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ABSTRACT Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and whole-genome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure. IMPORTANCE Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems. We employed a population genomics approach to investigate the genetic diversity and reproductive biology of the maize anthracnose pathogen (Colletotrichum graminicola) in 14 countries. We found that the populations are correlated with their geographical origin and that migration between countries is ongoing, possibly caused by the movement of infected plant material. This result has direct implications for disease management because migration can cause the movement of more virulent and/or fungicide-resistant genotypes. We conclude that genetic recombination is frequent (in contrast to the traditional view of C. graminicola being mainly asexual), which strongly impacts control measures and breeding programs aimed at controlling this disease.

This work describes the impact of long term consumption of an isoflavone-rich dietary daily supplement on the composition and diversity of the faecal microbiota of a menopausal, equol-producing woman. Sequencing of 16S rDNA amplicons was performed on faecal samples taken at 0, 1, 3 and 6 months of treatment. Additionally, and for comparative purposes, ethidium monoazide (EMA) was used to avoid detection of DNA from dead bacteria. Members of two genera of the family Coriobacteriaceae (Eggerthella and Collinsella) were found in greater proportions at all sampling points during isoflavone supplementation. Different genera of the family Ruminococcaceae (e.g., Ruminococcus and Faecalibacterium), as well as members of the family Lachnospiraceae (Coprococcus) also underwent significant increases. For this last genus a positive correlation with the levels of equol excretion in urine was found. Currently bacterial strains known to be involved in isoflavone metabolism and equol production have been assigned to these taxa. The use of EMA dye allowed us to unravel those bacterial gut linages (e.g., Lachnospiraceae) that were more susceptible to damage. Our study contributes to the identification of microorganisms possibly involved in the production of isoflavone-desirable metabolites (such as equol), which could ultimately be isolated and further used as probiotics by people who cannot naturally benefit from isoflavones.

Pesticide-resistant plant pathogens are an increasing threat to the global food supply and have generated a need for novel, efficacious agrochemicals. The current regulatory process for approving new agrochemicals is a tedious but necessary process. One way to accelerate the safety evaluation process is to utilize in vitro systems to demonstrate pesticide degradation by soil microbes prior to ex vivo soil evaluations. This approach may have the capability to generate metabolic profiles free of inhibitory substances, such as humic acids, commonly present in ex vivo soil systems. In this study, we used a packed-bed microbial bioreactor to assess the role of the natural soil microbial community during biodegradation of the triazolopyrimidine fungicide, ametoctradin. Metabolite profiles produced during in vitro ametoctradin degradation were similar to the metabolite profiles obtained during environmental fate studies and demonstrated the degradation of 81% of the parent compound in 72 h compared to a half-life of 2 weeks when ametoctradin was left in the soil. The microbial communities of four different soil locations and the bioreactor microbiome were compared using high throughput sequencing. It was found that biodegradation of ametoctradin in both ex vivo soils and in vitro in the bioreactor correlated with an increase in the relative abundance of Burkholderiales, well characterized microbial degraders of xenobiotic compounds.

Undernutrition in children commonly disrupts the structure and function of the small intestinal microbial community, leading to enteropathies, compromised metabolic health, and impaired growth and development. The mechanisms by which diet and microbes mediate the balance between commensal and pathogenic intestinal flora remain elusive. In a murine model of undernutrition, we investigated the direct interactions Giardia lamblia, a prevalent small intestinal pathogen, on indigenous microbiota and specifically on Lactobacillus strains known for their mucosal and growth homeostatic properties. Our research reveals that Giardia colonization shifts the balance of lactic acid bacteria, causing a relative decrease in Lactobacillus spp. and an increase in Bifidobacterium spp. This alteration corresponds with a decrease in multiple indicators of mucosal and nutritional homeostasis. Additionally, protein-deficient conditions coupled with Giardia infection exacerbate the rise of primary bile acids and susceptibility to bile acid-induced intestinal barrier damage. In epithelial cell monolayers, Lactobacillus spp. mitigated bile acid-induced permeability, showing strain-dependent protective effects. In vivo, L. plantarum, either alone or within a Lactobacillus spp consortium, facilitated growth in protein-deficient mice, an effect attenuated by Giardia, despite not inhibiting Lactobacillus colonization. These results highlight Giardia’s potential role as a disruptor of probiotic functional activity, underscoring the imperative for further research into the complex interactions between parasites and bacteria under conditions of nutritional deficiency.

