Comparison of Experimental Methodologies Based on Bulk-Metagenome and Virus-like Particle Enrichment: Pros and Cons for Representativeness and Reproducibility in the Study of the Fecal Human Virome.

Studies on the human virome based on the application of metagenomic approaches involve overcoming a series of challenges and limitations inherent not only to the biological features of viruses, but also to methodological pitfalls which different approaches have tried to minimize. These approaches fall into two main categories: bulk-metagenomes and virus-like particle (VLP) enrichment. In order to address issues associated with commonly used experimental procedures to assess the degree of reliability, representativeness, and reproducibility, we designed a comparative analysis applied to three experimental protocols, one based on bulk-metagenomes and two based on VLP enrichment. These protocols were applied to stool samples from 10 adult participants, including two replicas per protocol and subject. We evaluated the performances of the three methods, not only through the analysis of the resulting composition, abundance, and diversity of the virome via taxonomical classification and type of molecule (DNA versus RNA, single stranded vs. double stranded), but also according to how the a priori identical replicas differed from each other according to the extraction methods used. Our results highlight the strengths and weaknesses of each approach, offering valuable insights and tailored recommendations for drawing reliable conclusions based on specific research goals.

Almond (Prunus dulcis) Bagasse as a Source of Bioactive Compounds with Antioxidant Properties: An In Vitro Assessment.

The presence of components of nutritional interest makes fresh almond bagasse an interesting by-product for obtaining functional ingredients. Stabilization through a dehydration process is an interesting option for its integral use, ensuring its conservation and management. Subsequently, it can be turned into powder, facilitating its use as an ingredient. The aim of this paper was to determine the effects of hot air drying at 60 and 70 °C and lyophilization on the release of phenolic components and antiradical capacity in in vitro gastrointestinal digestion and colonic fermentation, as well as on growing microbiota composition by applying high throughput sequencing. The novelty of this study lies in this holistic approach; considering both technological and physiological aspects related to gastrointestinal digestion and colonic fermentation will provide the best conditions for functional foods. The results obtained showed that lyophilization provides a powder with a total phenol content and antiradical capacity higher than hot air drying. Furthermore, in dehydrated samples, both in vitro digestion and colonic fermentation revealed a phenol content and anti-radical capacity superior to those existing in undigested products. In addition, after colonic fermentation, beneficial bacteria species have been identified. Obtaining powders from almond bagasse is presented as an interesting opportunity for the valorization of this by-product.

Effects of different foods and cooking methods on the gut microbiota: an in vitro approach.

To support personalized diets targeting the gut microbiota, we employed an in vitro digestion-fermentation model and 16S rRNA gene sequencing to analyze the microbiota growing on representative foods of the Mediterranean and Western diets, as well as the influence of cooking methods. Plant- and animal-derived foods had significantly different impacts on the abundances of bacterial taxa. Animal and vegetable fats, fish and dairy products led to increases in many taxa, mainly within the Lachnospiraceae. In particular, fats favored increases in the beneficial bacteria Faecalibacterium, Blautia, and Roseburia. However, butter, as well as gouda cheese and fish, also resulted in the increase of Lachnoclostridium, associated to several diseases. Frying and boiling produced the most distinct effects on the microbiota, with members of the Lachnospiraceae and Ruminococcaceae responding the most to the cooking method employed. Nevertheless, cooking effects were highly individualized and food-dependent, challenging the investigation of their role in personalized diets.

Nutritional Supplementation to Increase Influenza Vaccine Response in Children Living With HIV: A Pilot Clinical Trial.

Aims: Vaccine response is poor among children living with HIV. The gut microbiota has been identified as a potential target to improve vaccine immunogenicity, but data are scarce in the context of HIV infection. Methods: Pilot, double-blind, randomized placebo-controlled trial in which 24 HIV-infected children were randomized to receive a mixture of symbiotics, omega-3/6 fatty acids, and amino acids or placebo for 4 weeks, each in combination with ART, and were then immunized against influenza. Vaccine response and safety of the nutritional supplementation were the primary outcomes. Results: Eighteen HIV-infected children completed the follow-up period (mean age 11.5 ± 4.14 years, 61% female). The nutritional supplement was safe but did not enhance the response to the influenza vaccine. A 4-fold rise in antibody titers was obtained in only 37.5% of participants in the intervention arm vs. 40% in the placebo. No immunological or inflammatory predictors of vaccine response were identified. Conclusions: In this exploratory study, a 4-week course of symbiotics did not increase influenza vaccine immunogenicity in HIV-infected children. Larger studies are warranted to address the potential of modulating the microbiome in children living with HIV.

