Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP), an antifolate drug with antimalarial and antibiotic activity, reproducibly improves birthweight across sub-Saharan Africa and the Western Pacific. This clinical protection is independent of SP's original malaria indication: it is not diminished by widespread antimalarial resistance or reduced transmission, and SP outperforms more potent non-antibiotic antimalarials (e.g., dihydroartemisinin-piperaquine, DP) for fetal growth. The biological mechanism is unexplained. We previously showed that gestational weight gain (GWG) is a significant component of this mechanism and mediates two-thirds of SP's overall birthweight benefit (NCT03009526). In the first longitudinal characterization of antifolate antibiotic effects on the pregnant gut microbiome, we show that ~45% of SP's GWG advantage over DP is explained by gut microbial changes consistent with its pharmacology. Microbiome-mediated GWG coincided with 126g higher birthweight in SP but not DP recipients (95%CI 22.6-229.3g; p=0.019). Relative to DP, SP suppressed gastrointestinal pathobionts and enriched anaerobic commensals with recognized roles in mucosal immunity and host metabolism, a microbiome-sparing pattern distinct from conventional antibiotic-associated dysbiosis.

The human microbiome is a foundational and dynamic foundation for several health-related functions and disease processes. Advances in microbiome sequencing have enabled the characterization of microbial communities in several niches. Longitudinal microbiome studies further strive to discover clinically informative microbial community trajectories. However, these data are fraught with dropout events, high noise, and irregular sampling that limit and prevent the use of many available longitudinal analysis tools. To address these challenges, we introduce Bidirectional GRU-ODE-Bayes (BGOB), a deep learning framework developed for longitudinal microbiome interpolation. BGOB combines bidirectional information flow and ODE-based continuous modeling to jointly interpolate and smoothen trends across individual participants, providing uniform, denoised time intervals across patients. BGOB enables vastly improved performance in differential abundance testing and time-to-event analysis, and makes possible longitudinal analyses requiring uniformity, such as lead-lag detection and temporal clustering. After interpolation, previously low-powered datasets are able to broadly recapitulate known microbiology and elucidate interacting microbial communities. We highlight several associations between microbial taxa and disease, including novel species associated with Early Childhood Caries and disruption of key healthy gut microbiota in Inflammatory Bowel Disease. The BGOB package is publicly available at https://github.com/Rachel-Lyu/BGOB\_n\_test ### Competing Interest Statement The authors have declared no competing interest.

Preserving a functional intestinal barrier is crucial for overall host health, as increased permeability can lead to systemic pathologies, including a dysregulated immune system and increased susceptibility to infections. Pure β (1-4) galacto-oligosaccharides (GOS) and Type 2 LacNAc-enriched β (1-4) GOS (humanized GOS, hGOS) modulate the gut microbiome, increasing the abundance of beneficial microorganisms, including Bifidobacterium, Akkermansia, and Lactobacillus. In this study, we report that direct exposure of monolayers of human primary colonic cells to β(1-4) GOS and β(1-4) hGOS enhances barrier integrity by significantly upregulating MUC2 (2-5 fold) and tight junction genes (1.5-8 fold) (p<0.05). RNA sequencing revealed that β(1-4) GOS and β(1-4) hGOS activated the Aryl Hydrocarbon Receptor (AHR) pathway, with GOS specifically inducing CYP1A1 (log2FC = 3.57) and TIPARP, while hGOS enriched pathways related to apical junction integrity. This suggests that AHR activation may enhance gut barrier function, in part, by repressing IL-1β-mediated inflammation. These findings were validated in vivo in young and old C57BL/6 mice, where GOS and hGOS enhanced intestinal permeability by increasing the expression of Muc2 and promoting mucus production, resulting in a thicker mucus layer (GOS: 95% CI 0.054-0.146; hGOS: 95% CI 0.080-0.176). Tight junction integrity genes were also upregulated by the prebiotics in our study (two-way ANOVA, p < 0.05). Our findings suggest that, beyond their modulation of the gut microbiome, GOS and hGOS enhance intestinal barrier function by directly inducing the expression of mucin and tight junction genes, likely through the AHR pathway. These results highlight the potential of prebiotic supplementation in improving gut barrier function and maintaining intestinal health.

