[This corrects the article DOI: 10.1371/journal.pone.0053838.].

Targeted amplicon sequencing is widely used to profile genetic variation in defined genomic regions. In microbial ecology, for example, amplicon sequencing of the 16S and 18S ribosomal RNA genes has been transformative for characterizing microbial communities. However, on high-capacity sequencing platforms with patterned flow cells, throughput is constrained by the requirement for unique dual indexes (UDIs), which increases primer costs and limits the number of samples that can be pooled per sequencing run. Here, we introduce CUPID-seq (Combinatorial, Unique, Phased, In-line Dual-indexed sequencing), a highly multiplexed amplicon-sequencing strategy that increases scalability through combinatorial indexing across two rounds of PCR. CUPID-seq introduces phased, in-line UDIs during Round 1 gene-specific amplification, enabling multiple samples to share the same Illumina UDI during Round 2 PCR while remaining uniquely identifiable. This design reduces upfront costs by up to 85% and reduces library preparation time and reagent use by up to 40%. We develop and validate CUPID-seq primers targeting the 16S V4 region and provide a computational workflow for demultiplexing in-line indexes. Although optimized here for 16S-based profiling, CUPID-seq can be readily adapted to other user-defined amplicons. By reducing cost and increasing multiplexing capacity, CUPID-seq enables users to leverage high-throughput sequencing platforms more effectively across diverse biological contexts.

The human small intestine (SI) plays a central role in nutrient processing, host-microbe interactions, and immune regulation, yet remains poorly characterized due to the lack of minimally disruptive sampling methods. Here, we present a protocol for deploying, recovering, and analyzing samples collected using an ingestible device that enables multi-region, lumen-targeted SI sampling during normal digestion. The device incorporates a ∼30-cm collapsible tube wound into pH- or time-responsive layers that sequentially unfurl in situ , typically capturing three spatially ordered samples with high yield and reliable retrieval. This protocol outlines study design, participant handling, device recovery, contamination control, and standardized workflows for analyses, including cell quantification, culturomics, sequencing, and metabolomics. We further describe benchmarking approaches for evaluating spatial resolution and strategies for assay prioritization when sample volume is limiting. By reducing participant burden and facilitating integration with stool, saliva, and clinical metadata, this approach enables longitudinal and large-cohort studies linking SI microbial ecology and host physiology to human health.

Bacterial vaginosis (BV) is characterized by high microbial diversity. High recurrence rates following antibiotics may stem from poor recolonization by protective Lactobacillus species. This phase 1 randomized trial in the United States and South Africa evaluated two vaginally delivered live biotherapeutic products (LBPs) containing multiple Lactobacillus crispatus strains. After metronidazole treatment for BV, participants received either a placebo or 3 or 7 days of active LBPs. LBP strains were detected by metagenomics in 66.1% (47/71) of participants in the active arms in the first 5 weeks. Among those, nearly half (49%, 23/47) remained colonized at 12 weeks despite the short initial treatment course. Participants were most often colonized by one of three component strains, with no geographic differences in strain colonization observed. LBPs were safe, acceptable, and well tolerated, with no serious adverse events (AEs) reported. These results provide a foundation for the development of transformational interventions aimed at optimizing the vaginal microbiome.

Despite the importance of the cervicovaginal microbiome, the mechanisms that govern its composition and drive its impact on host physiology remain poorly understood. With the aim to expand our understanding of the function and ecology of the vaginal microbiome, we present VIRGO2, an enhanced non-redundant gene catalog comprising over 1.7 million well-annotated genes from body-site specific microbes and viruses. Analyses using VIRGO2 reveal insights such as including the identification of previously uncharacterized vaginal bacteria, features of the vaginal mycobiome and phageome, and differential expression of bacterial carbohydrate catabolic genes. Constructed from over 2500 metagenomes and 4000 bacterial genomes, VIRGO2 broadens geographic representation and microbial diversity compared to its predecessor. This updated catalog enables more precise profiling of taxonomic and functional composition from metagenomic and metatranscriptomic datasets. VIRGO2 is a critical resource for integrative analyses of vaginal microbial communities and their interactions with host tissues, thereby enhancing our mechanistic understanding of vaginal health and disease.

