNA signatures at one site, culturable cells were not detected. Sequencing identified Pseudomonas fluorescens as the dominant non-starter bacterium, suggesting possible biofilm formation. Parallel laboratory-scale studies of P. fluorescens biofilm removal using a CDC Biofilm Reactor (R) coupled with Scanning Electron Microscopy (SEM) compared caustic and enzymatic CIP. Caustic treatment completely removed the biofilm matrix but left adherent cells, while enzymatic treatments degraded the matrix but showed residual matrix fragments and adherent cells. Plate counts confirmed significant differences in residual biofilm populations, with average reductions of 3.75 log, 2.56 log, and 1.87 log after caustic, enzyme cocktail, and protease treatments, respectively (p < 0.0001), underscoring the incomplete nature of these approaches. Caustic CIP improves microbial reduction, while enzymes disrupt biofilm matrices. However, neither alone achieves complete removal, supporting combined strategies to enhance cleaning efficacy and sustainability in dairy processing.

Metabolic health improvements in response to exercise and energy restriction may be mediated by the gut microbiome, yet causal evidence in humans remains limited. We used a 3-week exercise and energy restriction intervention to examine changes to the gut microbiome in otherwise healthy sedentary men and postmenopausal women with overweight/obesity. Intervention participants (n = 18) reduced habitual energy intake by 5000 kcal/week and expended 2000 kcal/week in addition to habitual physical activity through treadmill walking at 70% V̇O2Peak. Control participants (n = 12) maintained their usual lifestyle. Participants underwent dual-energy X-ray absorptiometry (DEXA), and samples of faeces, fasted venous blood, subcutaneous adipose tissue and skeletal muscle were collected. Faecal DNA was sequenced and profiled using shotgun metagenomics, Kraken2/Bracken and Human Microbiome Project Unified Metabolic Analysis Network 2 (HUMAnN2). The intervention significantly reduced body mass (mean Δ ± SD: -2.6 ± 1.5 kg), fat mass (-1.5 ± 1.3 kg), fasted insulin (-23.5 ± 38.1 pmol/l), leptin (-10.6 ± 7.3 ng/ml) and total cholesterol (-0.70 ± 0.42 mmol/l) concentrations, and also improved insulin sensitivity (HOMA2%S (homeostatic model of assessment)). Despite these significant metabolic changes the gut microbiome was unchanged in terms of α and β diversity and relative abundance. Thus, despite clinically meaningful improvements in body composition and metabolic health, we found no evidence for changes to the gut microbiome. In conclusion early metabolic changes with weight loss in humans are unlikely to be mediated by changes to the gut microbiome. KEY POINTS: Changes to the gut microbiome could contribute to metabolic improvements associated with weight loss in humans, but there have been limited attempts to address this question using robust randomised controlled trials (RCTs). We used a parallel-group RCT to examine whether a 3-week combined energy intake restriction and vigorous-intensity exercise intervention in people with overweight and obesity was temporally associated with changes to gut microbiome taxonomic composition and functional potential, short-chain fatty acid concentrations and expression of genes related to host-microbiome interactions in skeletal muscle and subcutaneous adipose tissue. We found that the human gut microbiome remains unchanged in the face of an intensive energy intake restriction and vigorous exercise intervention that significantly improved body composition and metabolic health in people with overweight/obesity. These findings indicate that early metabolic changes with weight loss in humans are unlikely to be mediated by changes to the gut microbiome.

Feeding waste milk, a common practice in dairy farming, exposes calves to subtherapeutic levels of antimicrobials, potentially contributing to antibiotic resistance-a growing concern globally. Many dairy farmers, including those in Ireland, continue this practice, feeding waste milk from antibiotic-treated cows to calves. Although previous studies have linked waste milk feeding to changes in calf growth and health during the preweaning period, its effects postweaning remain unclear. This study examined how the duration of antimicrobial exposure at levels equivalent to those found in waste milk influences health and growth outcomes of dairy heifer calves both before and after weaning. It also assessed the prevalence of extended-spectrum β-lactamase (ESBL)-producing antimicrobial-resistant Escherichia coli in feces and changes in the fecal microbiota over time. To mimic waste milk, as derived from a cow treated with an intramammary suspension of antibiotics, a simulated waste milk (SWM) was prepared by adding amoxicillin (1.68 mg/L) and neomycin (2.28 mg/L) to a conventional milk replacer (MR). The study employed a randomized block design with 87 dairy heifer calves assigned to 1 of 3 treatments: (1) long-term antibiotic (LTA), with calves fed SWM until weaning at 12 wk; (2) short-term antibiotic (STA), with SWM fed from 3 to 5 wk; and (3) control (CONT), with calves fed antibiotic-free MR. Calves were weighed weekly, and health scores, including fecal scores (tail and hindquarters cleanliness as diarrhea indicator), were recorded twice per week. Fecal and blood samples were collected to analyze microbiome changes and the shedding of antimicrobial resistance. Blood samples were taken to measure systemic inflammation, using serum amyloid A as a biomarker. Results indicated that SWM feeding did not affect average daily gains before or after weaning. However, higher fecal scores were observed in the LTA group during weaning and after weaning in the STA group. Antibiotic-resistant isolates were present in all groups, with the highest prevalence in LTA. Fecal microbiota analysis revealed treatment-specific microbial community variations, with an increase of Enterococcus faecium genes resistant to macrolide, aminoglycoside, and tetracycline antibiotics in LTA and STA compared with CONT. In summary, SWM feeding did not significantly affect growth or overall health, but it was associated with increased fecal shedding of resistant bacteria and some changes in the microbiota, indicating potential long-term implications for antimicrobial resistance in dairy herds.

