isolateR: an R package for generating microbial libraries from Sanger sequencing data.

MOTIVATION: Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. RESULTS: isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. AVAILABILITY AND IMPLEMENTATION: isolateR is available at: https://github.com/bdaisley/isolateR.

Apirhabdus apintestini gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the western honey bee Apis mellifera.

A Gram-negative, motile, rod-shaped bacterial strain, CA-0114T, was isolated from the midgut of a western honey bee, Apis mellifera. The isolate exhibited ≤96.43 % 16S rRNA gene sequence identity (1540 bp) to members of the families Enterobacteriaceae and Erwiniaceae. Phylogenetic trees based on genome blast distance phylogeny and concatenated protein sequences encoded by conserved genes atpD, fusA, gyrB, infB, leuS, pyrG and rpoB separated the isolate from other genera forming a distinct lineage in the Enterobacteriaceae. In both trees, the closest relatives were Tenebrionicola larvae YMB-R21T and Tenebrionibacter intestinalis BIT-L3T, which were isolated previously from Tenebrio molitor L., a plastic-eating mealworm. Digital DNA-DNA hybridization, orthologous average nucleotide identity and average amino acid identity values between strain CA-0114T and the closest related members within the Enterobacteriaceae were ≤23.1, 75.45 and 76.04 %, respectively. The complete genome of strain CA-0114T was 4 451669 bp with a G+C content of 52.12 mol%. Notably, the apparent inability of strain CA-0114T to ferment d-glucose, inositol and l-rhamnose in the API 20E system is unique among closely related members of the Enterobacteriaceae. Based on the results obtained through genotypic and phenotypic analysis, we propose that strain CA-0114T represents a novel species and genus within the family Enterobacteriaceae, for which we propose the name Apirhabdus apintestini gen. nov., sp. nov. (type strain CA-0114T=ATCC TSD-396T=DSM 116385T).

Capacity of a Microbial Synbiotic To Rescue the In Vitro Metabolic Activity of the Gut Microbiome following Perturbation with Alcohol or Antibiotics.

The human gut microbiome contributes crucial bioactive metabolites that support human health and is sensitive to perturbations from the ingestion of alcohol and antibiotics. We interrogated the response and recovery of human gut microbes after acute alcohol or broad-spectrum antibiotic administration in a gut model simulating the luminal and mucosal colonic environment with an inoculated human microbiome. Both alcohol and antibiotic treatments reduced the production of major short-chain fatty acids (SCFAs) (acetate, propionate, and butyrate), which are established modulators of human health. Treatment with a microbial synbiotic restored and enhanced gut function. Butyrate and acetate production increased by up to 29.7% and 18.6%, respectively, relative to untreated, dysbiotic samples. In parallel, treatment led to increases in the relative abundances of beneficial commensal organisms not found in the synbiotic (e.g., Faecalibacterium prausnitzii and the urolithin-producing organism Gordonibacter pamelaeae) as well as species present in the synbiotic (e.g., Bifidobacterium infantis), suggesting synergistic interactions between supplemented and native microorganisms. These results lead us to conclude that functional shifts in the microbiome, evaluated by both metabolite production and specific taxonomic compositional changes, are an appropriate metric to assess microbiome "recovery" following a dysbiosis-inducing disruption. Overall, these findings support the execution of randomized clinical studies to determine whether a microbial synbiotic can help restore microbiome function after a disruption. IMPORTANCE The human gut microbiome is sensitive to disruptions by common stressors such as alcohol consumption and antibiotic treatment. In this study, we used an in vitro system modeling the gut microbiome to investigate whether treatment with a microbial synbiotic can help restore microbiome function after stress. We find that a complex gut community treated with alcohol or antibiotics showed reduced levels of production of short-chain fatty acids, which are critical beneficial molecules produced by a healthy gut microbiota. Treatment of stressed communities with a microbial synbiotic resulted in the recovery of SCFA production as well as an increase in the abundance of beneficial commensal organisms. Our results suggest that treatment with a microbial synbiotic has the potential to restore healthy gut microbiome function after stress and merits further investigation in clinical studies.

Probiotics and in-hive fermentation as a source of beneficial microbes to support the gut microbial health of honey bees.