Abstract Streptococcus mutans has been implicated as the primary pathogen in childhood caries (tooth decay). While the role of polymicrobial communities is appreciated, it remains unclear whether other microorganisms are active contributors or interact with pathogens. Here, we integrate multi-omics of supragingival biofilm (dental plaque) from 416 preschool-age children (208 males and 208 females) in a discovery-validation pipeline to identify disease-relevant inter-species interactions. Sixteen taxa associate with childhood caries in metagenomics-metatranscriptomics analyses. Using multiscale/computational imaging and virulence assays, we examine biofilm formation dynamics, spatial arrangement, and metabolic activity of Selenomonas sputigena, Prevotella salivae and Leptotrichia wadei, either individually or with S. mutans. We show that S. sputigena, a flagellated anaerobe with previously unknown role in supragingival biofilm, becomes trapped in streptococcal exoglucans, loses motility but actively proliferates to build a honeycomb-like multicellular-superstructure encapsulating S. mutans, enhancing acidogenesis. Rodent model experiments reveal an unrecognized ability of S. sputigena to colonize supragingival tooth surfaces. While incapable of causing caries on its own, when co-infected with S. mutans, S. sputigena causes extensive tooth enamel lesions and exacerbates disease severity in vivo. In summary, we discover a pathobiont cooperating with a known pathogen to build a unique spatial structure and heighten biofilm virulence in a prevalent human disease.

The gut microbiome is widely recognized for its significant contribution to maintaining human health across all life stages, from infancy to adulthood and beyond. This perspective article focuses on the impacts of well-supported microbiome research on global caesarean delivery rates, breastfeeding practices, and antimicrobial use. The article also explores the impact of dietary choices, particularly those involving ultra-processed foods, on the gut microbiota and their potential contribution to conditions like obesity, metabolic syndrome, and inflammatory diseases. This perspective aims to emphasize the need for updated guidelines and policy interventions to address the increasing global trends of caesarean deliveries, reduced breastfeeding, overuse of antibiotics, and consumption of highly processed foods to counter their adverse effects on gut health.

Aged companion dogs have a high prevalence of periodontal disease and canine cognitive dysfunction syndrome (CCDS) and the two disorders are correlated. Similarly, periodontal disease and Alzheimer’s Disease are correlated in people. However, little is known about the oral microbiota of aging dogs. The goal of this project was to characterize the longitudinal changes in oral microbiota in aged dogs. Oral swabs were taken from ten senior client-owned dogs on 2–3 occasions spanning 24 months and they underwent whole genome shotgun (WGS) sequencing. Cognitive status was established at each sampling time. A statistically significant increase in alpha diversity for bacterial and fungal species was observed between the first and last study visits. Bacteroidetes and proteobacteria were the most abundant bacterial phyla. Porphyromonas gulae was the most abundant bacterial species (11.6% of total reads). The species Lactobacillus gasseri had a statistically significant increase in relative abundance with age whereas Leptotrichia sp. oral taxon 212 had a statistically significant positive longitudinal association with cognition score. There is an increased fungal and bacterial alpha diversity in aging dogs over time and nearly universal oral dysbiosis. The role of the oral microbiota, particularly Leptotrichia and P. gulae and P. gingivalis, in aging and CCDS warrants further investigation.