The long-term success of introduced populations depends on both their initial size and ability to compete against existing residents, but it remains unclear how these factors collectively shape colonization dynamics. Here, we investigate how initial population (propagule) size shapes the outcome of community coalescence by systematically mixing eight pairs of in vitro microbial communities at ratios that vary over six orders of magnitude, and we compare our results to neutral ecological theory. Although the composition of the resulting cocultures deviated substantially from neutral expectations, each coculture contained species whose relative abundance depended on propagule size even after ~40 generations of growth. Using a consumer–resource model, we show that this dose-dependent colonization can arise when resident and introduced species have high niche overlap and consume shared resources at similar rates. Strain isolates displayed longer-lasting dose dependence when introduced into diverse communities than in pairwise cocultures, consistent with our model’s prediction that propagule size should have larger, more persistent effects in diverse communities. Our model also successfully predicted that species with similar resource-utilization profiles, as inferred from growth in spent media and untargeted metabolomics, would show stronger dose dependence in pairwise coculture. This work demonstrates that transient, dose-dependent colonization dynamics can emerge from resource competition and exert long-term effects on the outcomes of community coalescence.

Abstract Parent-offspring interactions constitute the first contact of many newborns with their environment, priming community assembly of microbes through priority effects. Early exposure to microbes can have lasting influences on the assembly and functionality of the host’s microbiota, leaving a life-long imprint on host health and disease. Studies of the role played by parental care in microbial acquisition have primarily focused on humans and hosts with agricultural relevance. Anuran vertebrates offer the opportunity to examine microbial community composition across life stages as a function of parental investment. In this study, we investigate vertical transmission of microbiota during parental care in a poison frog (Family Dendrobatidae), where fathers transport their offspring piggyback-style from terrestrial clutches to aquatic nurseries. We found that substantial bacterial colonization of the embryo begins after hatching from the vitelline envelope, emphasizing its potential role as microbial barrier during early development. Using a laboratory cross-foster experiment, we demonstrated that poison frogs performing tadpole transport serve as a source of skin microbes for tadpoles on their back. To study how transport impacts the microbial skin communities of tadpoles in an ecologically relevant setting, we sampled frogs and tadpoles of sympatric species that do or do not exhibit tadpole transport in their natural habitat. We found more diverse microbial communities associated with tadpoles of transporting species compared to a non-transporting frog. However, we detected no difference in the degree of similarity between adult and tadpole skin microbiotas, based on whether the frog species exhibits transporting behavior or not. Using a field experiment, we confirmed that tadpole transport can result in the persistent colonization of tadpoles by isolated microbial taxa associated with the caregiver’s skin, albeit often at low prevalence. This is the first study to describe vertical transmission of skin microbes in anuran amphibians, showing that offspring transport may serve as a mechanism for transmission of parental skin microbes. Overall, these findings provide a foundation for further research on how vertical transmission in this order impacts host-associated microbiota and physiology.

Understanding the relationship between antibiotic use and the evolution of antimicrobial resistance is vital for effective antibiotic stewardship. Yet, animal models and in vitro experiments poorly replicate real-world conditions1. To explain how resistance evolves in vivo, we exposed 60 human participants to ciprofloxacin and used longitudinal stool samples and a new computational method to assemble the genomes of 5,665 populations of commensal bacterial species within participants. Analysis of 2.3 million polymorphic sequence variants revealed 513 populations that underwent selective sweeps. We found convergent evolution focused on DNA gyrase and evidence of dispersed selective pressure at other genomic loci. Roughly 10% of susceptible bacterial populations evolved towards resistance through sweeps that involved substitutions at a specific amino acid in gyrase. The evolution of gyrase was associated with large populations that decreased in relative abundance during exposure. Sweeps persisted for more than 10 weeks in most cases and were not projected to revert within a year. Targeted amplification showed that gyrase mutations arose de novo within the participants and exhibited no measurable fitness cost. These findings revealed that brief ciprofloxacin exposure drives the evolution of resistance in gut commensals, with mutations persisting long after exposure. This study underscores the capacity of the human gut to promote the evolution of resistance and identifies key genomic and ecological factors that shape bacterial adaptation in vivo.