Managed populations of honey bees (Apis mellifera Linnaeus; Hymenoptera: Apidae) are regularly exposed to infectious diseases. Good hive management including the occasional application of antibiotics can help mitigate infectious outbreaks, but new beekeeping tools and techniques that bolster immunity and help control disease transmission are welcome. In this review, we focus on the applications of beneficial microbes for disease management as well as to support hive health and sustainability within the apicultural industry. We draw attention to the latest advances in probiotic approaches as well as the integration of fermented foods (such as water kefir) with disease-fighting properties that might ultimately be delivered to hives as an alternative or partial antidote to antibiotics. There is substantial evidence from in vitro laboratory studies that suggest beneficial microbes could be an effective method for improving disease resistance in honey bees. However, colony level evidence is lacking and there is urgent need for further validation via controlled field trials experimentally designed to test defined microbial compositions against specific diseases of interest.

In vitro assessment of histamine and lactate production by a multi-strain synbiotic.

Recent studies suggest histamine and d-lactate may negatively impact host health. As excess histamine is deleterious to the host, the identification of bacterial producers has contributed to concerns over the consumption of probiotics or live microorganisms in fermented food items. Some probiotic products have been suspected of inducing d-lactic-acidosis; an illness associated with neurocognitive symptoms such as ataxia. The goals of the present study were to test the in vitro production of histamine and d-lactate by a 24-strain daily synbiotic and to outline methods that others can use to test for their production. Using enzymatic based assays, no significant production of histamine was observed compared to controls (P > 0.05), while d-lactate production was comparable to a commercially available probiotic with no associated health risk. These assays provide a means to add to the safety profile of synbiotic and probiotic products.

Microbiota-Mediated Modulation of Organophosphate Insecticide Toxicity by Species-Dependent Interactions with Lactobacilli in a Drosophila melanogaster Insect Model.

Despite the benefits to the global food supply and agricultural economies, pesticides are believed to pose a threat to the health of both humans and wildlife. Chlorpyrifos (CP), a commonly used organophosphate insecticide, has poor target specificity and causes acute neurotoxicity in a wide range of species via the suppression of acetylcholinesterase. This effect is exacerbated 10- to 100-fold by chlorpyrifos oxon (CPO), a principal metabolite of CP. Since many animal-associated symbiont microorganisms are known to hydrolyze CP into CPO, we used a Drosophila melanogaster insect model to investigate the hypothesis that indigenous and probiotic bacteria could affect CP metabolism and toxicity. Antibiotic-treated and germfree D. melanogaster insects lived significantly longer than their conventionally reared counterparts when exposed to 10 µM CP. Drosophila melanogaster gut-derived Lactobacillus plantarum, but not Acetobacterindonesiensis, was shown to metabolize CP. Liquid chromatography tandem-mass spectrometry confirmed that the L. plantarum isolate preferentially metabolized CP into CPO when grown in CP-spiked culture medium. Further experiments showed that monoassociating germfree D. melanogaster with the L. plantarum isolate could reestablish a conventional-like sensitivity to CP. Interestingly, supplementation with the human probiotic Lactobacillus rhamnosus GG (a strain that binds but does not metabolize CP) significantly increased the survival of the CP-exposed germfree D. melanogaster This suggests strain-specific differences in CP metabolism may exist among lactobacilli and emphasizes the need for further investigation. In summary, these results suggest that (i) CPO formation by the gut microbiota can have biologically relevant consequences for the host, and (ii) probiotic lactobacilli may be beneficial in reducing in vivo CP toxicity.IMPORTANCE An understudied area of research is how the microbiota (microorganisms living in/on an animal) affects the metabolism and toxic outcomes of environmental pollutants such as pesticides. This study focused specifically on how the microbial biotransformation of chlorpyrifos (CP; a common organophosphate insecticide) affected host exposure and toxicity parameters in a Drosophila melanogaster insect model. Our results demonstrate that the biotransformation of CP by the gut microbiota had biologically relevant and toxic consequences on host health and that certain probiotic lactobacilli may be beneficial in reducing CP toxicity. Since inadvertent pesticide exposure is suspected to negatively impact the health of off-target species, these findings may provide useful information for wildlife conservation and environmental sustainability planning. Furthermore, the results highlight the need to consider microbiota composition differences between beneficial and pest insects in future insecticide designs. More broadly, this study supports the use of beneficial microorganisms to modulate the microbiota-mediated biotransformation of xenobiotics.