Objective: To examine the plasma microbiome for differences between obese individuals with and without osteoarthritis (OA) and its association with serum lipopolysaccharide (LPS). Design: Blood samples from 70 participants with body mass index (BMI) ​≥ ​30kg/m2 and age ≥55 years, with (cases) or without (controls) hand plus knee OA, were analyzed for serum LPS and composition of the plasma microbiome. The Dirichlet-multinominal recursive partitioning model (DM-RPart) was applied to microbiome compositional data to test the hypothesis that LPS levels distinguish plasma microbiome, accounting for BMI and age. Results: No significant differences in alpha diversity, or compositional differences between groups at the genus level, were seen between cases and controls (p ​= ​0.11). β-Diversity was significantly associated with serum LPS levels (p ​= ​0.01). DM-RPart resulted in an optimal tree with 3 divisions: 1) based on age (split at 69 years); 2) those older than 69 were split based on BMI; 3) those with BMI

IntroductionDiarrhea is the second most common cause of mortality in shelter kittens. Studies examining prevention strategies in this population are lacking. Probiotics are of particular interest but studies in cats are largely limited to healthy adults or those with induced disease. Only one study in domestic cats describes the use of host-derived bacteria as a probiotic. We previously identified Enterococcus hirae as a dominant species colonizing the small intestinal mucosa in healthy shelter kittens. Oral administration of a probiotic formulation of kitten-origin E. hirae (strain 1002-2) mitigated the increase in intestinal permeability and fecal water loss resulting from experimental enteropathogenic E. coli infection in purpose-bred kittens. Based on these findings, we hypothesized that administration of kitten-origin E. hirae to weaned fostered shelter kittens could provide a measurable preventative health benefit.MethodsWe conducted a randomized, placebo-controlled, blinded clinical trial to determine the impact of a freeze-dried E. hirae probiotic on body weight gain, incidence of diarrhea, carriage of potential diarrheal pathogens, and composition of the intestinal microbiota in weaned fostered shelter kittens.ResultsOne-hundred thirty kittens completed the study. Fifty-eight kittens received the probiotic and 72 received the placebo. There were no significant differences in age, weight upon initiation of the study, number of days in the study, average daily gain in body weight, or weight at completion of the study. Kittens treated with E. hirae were 3.4 times less likely to develop diarrhea compared to kittens treated with placebo (odds ratio = 0.294, 95% CI 0.109–0.792, p = 0.022). A significant impact of E. hirae was not observed on the presence or abundance of 30 different bacterial, viral, protozoal, fungal, algal, and parasitic agents in feces examined by qPCR. With exception to a decrease in Megamonas, administration of the E. hirae probiotic did not alter the predominant bacterial phyla present in feces based on 16S rRNA gene amplicon sequencing.DiscussionDecreased incidence of diarrhea associated with preventative administration of E. hirae to foster kittens supports a rationale for use of E. hirae for disease prevention in this young population at high risk for intestinal disease though additional studies are warranted.

Abstract The gut microbiota plays an important role in human health and disease. Stool, rectal swab and rectal mucosal tissue samples have been used in individual studies to survey the microbial community but the consequences of using these different sample types are not completely understood. In this study, we report differences in stool, rectal swab and rectal mucosal tissue microbial communities with shotgun metagenome sequencing of 1397 stool, swab and mucosal tissue samples from 240 participants. The taxonomic composition of stool and swab samples was distinct, but less different to each other than mucosal tissue samples. Functional profile differences between stool and swab samples are smaller, but mucosal tissue samples remained distinct from the other two types. When the taxonomic and functional profiles were used for inference in association with host phenotypes of age, sex, body mass index (BMI), antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs) use, hypothesis testing using either stool or rectal swab gave broadly significantly correlated results, but inference performed on mucosal tissue samples gave results that were generally less consistent with either stool or swab. Our study represents an important resource for determination of how inference can change for taxa and pathways depending on the choice of where to sample within the human gut.