Parent-offspring interactions constitute the first contact of many newborns with their environment, priming community assembly of microbes through priority effects and shaping host health and disease. Microbe acquisition during parental care is well studied in humans and agriculturally relevant species but remains poorly understood in other vertebrate groups, such as amphibians. Here, we investigate vertical transmission of skin microbiota in poison frogs (Dendrobatidae), where fathers transport tadpoles piggyback-style from terrestrial clutches to aquatic nurseries. We found that substantial bacterial colonization of embryos begins after hatching, suggesting that the vitelline envelope acts as a microbial barrier. A cross-foster experiment demonstrated that poison frogs performing tadpole transport serve as a source of skin microbes for tadpoles on their back. To study how transport impacts skin communities of tadpoles in an ecologically relevant setting, we sampled sympatric species that do or do not exhibit tadpole transport in their natural habitat. We did not find a higher degree of similarity between microbial communities of tadpoles and adults in species that transport their offspring compared to those that do not. Similarly, communities of tadpoles were no more similar to their caregiver than to unrelated adults, indicating that most caregiver-associated microbes do not remain in tadpole communities long-term. Nonetheless, some taxa persisted on tadpoles over development. This study is the first to demonstrate that offspring transport facilitates transmission of parental skin microbes in anurans.

The gut microbiome is crucial for health and greatly affected by lifestyle. Many microbes common in non-industrialized populations are disappearing or extinct in industrialized populations. Understanding which microbes have been long-term residents of the human gut, and may have co-evolved with humans, has implications for the importance of microbial biodiversity loss for health. However, the genetic complexities of microbial evolution and the plasticity of gut microbiome composition have made it challenging to define these long-term associations. Here, we performed deep metagenomic sequencing of the Tsimane horticulturalists of Bolivia and compared their gut microbiomes with the Hadza hunter-gatherers of Tanzania. These two populations, whose ancestors were separated for tens of thousands of years, share 1,231 microbial species, most of which are absent in industrialized populations. Population genetic analyses in 636 of these shared species revealed patterns of microbial divergence and gene flow consistent with prehistoric human co-migration, with estimated split times that approximately align with human migration out of Africa and into the Americas. Our findings indicate that a diverse gut microbiome co-migrated with humans around the globe, persisting over millennia. However, many of these species are now vanishing in industrialized populations, and the consequences for human health remain uncertain. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R01-AG054442, R35-GM146949 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-17-EURE-0010 Chan Zuckerberg Initiative (United States) Open Philanthropy Project, https://ror.org/004d1k391

United States and global guidance documents and codes of conduct urge life scientists to practice a "culture of responsibility" by proactively managing the potential biosafety, biosecurity, and dual-use information risks of their work. However, research suggests that many life scientists are unfamiliar with or disengaged from aspects of biorisk management. To better understand life scientists' beliefs and attitudes about biorisk management, we conducted a survey with 127 researchers at a prestigious U.S. university who directly manipulate DNA or RNA in living organisms, cells, and/or viruses. We found that while participants were broadly positive about their efforts to address risks and expressed a sense of responsibility to do so, most failed to meet the expectations that they hold for how often a typical scientist in their research community should consider biosafety, biosecurity, or dual-use information risks. Faculty were more likely to meet their expectations than non-faculty, and all participants were more likely to meet their expectations considering biosafety risks compared with biosecurity or dual-use information risks. Most non-faculty said that they have "never or almost never" considered the risk of deliberate misuse or information release with their lab. Outside of mandatory biosafety training, few had received formal education about biorisks or discussed them at lab meetings. Career incentives and the logistical burdens of biorisk management were noted as reasons for disengagement. Our results suggest that by their own standards, U.S. life scientists have significant room for improvement in their capacity for proactive biorisk management, particularly regarding biosecurity and dual-use information risks.

