Advancing the microbiome research community.

The human microbiome has become a recognized factor in promoting and maintaining health. We outline opportunities in interdisciplinary research, analytical rigor, standardization, and policy development for this relatively new and rapidly developing field. Advances in these aspects of the research community may in turn advance our understanding of human microbiome biology.

Sex, puberty, and the gut microbiome.

In brief: Sex differences in the gut microbiome may impact multiple aspects of human health and disease. In this study, we review the evidence for microbial sex differences in puberty and adulthood and discuss potential mechanisms driving differentiation of the sex-specific gut microbiome. Abstract: In humans, the gut microbiome is strongly implicated in numerous sex-specific physiological processes and diseases. Given this, it is important to understand how sex differentiation of the gut microbiome occurs and how these differences contribute to host health and disease. While it is commonly believed that the gut microbiome stabilizes after 3 years of age, our review of the literature found considerable evidence that the gut microbiome continues to mature during and after puberty in a sex-dependent manner. We also review the intriguing, though sparse, literature on potential mechanisms by which host sex may influence the gut microbiome, and vice versa, via sex steroids, bile acids, and the immune system. We conclude that the evidence for the existence of a sex-specific gut microbiome is strong but that there is a dearth of research on how host-microbe interactions lead to this differentiation. Finally, we discuss the types of future studies needed to understand the processes driving the maturation of sex-specific microbial communities and the interplay between gut microbiota, host sex, and human health.

Altered microbiomes in thirdhand smoke-exposed children and their home environments.

INTRODUCTION: Tobacco smoke contains numerous toxic chemicals that accumulate in indoor environments creating thirdhand smoke (THS). We investigated if THS-polluted homes differed in children's human and built-environment microbiomes as compared to THS-free homes. METHODS: Participants were n = 19 THS-exposed children and n = 10 unexposed children (≤5 years) and their caregivers. Environmental and biological samples were analyzed for THS pollutants and exposure. Swab samples were collected from the built-environment (floor, table, armrest, bed frame) and child (finger, nose, mouth, and ear canal), and 16S ribosomal RNA genes were analyzed for bacterial taxa using high-throughput DNA sequencing. RESULTS: Phylogenetic α-diversity was significantly higher for the built-environment microbiomes in THS-polluted homes compared to THS-free homes (p < 0.014). Log2-fold comparison found differences between THS-polluted and THS-free homes for specific genera in samples from the built-environment (e.g., Acinetobacter, Bradyrhizobium, Corynebacterium, Gemella, Neisseria, Staphylococcus, Streptococcus, and Veillonella) and in samples from children (esp. Corynebacterium, Gemella, Lautropia, Neisseria, Rothia, Staphylococcus, and Veillonella). CONCLUSION: When exposed to THS, indoor and children microbiomes are altered in an environment-specific manner. Changes are similar to those reported in previous studies for smokers and secondhand smoke-exposed persons. THS-induced changes in child and built-environmental microbiomes may play a role in clinical outcomes in children. IMPACT: Despite smoking bans, children can be exposed to tobacco smoke residue (i.e., thirdhand smoke) that lingers on surfaces and in settled house dust. Thirdhand smoke exposure is associated with changes in the microbiomes of the home environment and of the children living in these homes. Thirdhand smoke is associated with increased phylogenetic diversity of the home environment and changes in the abundances of several genera of the child microbiome known to be affected by active smoking and secondhand smoke (e.g., Corynebacterium, Staphylococcus, Streptococcus). Thirdhand smoke exposure by itself may induce alterations in the microbiome that play a role in childhood pathologies.

Quantitative profiling of built environment bacterial and fungal communities reveals dynamic material dependent growth patterns and microbial interactions.