Background: Iron deficiency is the top leading cause of anaemia, whose treatment has been shown to deteriorate gut health. However, a comprehensive analysis of the intestinal barrier and the gut microbiome during iron deficiency anemia (IDA) has not been performed to date. This study aims to delve further into the analysis of these two aspects, which will mean a step forward minimising the negative impact of iron supplements on intestinal health. Methods: IDA was experimentally induced in an animal model. Shotgun sequencing was used to analyse the gut microbiome in the colonic region, while the intestinal barrier was studied through histological analyses, mRNA sequencing (RNA-Seq), qPCR and immunofluorescence assays. Determinations of lipopolysaccharide (LPS) and bacteria-specific immunoglobulins were performed to assess microbial translocation. Results: Microbial metabolism in the colon shifted towards an increased production of certain amino acids, short chain fatty acids and nucleotides, with Clostridium species being enriched during IDA. Structural alterations of the colonic epithelium were shown by histological analysis. RNA-Seq revealed a downregulation of extracellular matrix-associated genes and proteins and an overall underdeveloped epithelium. Increased levels of serum LPS and an increased immune response against dysbiotic bacteria support an impairment in the integrity of the gut barrier during IDA. Conclusions: IDA negatively impacts the gut microbiome and the intestinal barrier, triggering an increased microbial translocation. This study emphasizes the deterioration of gut health during IDA and the fact that it should be addressed when treating the disease.

Restricted sugar and ketogenic diets can alter energy balance/metabolism, but decreased energy intake may be compensated by reduced expenditure. In healthy adults, randomization to restricting free sugars or overall carbohydrates (ketogenic diet) for 12 weeks reduces fat mass without changing energy expenditure versus control. Free-sugar restriction minimally affects metabolism or gut microbiome but decreases low- density lipoprotein cholesterol (LDL-C). In contrast, a ketogenic diet decreases glucose tolerance, increases skeletal muscle PDK4, and reduces AMPK and GLUT4 levels. By week 4, the ketogenic diet reduces fasting glucose and increases apolipoprotein B, C-reactive protein, and postprandial glycerol concentrations. However, despite sustained ketosis, these effects are no longer apparent by week 12, when gut microbial beta diversity is altered, possibly reflective of longer-term adjustments to the ketogenic diet and/or energy balance. These data demonstrate that restricting free sugars or overall carbohydrates reduces energy intake without altering physical activity, but with divergent effects on glucose tolerance, lipoprotein profiles, and gut microbiome.

Alterations in the human Gut Bacteriome (GB) can be associated with human health issues, such as type-2 diabetes and obesity. Both external and internal factors can drive changes in the composition and in interactions of the human GB, impacting negatively on the host cells. This paper focuses on the human GB metabolism and proposes a two-layer network system to investigate its dynamics. Furthermore, we develop an in-silico simulation model (virtual GB), allowing us to study the impact of the metabolite exchange through molecular communications in the human GB network system. Our results show that regulation of molecular inputs strongly affects bacterial population growth and creates an unbalanced network, as shown by shifts in the node weights based on the produced molecular signals. Additionally, we show that the metabolite molecular communication production is greatly affected when directly manipulating the composition of the human GB network in the virtual GB. These results indicate that our human GB interaction model can help to identify hidden behaviours of the human GB depending on molecular signal interactions. Moreover, the virtual GB can support the research and development of novel medical treatments based on the accurate control of bacterial population growth and exchange of metabolites.

Parkinson's disease (PD) is a chronic neurological disorder associated with the misfolding of alpha-synuclein (α-syn) into aggregates within nerve cells that contribute to their neurodegeneration. Recent evidence suggests α-syn aggregation may begin in the gut and travel to the brain along the vagus nerve, with microbes potentially a trigger initiating α-syn misfolding. However, the effects α-syn alterations on the gut virome have not been investigated. In this study, we show longitudinal faecal virome changes in rats administered either monomeric or preformed fibrils (PFF) of α-syn directly into their enteric nervous system. Differential changes in rat viromes were observed when comparing monomeric and PFF α-syn, with alterations compounded by the addition of LPS. Changes in rat faecal viromes were observed after one month and did not resolve within the study's five-month observational period. These results suggest that virome alterations may be reactive to host α-syn changes that are associated with PD development.