Experimental manipulation of the gut microbiome in animal models impacts fear behaviours. Here, the authors show in a pilot study that features of the human infant gut microbiome are associated with non-social fear behaviours during a laboratory based assessment.

Abstract Background Prebiotic galacto-oligosaccharides (GOS) have an extensively demonstrated beneficial impact on intestinal health. In this study, we determined the impact of GOS diets on hallmarks of gut aging: microbiome dysbiosis, inflammation, and intestinal barrier defects (“leaky gut”). We also evaluated if short-term GOS feeding influenced how the aging gut responded to antibiotic challenges in a mouse model of Clostridioides difficile infection. Finally, we assessed if colonic organoids could reproduce the GOS responder—non-responder phenotypes observed in vivo. Results Old animals had a distinct microbiome characterized by increased ratios of non-saccharolytic versus saccharolytic bacteria and, correspondingly, a lower abundance of β-galactosidases compared to young animals. GOS reduced the overall diversity, increased the abundance of specific saccharolytic bacteria (species of Bacteroides and Lactobacillus), increased the abundance of β-galactosidases in young and old animals, and increased the non-saccharolytic organisms; however, a robust, homogeneous bifidogenic effect was not observed. GOS reduced age-associated increased intestinal permeability and increased MUC2 expression and mucus thickness in old mice. Clyndamicin reduced the abundance Bifidobacterium while increasing Akkermansia, Clostridium, Coprococcus, Bacillus, Bacteroides, and Ruminococcus in old mice. The antibiotics were more impactful than GOS on modulating serum markers of inflammation. Higher serum levels of IL-17 and IL-6 were observed in control and GOS diets in the antibiotic groups, and within those groups, levels of IL-6 were higher in the GOS groups, regardless of age, and higher in the old compared to young animals in the control diet groups. RTqPCR revealed significantly increased gene expression of TNFα in distal colon tissue of old mice, which was decreased by the GOS diet. Colon transcriptomics analysis of mice fed GOS showed increased expression of genes involved in small-molecule metabolic processes and specifically the respirasome in old animals, which could indicate an increased oxidative metabolism and energetic efficiency. In young mice, GOS induced the expression of binding-related genes. The galectin gene Lgals1, a β-galactosyl-binding lectin that bridges molecules by their sugar moieties and is an important modulator of the immune response, and the PI3K-Akt and ECM-receptor interaction pathways were also induced in young mice. Stools from mice exhibiting variable bifidogenic response to GOS injected into colon organoids in the presence of prebiotics reproduced the response and non-response phenotypes observed in vivo suggesting that the composition and functionality of the microbiota are the main contributors to the phenotype. Conclusions Dietary GOS modulated homeostasis of the aging gut by promoting changes in microbiome composition and host gene expression, which was translated into decreased intestinal permeability and increased mucus production. Age was a determining factor on how prebiotics impacted the microbiome and expression of intestinal epithelial cells, especially apparent from the induction of galectin-1 in young but not old mice. Video abstract

An amendment to this paper has been published and can be accessed via the original article.

