Towards modulating the gut microbiota to enhance the efficacy of immune-checkpoint inhibitors.

The gut microbiota modulates immune processes both locally and systemically. This includes whether and how the immune system reacts to emerging tumours, whether antitumour immune responses are reactivated during treatment with immune-checkpoint inhibitors (ICIs), and whether unintended destructive immune pathologies accompany such treatment. Advances over the past decade have established that the gut microbiota is a promising target and that modulation of the microbiota might overcome resistance to ICIs and/or improve the safety of treatment. However, the specific mechanisms through which the microbiota modulates antitumour immunity remain unclear. Understanding the biology underpinning microbial associations with clinical outcomes in patients receiving ICIs, as well as the landscape of a 'healthy' microbiota would provide a critical foundation to facilitate opportunities to effectively manipulate the microbiota and thus improve patient outcomes. In this Review, we explore the role of diet and the gut microbiota in shaping immune responses during treatment with ICIs and highlight the key challenges in attempting to leverage the gut microbiome as a practical tool for the clinical management of patients with cancer.

Low Levels of Hive Stress Are Associated with Decreased Honey Activity and Changes to the Gut Microbiome of Resident Honey Bees.

Honey bees (Apis mellifera) face increasing threats to their health, particularly from the degradation of floral resources and chronic pesticide exposure. The properties of honey and the bee gut microbiome are known to both affect and be affected by bee health. Using samples from healthy hives and hives showing signs of stress from a single apiary with access to the same floral resources, we profiled the antimicrobial activity and chemical properties of honey and determined the bacterial and fungal microbiome of the bee gut and the hive environment. We found honey from healthy hives was significantly more active than honey from stressed hives, with increased phenolics and antioxidant content linked to higher antimicrobial activity. The bacterial microbiome was more diverse in stressed hives, suggesting they may have less capacity to exclude potential pathogens. Finally, bees from healthy and stressed hives had significant differences in core and opportunistically pathogenic taxa in gut samples. Our results emphasize the need for understanding and proactively managing bee health. IMPORTANCE Honey bees serve as pollinators for many plants and crops worldwide and produce valuable hive products such as honey and wax. Various sources of stress can disrupt honey bee colonies, affecting their health and productivity. Growing evidence suggests that honey is vitally important to hive functioning and overall health. In this study, we determined the antimicrobial activity and chemical properties of honey from healthy hives and hives showing signs of stress, finding that honey from healthy hives was significantly more antimicrobial, with increased phenolics and antioxidant content. We next profiled the bacterial and fungal microbiome of the bee gut and the hive environment, finding significant differences between healthy and stressed hives. Our results underscore the need for greater understanding in this area, as we found even apparently minor stress can have implications for overall hive fitness as well as the economic potential of hive products.

Alterations to the duodenal microbiota are linked to gastric emptying and symptoms in functional dyspepsia.

OBJECTIVE: Functional dyspepsia (FD) is a complex disorder, with debilitating epigastric symptoms. Evidence suggests alterations in gastrointestinal (GI) motility, visceral hypersensitivity, permeability and low-level immune activation in the duodenum may play a role. However, we still have a relatively poor understanding of how these factors interact to precipitate the onset of FD symptoms which are frequently meal related. The duodenal microbiota, in combination with specific dietary substrates, may be important mediators in disease pathophysiology; however, these interlinked factors have not been thoroughly investigated in FD. DESIGN: Eighty-six individuals (56 FD, 30 controls) undergoing endoscopy were consecutively recruited and underwent detailed clinical assessment, including upper GI symptoms, gastric emptying and dietary assessment. Duodenal biopsies were obtained aseptically, and the mucosa-associated microbiota (MAM) analysed via 16S rRNA gene amplicon sequencing. RESULTS: The relative abundances of predominant members of the Firmicutes, Bacteroidota and Fusobacteriota phyla were linked to symptom burden in FD. Inverse relationships between the relative abundances of Streptococcus and Prevotella, and the relative abundance of Veillonella spp with gastric emptying time, were also observed. No significant differences in long-term nutrient intake or diet quality were found between FD and controls, and there appeared to be limited association between habitual diet and duodenal MAM profiles. CONCLUSION: This study suggests a link between the duodenal MAM, gastric emptying and FD symptoms, and this is largely independent of long-term dietary intake.

The duodenal mucosa associated microbiome, visceral sensory function, immune activation and psychological comorbidities in functional gastrointestinal disorders with and without self-reported non-celiac wheat sensitivity.