Indoor microbial communities vary in composition and diversity depending on material type, moisture levels, and occupancy. In this study, we integrated bacterial cell counting, fungal biomass estimation, and fluorescence-assisted cell sorting (FACS) with amplicon sequencing of bacterial (16S rRNA) and fungal (ITS) communities to investigate the influence of wetting on medium density fiberboard (MDF) and gypsum wallboard. Surface samples were collected longitudinally from wetted materials maintained at high relative humidity (~95%). Bacterial and fungal growth patterns were strongly time-dependent and material-specific. Fungal growth phenotypes differed between materials: spores dominated MDF surfaces while fungi transitioned from spores to hyphae on gypsum. FACS confirmed that most of the bacterial cells were intact (viable) on both materials over the course of the study. Integrated cell count and biomass data (quantitative profiling) revealed that small changes in relative abundance often resulted from large changes in absolute abundance, while negative correlations in relative abundances were explained by rapid growth of only one group of bacteria or fungi. Comparisons of bacterial-bacterial and fungal-bacterial networks suggested a top-down control of fungi on bacterial growth, possibly via antibiotic production. In conclusion, quantitative profiling provides novel insights into microbial growth dynamics on building materials with potential implications for human health.

Letrozole treatment of adult female mice results in a similar reproductive phenotype but distinct changes in metabolism and the gut microbiome compared to pubertal mice.

BACKGROUND: A majority of women with polycystic ovary syndrome (PCOS) have metabolic dysfunction that results in an increased risk of type 2 diabetes. We previously developed a pubertal mouse model using the aromatase inhibitor, letrozole, which recapitulates many of the reproductive and metabolic features of PCOS. To further our understanding of the effects of androgen excess, we compared the effects of letrozole treatment initiated in puberty versus adulthood on reproductive and metabolic phenotypes as well as on the gut microbiome. RESULTS: Letrozole treatment of both pubertal and adult female mice resulted in reproductive hallmarks of PCOS, including hyperandrogenemia, anovulation and polycystic ovaries. However, unlike pubertal mice, treatment of adult female mice resulted in modest weight gain and abdominal adiposity, minimal elevation in fasting blood glucose and insulin levels, and no detectable insulin resistance. In addition, letrozole treatment of adult mice was associated with a distinct shift in gut microbial diversity compared to letrozole treatment of pubertal mice. CONCLUSIONS: Our results indicate that dysregulation of metabolism and the gut microbiome in PCOS may be influenced by the timing of androgen exposure. In addition, the minimal weight gain and lack of insulin resistance in adult female mice after letrozole treatment indicates that this model may be useful for investigating the effects of hyperandrogenemia on the hypothalamic-pituitary-gonadal axis and the periphery without the influence of substantial metabolic dysregulation.

Discovery of a Novel Periodontal Disease-Associated Bacterium.

One of the world's most common infectious disease, periodontitis (PD), derives from largely uncharacterized communities of oral bacteria growing as biofilms (a.k.a. plaque) on teeth and gum surfaces in periodontal pockets. Bacteria associated with periodontal disease trigger inflammatory responses in immune cells, which in later stages of the disease cause loss of both soft and hard tissue structures supporting teeth. Thus far, only a handful of bacteria have been characterized as infectious agents of PD. Although deep sequencing technologies, such as whole community shotgun sequencing have the potential to capture a detailed picture of highly complex bacterial communities in any given environment, we still lack major reference genomes for the oral microbiome associated with PD and other diseases. In recent work, by using a combination of supervised machine learning and genome assembly, we identified a genome from a novel member of the Bacteroidetes phylum in periodontal samples. Here, by applying a comparative metagenomics read-classification approach, including 272 metagenomes from various human body sites, and our previously assembled draft genome of the uncultivated Candidatus Bacteroides periocalifornicus (CBP) bacterium, we show CBP's ubiquitous distribution in dental plaque, as well as its strong association with the well-known pathogenic "red complex" that resides in deep periodontal pockets.

Effects of moderate, voluntary ethanol consumption on the rat and human gut microbiome.

Many alcohol-induced health complications are directly attributable to the toxicity of alcohol or its metabolites, but another potential health impact of alcohol may be on the microbial communities of the human gut. Clear distinctions between healthy and diseased-state gut microbiota have been observed in subjects with metabolic diseases, and recent studies suggest that chronic alcoholism is linked to gut microbiome dysbiosis. Here, we investigated the effects of moderate levels of alcohol consumption on the gut microbiome in both rats and humans. The gut microbiota of rats voluntarily consuming a 20 percent ethanol solution, on alternate days, were compared with a non-exposed control group to identify differential taxonomic and functional profiles. Gut microbial diversity profiles were determined using culture-independent amplification, next-generation sequencing and bioinformatic analysis of bacterial 16S ribosomal RNA gene sequence libraries. Our results showed that, compared with controls, ethanol-consuming rats experienced a significant decline in the biodiversity of their gut microbiomes, a state generally associated with dysbiosis. We also observed significant shifts in the overall diversity of the gut microbial communities and a dramatic change in the relative abundance of particular microbes, such as the Lactobacilli. We also compared our results to human fecal microbiome data collected as part of the citizen science American Gut Project. In contrast to the rat data, human drinkers had significantly higher gut microbial biodiversity than non-drinkers. However, we also observed that microbes that differed among the human subjects displayed similar trends in the rat model, including bacteria implicated in metabolic disease.