Complex dietary carbohydrate structures including β(1–4) galacto-oligosaccharides (GOS) are resistant to digestion in the upper gastrointestinal (GI) tract and arrive intact to the colon where they benefit the host by selectively stimulating microbial growth. Studies have reported the beneficial impact of GOS (alone or in combination with other prebiotics) by serving as metabolic substrates for modulating the assembly of the infant gut microbiome while reducing GI infections. N-Acetyl-D-lactosamine (LacNAc, Galβ1,4GlcNAc) is found in breast milk as a free disaccharide. This compound is also found as a component of human milk oligosaccharides (HMOs), which have repeating and variably branched lactose and/or LacNAc units, often attached to sialic acid and fucose monosaccharides. Human glycosyl-hydrolases do not degrade most HMOs, indicating that these structures have evolved as natural prebiotics to drive the proper assembly of the infant healthy gut microbiota. Here, we sought to develop a novel enzymatic method for generating LacNAc-enriched GOS, which we refer to as humanized GOS (hGOS). We showed that the membrane-bound β-hexosyl transferase (rBHT) from Hamamotoa (Sporobolomyces) singularis was able to generate GOS and hGOS from lactose and N-Acetyl-glucosamine (GlcNAc). The enzyme catalyzed the regio-selective, repeated addition of galactose from lactose to GlcNAc forming the β-galactosyl linkage at the 4-position of the GlcNAc and at the 1-position of D-galactose generating, in addition to GOS, LacNAc, and Galactosyl-LacNAc trisaccharides which were produced by two sequential transgalactosylations. Humanized GOS is chemically distinct from HMOs, and its effects in vivo have yet to be determined. Thus, we evaluated its safety and demonstrated the prebiotic's ability to modulate the gut microbiome in 6-week-old C57BL/6J mice. Longitudinal analysis of gut microbiome composition of stool samples collected from mice fed a diet containing hGOS for 5 weeks showed a transient reduction in alpha diversity. Differences in microbiome community composition mostly within the Firmicutes phylum were observed between hGOS and GOS, compared to control-fed animals. In sum, our study demonstrated the biological synthesis of hGOS, and signaled its safety and ability to modulate the gut microbiome in vivo, promoting the growth of beneficial microorganisms, including Bifidobacterium and Akkermansia.

Aim: This in vivo experimental study investigated bacterial microbiome and metabolome longitudinal changes associated with enamel caries lesion progression and arrest. Methods: We induced natural caries activity in three caries-free volunteers prior to four premolar extractions for orthodontic reasons. The experimental model included placement of a modified orthodontic band on smooth surfaces and a mesh on occlusal surfaces. We applied the caries-inducing protocol for 4- and 6-weeks, and subsequently promoted caries lesion arrest via a 2-week toothbrushing period. Lesions were verified clinically and quantitated via micro-CT enamel density measurements. The biofilm microbial composition was determined via 16S rRNA gene Illumina sequencing and NMR spectrometry was used for metabolomics. Results: Biofilm maturation and caries lesion progression were characterized by an increase in Gram-negative anaerobes, including Veillonella and Prevotella. Streptococcus was associated caries lesion progression, while a more equal distribution of Streptococcus, Bifidobacterium, Atopobium, Prevotella, Veillonella, and Saccharibacteria (TM7) characterized arrest. Lactate, acetate, pyruvate, alanine, valine, and sugars were more abundant in mature biofilms compared to newly formed biofilms. Conclusions: These longitudinal bacterial microbiome and metabolome results provide novel mechanistic insights into the role of the biofilm in caries progression and arrest and offer promising candidate biomarkers for validation in future studies.

Food allergy is a potentially fatal disease affecting 8% of children and has become increasingly common in the past two decades. Despite the prevalence and severe nature of the disease, the mechanisms underlying sensitization remain to be further elucidated. The Collaborative Cross is a genetically diverse panel of inbred mice that were specifically developed to study the influence of genetics on complex diseases. Using this panel of mouse strains, we previously demonstrated CC027/GeniUnc mice, but not C3H/HeJ mice, develop peanut allergy after oral exposure to peanut in the absence of a Th2-skewing adjuvant. Here, we investigated factors associated with sensitization in CC027/GeniUnc mice following oral exposure to peanut, walnut, milk, or egg. CC027/GeniUnc mice mounted antigen-specific IgE responses to peanut, walnut and egg, but not milk, while C3H/HeJ mice were not sensitized to any antigen. Naïve CC027/GeniUnc mice had markedly lower total fecal IgA compared to C3H/HeJ, which was accompanied by stark differences in gut microbiome composition. Sensitized CC027/GeniUnc mice had significantly fewer CD3+ T cells but higher numbers of CXCR5+ B cells and T follicular helper cells in the mesenteric lymph nodes compared to C3H/HeJ mice, which is consistent with their relative immunoglobulin production. After oral challenge to the corresponding food, peanut- and walnut-sensitized CC027/GeniUnc mice experienced anaphylaxis, whereas mice exposed to milk and egg did not. Ara h 2 was detected in serum collected post-challenge from peanut-sensitized mice, indicating increased absorption of this allergen, while Bos d 5 and Gal d 2 were not detected in mice exposed to milk and egg, respectively. Machine learning on the change in gut microbiome composition as a result of food protein exposure identified a unique signature in CC027/GeniUnc mice that experienced anaphylaxis, including the depletion of Akkermansia. Overall, these results demonstrate several factors associated with enteral sensitization in CC027/GeniUnc mice, including diminished total fecal IgA, increased allergen absorption and altered gut microbiome composition. Furthermore, peanuts and tree nuts may have inherent properties distinct from milk and eggs that contribute to allergy.

