Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

Longitudinal profiling of the microbiome at four body sites reveals core stability and individualized dynamics during health and disease.

To understand the dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune, and clinical markers of microbiomes from four body sites in 86 participants over 6 years. We found that microbiome stability and individuality are body-site specific and heavily influenced by the host. The stool and oral microbiome are more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. We identify individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlate across body sites, suggesting systemic dynamics influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals show altered microbial stability and associations among microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease.

A serum-induced gene signature in hepatocytes is associated with pediatric nonalcoholic fatty liver disease.

OBJECTIVE: Pediatric nonalcoholic fatty liver disease (NAFLD) is a growing problem, but its underlying mechanisms are poorly understood. We used transcriptomic reporter cell assays to investigate differences in transcriptional signatures induced in hepatocyte reporter cells by the sera of children with and without NAFLD. METHODS: We studied serum samples from 45 children with NAFLD and 28 children without NAFLD. The sera were used to induce gene expression in cultured HepaRG cells and RNA-sequencing was used to determine gene expression. Computational techniques were used to compare gene expression patterns. RESULTS: Sera from children with NAFLD induced the expression of 195 genes that were significantly differentially expressed in hepatocytes compared to controls with obesity. NAFLD was associated with increased expression of genes promoting inflammation, collagen synthesis, and extracellular matrix remodeling. Additionally, there was lower expression of genes involved in endobiotic and xenobiotic metabolism, and downregulation of peroxisome function, oxidative phosphorylation, and xenobiotic, bile acid, and fatty acid metabolism. A 13-gene signature, including upregulation of TREM1 and MMP1 and downregulation of CYP2C9, was consistently associated with all diagnostic categories of pediatric NAFLD. CONCLUSION: The extracellular milieu of sera from children with NAFLD induced specific gene profiles distinguishable by a hepatocyte reporter system. Circulating factors may contribute to inflammation and extracellular matrix remodeling and impair xenobiotic and endobiotic metabolism in pediatric NAFLD.

Longitudinal profiling of the microbiome at four body sites reveals core stability and individualized dynamics during health and disease.

To understand dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune and clinical markers of microbiomes from four body sites in 86 participants over six years. We found that microbiome stability and individuality are body-site-specific and heavily influenced by the host. The stool and oral microbiome were more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. Also, we identified individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlated across body sites, suggesting systemic coordination influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals showed altered microbial stability and associations between microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease. Study Highlights: The stability of the human microbiome varies among individuals and body sites.Highly individualized microbial genera are more stable over time.At each of the four body sites, systematic interactions between the environment, the host and bacteria can be detected.Individuals with insulin resistance have lower microbiome stability, a more diversified skin microbiome, and significantly altered host-microbiome interactions.

Targeted metabolomics reveals plasma biomarkers and metabolic alterations of the aging process in healthy young and older adults.

With the exponential growth in the older population in the coming years, many studies have aimed to further investigate potential biomarkers associated with the aging process and its incumbent morbidities. Age is the largest risk factor for chronic disease, likely due to younger individuals possessing more competent adaptive metabolic networks that result in overall health and homeostasis. With aging, physiological alterations occur throughout the metabolic system that contribute to functional decline. In this cross-sectional analysis, a targeted metabolomic approach was applied to investigate the plasma metabolome of young (21-40y; n = 75) and older adults (65y + ; n = 76). A corrected general linear model (GLM) was generated, with covariates of gender, BMI, and chronic condition score (CCS), to compare the metabolome of the two populations. Among the 109 targeted metabolites, those associated with impaired fatty acid metabolism in the older population were found to be most significant: palmitic acid (p < 0.001), 3-hexenedioic acid (p < 0.001), stearic acid (p = 0.005), and decanoylcarnitine (p = 0.036). Derivatives of amino acid metabolism, 1-methlyhistidine (p = 0.035) and methylhistamine (p = 0.027), were found to be increased in the younger population and several novel metabolites were identified, such as cadaverine (p = 0.034) and 4-ethylbenzoic acid (p = 0.029). Principal component analysis was conducted and highlighted a shift in the metabolome for both groups. Receiver operating characteristic analyses of partial least squares-discriminant analysis models showed the candidate markers to be more powerful indicators of age than chronic disease. Pathway and enrichment analyses uncovered several pathways and enzymes predicted to underlie the aging process, and an integrated hypothesis describing functional characteristics of the aging process was synthesized. Compared to older participants, the young group displayed greater abundance of metabolites related to lipid and nucleotide synthesis; older participants displayed decreased fatty acid oxidation and reduced tryptophan metabolism, relative to the young group. As a result, we offer a better understanding of the aging metabolome and potentially reveal new biomarkers and predicted mechanisms for future study.