Probiotic Lactobacillus rhamnosus Reduces Organophosphate Pesticide Absorption and Toxicity to Drosophila melanogaster.

Organophosphate pesticides used in agriculture can pose health risks to humans and wildlife. We hypothesized that dietary supplementation with Lactobacillus, a genus of commensal bacteria, would reduce absorption and toxicity of consumed organophosphate pesticides (parathion and chlorpyrifos [CP]). Several Lactobacillus species were screened for toleration of 100 ppm of CP or parathion in MRS broth based on 24-h growth curves. Certain Lactobacillus strains were unable to reach stationary-phase culture maxima and displayed an abnormal culture morphology in response to pesticide. Further characterization of commonly used, pesticide-tolerant and pesticide-susceptible, probiotic Lactobacillus rhamnosus strain GG (LGG) and L. rhamnosus strain GR-1 (LGR-1), respectively, revealed that both strains could significantly sequester organophosphate pesticides from solution after 24-h coincubations. This effect was independent of metabolic activity, as L. rhamnosus GG did not hydrolyze CP and no difference in organophosphate sequestration was observed between live and heat-killed strains. Furthermore, LGR-1 and LGG reduced the absorption of 100 µM parathion or CP in a Caco-2 Transwell model of the small intestine epithelium. To determine the effect of sequestration on acute toxicity, newly eclosed Drosophila melanogaster flies were exposed to food containing 10 µM CP with or without supplementation with live LGG. Supplementation with LGG simultaneously, but not with administration of CP 3 days prior (prophylactically), mitigated CP-induced mortality. In summary, the results suggest that L. rhamnosus may be useful for reducing toxic organophosphate pesticide exposure via passive binding. These findings could be transferable to clinical and livestock applications due to affordability and practical ability to supplement products with food-grade bacteria. IMPORTANCE: The consequences of environmental pesticide pollution due to widespread usage in agriculture and soil leaching are becoming a major societal concern. Although the long-term effects of low-dose pesticide exposure for humans and wildlife remain largely unknown, logic suggests that these chemicals are not aligned with ecosystem health. This observation is most strongly supported by the agricultural losses associated with honeybee population declines, known as colony collapse disorder, in which pesticide usage is a likely trigger. Lactobacilli are bacteria used as beneficial microorganisms in fermented foods and have shown potentials to sequester and degrade environmental toxins. This study demonstrated that commonly used probiotic strains of lactobacilli could sequester, but not metabolize, organophosphate pesticides (parathion and chlorpyrifos). This Lactobacillus-mediated sequestration was associated with decreased intestinal absorption and insect toxicity in appropriate models. These findings hold promise for supplementing human, livestock, or apiary foods with probiotic microorganisms to reduce organophosphate pesticide exposure.

Microbes as medicine.

Over the last two decades, advancements in sequencing technologies have significantly deepened our understanding of the human microbiome's complexity, leading to increased concerns about the detrimental effects of antibiotics on these intricate microbial ecosystems. Concurrently, the rise in antimicrobial resistance has intensified the focus on how beneficial microbes can be harnessed to treat diseases and improve health and offer potentially promising alternatives to traditional antibiotic treatments. Here, we provide a comprehensive overview of both established and emerging microbe-centric therapies, from probiotics to advanced microbial ecosystem therapeutics, examine the sophisticated ways in which microbes are used medicinally, and consider their impacts on microbiome homeostasis and health outcomes through a microbial ecology lens. In addition, we explore the concept of rewilding the human microbiome by reintroducing "missing microbes" from nonindustrialized societies and personalizing microbiome modulation to fit individual microbial profiles-highlighting several promising directions for future research. Ultimately, the advancements in sequencing technologies combined with innovative microbial therapies and personalized approaches herald a new era in medicine poised to address antibiotic resistance and improve health outcomes through targeted microbiome management.

The untapped potential of cell culture in disentangling insect-microbial relationships.