We investigated how protein quantity (10%-30%) and quality (casein and whey) interact with dietary fat (20%-55%) to affect metabolic health in adult mice. Although dietary fat was the main driver of body weight gain and individual tissue weight, high (30%) casein intake accentuated and high whey intake reduced the negative metabolic aspects of high fat. Jejunum and liver transcriptomics revealed increased intestinal permeability, low-grade inflammation, altered lipid metabolism, and liver dysfunction in casein-fed but not whey-fed animals. These differential effects were accompanied by altered gut size and microbial functions related to amino acid degradation and lipid metabolism. Fecal microbiota transfer confirmed that the casein microbiota increases and the whey microbiota impedes weight gain. These data show that the effects of dietary fat on weight gain and tissue partitioning are further influenced by the quantity and quality of the associated protein, primarily via effects on the microbiota.

The prebiotic activity of a commercially available oat product and a novel oat ingredient, at similar β-glucan loads, was tested using a validated in vitro gut model (M-SHIME®). The novel oat ingredient was tested further at lower β-glucan loads in vitro, while the commercially available oat product was assessed in a randomised, single-blind, placebo-controlled, and cross-over human study. Both approaches focused on healthy individuals with mild hypercholesterolemia. In vitro analysis revealed that both oat products strongly stimulated Lactobacillaceae and Bifidobacteriaceae in the intestinal lumen and the simulated mucus layer, and corresponded with enhanced levels of acetate and lactate with cross-feeding interactions leading to an associated increase in propionate and butyrate production. The in vitro prebiotic activity of the novel oat ingredient remained at lower β-glucan levels, indicating the prebiotic potential of the novel oat product. Finally, the stimulation of Lactobacillus spp. was confirmed during the in vivo trial, where lactobacilli abundance significantly increased in the overall population at the end of the intervention period with the commercially available oat product relative to the control product, indicating the power of in vitro gut models in predicting in vivo response of the microbial community to dietary modulation.

The athlete gut microbiome differs from that of non-athletes in its composition and metabolic function. Short-term fitness improvement in sedentary adults does not replicate the microbiome characteristics of athletes. The objective of this study was to investigate whether sustained fitness improvement leads to pronounced alterations in the gut microbiome. This was achieved using a repeated-measures, case-study approach that examined the gut microbiome of two initially unfit volunteers undertaking progressive exercise training over a 6-month period. Samples were collected every two weeks, and microbiome, metabolome, diet, body composition, and cardiorespiratory fitness data were recorded. Training culminated in both participants completing their respective goals (a marathon or Olympic-distance triathlon) with improved body composition and fitness parameters. Increases in gut microbiota α-diversity occurred with sustained training and fluctuations occurred in response to training events (eg, injury, illness, and training peaks). Participants' BMI reduced during the study and was significantly associated with increased urinary measurements of N-methyl nicotinate and hippurate, and decreased phenylacetylglutamine. These results suggest that sustained fitness improvements support alterations to gut microbiota and physiologically-relevant metabolites. This study provides longitudinal analysis of the gut microbiome response to real-world events during progressive fitness training, including intercurrent illness and injury.

Improved control of dietary confounders, through the use of an objective dietary assessment score, has uncovered further insights into the complex, multifactorial relationship between diet, exercise, the gut microbiome, and metabolism. Each of the models pertaining to diet healthiness, physical exercise, or a combination of both, displayed a distinct metabolic and functional microbial signature.