The microbial community dynamics of cocaine sensitization in two behaviorally divergent strains of collaborative cross mice.

The gut-brain axis is increasingly recognized as an important pathway involved in cocaine use disorder. Microbial products of the murine gut have been shown to affect striatal gene expression, and depletion of the microbiome by antibiotic treatment alters cocaine-induced behavioral sensitization in C57BL/6J male mice. Some reports suggest that cocaine-induced behavioral sensitization is correlated with drug self-administration behavior in mice. Here, we profile the composition of the naïve microbiome and its response to cocaine sensitization in two collaborative cross (CC) strains. These strains display extremely divergent behavioral responses to cocaine sensitization. A high-responding strain, CC004/TauUncJ (CC04), has a gut microbiome that contains a greater amount of Lactobacillus than the cocaine-nonresponsive strain CC041/TauUncJ (CC41). The gut microbiome of CC41 is characterized by an abundance of Eisenbergella, Robinsonella and Ruminococcus. In response to cocaine, CC04 has an increased Barnsiella population, while the gut microbiome of CC41 displays no significant changes. PICRUSt functional analysis of the functional potential of the gut microbiome in CC04 shows a significant number of potential gut-brain modules altered after exposure to cocaine, specifically those encoding for tryptophan synthesis, glutamine metabolism, and menaquinone synthesis (vitamin K2). Depletion of the microbiome by antibiotic treatment revealed an altered cocaine-sensitization response following antibiotics in female CC04 mice. Depleting the microbiome by antibiotic treatment in males revealed increased infusions for CC04 during a cocaine intravenous self-administration dose-response curve. Together these data suggest that genetic differences in cocaine-related behaviors may involve the microbiome.

The Fungal Microbiome of the Upper Airway Is Associated With Future Loss of Asthma Control and Exacerbation Among Children With Asthma.

BACKGROUND: Accumulating evidence suggests that the upper airway bacterial microbiota is implicated in asthma inception, severity, and exacerbation. Unlike bacterial microbiota, the role of the upper airway fungal microbiome (mycobiome) in asthma control is poorly understood. RESEARCH QUESTION: What are the upper airway fungal colonization patterns among children with asthma and their relationship with subsequent loss of asthma control and exacerbation of asthma? STUDY DESIGN AND METHODS: The study was coupled with the Step Up Yellow Zone Inhaled Corticosteroids to Prevent Exacerbations (ClinicalTrials.gov Identifier: NCT02066129) clinical trial. The upper airway mycobiome was investigated using Internal transcribed spacer 1 (ITS1) sequencing of nasal blow samples collected from children with asthma when asthma was well controlled (baseline, n = 194) and during early signs of loss of asthma control (yellow zone [YZ], n = 107). RESULTS: At baseline, 499 fungal genera were detected in the upper airway samples, with two commensal fungal species, Malassezia globosa and Malassezia restricta, being most dominant. The relative abundance of Malassezia species varies by age, BMI, and race. Higher relative abundance of M globosa at baseline was associated with lower risk of future YZ episodes (P = .038) and longer time to development of first YZ episode (P = .022). Higher relative abundance of M globosa at YZ episode was associated with lower risk of progression from YZ episode to severe asthma exacerbation (P = .04). The upper airway mycobiome underwent significant changes from baseline to YZ episode, and increased fungal diversity was correlated highly with increased bacterial diversity (ρ = 0.41). INTERPRETATION: The upper airway commensal mycobiome is associated with future asthma control. This work highlights the importance of the mycobiota in asthma control and may contribute to the development of fungi-based markers to predict asthma exacerbation.

