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Competing Interests: The authors declare no conflicts of interest.

Among dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen by dnd and ssp gene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possessed dnd or ssp genes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis uncovered several putative new PT synthesis systems, including Type 4 Bacteriophage Exclusion (BREX) brx genes, which were genetically validated in Bacteroides salyersiae. Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessing dnd , ssp , and brx genes revealed 8 PT dinucleotide settings confirmed in 6 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.

Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we established a gut epithelium-microbe-immune (GuMI) microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), and CD4 + naive T cells circulating underneath the colonic epithelium. In GuMI-APC condition, multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines secreted into both apical and basolateral compartments compared to GuMI condition that lacks APC. In GuMI-APC with F. prausnitzii (GuMI-APC-FP), F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, without a significant effect on cytokine secretion, compared to the GuMI-APC without bacteria (GuMI-APC-NB). In contrast, in the presence of CD4 + naive T cells (GuMI-APCT-FP), TLR1, IFNA1, and IDO1 transcription levels decreased with a simultaneous increase in F. prausnitzii-induced secretion of pro-inflammatory cytokines (e.g., IL8) compared to GuMI-APC-FP that lacks T cells. These results highlight the contribution of individual innate immune cells in regulating the immune response triggered by the gut commensal F. prausnitzii. The integration of defined populations of immune cells in the gut microphysiological system demonstrated the usefulness of GuMI physiomimetic platform to study microbe-epithelial-immune interactions in healthy and disease conditions., (© 2024. The Author(s).)

Increasing levels of industrialization have been associated with changes in gut microbiome structure and loss of features thought to be crucial for maintaining gut ecological balance. The stability of gut microbial communities over time within individuals seems to be largely affected by these changes but has been overlooked among transitioning populations from low- to middle-income countries. Here, we used metagenomic sequencing to characterize the temporal dynamics in gut microbiomes of 24 individuals living an urban non-industrialized lifestyle in the Brazilian Amazon. We further contextualized our data with 165 matching longitudinal samples from an urban industrialized and a rural non-industrialized population. We show that gut microbiome composition and diversity have greater variability over time among non-industrialized individuals when compared to industrialized counterparts and that taxa may present diverse temporal dynamics across human populations. Enterotype classifications show that community types are generally stable over time despite shifts in microbiome structure. Furthermore, by tracking genomes over time, we show that levels of bacterial population replacements are more frequent among Amazonian individuals and that non-synonymous variants accumulate in genes associated with degradation of host dietary polysaccharides. Taken together, our results suggest that the stability of gut microbiomes is influenced by levels of industrialization and that tracking microbial population dynamics is important to understand how the microbiome will adapt to these transitions.IMPORTANCEThe transition from a rural or non-industrialized lifestyle to urbanization and industrialization has been linked to changes in the structure and function of the human gut microbiome. Understanding how the gut microbiomes changes over time is crucial to define healthy states and to grasp how the gut microbiome interacts with the host environment. Here, we investigate the temporal dynamics of gut microbiomes from an urban and non-industrialized population in the Amazon, as well as metagenomic data sets from urban United States and rural Tanzania. We showed that healthy non-industrialized microbiomes experience greater compositional shifts over time compared to industrialized individuals. Furthermore, bacterial strain populations are more frequently replaced in non-industrialized microbiomes, and most non-synonymous mutations accumulate in genes associated with the degradation of host dietary components. This indicates that microbiome stability is affected by transitions to industrialization, and that strain tracking can elucidate the ecological dynamics behind such transitions., Competing Interests: E.J.A. is a co-founder and shareholder of Finch Therapeutics, a company that specializes in microbiome-targeted therapeutics.

Neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's disease (AD), the prevalence of which is rapidly rising due to an aging world population and westernization of lifestyles, are expected to put a strong socioeconomic burden on health systems worldwide. Clinical trials of therapies against PD and AD have only shown limited success so far. Therefore, research has extended its scope to a systems medicine point of view, with a particular focus on the gastrointestinal-brain axis as a potential main actor in disease development and progression. Microbiome and metabolome studies have already revealed important insights into disease mechanisms. Both the microbiome and metabolome can be easily manipulated by dietary and lifestyle interventions, and might thus offer novel, readily available therapeutic options to prevent the onset as well as the progression of PD and AD. This review summarizes our current knowledge on the interplay between microbiota, metabolites, and neurodegeneration along the gastrointestinal-brain axis. We further illustrate state-of-the art methods of microbiome and metabolome research as well as metabolic modeling that facilitate the identification of disease pathomechanisms. We conclude with therapeutic options to modulate microbiome composition to prevent or delay neurodegeneration and illustrate potential future research directions to fight PD and AD.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4 + T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4 + T cells that can differentiate into CD4IELs remains unknown. We identified β-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, β-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.

Antibiotic resistance genes (ARGs) are widespread among bacteria. However, not all ARGs pose serious threats to public health, highlighting the importance of identifying those that are high-risk. Here, we developed an 'omics-based' framework to evaluate ARG risk considering human-associated-enrichment, gene mobility, and host pathogenicity. Our framework classifies human-associated, mobile ARGs (3.6% of all ARGs) as the highest risk, which we further differentiate as 'current threats' (Rank I; 3%) - already present among pathogens - and 'future threats' (Rank II; 0.6%) - novel resistance emerging from non-pathogens. Our framework identified 73 'current threat' ARG families. Of these, 35 were among the 37 high-risk ARGs proposed by the World Health Organization and other literature; the remaining 38 were significantly enriched in hospital plasmids. By evaluating all pathogen genomes released since framework construction, we confirmed that ARGs that recently transferred into pathogens were significantly enriched in Rank II ('future threats'). Lastly, we applied the framework to gut microbiome genomes from fecal microbiota transplantation donors. We found that although ARGs were widespread (73% of genomes), only 8.9% of genomes contained high-risk ARGs. Our framework provides an easy-to-implement approach to identify current and future antimicrobial resistance threats, with potential clinical applications including reducing risk of microbiome-based interventions., (© 2021. The Author(s).)

Industrialization has impacted the human gut ecosystem, resulting in altered microbiome composition and diversity. Whether bacterial genomes may also adapt to the industrialization of their host populations remains largely unexplored. Here, we investigate the extent to which the rates and targets of horizontal gene transfer (HGT) vary across thousands of bacterial strains from 15 human populations spanning a range of industrialization. We show that HGTs have accumulated in the microbiome over recent host generations and that HGT occurs at high frequency within individuals. Comparison across human populations reveals that industrialized lifestyles are associated with higher HGT rates and that the functions of HGTs are related to the level of host industrialization. Our results suggest that gut bacteria continuously acquire new functionality based on host lifestyle and that high rates of HGT may be a recent development in human history linked to industrialization., Competing Interests: Declaration of interests R.J.X. is a consultant to Novartis and Nestle. E.J.A. is a co-founder and shareholder of Finch Therapeutics, a company that specializes in microbiome-targeted therapeutics., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Wastewater surveillance represents a complementary approach to clinical surveillance to measure the presence and prevalence of emerging infectious diseases like the novel coronavirus SARS-CoV-2. This innovative data source can improve the precision of epidemiological modeling to understand the penetrance of SARS-CoV-2 in specific vulnerable communities. Here, we tested wastewater collected at a major urban treatment facility in Massachusetts and detected SARS-CoV-2 RNA from the N gene at significant titers (57 to 303 copies per ml of sewage) in the period from 18 to 25 March 2020 using RT-qPCR. We validated detection of SARS-CoV-2 by Sanger sequencing the PCR product from the S gene. Viral titers observed were significantly higher than expected based on clinically confirmed cases in Massachusetts as of 25 March. Our approach is scalable and may be useful in modeling the SARS-CoV-2 pandemic and future outbreaks. IMPORTANCE Wastewater-based surveillance is a promising approach for proactive outbreak monitoring. SARS-CoV-2 is shed in stool early in the clinical course and infects a large asymptomatic population, making it an ideal target for wastewater-based monitoring. In this study, we develop a laboratory protocol to quantify viral titers in raw sewage via qPCR analysis and validate results with sequencing analysis. Our results suggest that the number of positive cases estimated from wastewater viral titers is orders of magnitude greater than the number of confirmed clinical cases and therefore may significantly impact efforts to understand the case fatality rate and progression of disease. These data may help inform decisions surrounding the advancement or scale-back of social distancing and quarantine efforts based on dynamic wastewater catchment-level estimations of prevalence., (Copyright © 2020 Wu et al.)