Cell culture is a powerful technique for the investigation of molecular mechanisms fundamental to health and disease in a diverse array of organisms. Cell lines offer several advantages, namely their simplistic approach and high degree of reproducibility. One field where cell culture has proven particularly useful is the study of the microbiome, where cell culture has led to the illumination of microbial influences on host immunity, nutrition, and physiology. Thus far, researchers have focused cell culture work predominantly on humans, but the growing field of insect microbiome research stands to benefit greatly from its application. Insects constitute one of Earth's most diverse and ancient life forms and, just as with humans, possess microbiomes with great significance to their health. Insects, which play critical roles in supporting food security and ecological stability, are facing increasing threats from agricultural intensification, climate change, and pesticide use. As the microbiome is closely tied to host health, gaining a more robust understanding is of increasing importance. In this review, we assert that the cultivation and utilization of insect gut cell lines in microbiome research will bridge critical knowledge gaps essential for informing insect management practices in a world under pressure.

Assessing phage-host population dynamics by reintroducing virulent viruses to synthetic microbiomes.

Microbiomes feature complex interactions between diverse bacteria and bacteriophages. Synthetic microbiomes offer a powerful way to study these interactions; however, a major challenge is obtaining a representative bacteriophage population during the bacterial isolation process. We demonstrate that colony isolation reliably excludes virulent viruses from sample sources with low virion-to-bacteria ratios such as feces, creating "virulent virus-free" controls. When the virulent dsDNA virome is reintroduced to a 73-strain synthetic gut microbiome in a bioreactor model of the human colon, virulent viruses target susceptible strains without significantly altering community structure or metabolism. In addition, we detected signals of prophage induction that associate with virulent predation. Overall, our findings indicate that dilution-based isolation methods generate synthetic gut microbiomes that are heavily depleted, if not devoid, of virulent viruses and that such viruses, if reintroduced, have a targeted effect on community assembly, metabolism, and prophage replication.

Dissecting mechanisms of fecal microbiota transplantation efficacy in disease.

Fecal microbiota transplantation (FMT) has emerged as an alternative or adjunct experimental therapy for microbiome-associated diseases following its success in the treatment of recurrent Clostridioides difficile infections (rCDIs). However, the mechanisms of action involved remain relatively unknown. The term 'dysbiosis' has been used to describe microbial imbalances in relation to disease, but this traditional definition fails to consider the complex cross-feeding networks that define the stability of the microbiome. Emerging research transitions toward the targeted restoration of microbial functional networks in treating different diseases. In this review, we explore potential mechanisms responsible for the efficacy of FMT and future therapeutic applications, while revisiting definitions of 'dysbiosis' in favor of functional network restoration in rCDI, inflammatory bowel diseases (IBDs), metabolic diseases, and cancer.

Disentangling the microbial ecological factors impacting honey bee susceptibility to Paenibacillus larvae infection.

Paenibacillus larvae is a spore-forming bacterial entomopathogen and causal agent of the important honey bee larval disease, American foulbrood (AFB). Active infections by vegetative P. larvae are often deadly, highly transmissible, and incurable for colonies but, when dormant, the spore form of this pathogen can persist asymptomatically for years. Despite intensive investigation over the past century, this process has remained enigmatic. Here, we provide an up-to-date synthesis on the often overlooked microbiota factors involved in the spore-to-vegetative growth transition (corresponding with the onset of AFB disease symptoms) and offer a novel outlook on AFB pathogenesis by focusing on the 'collaborative' and 'competitive' interactions between P. larvae and other honey bee-adapted microorganisms. Furthermore, we discuss the health trade-offs associated with chronic antibiotic exposure and propose new avenues for the sustainable control of AFB via probiotic and microbiota management strategies.

The Robogut: A Bioreactor Model of the Human Colon for Evaluation of Gut Microbial Community Ecology and Function.

The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles.

Multi-site microbiota alteration is a hallmark of kidney stone formation.