Interactions among the mycobiome, bacteriome, inflammation, and diet in people living with HIV.

While the intestinal microbiome seems a major driver of persistent immune defects in people with HIV (PWH), little is known about its fungal component, the mycobiome. We assessed the inter-kingdom mycobiome-bacteriome interactions, the impact of diet, and the association with the innate and adaptive immunity in PWH on antiretroviral therapy. We included 24 PWH individuals and 12 healthy controls. We sequenced the Internal Transcribed Spacer 2 amplicons, determined amplicon sequence variants, measured biomarkers of the innate and adaptive immunity in blood and relations with diet. Compared to healthy controls, PWH subjects exhibited a distinct and richer mycobiome and an enrichment for Debaryomyces hansenii, Candida albicans, and Candida parapsilosis. In PWH, Candida and Pichia species were strongly correlated with several bacterial genera, including Faecalibacterium genus. Regarding the links between the mycobiome and systemic immunology, we found a positive correlation between Candida species and the levels of proinflammatory cytokines (sTNF-R2 and IL-17), interleukin 22 (a cytokine implicated in the regulation of mucosal immunity), and CD8+ T cell counts. This suggests an important role of the yeasts in systemic innate and adaptive immune responses. Finally, we identified inter-kingdom interactions implicated in fiber degradation, short-chain fatty acid production, and lipid metabolism, and an effect of vegetable and fiber intake on the mycobiome. Therefore, despite the great differences in abundance and diversity between the bacterial and fungal communities of the gut, we defined the changes associated with HIV, determined several different inter-kingdom associations, and found links between the mycobiome, nutrient metabolism, and systemic immunity.

Evaluation of the Effects of a Short Supplementation With Tannins on the Gut Microbiota of Healthy Subjects.

Western diet, high in fats and sugars and low in greens, contributes to dysbiosis of the gut microbiota, which can lead to a variety of chronic diseases related with inflammation. Supplementation with bioactive compounds can help to maintain a healthy eubiotic state. Thus, we performed a 4-weeks nutritional intervention on healthy volunteers to investigate whether a blend of natural tannin extracts could induce healthy changes in the microbial intestinal ecosystem. Changes in the composition and functionality of the microbiota could be observed from the first two weeks onward. 16S rRNA amplicon next-generation sequencing (NGS) revealed a significant increase in microbial diversity at the end of the intervention, as well as trends toward increases in the relative abundances of several beneficial taxa, such as Ruminococcus bicirculans, Faecalibacterium prausnitzii, Lachnospiraceae UCG 010, Lachnospiraceae NK4A136, Bacteroides thetaiotaomicron and B. uniformis. Remarkably, some of the identified taxa were also identified as responsible for an increase in the production of short-chain fatty acids (SCFAs), microbial metabolites that contribute to the modulation of the immune system and have various other anti-inflammatory functions in the gut. Taken together, these results suggest that the tannin supplementation could exert a prebiotic effect by selectively stimulating the growth and the activity of bacteria that are advantageous for the host.

Targeting the Gut Microbiota of Vertically HIV-Infected Children to Decrease Inflammation and Immunoactivation: A Pilot Clinical Trial.

Aims: Children with HIV exhibit chronic inflammation and immune dysfunction despite antiretroviral therapy (ART). Strategies targeting persistent inflammation are needed to improve health in people living with HIV. The gut microbiota likely interacts with the immune system, but the clinical implications of modulating the dysbiosis by nutritional supplementation are unclear. Methods: Pilot, double-blind, randomized placebo-controlled trial in which 24 HIV-infected on ART were randomized to supplementation with a daily mixture of symbiotics, omega-3/6 fatty acids and amino acids, or placebo four weeks, in combination with ART. We analyzed inflammatory markers and T-cell activation changes and their correlations with shifts in fecal microbiota. Results: Twenty-four HIV-infected children were recruited and randomized to receive a symbiotic nutritional supplement or placebo. Mean age was 12 ± 3.9 years, 62.5% were female. All were on ART and had HIV RNA < 50/mL. We did not detect changes in inflammatory (IL-6, IL-7, IP-10), microbial translocation (sCD14), mucosal integrity markers (IFABP, zonulin) or the kynurenine to tryptophan ratio, or changes in markers of the adaptive immune response in relation to the intervention. However, we found correlations between several key bacteria and the assessed inflammatory and immunological parameters, supporting a role of the microbiota in immune modulation in children with HIV. Conclusions: In this exploratory study, a four-week nutritional supplementation had no significant effects in terms of decreasing inflammation, microbial translocation, or T-cell activation in HIV-infected children. However, the correlations found support the interaction between gut microbiota and the immune system.