The endangered Southern Resident killer whale ( Orcinus orca ) (SRKW) population is burdened by multiple anthropogenic stressors with limited non-invasive approaches for health surveillance. The gut microbiome is interconnected with host physiology and can be characterized using remotely collected fecal samples, creating unique opportunities to integrate environmental and individualized host-associated features of health. In this study, we used fecal samples collected over a 15-year period (2005-2019) from 77% of the living, wild SRKW population (56 individuals, 1-17 time points per individual), as well as fecal samples from the conspecific Northern and Alaska resident killer whale populations, to characterize distal gut microbiota structure and genomic composition. SRKW microbiotas were individualized and distinct from those of the nearby Northern and Alaska Resident populations, both of which exhibit consistently higher fecundity and survivorship. During the study period, SRKW fecal microbiota species richness declined, despite stability over periods of time less than or equal to 1 year. Several potential bacterial pathogens, such as Fusobacterium spp., achieved dominance in the fecal microbiotas of SRKW individuals sampled within 6 months of death. These findings demonstrate the feasibility and value of harnessing non-invasively collected fecal samples and microbiome profiles for longitudinal killer whale health surveillance. ### Competing Interest Statement The authors have declared no competing interest. Fisheries and Oceans Canada, https://ror.org/02qa1x782, MEQ-2023-24-009 National Oceanic and Atmospheric Administration, https://ror.org/02z5nhe81 Exxon Valdez Oil Spill Trustee Council, https://ror.org/02t378715 Marine Mammal Commission, https://ror.org/05sn2fr43

The vaginal ecosystem is closely tied to human health and reproductive outcomes, yet its dynamics in the wake of childbirth remain poorly characterized. Here, we profile the vaginal microbiota and cytokine milieu of participants sampled longitudinally throughout pregnancy and for at least one year postpartum. We show that delivery, regardless of mode, is associated with a vaginal pro-inflammatory cytokine response and the loss of Lactobacillus dominance. By contrast, neither the progression of gestation nor the approach of labor strongly altered the vaginal ecosystem. At 9.5-months postpartum—the latest timepoint at which cytokines were assessed—elevated inflammation coincided with vaginal bacterial communities that had remained perturbed (highly diverse) from the time of delivery. Time-to-event analysis indicated a one-year postpartum probability of transitioning to Lactobacillus dominance of 49.4%. As diversity and inflammation declined during the postpartum period, dominance by L. crispatus , the quintessential health-associated commensal, failed to return: its prevalence before, immediately after, and one year after delivery was 41%, 4%, and 9%, respectively. Revisiting our pre-delivery data, we found that a prior live birth was associated with a lower odds of L. crispatus dominance in pregnant participants—an outcome modestly tempered by a longer ( > 18-month) interpregnancy interval. Our results suggest that reproductive history and childbirth in particular remodel the vaginal ecosystem and that the timing and degree of recovery from delivery may help determine the subsequent health of the woman and of future pregnancies.

The rapid advancement of artificial intelligence (AI) in biomedical research presents considerable potential for misuse, including authoritarian surveillance, data misuse, bioweapon development, increase in inequity and abuse of privacy. We propose a multi-pronged framework for researchers to mitigate these risks, looking first to existing ethical frameworks and regulatory measures researchers can adapt to their own work, next to off-the-shelf AI solutions, then to design-specific solutions researchers can build into their AI to mitigate misuse. When researchers remain unable to address the potential for harmful misuse, and the risks outweigh potential benefits, we recommend researchers consider a different approach to answering their research question, or a new research question if the risks remain too great. We apply this framework to three different domains of AI research where misuse is likely to be problematic: (1) AI for drug and chemical discovery; (2) generative models for synthetic data; (3) ambient intelligence.