BACKGROUND: Inquiry of microbiota involvement in kidney stone disease (KSD) has largely focussed on potential oxalate handling abilities by gut bacteria and the increased association with antibiotic exposure. By systematically comparing the gut, urinary, and oral microbiota of 83 stone formers (SF) and 30 healthy controls (HC), we provide a unified assessment of the bacterial contribution to KSD. RESULTS: Amplicon and shotgun metagenomic sequencing approaches were consistent in identifying multi-site microbiota disturbances in SF relative to HC. Biomarker taxa, reduced taxonomic and functional diversity, functional replacement of core bioenergetic pathways with virulence-associated gene markers, and community network collapse defined SF, but differences between cohorts did not extend to oxalate metabolism. CONCLUSIONS: We conclude that multi-site microbiota alteration is a hallmark of SF, and KSD treatment should consider microbial functional restoration and the avoidance of aberrant modulators such as poor diet and antibiotics where applicable to prevent stone recurrence. Video Abstract.

Delivery mechanism can enhance probiotic activity against honey bee pathogens.

Managed honey bee (Apis mellifera) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems-standard pollen patty infusion and a novel spray-based formulation-affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus, Bifidobacterium, Lactobacillus, and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.

Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial.

Fecal microbiota transplantation (FMT) represents a potential strategy to overcome resistance to immune checkpoint inhibitors in patients with refractory melanoma; however, the role of FMT in first-line treatment settings has not been evaluated. We conducted a multicenter phase I trial combining healthy donor FMT with the PD-1 inhibitors nivolumab or pembrolizumab in 20 previously untreated patients with advanced melanoma. The primary end point was safety. No grade 3 adverse events were reported from FMT alone. Five patients (25%) experienced grade 3 immune-related adverse events from combination therapy. Key secondary end points were objective response rate, changes in gut microbiome composition and systemic immune and metabolomics analyses. The objective response rate was 65% (13 of 20), including four (20%) complete responses. Longitudinal microbiome profiling revealed that all patients engrafted strains from their respective donors; however, the acquired similarity between donor and patient microbiomes only increased over time in responders. Responders experienced an enrichment of immunogenic and a loss of deleterious bacteria following FMT. Avatar mouse models confirmed the role of healthy donor feces in increasing anti-PD-1 efficacy. Our results show that FMT from healthy donors is safe in the first-line setting and warrants further investigation in combination with immune checkpoint inhibitors. ClinicalTrials.gov identifier NCT03772899 .

Deteriorating microbiomes in agriculture - the unintended effects of pesticides on microbial life.

There is emerging concern regarding the unintentional and often unrecognized antimicrobial properties of "non-antimicrobial" pesticides. This includes insecticides, herbicides, and fungicides commonly used in agriculture that are known to produce broad ranging, off-target effects on beneficial wildlife, even at seemingly non-toxic low dose exposures. Notably, these obscure adverse interactions may be related to host-associated microbiome damage occurring from antimicrobial effects, rather than the presumed toxic effects of pesticides on host tissue. Here, we critically review the literature on this topic as it pertains to the rhizosphere microbiome of crop plants and gut microbiome of pollinator insects (namely managed populations of the western honey bee, Apis mellifera), since both are frequent recipients of chronic pesticide exposure. Clear linkages between pesticide mode of action and host-specific microbiome functionalities are identified in relation to potential antimicrobial risks. For example, inherent differences in nitrogen metabolism of plant- and insect-associated microbiomes may dictate whether neonicotinoid-based insecticides ultimately exert antimicrobial activities or not. Several other context-dependent scenarios are discussed. In addition to direct effects (e.g., microbicidal action of the parent compound or breakdown metabolites), pesticides may indirectly alter the trajectory of host-microbiome coevolution in honey bees via modulation of social behaviours and the insect gut-brain axis - conceivably with consequences on plant-pollinator symbiosis as well. In summary, current evidence suggests: (1) immediate action is needed by regulatory authorities in amending safety assessments for "non-antimicrobial" pesticides; and (2) that the development of host-free microbiome model systems could be useful for rapidly screening pesticides against functionally distinct microbial catalogues of interest.

Harnessing the microbiome to prevent global biodiversity loss.