An extended reconstruction of human gut microbiota metabolism of dietary compounds.

Understanding how diet and gut microbiota interact in the context of human health is a key question in personalized nutrition. Genome-scale metabolic networks and constraint-based modeling approaches are promising to systematically address this complex problem. However, when applied to nutritional questions, a major issue in existing reconstructions is the limited information about compounds in the diet that are metabolized by the gut microbiota. Here, we present AGREDA, an extended reconstruction of diet metabolism in the human gut microbiota. AGREDA adds the degradation pathways of 209 compounds present in the human diet, mainly phenolic compounds, a family of metabolites highly relevant for human health and nutrition. We show that AGREDA outperforms existing reconstructions in predicting diet-specific output metabolites from the gut microbiota. Using 16S rRNA gene sequencing data of faecal samples from Spanish children representing different clinical conditions, we illustrate the potential of AGREDA to establish relevant metabolic interactions between diet and gut microbiota.

Gut Microbiota Profile and Its Association with Clinical Variables and Dietary Intake in Overweight/Obese and Lean Subjects: A Cross-Sectional Study.

We aimed to differentiate gut microbiota composition of overweight/obese and lean subjects and to determine its association with clinical variables and dietary intake. A cross-sectional study was performed with 96 overweight/obese subjects and 32 lean subjects. Anthropometric parameters were positively associated with Collinsella aerofaciens, Dorea formicigenerans and Dorea longicatena, which had higher abundance the overweight/obese subjects. Moreover, different genera of Lachnospiraceae were negatively associated with body fat, LDL and total cholesterol. Saturated fatty acids (SFAs) were negatively associated with the genus Intestinimonas, a biomarker of the overweight/obese group, whereas SFAs were positively associated with Roseburia, a biomarker for the lean group. In conclusion, Dorea formicigenerans, Dorea longicatena and Collinsella aerofaciens could be considered obesity biomarkers, Lachnospiraceae is associated with lipid cardiovascular risk factors. SFAs exhibited opposite association profiles with butyrate-producing bacteria depending on the BMI. Thus, the relationship between diet and microbiota opens new tools for the management of obesity.

Nutrigenetic Interactions Might Modulate the Antioxidant and Anti-Inflammatory Status in Mastiha-Supplemented Patients With NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease with no therapeutic consensus. Oxidation and inflammation are hallmarks in the progression of this complex disease, which also involves interactions between the genetic background and the environment. Mastiha is a natural nutritional supplement known to possess antioxidant and anti-inflammatory properties. This study investigated how a 6-month Mastiha supplementation (2.1 g/day) could impact the antioxidant and inflammatory status of patients with NAFLD, and whether genetic variants significantly mediate these effects. We recruited 98 patients with obesity (BMI ≥ 30 kg/m2) and NAFLD and randomly allocated them to either the Mastiha or the placebo group for 6 months. The anti-oxidative and inflammatory status was assessed at baseline and post-treatment. Genome-wide genetic data was also obtained from all participants, to investigate gene-by-Mastiha interactions. NAFLD patients with severe obesity (BMI > 35kg/m2) taking the Mastiha had significantly higher total antioxidant status (TAS) compared to the corresponding placebo group (P value=0.008). We did not observe any other significant change in the investigated biomarkers as a result of Mastiha supplementation alone. We identified several novel gene-by-Mastiha interaction associations with levels of cytokines and antioxidant biomarkers. Some of the identified genetic loci are implicated in the pathological pathways of NAFLD, including the lanosterol synthase gene (LSS) associated with glutathione peroxidase activity (Gpx) levels, the mitochondrial pyruvate carrier-1 gene (MPC1) and the sphingolipid transporter-1 gene (SPNS1) associated with hemoglobin levels, the transforming growth factor-beta-induced gene (TGFBI) and the micro-RNA 129-1 (MIR129-1) associated with IL-6 and the granzyme B gene (GZMB) associated with IL-10 levels. Within the MAST4HEALTH randomized clinical trial (NCT03135873, www.clinicaltrials.gov) Mastiha supplementation improved the TAS levels among NAFLD patients with severe obesity. We identified several novel genome-wide significant nutrigenetic interactions, influencing the antioxidant and inflammatory status in NAFLD. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03135873.