Background:Gut microbiome diversity and composition have been associated with type 1 diabetes (T1D) onset and progression. These features have not been explored among the growing number of older adults with T1D, including how they may differ by diet. Objectives:In the present observational study, we investigated differences in gut microbiome diversity and composition across quantiles of high and low macronutrient, fiber, and simple sugar intake. Methods:We collected observational data on routine diabetes management, diet, and other health indicators from older adults with T1D over 7-9 d, including 2 24-h dietary recall interviews, diabetes technology, and medical record data. Stool samples were collected for gut microbiome characterization using high-throughput sequencing of the 16S ribosomal RNA gene. Gut microbiome characteristics were assessed by Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC2), Shannon index, and Bray-Curtis distances. Results:Among 17 older adults [mean (SD) age 73.5 (6.2) y; diabetes duration: 34.0 (16.0) y, and glycated hemoglobin: 7.0 (0.9) %], increased fat intake was associated with enriched genus Lactococcus, increased fiber intake was associated with enriched genus Hemophilus and unclassified Clostridium within family CAG:508, and increased fructose intake was associated with enrichment of unclassified genus SFMI01 within family Christensenellaceae. Greater carbohydrate intake was associated with depletion of the family Enterobacteriaceae. Microbial diversity differed significantly with greater fiber and fructose intake. Conclusions:We found statistically significant evidence suggesting that the gut microbiome of older adults with T1D may be modulated by macronutrient, fiber, and fructose intake. Similar trends between fiber intake differences and significantly different microbial community structure have been found in younger populations. Together with the literature, our other findings suggest that macronutrient, fiber, and fructose intakes may impact the gut microbiome differently in older adults with T1D.

Mucus-digesting Ruminococcus gnavus (Rg), adherent-invasive E. coli (AIEC) and fecal hydrogen sulfide (H2S) are increased in active Crohn’s disease (CD) compared to healthy subjects. We established a link between Rg and ileal CD AIEC strain LF82 by showing that dual colonization of ex-germ-free (GF) IL10-/- mice potentiated colitis and cecal H2S production. Rg precultured mucus increased LF82 growth and H2S production, H2S supported in vitro Rg survival in minimal medium, and H2S enhanced LF82 adhesion and invasion both in vitro and in vivo (DDW 2023). Rg’s capsule contains glucose and has an enzyme that cleaves off sialic acid (Neu5Ac), one of the terminal carbohydrates of mucus. Because inflamed colons become microaerophilic to some extent, we focused on Rg-derived glucose and mucus-derived Neu5Ac to explore expression of LF82 molecular pathways under aerobic and anaerobic conditions. AIEC LF82 genes regulating growth, H2S production and virulence are differentially expressed with glucose or Neu5Ac substrates in aerobic vs. anaerobic conditions. LF82 was cultured in M9 minimal medium supplemented with Rg-/mucus-derived carbohydrates or gut-relevant sulfur substrates for 16 hours in aerobic or anaerobic conditions. LF82 RNA cultured with glucose or Neu5Ac medium in each condition was isolated for RNA sequencing. We compared aerobic vs. anaerobic results for each medium with |Log2FC| > 3.0 considered significant change. Rg associated mucus carbohydrates and gut-relevant sulfur substrates variably enhanced LF82 growth. LF82 grew better in Neu5Ac than glucose in aerobic and vice versa in anaerobic conditions (Fig.1). Neu5Ac metabolism pathways involving nanE (N-acetylmannosamine-6-phosphate 2-epimerase) and dnaA (chromosomal replication initiator), directly involved in cell replication, were overexpressed in aerobic vs. anaerobic conditions in Neu5Ac medium. Additionally, expression of other Neu5Ac metabolic pathway genes, nanT (sialic transporter) and nanA (N-acetylneuraminate lyase) were slightly increased. Porin OmpC, a non-specific glucose transporter, and dnaA were more highly expressed in anaerobic than aerobic conditions in glucose medium (Table 1A). Moreover, LF82 genes involved in H2S production, biofilm formation and antimicrobial drug resistance were overexpressed in glucose vs. Neu5Ac with aerobic culture (Table 1B). AIEC LF82 differentially uses R. gnavus released mucus components for growth in aerobic vs. anaerobic conditions, and sialic acid and glucose activate distinct molecular pathways in the two conditions. Confirmatory functional experiments are underway. Glucose influences on LF82 H2S production and biofilm formation would provide strong evidence of cross-feeding between LF82 and R. gnavus, leading to potential diagnostic/treatment clues for further investigation.