Microbial glutamate metabolism predicts intravenous cocaine self-administration in diversity outbred mice.

The gut microbiome is thought to play a critical role in the onset and development of psychiatric disorders, including depression and substance use disorder (SUD). To test the hypothesis that the microbiome affects addiction predisposing behaviors and cocaine intravenous self-administration (IVSA) and to identify specific microbes involved in the relationship, we performed 16S rRNA gene sequencing on feces from 228 diversity outbred mice. Twelve open field measures, two light-dark assay measures, one hole board and novelty place preference measure significantly differed between mice that acquired cocaine IVSA (ACQ) and those that failed to acquire IVSA (FACQ). We found that ACQ mice are more active and exploratory and display decreased fear than FACQ mice. The microbial abundances that differentiated ACQ from FACQ mice were an increased abundance of Barnesiella, Ruminococcus, and Robinsoniella and decreased Clostridium IV in ACQ mice. There was a sex-specific correlation between ACQ and microbial abundance, a reduced Lactobacillus abundance in ACQ male mice, and a decreased Blautia abundance in female ACQ mice. The abundance of Robinsoniella was correlated, and Clostridium IV inversely correlated with the number of doses of cocaine self-administered during acquisition. Functional analysis of the microbiome composition of a subset of mice suggested that gut-brain modules encoding glutamate metabolism genes are associated with the propensity to self-administer cocaine. These findings establish associations between the microbiome composition and glutamate metabolic potential and the ability to acquire cocaine IVSA thus indicating the potential translational impact of targeting the gut microbiome or microbial metabolites for treatment of SUD. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

Exploratory studies of oral and fecal microbiome in healthy human aging.

Growing evidence has linked an altered host fecal microbiome composition with health status, common chronic diseases, and institutionalization in vulnerable older adults. However, fewer studies have described microbiome changes in healthy older adults without major confounding diseases or conditions, and the impact of aging on the microbiome across different body sites remains unknown. Using 16S ribosomal RNA gene sequencing, we reconstructed the composition of oral and fecal microbiomes in young (23-32; mean = 25 years old) and older (69-94; mean = 77 years old) healthy community-dwelling research subjects. In both body sites, we identified changes in minor bacterial operational taxonomic units (OTUs) between young and older subjects. However, the composition of the predominant bacterial species of the healthy older group in both microbiomes was not significantly different from that of the young cohort, which suggests that dominant bacterial species are relatively stable with healthy aging. In addition, the relative abundance of potentially pathogenic genera, such as Rothia and Mycoplasma, was enriched in the oral microbiome of the healthy older group relative to the young cohort. We also identified several OTUs with a prevalence above 40% and some were more common in young and others in healthy older adults. Differences with aging varied for oral and fecal samples, which suggests that members of the microbiome may be differentially affected by aging in a tissue-specific fashion. This is the first study to investigate both oral and fecal microbiomes in the context of human aging, and provides new insights into interactions between aging and the microbiome within two different clinically relevant sites.

Bifidobacterial carbohydrate/nucleoside metabolism enhances oxidative phosphorylation in white adipose tissue to protect against diet-induced obesity.

BACKGROUND: Comparisons of the gut microbiome of lean and obese humans have revealed that obesity is associated with the gut microbiome plus changes in numerous environmental factors, including high-fat diet (HFD). Here, we report that two species of Bifidobacterium are crucial to controlling metabolic parameters in the Korean population. RESULTS: Based on gut microbial analysis from 99 Korean individuals, we observed the abundance of Bifidobacterium longum and Bifidobacterium bifidum was markedly reduced in individuals with increased visceral adipose tissue (VAT), body mass index (BMI), blood triglyceride (TG), and fatty liver. Bacterial transcriptomic analysis revealed that carbohydrate/nucleoside metabolic processes of Bifidobacterium longum and Bifidobacterium bifidum were associated with protecting against diet-induced obesity. Oral treatment of specific commercial Bifidobacterium longum and Bifidobacterium bifidum enhanced bile acid signaling contributing to potentiate oxidative phosphorylation (OXPHOS) in adipose tissues, leading to reduction of body weight gain and improvement in hepatic steatosis and glucose homeostasis. Bifidobacterium longum or Bifidobacterium bifidum manipulated intestinal sterol biosynthetic processes to protect against diet-induced obesity in germ-free mice. CONCLUSIONS: Our findings support the notion that treatment of carbohydrate/nucleoside metabolic processes-enriched Bifidobacterium longum and Bifidobacterium bifidum would be a novel therapeutic strategy for reprograming the host metabolic homeostasis to protect against metabolic syndromes, including diet-induced obesity. Video Abstract.