Enrichment of Food With Tannin Extracts Promotes Healthy Changes in the Human Gut Microbiota.

Food and food bioactive components are major drivers of modulation of the human gut microbiota. Tannin extracts consist of a mix of bioactive compounds, which are already exploited in the food industry for their chemical and sensorial properties. The aim of our study was to explore the viability of associations between tannin wood extracts of different origin and food as gut microbiota modulators. 16S rRNA amplicon next-generation sequencing (NGS) was used to test the effects on the gut microbiota of tannin extracts from quebracho, chestnut, and tara associated with commercial food products with different composition in macronutrients. The different tannin-enriched and non-enriched foods were submitted to in vitro digestion and fermentation by the gut microbiota of healthy subjects. The profile of the short chain fatty acids (SCFAs) produced by the microbiota was also investigated. The presence of tannin extracts in food promoted an increase of the relative abundance of the genus Akkermansia, recognized as a marker of a healthy gut, and of various members of the Lachnospiraceae and Ruminococcaceae families, involved in SCFA production. The enrichment of foods with tannin extracts had a booster effect on the production of SCFAs, without altering the profile given by the foods alone. These preliminary results suggest a positive modulation of the gut microbiota with potential benefits for human health through the enrichment of foods with tannin extracts.

Effect of a Nutritional Intervention on the Intestinal Microbiota of Vertically HIV-Infected Children: The Pediabiota Study.

AIMS: The gut microbiota exerts a critical influence in the immune system. The gut microbiota of human virus immunodeficiency (HIV)-infected children remains barely explored. We aimed to characterize the fecal microbiota in vertically HIV-infected children and to explore the effects of its modulation with a symbiotic nutritional intervention. METHODS: a pilot, double blind, randomized placebo-controlled study including HIV-infected children who were randomized to receive a nutritional supplementation including prebiotics and probiotics or placebo for four weeks. HIV-uninfected siblings were recruited as controls. The V3-V4 region of the 16S rRNA gene was sequenced in fecal samples. RESULTS: 22 HIV-infected children on antiretroviral therapy (ART) and with viral load (VL) <50/mL completed the follow-up period. Mean age was 11.4 ± 3.4 years, eight (32%) were male. Their microbiota showed reduced alpha diversity compared to controls and distinct beta diversity at the genus level (Adonis p = 0.042). Patients showed decreased abundance of commensals Faecalibacterium and an increase in Prevotella, Akkermansia and Escherichia. The nutritional intervention shaped the microbiota towards the control group, without a clear directionality. CONCLUSIONS: Vertical HIV infection is characterized by changes in gut microbiota structure, distinct at the compositional level from the findings reported in adults. A short nutritional intervention attenuated bacterial dysbiosis, without clear changes at the community level. SUMMARY: In a group of 24 vertically HIV-infected children, in comparison to 11 uninfected controls, intestinal dysbiosis was observed despite effective ART. Although not fully effective to restore the microbiota, a short intervention with pre/probiotics attenuated bacterial dysbiosis.

Valorization of Persimmon and Blueberry Byproducts to Obtain Functional Powders: In Vitro Digestion and Fermentation by Gut Microbiota.

Globalization of fruit and vegetable markets generates overproduction, surpluses, and potentially valuable residues. The valorization of these byproducts constitutes a challenge, to ensure sustainability and reintroduce them into the food chain. This work focuses on blueberry and persimmon residues, rich in polyphenols and carotenoids, to obtain powders with high added value to be used as ingredients in food formulation. These powders have been characterized, and the changes in the bioactive compounds in in vitro gastrointestinal digestion have been evaluated. The results indicated that the type of residue, the drying process, as well as the content and type of fiber determine the release of antioxidants during digestion. In vitro colonic fermentations were also performed, and it was observed that the characteristics of digested powders had an effect on the composition of the growing microbial community. Thus, carotenoids and anthocyanins maintain an interplay with microbiota that could be beneficial for human health.

Gut metagenomic and short chain fatty acids signature in hypertension: a cross-sectional study.