INTRODUCTION:Common oral diseases are known to be associated with dysbiotic shifts in the supragingival microbiome, yet most oral microbiome associations with clinical end points emanate from cross-sectional studies. Orthodontic treatment is an elective procedure that can be exploited to prospectively examine clinically relevant longitudinal changes in the composition and function of the supragingival microbiome.METHODS:A longitudinal cohort study was conducted among 24 adolescent orthodontic patients who underwent saliva and plaque sampling and clinical examinations at time points: before fixed appliance bonding and at 1, 6, and 12 wk thereafter. Clinical indices included bleeding on probing (BOP), mean gingival index (GI), probing depths (PDs), and plaque index (PI). To study the biologically (i.e., transcriptionally) active microbial communities, RNA was extracted from plaque and saliva for RNA sequencing and microbiome bioinformatics analysis. Longitudinal changes in microbiome beta diversity were examined using PERMANOVA tests, and the relative abundance of microbial taxa was measured using Kruskal-Wallis tests, Wilcoxon rank-sum tests, and negative binomial and zero-inflated mixed models.RESULTS:Clinical measures of oral health deteriorated over time-the proportion of sites with GI and PI ≥1 increased by over 70% between prebonding and 12 wk postbonding while the proportion of sites with PD ≥4 mm increased 2.5-fold. Streptococcus sanguinis, a health-associated species that antagonizes cariogenic pathogens, showed a lasting decrease in relative abundance during orthodontic treatment. Contrarily, caries- and periodontal disease-associated taxa, including Selenomonas sputigena, Leptotrichia wadei, and Lachnoanaerobaculum saburreum, increased in abundance after bonding. Relative abundances of Stomatobaculum longum and Mogibacterium diversum in prebonding saliva predicted elevated BOP 12 wk postbonding, whereas Neisseria subflava was associated with lower BOP.CONCLUSIONS:This study offers insights into longitudinal community and species-specific changes in the supragingival microbiome transcriptome during fixed orthodontic treatment, advancing our understanding of microbial dysbioses and identifying targets of future health-promoting clinical investigations.KNOWLEDGE TRANSFER STATEMENT:Bonding braces was associated with subsequent changes in the oral microbiome characterized by increases in disease-associated species, decreases in health-associated species, and worsened clinical measures of oral health.

Objectives: Galacto-oligosaccharides (GOS) are prebiotics that are metabolized by the microbes in the gut to confer beneficial effects to the host. GOS have been shown to protect against pathogen infections by structurally mimicking intestinal binding sites and preventing epithelial barrier dysfunction. Its anti-adhesive properties have been shown to inhibit Escherichia coli and Salmonella enterica serovar Typhimurium-associated enteric infections. The goal of this study was to explore the effects of GOS, along with a beneficial bacterial consortium or fecal microbiota transplantation (FMT) in a mouse model of Clostridioides difficile (C. difficile) infection. Methods: C57BL/6J mice(4–6 weeks old) were given either a Control or GOS diet, received either the bacterial consortium or FMT and were infected with C. difficile R20291. Intestinal permeability was determined by measuring FITC-dextran concentrations in serum at the end of the experiment and C. difficile clearance over time was determined by qPCR. Microbiome analysis was conducted via 16S rRNA amplicon sequencing. Results: The GOS diet favored the resistance to C. difficile colonization but did not impact clearance rates. Mice that received the bacterial consortium and FMT had increased permeability but a lower abundance of C. difficile at the end of the study. Conclusions: Our results suggest that GOS supplementation may create an environment that aids in the prevention of pathogen colonization, leading to future clinical interventions to explore prebiotic supplementation as a preventative treatment for infection. Funding Sources: UNC Microbiome Core, Center for Gastrointestinal Biology and Disease (CGIBD), Nutrition Obesity Research Center (NORC).