Ecological succession and viability of human-associated microbiota on restroom surfaces.

Human-associated bacteria dominate the built environment (BE). Following decontamination of floors, toilet seats, and soap dispensers in four public restrooms, in situ bacterial communities were characterized hourly, daily, and weekly to determine their successional ecology. The viability of cultivable bacteria, following the removal of dispersal agents (humans), was also assessed hourly. A late-successional community developed within 5 to 8 h on restroom floors and showed remarkable stability over weeks to months. Despite late-successional dominance by skin- and outdoor-associated bacteria, the most ubiquitous organisms were predominantly gut-associated taxa, which persisted following exclusion of humans. Staphylococcus represented the majority of the cultivable community, even after several hours of human exclusion. Methicillin-resistant Staphylococcus aureus (MRSA)-associated virulence genes were found on floors but were not present in assembled Staphylococcus pan-genomes. Viral abundances, which were predominantly enterophages, human papilloma virus, and herpesviruses, were significantly correlated with bacterial abundances and showed an unexpectedly low virus-to-bacterium ratio in surface-associated samples, suggesting that bacterial hosts are mostly dormant on BE surfaces.

Bayesian community-wide culture-independent microbial source tracking.

Contamination is a critical issue in high-throughput metagenomic studies, yet progress toward a comprehensive solution has been limited. We present SourceTracker, a Bayesian approach to estimate the proportion of contaminants in a given community that come from possible source environments. We applied SourceTracker to microbial surveys from neonatal intensive care units (NICUs), offices and molecular biology laboratories, and provide a database of known contaminants for future testing.

Data-driven mathematical and visualization approaches for removing rare features for Compositional Data Analysis (CoDA).

Sparse feature tables, in which many features are present in very few samples, are common in big biological data (e.g. metagenomics). Ignoring issues of zero-laden datasets can result in biased statistical estimates and decreased power in downstream analyses. Zeros are also a particular issue for compositional data analysis using log-ratios since the log of zero is undefined. Researchers typically deal with this issue by removing low frequency features, but the thresholds for removal differ markedly between studies with little or no justification. Here, we present CurvCut, an unsupervised data-driven approach with human confirmation for rare-feature removal. CurvCut implements two distinct approaches for determining natural breaks in the feature distributions: a method based on curvature analysis borrowed from thermodynamics and the Fisher-Jenks statistical method. Our results show that CurvCut rapidly identifies data-specific breaks in these distributions that can be used as cutoff points for low-frequency feature removal that maximizes feature retention. We show that CurvCut works across different biological data types and rapidly generates clear visual results that allow researchers to confirm and apply feature removal cutoffs to individual datasets.

Metagenomic analysis of microbial communities and antibiotic resistant genes in the Tijuana river, and potential sources.

The Tijuana River is a transborder river that flows northwest across the border from Baja California in Mexico into Southern California before discharging into the Pacific Ocean. The river is frequently contaminated with raw sewage due to inadequate sanitary infrastructure in Tijuana. To assess the type and degree of microbial contamination, water samples were collected monthly from a near-border and an estuarine site from August 2020 until May 2021. A portion of each sample was used for epifluorescent microscopy and DNA was extracted directly from the rest for shotgun metagenomic sequencing. After sequence quality checking and processing, we used the rapid taxonomic identifier tool Kaiju to characterize the microbial diversity of the metagenomes and matched the sequences against the Comprehensive Antibiotic Resistance Database (CARD) to examine antimicrobial resistance genes (ARGs). Bacterial and viral-like particle (VLP) abundance was consistently higher in the near-border samples than in the estuarine samples, while alpha diversity (within sample biodiversity) was higher in estuarine samples. Beta-diversity analysis found clear compositional separation between samples from the two sites, and the near-border samples were more dissimilar to one another than were the estuarine sites. Near-border samples were dominated by fecal-associated bacteria and bacteria associated with sewage sludge, while estuarine sites were dominated by marine bacteria. ARGs were more abundant at the near-border site, but were also readily detectable in the estuarine samples, and the most abundant ARGs had multi-resistance to beta-lactam antibiotics. SourceTracker analysis identified human feces and sewage sludge to be the largest contributors to the near-border samples, while marine waters dominated estuarine samples except for two sewage overflow dates with high fecal contamination. Overall, our research determined human sewage microbes to be common in the Tijuana River, and the prevalence of ARGs confirms the importance of planned infrastructure treatment upgrades for environmental health.