Hypertension is an independent and preventable risk factor for the development of cardiovascular diseases, however, little is known about the impact of gut microbiota composition in its development. We carried out comprehensive gut microbiota analysis and targeted metabolomics in a cross-sectional study of 29 non-treated hypertensive (HT) and 32 normotensive (NT) subjects. We determined fecal microbiota composition by 16S rRNA gene sequencing and bacterial functions by metagenomic analysis. The microbial metabolites analysed were short chain fatty acids (SCFA) both in plasma and feces, and trimethylamine N-oxide (TMAO) in plasma. The overall bacterial composition and diversity of bacterial community in the two groups were not significantly different. However, Ruminococcaceae NK4A214, Ruminococcaceae_UCG-010, Christensenellaceae_R-7, Faecalibacterium prausnitzii and Roseburia hominis were found to be significantly enriched in NT group, whereas, Bacteroides coprocola, Bacteroides plebeius and genera of Lachnospiraceae were increased in HT patients. We found a positive correlation between the HT-associated species and systolic and diastolic blood pressure after adjusted for measured confounders. SCFA showed antagonistic results in plasma and feces, detecting in HT subjects significant higher levels in feces and lower levels in plasma, which could indicate a less efficient SCFA absorption. Overall, our results present a disease classifier based on microbiota and bacterial metabolites to discriminate HT individuals from NT controls in a first disease grade prior to drug treatment.

Mastiha (Pistacia lentiscus) Improves Gut Microbiota Diversity, Hepatic Steatosis, and Disease Activity in a Biopsy-Confirmed Mouse Model of Advanced Non-Alcoholic Steatohepatitis and Fibrosis.

SCOPE: As a result of the obesity epidemic, the prevalence of non-alcoholic steatohepatitis (NASH) is increasing. No drug is approved for the treatment of NASH. In this study, the effect of a nutritional supplement, Mastiha or Chios mastic gum, on metabolic and histological parameters and on the gut microbiome in mice with NASH and fibrosis was investigated. METHODS AND RESULTS: Advanced NASH was induced by feeding C57BL/6J mice a diet rich in fat, sucrose, and cholesterol for 41 weeks. After randomization, animals received the NASH-inducing diet with or without 0.2% (w/w) Mastiha for a further 8 weeks. Disease activity was assessed by liver histology and determination of plasma transaminase activities. Fecal microbiota DNA extraction and 16S rRNA amplicon sequencing were used to determine the composition of the gut microbiome. Mastiha supplementation led to a significant reduction in circulating alanine aminotransferase (ALT) activity, improvement in hepatic steatosis and collagen content, and a reduction in NAFLD activity score. Furthermore, it resulted in a partial but significant recovery of gut microbiota diversity and changes in identity and abundance of specific taxa. CONCLUSION: This is the first study demonstrating an improvement in disease activity in mice with advanced NASH with fibrosis by a diet containing Mastiha.

Modulation of Saliva Microbiota through Prebiotic Intervention in HIV-Infected Individuals.

Human immunodeficiency virus (HIV) infection is characterized by an early depletion of the mucosal associated T helper (CD4+) cells that impair the host immunity and impact the oral and gut microbiomes. Although, the HIV-associated gut microbiota was studied in depth, few works addressed the dysbiosis of oral microbiota in HIV infection and, to our knowledge, no studies on intervention with prebiotics were performed. We studied the effect of a six-week-long prebiotic administration on the salivary microbiota in HIV patients and healthy subjects. Also, the co-occurrence of saliva microorganisms in the fecal bacteria community was explored. We assessed salivary and feces microbiota composition using deep 16S ribosomal RNA (rRNA) gene sequencing with Illumina methodology. At baseline, the different groups shared the same most abundant genera, but the HIV status had an impact on the saliva microbiota composition and diversity parameters. After the intervention with prebiotics, we found a drastic decrease in alpha diversity parameters, as well as a change of beta diversity, without a clear directionality toward a healthy microbiota. Interestingly, we found a differential response to the prebiotics, depending on the initial microbiota. On the basis of 100% identity clustering, we detected saliva sequences in the feces datasets, suggesting a drag of microorganisms from the upper to the lower gastrointestinal tract.

Spent Coffee Grounds Extract, Rich in Mannooligosaccharides, Promotes a Healthier Gut Microbial Community in a Dose-Dependent Manner.