Urban populations from highly industrialized countries are characterized by a lower gut bacterial diversity as well as by changes in composition compared to rural populations from less industrialized countries. To unveil the mechanisms and factors leading to this diversity loss, it is necessary to identify the factors associated with urbanization-induced shifts at a smaller geographical scale, especially in less industrialized countries. To do so, we investigated potential associations between a variety of dietary, medical, parasitological and socio-cultural factors and the gut and saliva microbiomes of 147 individuals from three populations along an urbanization gradient in Cameroon. We found that the presence of Entamoeba sp., a commensal gut protozoan, followed by stool consistency, were major determinants of the gut microbiome diversity and composition. Interestingly, urban individuals have retained most of their gut eukaryotic and bacterial diversity despite significant changes in diet compared to the rural areas, suggesting that the loss of bacterial microbiome diversity observed in industrialized areas is likely associated with medication. Finally, we observed a weak positive correlation between the gut and the saliva microbiome diversity and composition, even though the saliva microbiome is mainly shaped by habitat-related factors.

Natural selection shapes bacterial evolution in all environments. However, the extent to which commensal bacteria diversify and adapt within the human gut remains unclear. Here, we combine culture-based population genomics and metagenomics to investigate the within-microbiome evolution of Bacteroides fragilis. We find that intra-individual B. fragilis populations contain substantial de novo nucleotide and mobile element diversity, preserving years of within-person history. This history reveals multiple signatures of within-person adaptation, including parallel evolution in sixteen genes. Many of these genes are implicated in cell-envelope biosynthesis and polysaccharide utilization. Tracking evolutionary trajectories using near-daily metagenomic sampling, we find evidence for years-long coexistence in one subject despite adaptive dynamics. We used public metagenomes to investigate one adaptive mutation common in our cohort and found that it emerges frequently in Western, but not Chinese, microbiomes. Collectively, these results demonstrate that B. fragilis adapts within individual microbiomes, pointing to factors that promote long-term gut colonization., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Phase variation, the reversible alternation between genetic states, enables infection by pathogens and colonization by commensals. However, the diversity of phase variation remains underexplored. We developed the PhaseFinder algorithm to quantify DNA inversion-mediated phase variation. A systematic search of 54,875 bacterial genomes identified 4686 intergenic invertible DNA regions (invertons), revealing an enrichment in host-associated bacteria. Invertons containing promoters often regulate extracellular products, underscoring the importance of surface diversity for gut colonization. We found invertons containing promoters regulating antibiotic resistance genes that shift to the ON orientation after antibiotic treatment in human metagenomic data and in vitro, thereby mitigating the cost of antibiotic resistance. We observed that the orientations of some invertons diverge after fecal microbiota transplant, potentially as a result of individual-specific selective forces., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Endospore-formers in the human microbiota are well adapted for host-to-host transmission, and an emerging consensus points to their role in determining health and disease states in the gut. The human gut, more than any other environment, encourages the maintenance of endospore formation, with recent culture-based work suggesting that over 50% of genera in the microbiome carry genes attributed to this trait. However, there has been limited work on the ecological role of endospores and other stress-resistant cellular states in the human gut. In fact, there is no data to indicate whether organisms with the genetic potential to form endospores actually form endospores in situ and how sporulation varies across individuals and over time. Here we applied a culture-independent protocol to enrich for endospores and other stress-resistant cells in human feces to identify variation in these states across people and within an individual over time. We see that cells with resistant states are more likely than those without to be shared among multiple individuals, which suggests that these resistant states are particularly adapted for cross-host dissemination. Furthermore, we use untargeted fecal metabolomics in 24 individuals and within a person over time to show that these organisms respond to shared environmental signals, and in particular, dietary fatty acids, that likely mediate colonization of recently disturbed human guts.