Murine Model for Measuring Effects of Humanized-Dosing of Antibiotics on the Gut Microbiome.

There is a current need for enhancing our insight in the effects of antimicrobial treatment on the composition of human microbiota. Also, the spontaneous restoration of the microbiota after antimicrobial treatment requires better understanding. This is best addressed in well-defined animal models. We here present a model in which immune-competent or neutropenic mice were administered piperacillin-tazobactam (TZP) according to human treatment schedules. Before, during and after the TZP treatment, fecal specimens were longitudinally collected at established intervals over several weeks. Gut microbial taxonomic distribution and abundance were assessed through culture and molecular means during all periods. Non-targeted metabolomics analyses of stool samples using Quadrupole Time of Flight mass spectrometry (QTOF MS) were also applied to determine if a metabolic fingerprint correlated with antibiotic use, immune status, and microbial abundance. TZP treatment led to a 5-10-fold decrease in bacterial fecal viability counts which were not fully restored during post-antibiotic follow up. Two distinct, relatively uniform and reproducible restoration scenarios of microbiota changes were seen in post TZP-treatment mice. Post-antibiotic flora could consist of predominantly Firmicutes or, alternatively, a more diverse mix of taxa. In general, the pre-treatment microbial communities were not fully restored within the screening periods applied. A new species, closely related to Eubacterium siraeum, Mageeibacillus indolicus, and Saccharofermentans acetigenes, became predominant post-treatment in a significant proportion of mice, identified by 16S rRNA gene sequencing. Principal component analysis of QTOF MS of mouse feces successfully distinguished treated from non-treated mice as well as immunocompetent from neutropenic mice. We observe dynamic but distinct and reproducible responses in the mouse gut microbiota during and after TZP treatment and propose the current murine model as a useful tool for defining the more general post-antibiotic effects in the gastro-intestinal ecosystem where humanized antibiotic dosing may ultimately facilitate extrapolation to humans.

Alterations in subgingival microbiota during full-fixed appliance orthodontic treatment-A prospective study.

BACKGROUND: Full-fixed appliance orthodontic treatment (commonly called braces) increases plaque accumulation and the risk of gingivitis and periodontitis. However, little consensus exists on changes to subgingival microbiota and specific periodontopathogens during treatment with braces. Prior studies have been hampered by selection biases due to dependence on culture conditions, candidate-based PCR and shallow sequencing methods. OBJECTIVE: The objective was to provide the first longitudinal, culture-free and deep-sequence profiling of subgingival bacteria in subjects during early stages of full-fixed orthodontic treatment. METHODS: We performed 16S rRNA next-generation sequencing (NGS) on 168 subgingival samples collected at 4 distinct mandibular tooth sites per subject before (0 weeks) and during (6 and 12 weeks) orthodontic intervention in 9 experimental and 5 control subjects not undergoing treatment. RESULTS: Overall, we noted that orthodontic intervention led to increased microbial richness, accompanied by an increased incidence of localized gingivitis/mild periodontitis in subjects requiring orthodontic treatment compared to controls, as well as significant baseline variations in subgingival microbiomes in all subjects. Moreover, we confirmed individual- and site-dependent microbiome variability (in particular, the lingual site harboured higher microbiome diversity than buccal sites) that orthodontic bands may lead to more prolonged shifts in microbial changes compared to brackets, and evidence of adaptive enrichment of consensus bacteria with orthodontic intervention (12 novel, consensus bacterial species were identified). CONCLUSION: Our study, along with evolving global profiling methods and data analyses, builds a strong foundation for further analyses of subgingival microbiomes during full-fixed orthodontic treatment.

Metagenomics and chemotherapy-induced nausea: A roadmap for future research.