Fermented foods have been the focus of ever greater interest as a consequence of purported health benefits. Indeed, it has been suggested that the consumption of these foods that help to address the negative consequences of ‘industrialization’ of the human gut microbiota in Western society. However, as the mechanisms via which the microbes in fermented foods improve health are not understood, it is necessary to develop an understanding of the composition and functionality of the fermented food microbiota to better harness desirable traits. Here we considerably expand the understanding of fermented food microbiomes by employing shotgun metagenomic sequencing to provide a comprehensive insight into the microbial composition, diversity and functional potential (including antimicrobial resistance, carbohydrate-degrading and health-associated gene content) of a diverse range of 58 fermented foods from artisanal producers from around the Globe. Food type, i.e., dairy-, sugar- or brine-type fermented foods, was to be the primary driver of microbial composition, with dairy foods found to have the lowest microbial diversity. From the combined dataset, 127 high quality metagenome-assembled genomes (MAGs), including 10 MAGs representing putatively novel species of Acetobacter, Acidisphaera, Gluconobacter, Lactobacillus, Leuconostoc and Rouxiella , were generated. Potential health promoting attributes were more common in fermented foods than non-fermented equivalents, with waterkefirs, sauerkrauts and kvasses containing the greatest numbers of potentially health-associated gene clusters (PHAGCs). Ultimately, this study provides the most comprehensive insight into the microbiomes of fermented foods to date, and yields novel information regarding their relative health-promoting potential. Importance Fermented foods are regaining popularity in Western society due in part to an appreciation of the potential for fermented food microbiota to positively impact on health. Many previous studies have studied fermented microbiota using classical culture-based microbiological methods, older molecular techniques or, where deeper analyses have been performed, have involved a relatively small number of one specific food type. Here, we have used a state-of-the-art shotgun metagenomic approach to investigate 58 different fermented foods of different type and origin. Through this analysis, we were able to identify the differences in the microbiota across these foods, the factors that drove their microbial composition, and the relative potential functional benefits of these microbes. The information provided here will provide significant opportunities for the further optimisation of fermented food production and the harnessing of their health promoting potential.

Distinct microbial communities inhabit individuals as part of the human skin microbiome and are continually shed to the surrounding environment.Microbial communities from 17 skin sites of 10 sexually active cohabiting couples (20 individuals) were sampled to test whether cohabitation impacts an individual's skin microbiome, leading to shared skin microbiota among partner pairs.Amplified 16S rRNA genes of bacteria and archaea from a total of 340 skin swabs were analyzed by high-throughput sequencing, and the results demonstrated that cohabitation was significantly associated with microbial community composition, although this association was greatly exceeded by characteristics of body location and individuality.Random forest modeling demonstrated that the partners could be predicted 86% of the time (P Ͻ 0.001) based on their skin microbiome profiles, which was always greater than combinations of incorrectly matched partners.Cohabiting couples had the most similar overall microbial skin communities on their feet, according to Bray-Curtis distances.In contrast, thigh microbial communities were strongly associated with biological sex rather than cohabiting partner.Additional factors that were associated with the skin microbiome of specific body locations included the use of skin care products, pet ownership, allergies, and alcohol consumption.These baseline data identified links between the skin microbiome and daily interactions among cohabiting individuals, adding to known factors that shape the human microbiome and, by extension, its relation to human health.IMPORTANCE Our work characterizes the influence of cohabitation as a factor influencing the composition of the skin microbiome.Although the body site and sampled individual were stronger influences than other factors collected as metadata in this study, we show that modeling of detected microbial taxa can help with correct identifications of cohabiting partners based on skin microbiome profiles using machine learning approaches.These results show that a cohabiting partner can significantly influence our microbiota.Follow-up studies will be important for investigating the implications of shared microbiota on dermatological health and the contributions of cohabiting parents to the microbiome profiles of their infants.

Although culture-independent techniques have refuted lung sterility in health, controversy about contamination during bronchoscope passage through the upper respiratory tract (URT) has impeded research progress.We sought to establish whether bronchoscopic sampling accurately reflects the lung microbiome in health and to distinguish between two proposed routes of authentic microbial immigration, (i) dispersion along contiguous respiratory mucosa and (ii) subclinical microaspiration.During bronchoscopy of eight adult volunteers without lung disease, we performed seven protected specimen brushings (PSB) and bilateral bronchoalveolar lavages (BALs) per subject.We amplified, sequenced, and analyzed the bacterial 16S rRNA gene V4 regions by using the Illumina MiSeq platform.Rigorous attention was paid to eliminate potential sources of error or contamination, including a randomized processing order and the inclusion and analysis of exhaustive procedural and sequencing control specimens.Indices of mouth-lung immigration (mouth-lung community similarity, bacterial burden, and community richness) were all significantly greater in airway and alveolar specimens than in bronchoscope contamination control specimens, indicating minimal evidence of pharyngeal contamination.Ecological indices of mouth-lung immigration peaked at or near the carina, as predicted for a primary immigration route of microaspiration.Bacterial burden, diversity, and mouth-lung similarity were greater in BAL than PSB samples, reflecting differences in the sampled surface areas.(This study has been registered at ClinicalTrials-.gov under registration no.NCT02392182.

The human microbiome has been identified as having a key role in health and numerous diseases. Trillions of microbial cells and viral particles comprise the microbiome, each representing modifiable working elements of an intricate bioactive ecosystem. The significance of the human microbiome as it relates to human biology has progressed through culture-dependent (for example, media-based methods) and, more recently, molecular (for example, genetic sequencing and metabolomic analysis) techniques. The latter have become increasingly popular and evolved from being used for taxonomic identification of microbiota to elucidation of functional capacity (sequencing) and metabolic activity (metabolomics). This review summarises key elements of the human microbiome and its metabolic capabilities within the context of health and disease.