Global biodiversity loss and mass extinction of species are two of the most critical environmental issues the world is currently facing, resulting in the disruption of various ecosystems central to environmental functions and human health. Microbiome-targeted interventions, such as probiotics and microbiome transplants, are emerging as potential options to reverse deterioration of biodiversity and increase the resilience of wildlife and ecosystems. However, the implementation of these interventions is urgently needed. We summarize the current concepts, bottlenecks and ethical aspects encompassing the careful and responsible management of ecosystem resources using the microbiome (termed microbiome stewardship) to rehabilitate organisms and ecosystem functions. We propose a real-world application framework to guide environmental and wildlife probiotic applications. This framework details steps that must be taken in the upscaling process while weighing risks against the high toll of inaction. In doing so, we draw parallels with other aspects of contemporary science moving swiftly in the face of urgent global challenges.

BEExact: a Metataxonomic Database Tool for High-Resolution Inference of Bee-Associated Microbial Communities.

High-throughput 16S rRNA gene sequencing technologies have robust potential to improve our understanding of bee (Hymenoptera: Apoidea)-associated microbial communities and their impact on hive health and disease. Despite recent computation algorithms now permitting exact inferencing of high-resolution exact amplicon sequence variants (ASVs), the taxonomic classification of these ASVs remains a challenge due to inadequate reference databases. To address this, we assemble a comprehensive data set of all publicly available bee-associated 16S rRNA gene sequences, systematically annotate poorly resolved identities via inclusion of 618 placeholder labels for uncultivated microbial dark matter, and correct for phylogenetic inconsistencies using a complementary set of distance-based and maximum likelihood correction strategies. To benchmark the resultant database (BEExact), we compare performance against all existing reference databases in silico using a variety of classifier algorithms to produce probabilistic confidence scores. We also validate realistic classification rates on an independent set of ∼234 million short-read sequences derived from 32 studies encompassing 50 different bee types (36 eusocial and 14 solitary). Species-level classification rates on short-read ASVs range from 80 to 90% using BEExact (with ∼20% due to "bxid" placeholder names), whereas only ∼30% at best can be resolved with current universal databases. A series of data-driven recommendations are developed for future studies. We conclude that BEExact (https://github.com/bdaisley/BEExact) enables accurate and standardized microbiota profiling across a broad range of bee species-two factors of key importance to reproducibility and meaningful knowledge exchange within the scientific community that together, can enhance the overall utility and ecological relevance of routine 16S rRNA gene-based sequencing endeavors.IMPORTANCE The failure of current universal taxonomic databases to support the rapidly expanding field of bee microbiota research has led to many investigators relying on "in-house" reference sets or manual classification of sequence reads (usually based on BLAST searches), often with vague identity thresholds and subjective taxonomy choices. This time-consuming, error- and bias-prone process lacks standardization, cripples the potential for comparative cross-study analysis, and in many cases is likely to incorrectly sway study conclusions. BEExact is structured on and leverages several complementary bioinformatic techniques to enable refined inference of bee host-associated microbial communities without any other methodological modifications necessary. It also bridges the gap between current practical outcomes (i.e., phylotype-to-genus level constraints with 97% operational taxonomic units [OTUs]) and the theoretical resolution (i.e., species-to-strain level classification with 100% ASVs) attainable in future microbiota investigations. Other niche habitats could also likely benefit from customized database curation via implementation of the novel approaches introduced in this study.

Emerging connections between gut microbiome bioenergetics and chronic metabolic diseases.

The conventional viewpoint of single-celled microbial metabolism fails to adequately depict energy flow at the systems level in host-adapted microbial communities. Emerging paradigms instead support that distinct microbiomes develop interconnected and interdependent electron transport chains that rely on cooperative production and sharing of bioenergetic machinery (i.e., directly involved in generating ATP) in the extracellular space. These communal resources represent an important subset of the microbial metabolome, designated here as the "pantryome" (i.e., pantry or external storage compartment), that critically supports microbiome function and can exert multifunctional effects on host physiology. We review these interactions as they relate to human health by detailing the genomic-based sharing potential of gut-derived bacterial and archaeal reference strains. Aromatic amino acids, metabolic cofactors (B vitamins), menaquinones (vitamin K2), hemes, and short-chain fatty acids (with specific emphasis on acetate as a central regulator of symbiosis) are discussed in depth regarding their role in microbiome-related metabolic diseases.