Uncontrolled chemotherapy-induced nausea and vomiting can reduce patients' quality of life and may result in premature discontinuation of chemotherapy. Although nausea and vomiting are commonly grouped together, research has shown that antiemetics are clinically effective against chemotherapy-induced vomiting (CIV) but less so against chemotherapy-induced nausea (CIN). Nausea remains a problem for up to 68% of patients who are prescribed guideline-consistent antiemetics. Despite the high prevalence of CIN, relatively little is known regarding its etiology independent of CIV. This review summarizes a metagenomics approach to the study and treatment of CIN with the goal of encouraging future research. Metagenomics focuses on genetic risk factors and encompasses both human (ie, host) and gut microbial genetic variation. Little work to date has focused on metagenomics as a putative biological mechanism of CIN. Metagenomics has the potential to be a powerful tool in advancing scientific understanding of CIN by identifying new biological pathways and intervention targets. The investigation of metagenomics in the context of well-established demographic, clinical, and patient-reported risk factors may help to identify patients at risk and facilitate the prevention and management of CIN.

The Gut Microbiome and Substance Use Disorder.

Substance use disorders (SUDs) remain a significant public health challenge, affecting tens of millions of individuals worldwide each year. Often comorbid with other psychiatric disorders, SUD can be poly-drug and involve several different substances including cocaine, opiates, nicotine, and alcohol. SUD has a strong genetic component. Much of SUD research has focused on the neurologic and genetic facets of consumption behavior. There is now interest in the role of the gut microbiome in the pathogenesis of SUD. In this review, we summarize current animal and clinical evidence that the gut microbiome is involved in SUD, then address the underlying mechanisms by which the gut microbiome interacts with SUD through metabolomic, immune, neurological, and epigenetic mechanisms. Lastly, we discuss methods using various inbred and outbred mice models to gain an integrative understanding of the microbiome and host genetic controls in SUD.

Host genetic control of gut microbiome composition.

The gut microbiome plays a significant role in health and disease, and there is mounting evidence indicating that the microbial composition is regulated in part by host genetics. Heritability estimates for microbial abundance in mice and humans range from (0.05-0.45), indicating that 5-45% of inter-individual variation can be explained by genetics. Through twin studies, genetic association studies, systems genetics, and genome-wide association studies (GWAS), hundreds of specific host genetic loci have been shown to associate with the abundance of discrete gut microbes. Using genetically engineered knock-out mice, at least 30 specific genes have now been validated as having specific effects on the microbiome. The relationships among of host genetics, microbiome composition, and abundance, and disease is now beginning to be unraveled through experiments designed to test causality. The genetic control of disease and its relationship to the microbiome can manifest in multiple ways. First, a genetic variant may directly cause the disease phenotype, resulting in an altered microbiome as a consequence of the disease phenotype. Second, a genetic variant may alter gene expression in the host, which in turn alters the microbiome, producing the disease phenotype. Finally, the genetic variant may alter the microbiome directly, which can result in the disease phenotype. In order to understand the processes that underlie the onset and progression of certain diseases, future research must take into account the relationship among host genetics, microbiome, and disease phenotype, and the resources needed to study these relationships.

Circadian rhythms and the gut microbiome synchronize the host's metabolic response to diet.

The molecular circadian clock and symbiotic host-microbe relationships both evolved as mechanisms that enhance metabolic responses to environmental challenges. The gut microbiome benefits the host by breaking down diet-derived nutrients indigestible by the host and generating microbiota-derived metabolites that support host metabolism. Similarly, cellular circadian clocks optimize organismal physiology to the environment by influencing the timing and coordination of metabolic processes. Host-microbe interactions are influenced by dietary quality and timing, as well as daily light/dark cycles that entrain circadian rhythms in the host. Together, the gut microbiome and the molecular circadian clock play a coordinated role in neural processing, metabolism, adipogenesis, inflammation, and disease initiation and progression. This review examines the bidirectional interactions between the circadian clock, gut microbiota, and host metabolic systems and their effects on obesity and energy homeostasis. Directions for future research and the development of therapies that leverage these systems to address metabolic disease are highlighted.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.