Cognitive decline is common in older adults and more than 5 million Americans suffer from Alzheimer's disease (AD). A number of physiological processes including systemic inflammation, excess adiposity, and impaired glucoregulation arise from a combination of genetic and behavioral factors and increase risk for developing AD or other forms of dementia. Recent research suggests that the gut microbiome may moderate these pathological processes and possibly influence cognitive outcomes. This paper reviews the methodology for a double-blind, randomized clinical trial examining the influence of Lactobacillus GG (LGG) probiotic supplementation on mood and cognitive functioning in middle-aged and older adults. Our two primary hypotheses include: 1) Participants randomized to the probiotic group will show greater improvements in psychological status compared to participants in the placebo group; 2) Participants randomized to the probiotic group will show greater improvements in executive functioning and processing speed, as evidenced through performance on neuropsychological testing, than participants in the placebo group. We anticipate these results will inform future efforts on the feasibility of LGG probiotic supplementation as an intervention for psychological status and cognitive functioning and further elucidate the link between the gut microbiome and cognitive health. Keywords: Probiotics, Lactobacillus rhamnosus, Cognitive function, Executive function, Alzheimer's disease, Depression

Studies on the effects of breastfeeding on the infant gut microbiota have provided inconsistent results. Here, Ho et al. perform a meta-analysis of seven studies across different populations, supporting that exclusive breastfeeding is associated with short-term and long-term alterations in the infant gut microbiota.

Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota. Keywords: Organoid, Microinjection, High-Throughput, Fecal Microbiota, Anaerobic, Barrier Function, High-Content Sampling

Abstract Background Advancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines. Results Chicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150–199 for GS FLX+ and bases 90–99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME. Conclusions The three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.

Knowledge of the intra-individual spatial and regional distribution of intestinal microbial populations is essential to understand gut host–microbial interactions. In this study, we performed a compositional analysis of luminal and mucosal samples from the small and large intestine of four organ donors by 16S rRNA amplicon sequencing and high-throughput quantitative polymerase chain reaction. Since the human microbiota is subject to selection pressure at lower taxonomic levels, we isolated over 400 bacterial strains and investigated strain-level variation of 11 Lactobacillus rhamnosus from different intestinal regions. Results substantiate reported inter-individual variability as well as intra-individual differences along the gastrointestinal tract. Although the luminal and mucosal-associated communities were similar within individuals, relative abundance reflected the donors’ demographic and potential pathologies. The total bacterial load of all donors increased from small intestine to colon, while Bifidobacterium was in greater abundance in the small intestine. Comparative genomic analysis of L. rhamnosus showed the strains segregated into two distinct clusters and identified no features specific to location. Analysis revealed genetic differences for exopolysaccharide production, carbohydrate utilization, pilus formation and vitamin K biosynthesis between clusters. This study contributes to the understanding of niche-specific microbial communities, encouraging subsequent studies to better understand microbial signatures at lower taxonomic levels.