AutoPhy: Automated phylogenetic identification of novel protein subfamilies.

Phylogenetic analysis of protein sequences provides a powerful means of identifying novel protein functions and subfamilies, and for identifying and resolving annotation errors. However, automation of functional clustering based on phylogenetic trees has been challenging and most of it is done manually. Clustering phylogenetic trees usually requires the delineation of tree-based thresholds (e.g., distances), leading to an ad hoc problem. We propose a new phylogenetic clustering approach that identifies clusters without using ad hoc distances or other pre-defined values. Our workflow combines uniform manifold approximation and projection (UMAP) with Gaussian mixture models as a k-means like procedure to automatically group sequences into clusters. We then apply a "second pass" clade identification algorithm to resolve non-monophyletic groups. We tested our approach with several well-curated protein families (outer membrane porins, acyltransferase, and nuclear receptors) and showed our automated methods recapitulated known subfamilies. We also applied our methods to a broad range of different protein families from multiple databases, including Pfam, PANTHER, and UniProt, and to alignments of RNA viral genomes. Our results showed that AutoPhy rapidly generated monophyletic clusters (subfamilies) within phylogenetic trees evolving at very different rates both within and among phylogenies. The phylogenetic clusters generated by AutoPhy resolved misannotations and identified new protein functional groups and novel viral strains.

Metabolic dysregulation and gut dysbiosis linked to hyperandrogenism in female mice.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a common endocrine pathology in women. In addition to infertility, women with PCOS have metabolic dysregulation which predisposes them to Type 2 diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Moreover, women with PCOS have changes in their gut microbial community that may be indicative of dysbiosis. While hyperandrogenism is associated with both the development of metabolic dysfunction and gut dysbiosis in females, the mechanisms involved are not well understood. METHODS: We used dihydrotestosterone (DHT) and ovariectomy (OVX) mouse models coupled with metabolic assessments and 16S rRNA gene sequencing to explore the contributions of hyperandrogenism and oestrogen deficiency to the development of insulin resistance and gut microbial dysbiosis in pubertal female mice. RESULTS: We demonstrated that, while DHT treatment or OVX alone were insufficient to induce insulin resistance during the pubertal-to-adult transition, combining OVX with DHT resulted in insulin resistance similar to that observed in letrozole-treated mice with elevated testosterone and decreased oestrogen levels. In addition, our results showed that OVX and DHT in combination resulted in a distinct shift in the gut microbiome compared to DHT or OVX alone, suggesting that the substantial metabolic dysregulation occurring in the OVX + DHT model was accompanied by unique changes in the abundances of gut bacteria including S24-7, Rikenellaceae and Mucispirillum schaedleri. CONCLUSIONS: While hyperandrogenism plays an important role in the development of metabolic dysregulation in female mice, our results indicate that investigation into additional factors influencing insulin resistance and the gut microbiome during the pubertal-to-adult transition could provide additional insight into the pathophysiology of PCOS.

Association of phosphodiesterase 4 polymorphism (rs702553) with blood pressure in the African American Study of Kidney Disease and Hypertension Genomics Study.