The vaginal microbiome has been linked to negative health outcomes including preterm birth. Specific taxa, including Gardnerella spp., have been identified as risk factors for these conditions. Historically, microbiome analysis methods have treated all Gardnerella spp. as one species, but the broad diversity of Gardnerella has become more apparent. We explore the diversity of Gardnerella clades and genomic species in the vaginal microbiome of pregnant women and their associations with microbiome composition and preterm birth. Relative abundance of Gardnerella clades and genomic species and other taxa was quantified in shotgun metagenomic sequencing data from three distinct cohorts of pregnant women. We also assessed the diversity and abundance of Gardnerella variants in 16S rRNA gene amplicon sequencing data from seven previously conducted studies in differing populations. Individual microbiomes often contained multiple Gardnerella variants, and the number of clades was associated with increased microbial load, or the ratio of non-human reads to human reads. Taxon co-occurrence patterns were largely consistent across Gardnerella clades and among cohorts. Some variants previously described as rare were prevalent in other cohorts, highlighting the importance of surveying a diverse set of populations to fully capture the diversity of Gardnerella. The diversity of Gardnerella both across populations and within individual vaginal microbiomes has long been unappreciated, as has been the intra-species diversity of many other members of the vaginal microbiome. The broad genomic diversity of Gardnerella has led to its reclassification as multiple species; here we demonstrate the diversity of Gardnerella found within and between vaginal microbiomes.IMPORTANCEThe present study shows that single microbiomes can contain all currently known species of Gardnerella and that multiple similar species can exist within the same environment. Furthermore, surveys of demographically distinct populations suggest that some species appear more commonly in certain populations. Further studies in broad and diverse populations will be necessary to fully understand the ecological roles of each Gardnerella sp., how they can co-exist, and their distinct impacts on microbial communities, preterm birth, and other health outcomes.

Background: Lifestyle plays an important role in shaping the gut microbiome. However, its contributions to the oral microbiome remains less clear, due to the confounding effects of geography and methodology in investigations of populations studied to date. Furthermore, while the oral microbiome seems to differ between foraging and industrialized populations, we lack insight into whether transitions to and away from agrarian lifestyles shape the oral microbiota. Given the growing interest in so-called 'vanishing microbiomes' potentially being a risk factor for increased disease prevalence in industrialized populations, it is important that we distinguish lifestyle from geography in the study of microbiomes across populations. Results: Here, we investigate salivary microbiomes of 63 Nepali individuals representing a spectrum of lifestyles: foraging, subsistence farming (individuals that transitioned from foraging to farming within the last 50 years), agriculturalists (individuals that have transitioned to farming for at least 300 years), and industrialists (expatriates that immigrated to the United States within the last 20 years). We characterize the role of lifestyle in microbial diversity, identify microbes that differ between lifestyles, and pinpoint specific lifestyle factors that may be contributing to differences in the microbiomes across populations. Contrary to prevailing views, when geography is controlled for, oral microbiome alpha diversity does not differ significantly across lifestyles. Microbiome composition, however, follows the gradient of lifestyles from foraging through agrarianism to industrialism, supporting the notion that lifestyle indeed plays a role in the oral microbiome. Relative abundances of several individual taxa, including Streptobacillus and an unclassified Porphyromonadaceae genus, also mirror lifestyle. Finally, we identify specific lifestyle factors associated with microbiome composition across the gradient of lifestyles, including smoking and grain source. Conclusion: Our findings demonstrate that by controlling for geography, we can isolate an important role for lifestyle in determining oral microbiome composition. In doing so, we highlight the potential contributions of several lifestyle factors, underlining the importance of carefully examining the oral microbiome across lifestyles to improve our understanding of global microbiomes.

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Diverse and non-Lactobacillus-dominated vaginal microbial communities are associated with adverse health outcomes such as preterm birth and the acquisition of sexually transmitted infections. Despite the importance of recognizing and understanding the key risk-associated features of these communities, their heterogeneous structure and properties remain ill-defined. Clustering approaches are commonly used to characterize vaginal communities, but they lack sensitivity and robustness in resolving substructures and revealing transitions between potential sub-communities. Here, we address this need with an approach based on mixed membership topic models. Using longitudinal data from cohorts of pregnant and non-pregnant study participants, we show that topic models more accurately describe sample composition, longitudinal changes, and better predict the loss of Lactobacillus dominance. We identify several non-Lactobacillus-dominated sub-communities common to both cohorts and independent of reproductive status. In non-pregnant individuals, we find that the menstrual cycle modulates transitions between and within sub-communities, as well as the concentrations of half of the cytokines and 18% of metabolites. Overall, our analyses based on mixed membership models reveal substructures of vaginal ecosystems which may have important clinical and biological associations.