Coffee is one of the most consumed beverages around the world, and as a consequence, spent coffee grounds are a massively produced residue that is causing environmental problems. Reusing them is a major focus of interest presently. We extracted mannooligosaccharides (MOS) from spent coffee grounds and submitted them to an in vitro fermentation with human feces. Results obtained suggest that MOS are able to exert a prebiotic effect on gut microbiota by stimulating the growth of some beneficial genera, such as Barnesiella, Odoribacter, Coprococcus, Butyricicoccus, Intestinimonas, Pseudoflavonifractor, and Veillonella. Moreover, short-chain fatty acids (SCFA) production also increased in a dose-dependent manner. However, we observed that 5-(hydroxymethyl)furfural, furfural, and polyphenols (which are either produced or released from the spent coffee grounds matrix during hydrolysis) could have an inhibitory effect on other beneficial genera, such as Faecalibacterium, Ruminococcus, Blautia, Butyricimonas, Dialister, Collinsella, and Anaerostipes, which could negatively affect the prebiotic activity of MOS.

Metabolic adaptation in the human gut microbiota during pregnancy and the first year of life.

BACKGROUND: The relationship between the gut microbiome and the human host is dynamic and we may expect adjustments in microbiome function if host physiology changes. Metatranscriptomic approaches should be key in unraveling how such adjustments occur. METHODS: We employ metatranscriptomic sequencing analyses to study gene expression in the gut microbiota of infants through their first year of life, and of their mothers days before delivery and one year afterwards. FINDINGS: In infants, hallmarks of aerobic metabolism disappear from the microbial metatranscriptome as development proceeds, while the expression of functions related to carbohydrate transport and metabolism increases and diversifies, approaching that observed in non-pregnant women. Butyrate synthesis enzymes are overexpressed at three months of age, even though most butyrate-producing organisms are still rare. In late pregnancy, the microbiota readjusts the expression of carbohydrate-related functions in a manner consistent with a high availability of glucose. INTERPRETATION: Our findings suggest that butyrate production may be ensured in the gut of young infants before the typical butyrate synthesizers of the adult gut become abundant. The late pregnancy gut microbiota may be able to access the high levels of blood glucose characteristic of this period. Moreover, late pregnancy gut bacteria may reach stationary phase, which may affect their likelihood of translocating across the intestinal epithelium. FUNDS: This work was supported by grants CSD2009-00006 (CONSOLIDER Program) and SAF2009-13032-C02-02 from MICINN (Ministry of Science and Innovation, Spain), and by grant SAF2012-31187 from MINECO (Ministry of Economics and Competitiveness, Spain).

Effect of Food Thermal Processing on the Composition of the Gut Microbiota.

Cooking modifies food composition due to chemical reactions. Additionally, food composition shapes the human gut microbiota. Thus, the objective of this research was to unravel the effect of different food cooking methods on the structure and functionality of the gut microbiota. Common culinary techniques were applied to five foods, which were submitted to in vitro digestion-fermentation. Furosine, 5-(hydroxymethyl)furfural, and furfural were used as Maillard reaction indicators to control the heat treatment. Short-chain fatty acids production was quantified as indicator of healthy metabolic output. Gut microbial community structure was analyzed through 16S rRNA. Both food composition and cooking methods modified the microbiota composition and released short-chain fatty acids. In general, intense cooking technologies (roasting and grilling) increased the abundance of beneficial bacteria like Ruminococcus spp. or Bifidobacterium spp. compared to milder treatments (boiling). However, for some foods (banana or bread), intense cooking decreased the levels of healthy bacteria.

Interplay between gut microbiota metabolism and inflammation in HIV infection.

HIV infection causes a disruption of gut-associated lymphoid tissue, driving a shift in the composition of gut microbiota. A deeper understanding of the metabolic changes and how they affect the interplay with the host is needed. Here, we assessed functional modifications of HIV-associated microbiota by combining metagenomic and metatranscriptomic analyses. The transcriptionally active microbiota was well-adapted to the inflamed environment, overexpressing pathways related to resistance to oxidative stress. Furthermore, gut inflammation was maintained by the Gram-negative nature of the HIV-associated microbiota and underexpression of anti-inflammatory processes, such as short chain fatty acid biosynthesis or indole production. We performed co-occurrence and metabolic network analyses that showed relevance in the microbiota structure of both taxonomic and metabolic HIV-associated biomarkers. The Bayesian network revealed the most determinant pathways for maintaining the structure stability of the bacterial community. In addition, we identified the taxa's contribution to metabolic activities and their interactions with host health.