Hypertension is a risk factor for diseases such as stroke, heart and kidney disease. Phosphodiesterase (PDE) enzymes play a significant role in regulating inflammation and there is some association between increased inflammatory cell mediators and development of hypertension. Previous research has shown the single nucleotide polymorphism (rs702553) on the PDE4D gene to be associated with stroke and carotid atherosclerosis. This research analyzed the association of rs702553 with baseline mean arterial blood pressure (MAP) in a subset of the African American Study of Kidney Disease and Hypertension Cohort. Data analysis identified baseline diuretic use as an interaction term in the association between this polymorphism and MAP. Compared with participants with AA/AT genotypes, those with a TT genotype at rs702553 had significantly lower baseline MAP among study participants on a diuretic (P=0.02). To our knowledge, the influence of rs702553 on final PDE4D gene expression has not yet been studied. Additional clinical and in-vitro studies are needed to better understand the biological mechanisms from gene expression to enzyme translation that affect blood pressure.

Metabolic reconstruction for metagenomic data and its application to the human microbiome.

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at http://huttenhower.sph.harvard.edu/humann. This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.

Metagenome-Assembled Genomes from Murine Fecal Microbiomes Dominated by Uncharacterized Bacteria.

The laboratory mouse gut microbiome has been extensively studied, but our understanding of its diversity remains incomplete. We report the assembly of 51 draft metagenome-assembled genomes (MAGs) from murine fecal samples dominated by uncharacterized bacteria. These MAGs add to our understanding of gut microbial diversity in this critical model organism.

Taxonomic and functional restoration of tallgrass prairie soil microbial communities in comparison to remnant and agricultural soils.

Restoring ecosystems requires the re-establishment of diverse soil microbial communities that drive critical ecosystem functions. In grasslands, restoration and management require the application of disturbances like fire and grazing. Disturbances can shape microbial taxonomic composition and potentially functional composition as well. We characterized taxonomic and functional gene composition of soil communities using whole genome shotgun metagenomic sequencing to determine how restored soil communities differed from pre-restoration agricultural soils and original remnant soils, how management affects soil microbes, and whether restoration and management affect the number of microbial genes associated with carbohydrate degradation. We found distinct differences in both taxonomic and functional diversity and composition among restored, remnant, and agricultural soils. Remnant soils had low taxonomic and functional richness and diversity, as well as distinct composition, indicating that restoration of agricultural soils does not re-create soil microbial communities that match remnants. Prescribed fire management increased functional diversity, which also was higher in more recently planted restorations. Finally, restored and post-fire soils included high abundances of genes encoding cellulose-degrading enzymes, so restorations and their ongoing management can potentially support functions important in carbon cycling.

Genetic hypogonadal mouse model reveals niche-specific influence of reproductive axis and sex on intestinal microbial communities.

BACKGROUND: The gut microbiome has been linked to many diseases with sex bias including autoimmune, metabolic, neurological, and reproductive disorders. While numerous studies report sex differences in fecal microbial communities, the role of the reproductive axis in this differentiation is unclear and it is unknown how sex differentiation affects microbial diversity in specific regions of the small and large intestine. METHODS: We used a genetic hypogonadal mouse model that does not produce sex steroids or go through puberty to investigate how sex and the reproductive axis impact bacterial diversity within the intestine. Using 16S rRNA gene sequencing, we analyzed alpha and beta diversity and taxonomic composition of fecal and intestinal communities from the lumen and mucosa of the duodenum, ileum, and cecum from adult female (n = 20) and male (n = 20) wild-type mice and female (n = 17) and male (n = 20) hypogonadal mice. RESULTS: Both sex and reproductive axis inactivation altered bacterial composition in an intestinal section and niche-specific manner. Hypogonadism was significantly associated with bacteria from the Bacteroidaceae, Eggerthellaceae, Muribaculaceae, and Rikenellaceae families, which have genes for bile acid metabolism and mucin degradation. Microbial balances between males and females and between hypogonadal and wild-type mice were also intestinal section-specific. In addition, we identified 3 bacterial genera (Escherichia Shigella, Lachnoclostridium, and Eggerthellaceae genus) with higher abundance in wild-type female mice throughout the intestinal tract compared to both wild-type male and hypogonadal female mice, indicating that activation of the reproductive axis leads to female-specific differentiation of the gut microbiome. Our results also implicated factors independent of the reproductive axis (i.e., sex chromosomes) in shaping sex differences in intestinal communities. Additionally, our detailed profile of intestinal communities showed that fecal samples do not reflect bacterial diversity in the small intestine. CONCLUSIONS: Our results indicate that sex differences in the gut microbiome are intestinal niche-specific and that sampling feces or the large intestine may miss significant sex effects in the small intestine. These results strongly support the need to consider both sex and reproductive status when studying the gut microbiome and while developing microbial-based therapies.