Background and Aims: Primary sclerosing cholangitis (PSC) associated inflammatory bowel diseases (IBD) increase colorectal dysplasia and malignancy risk. Current mouse models do not adequately replicate human PSC-IBD, limiting mechanistic understanding and therapeutic development. This study uses Mdr2/Il10 double knockout (DKO) mice to examine microbiota roles in mediating colitis, colitis-associated colorectal dysplasia and hepatobiliary inflammation/fibrosis. Goal: Develop and phenotype a chronic spontaneous PSC-IBD mouse model, emphasizing colitis, colonic dysplasia, hepatobiliary inflammation/ fibrosis and the functional roles of resident microbiota. Methods: We utilized germ-free (GF) and specific-pathogen-free Mdr2/Il10 DKO, Il10-/- and Mdr2-/- mice to model PSC-IBD. We monitored colonic dysplasia progression, colitis kinetics and severity by lipocalin-2, cytokine measurement, and tissue evaluations of colon and liver. We manipulated the microbiome to assess its functional effects. Results: DKO mice exhibited age- and region-specific accelerated colitis and spontaneous colonic dysplasia progressing to high-grade invasive adenocarcinomas. Despite aggressive colonic inflammation, DKO mice showed reduced hepatic fibrosis, increased hepatic reparative macrophages, and matrix metalloproteinase activity compared to Mdr2-/- mice. GF DKO had heightened liver inflammation and mortality with absent colitis and colonic dysplasia, reversed with microbial reconstitution from DKO mice. Changes in DKO primary/secondary bile acid profiles mirrored those in PSC-IBD. Conclusion: The Mdr2/Il10 DKO model mirrors key factors in PSC-IBD patients in terms of inflammation and carcinogenesis. We found an important role for the dysbiotic microbiota in DKO mice for disease onset and progression. Targeting microbiota and bile acid metabolism may provide promising strategies for developing effective PSC-IBD therapies. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVE:Despite repeated public health interventions, anemia prevalence among children remains a concern. We use an evolutionary medicine perspective to examine the intestinal microbiome as a pathway underlying the efficacy of iron-sulfate treatment. This study explores whether gut microbiota composition differs between anemic children who respond and do not respond to treatment at baseline and posttreatment and if specific microbiota taxa remain associated with response to iron supplementation after controlling for relevant inflammatory and pathogenic variables. METHODS:Data come from 49 pre-school-aged anemic children living in San Juan de Lurigancho, Lima, Peru. We tested for differences in alpha and beta diversity using QIIME 2 and performed differential abundance testing in DESeq2 in R. We ran multivariate regression models to assess associations between abundance of specific taxa and response while controlling for relevant variables in Stata 17. RESULTS:While we found no evidence for gut microbiota diversity associated with child response to iron treatment, we observed several differential abundance patterns between responders and non-responders at both timepoints. Additionally, we present support for a nonzero relationship between lower relative abundance of Barnesiellaceae and response to iron supplementation in samples collected before and after treatment. CONCLUSION:While larger studies and more specific approaches are needed to understand the relationship between microbes and anemia in an epidemiological context, this study suggests that investigating nutritional status and pathogen exposure is key to better understanding the gut microbiome and impact of iron fortification.

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.

Integration of multi-omics data is a challenging but necessary step to advance our understanding of the biology underlying human health and disease processes. To date, investigations seeking to integrate multi-omics (e.g., microbiome and metabolome) employ simple correlation-based network analyses; however, these methods are not always well-suited for microbiome analyses because they do not accommodate the excess zeros typically present in these data. In this paper, we introduce a bivariate zero-inflated negative binomial (BZINB) model-based network and module analysis method that addresses this limitation and improves microbiome–metabolome correlation-based model fitting by accommodating excess zeros. We use real and simulated data based on a multi-omics study of childhood oral health (ZOE 2.0; investigating early childhood dental caries, ECC) and find that the accuracy of the BZINB model-based correlation method is superior compared to Spearman’s rank and Pearson correlations in terms of approximating the underlying relationships between microbial taxa and metabolites. The new method, BZINB-iMMPath, facilitates the construction of metabolite–species and species–species correlation networks using BZINB and identifies modules of (i.e., correlated) species by combining BZINB and similarity-based clustering. Perturbations in correlation networks and modules can be efficiently tested between groups (i.e., healthy and diseased study participants). Upon application of the new method in the ZOE 2.0 study microbiome–metabolome data, we identify that several biologically-relevant correlations of ECC-associated microbial taxa with carbohydrate metabolites differ between healthy and dental caries-affected participants. In sum, we find that the BZINB model is a useful alternative to Spearman or Pearson correlations for estimating the underlying correlation of zero-inflated bivariate count data and thus is suitable for integrative analyses of multi-omics data such as those encountered in microbiome and metabolome studies.

Table S4 includes immune signature and iCluster results.

Table S7 contains microbe screening results.

Table S1 contains cohort description, Master Patient Table and MutSigCV results.

Table S2 contains BAP1 analysis results, as well as detailed lists of YY1 and IRF8 target genes.

Table S6 contains results from the analysis of DNA methylation in SETD2 mutated and BAP1 inactivated samples.

Supplementary materials and methods, as well as supplementary figures S1-S7.

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.

Table S3 contains the karyotypes of 16 genome-wide LOH MPM cases from the BWH cohort.

Table S5 contains detailed miRs and lncRNAs results.