Large-scale microbiome studies have established that most of the diversity contained in the gastrointestinal tract is represented at the strain level; however, exhaustive genomic and physiological characterization of human isolates is still lacking. With increased use of probiotics as interventions for gastrointestinal disorders, genomic and functional characterization of novel microorganisms becomes essential. In this study, we explored the impact of strain-level genomic variability on bacterial physiology of two novel human Lactobacillus rhamnosus strains (AMC143 and AMC010) of probiotic potential in relation to stress resistance. The strains showed differences with known probiotic strains (L. rhamnosus GG, Lc705, and HN001) at the genomic level, including nucleotide polymorphisms, mutations in non-coding regulatory regions, and rearrangements of genomic architecture. Transcriptomics analysis revealed that gene expression profiles differed between strains when exposed to simulated gastrointestinal stresses, suggesting the presence of unique regulatory systems in each strain. In vitro physiological assays to test resistance to conditions mimicking the gut environment (acid, alkali, and bile stress) showed that growth of L. rhamnosus AMC143 was inhibited upon exposure to alkaline pH, while AMC010 and control strain LGG were unaffected. AMC143 also showed a significant survival advantage compared to the other strains upon bile exposure. Reverse transcription qPCR targeting the bile salt hydrolase gene (bsh) revealed that AMC143 expressed bsh poorly (a consequence of a deletion in the bsh promoter and truncation of bsh gene in AMC143), while AMC010 had significantly higher expression levels than AMC143 or LGG. Insertional inactivation of the bsh gene in AMC010 suggested that bsh could be detrimental to bacterial survival during bile stress. Together, these findings show that coupling of classical microbiology with functional genomics methods for the characterization of bacterial strains is critical for the development of novel probiotics, as variability between strains can dramatically alter bacterial physiology and functionality.

Prebiotics are ingredients selectively fermented by the intestinal microbiota that promote changes in its structure and/or metabolism, conferring health benefits to the host. Studies show that β (1-4) galacto-oligosaccharides [β (1-4) GOS], lactulose and fructo-oligosaccharides increase intestinal concentration of lactate and short chain fatty acids, and stool frequency and weight, and they decrease fecal concentration of secondary bile acids, fecal pH, and nitroreductase and β-glucuronidase activities suggesting a clear role in colorectal cancer (CRC) prevention. This review summarizes research on prebiotics bioassimilation, specifically β (1-4) GOS, and their potential role in CRC. We also evaluate research that shows that the impact of prebiotics on host physiology can be direct or through modulation of the gut intestinal microbiome, specifically the probiome (autochtonous beneficial bacteria). We present studies on a potential role in CRC progression to finally describe the current state of β (1-4) GOS generation for industrial production.

Diet is one of the main factors shaping the human microbiome, yet our understanding of how specific dietary components influence microbial consortia assembly and subsequent stability in response to press disturbances – such as increasing resource availability (feeding rate) – is still incomplete. This study explores the reproducible re-assembly, metabolic interplay, and compositional stability within microbial consortia derived from pooled stool samples of three healthy infants. Using a single-step packed-bed reactor (PBR) system, we assessed the reassembly and metabolic output of consortia exposed to lactose, glucose, galacto-oligosaccharides (GOS), and humanized GOS (hGOS). Our findings reveal that complex carbohydrates, especially those containing low inclusion (~1.25 gL−1) components present in human milk, such as N-acetyl-lactosamine (LacNAc), promote taxonomic, and metabolic stability under varying feeding rates, as shown by diversity metrics and network analysis. Targeted metabolomics highlighted distinct metabolic responses to different carbohydrates: GOS was linked to increased lactate, lactose to propionate, sucrose to butyrate, and CO2, and the introduction of bile salts with GOS or hGOS resulted in butyrate reduction and increased hydrogen production. This study validates the use of single-step PBRs for reliably studying microbial consortium stability and functionality in response to nutritional press disturbances, offering insights into the dietary modulation of microbial consortia and their ecological dynamics.