Microbial communities in the intestinal tract, known as the gut microbiome, regulate many critical aspects of host physiology. Previous studies have shown that the diversity of the gut microbiome differs between the sexes. There are also many diseases with a sex bias linked to the gut microbiome, including autoimmune, metabolic, neurological, and reproductive disorders. The gut microbiome differentiates during puberty, but it is unknown if the reproductive axis, the system responsible for sexual maturation and production of gonadal sex hormones, is critical for this process. Furthermore, since most studies use feces to examine the gut microbiome, it is unknown how sex influences the microbial communities within different segments of the small and large intestine. To address this gap in knowledge, we used DNA-based molecular methods to compare the intestinal-specific microbiomes of a mouse model with a genetically inactivated reproductive axis to that of wild-type mice. We found that both sex and the reproductive axis impacted gut microbial diversity in an intestinal section-specific manner. We also detected significant differences in intestinal microbial diversity between male and female mutant mice, suggesting that sex chromosome factors also affect the gut microbiome. We also showed that fecal samples were dissimilar to small intestine microbial communities, indicating that studies only sampling feces likely miss sex differences specific to the small intestine. Our results strongly support the need to consider both sex and reproductive status when studying the gut microbiome and while developing microbial-based therapies.

Metagenomic Analysis of Microbial Contamination in the U.S. Portion of the Tijuana River Watershed.

The Tijuana River watershed is binational, flowing from Tijuana, Mexico into San Diego and Imperial Beach, USA. Aging sewage and stormwater infrastructure in Tijuana has not kept pace with population growth, causing overflows into this watershed during major rainfall or equipment failures. The public health consequences of this impaired watershed on the surrounding communities remain unknown. Here, we performed untargeted metagenomic sequencing to better characterize the sewage contamination in the Tijuana River, identifying potential pathogens and molecular indicators of antibiotic resistance in surface waters. In 2019-2020, water samples were collected within 48 h of major rainfall events at five transborder flow sites and at the mouth of the river in the US portion of the Tijuana River and estuary. After filtration, DNA was extracted and sequenced, and sequences were run through the Kaiju taxonomic classification program. A pathogen profile of the most abundant disease-causing microbes and viruses present in each of the samples was constructed, and specific markers of fecal contamination were identified and linked to each site. Results from diversity analysis between the sites showed clear distinction as well as similarities between sites and dates, and antibiotic-resistant genes were found at each site. This serves as a baseline characterization of microbial exposures to these local communities.

Comparative analysis of microbial diversity in Longitarsus flea beetles (Coleoptera: Chrysomelidae).

Herbivorous beetles comprise a significant fraction of eukaryotic biodiversity and their plant-feeding adaptations make them notorious agricultural pests. Despite more than a century of research on their ecology and evolution, we know little about the diversity and function of their symbiotic microbial communities. Recent culture-independent molecular studies have shown that insects possess diverse gut microbial communities that appear critical for their survival. In this study, we combined culture-independent methods and high-throughput sequencing strategies to perform a comparative analysis of Longitarsus flea-beetles microbial community diversity (MCD). This genus of beetle herbivores contains host plant specialists and generalists that feed on a diverse array of toxic plants. Using a deep-sequencing approach, we characterized the MCD of eleven Longitarsus species across the genus, several of which represented independent shifts to the same host plant families. Database comparisons found that Longitarsus-associated microbes came from two habitat types: insect guts and the soil rhizosphere. Statistical clustering of the Longitarsus microbial communities found little correlation with the beetle phylogeny, and uncovered discrepancies between bacterial communities extracted directly from beetles and those from frass. A Principal Coordinates Analysis also found some correspondence between beetle MCD and host plant family. Collectively, our data suggest that environmental factors play a dominant role in shaping Longitarsus MCD and that the root-feeding beetle larvae of these insects are inoculated by soil rhizosphere microbes. Future studies will investigate MCD of select Longitarsus species across their geographic ranges and explore the connection between the soil rhizosphere and the beetle MCD.