Co-evolution of host-parasitoid interactions is determined by the costs of host resistance, which received empirical evidence, and the costs of parasitoid virulence, which have been mostly hypothesized. Asobara tabida is a parasitoid, which mainly parasitizes Drosophila melanogaster and D. subobscura, the first species being able to resist to the parasitoid development while the second species is not. To parasitize resistant hosts, including D. melanogaster, A. tabida develops sticky eggs, which prevent encapsulation, but this virulence mechanism may be costly. Interindividual and interpopulation variation in the proportion of sticky eggs respectively allowed us to (i) artificially select and compare life-history traits of a virulent and a nonvirulent laboratory strain, and (ii) compare a virulent and a nonvirulent field strain, to investigate the hypothetical costs of virulence. We observed strong differences between the 2 laboratory strains. The nonvirulent strain invested fewer resources in reproduction and walked less than the virulent one but lived longer. Concerning the field strains, we observed that the nonvirulent strain had larger wings while the virulent one walked more and faster. All together, our results suggest that virulence may not always be costly, but rather that different life histories associated with different levels of virulence may coexist at both intra- and interpopulation levels., (© 2016 Institute of Zoology, Chinese Academy of Sciences.)

Animal medication is a behavioral strategy to resist enemies based on the use of substances from the environment. While it has been observed in several animals, whether invasive species can use medication to resist new enemies during its expansion is unknown. Here, we show that the worldwide invasive pest Drosophila suzukii performs trans-generational prophylactic medication by adapting its oviposition behavior in the presence of enemies. We find that flies preferentially lay their eggs on media containing atropine - an entomotoxic alkaloid - in the presence of parasitoids. We further show that flies developing on atropine more efficiently resist parasitization by parasitoids. Finally, we find that developing in hosts reared on atropine strongly impacts the life-history traits of parasitoids. This protective behavior is reported for the first time in a pest and invasive species, and suggests that animal medication may be an important driver of population dynamics during invasions.

The Asiatic fruit fly Drosophila suzukii has recently invaded Europe and North and South America, causing severe damage to fruit production systems. Although agronomic host plants of that fly are now well documented, little is known about the suitability of wild and ornamental hosts in its exotic area. In order to study the potential trophic niche of D. suzukii with relation to fruit characteristics, fleshy fruits from 67 plant species were sampled in natural and anthropic ecosystems (forests, hedgerows, grasslands, coastal areas, gardens and urban areas) of the north of France and submitted to experimental infestations. A set of fruit traits (structure, colour, shape, skin texture, diameter and weight, phenology) potentially interacting with oviposition choices and development success of D. suzukii was measured. Almost half of the tested plant species belonging to 17 plant families allowed the full development of D. suzukii. This suggests that the extreme polyphagy of the fly and the very large reservoir of hosts producing fruits all year round ensure temporal continuity in resource availability and contribute to the persistence and the exceptional invasion success of D. suzukii in natural habitats and neighbouring cultivated systems. Nevertheless, this very plastic trophic niche is not systematically beneficial to the fly. Some of the tested plants attractive to D. suzukii gravid females stimulate oviposition but do not allow full larval development. Planted near sensitive crops, these "trap plants" may attract and lure D. suzukii, therefore contributing to the control of the invasive fly.