Frequently, patients with functional gastrointestinal disorders (FGIDs) report intolerance of wheat products. We compared gastrointestinal symptoms, sensory function, psychiatric comorbidities, gut-homing immune cells, and duodenal mucosa-associated microbiome (d-MAM) in FGID patients and controls with and without self-reported wheat sensitivity (SR-NCWS). We recruited 40 FGID patients and 20 controls referred by GPs for treatment. Gastrointestinal/extraintestinal symptoms, visceral sensory function, psychological comorbidities, and SR-NCWS were assessed in a standardized approach. Peripheral gut homing T-cells (CD4+α4+ß7+CCR9+/CD8+α4+ß7+CCR9+) were quantified, and the d-MAM was assessed by DNA sequencing for 46 subjects. Factors of bacterial genera were extracted utilizing factor analysis with varimax rotation and factors univariately associated with FGID or SR-NCWS included in a subsequent multivariate analysis of variance to identify statistically independent discriminators. Anxiety scores (p < .05) and increased symptom responses to a nutrient challenge (p < .05) were univariately associated with FGID. Gut homing T-cells were increased in FGID patients with SR-NCWS compared to other groups (p all <0.05). MANOVA revealed that anxiety (p = .03), visceral sensory function (p = 0.007), and a d-MAM factor comprise members of the Alloprevotella, Prevotella, Peptostreptococcus, Leptotrichia, and Veillonella lineages were significantly (p = .001) associated with FGID, while gut homing CD4+α4+ ß7+CCR9+ T-cells were associated (p = .002) with SR-NCWS. Compared to controls, patients with and without SR-NCWS show that there are shifts in the amplicon sequence variants within specific bacterial genera between the FGID subgroups (particularly Prevotella and Streptococcus) as well as distinct bacterial taxa discriminatory for the two different FGID subtypes. Compared to controls, both FGID patients with and without SR-NCWS have an increased symptom response to a standardized nutrient challenge and increased anxiety scores. The FGID patients with SR-NCWS - as compared to FGID without SR-NCWS (and controls without SR-NCWS) - have increased gut homing T-cells. The d-MAM profiles suggest species and strain-based variations between the two FGID subtypes and in comparison to controls.

Diet-driven microbial ecology underpins associations between cancer immunotherapy outcomes and the gut microbiome.

The gut microbiota shapes the response to immune checkpoint inhibitors (ICIs) in cancer, however dietary and geographic influences have not been well-studied in prospective trials. To address this, we prospectively profiled baseline gut (fecal) microbiota signatures and dietary patterns of 103 trial patients from Australia and the Netherlands treated with neoadjuvant ICIs for high risk resectable metastatic melanoma and performed an integrated analysis with data from 115 patients with melanoma treated with ICIs in the United States. We observed geographically distinct microbial signatures of response and immune-related adverse events (irAEs). Overall, response rates were higher in Ruminococcaceae-dominated microbiomes than in Bacteroidaceae-dominated microbiomes. Poor response was associated with lower fiber and omega 3 fatty acid consumption and elevated levels of C-reactive protein in the peripheral circulation at baseline. Together, these data provide insight into the relevance of native gut microbiota signatures, dietary intake and systemic inflammation in shaping the response to and toxicity from ICIs, prompting the need for further studies in this area.

Draft Genome Sequence of Streptococcus salivarius AGIRA0003, Isolated from Functional Gastrointestinal Disorder Duodenal Tissue.

Patients suffering functional dyspepsia symptoms have been shown to possess a greater relative abundance of Streptococcus compared to asymptomatic controls. Here, we describe the isolation and genomic features of a new Streptococcus isolate, from the duodenal tissue of a subject reporting dyspeptic symptoms, taxonomically assigned to Streptococcus salivarius and designated strain AGIRA0003.

Enabling rational gut microbiome manipulations by understanding gut ecology through experimentally-evidenced in silico models.

The gut microbiome has emerged as a contributing factor in non-communicable disease, rendering it a target of health-promoting interventions. Yet current understanding of the host-microbiome dynamic is insufficient to predict the variation in intervention outcomes across individuals. We explore the mechanisms that underpin the gut bacterial ecosystem and highlight how a more complete understanding of this ecology will enable improved intervention outcomes. This ecology varies within the gut over space and time. Interventions disrupt these processes, with cascading consequences throughout the ecosystem. In vivo studies cannot isolate and probe these processes at the required spatiotemporal resolutions, and in vitro studies lack the representative complexity required. However, we highlight that, together, both approaches can inform in silico models that integrate cellular-level dynamics, can extrapolate to explain bacterial community outcomes, permit experimentation and observation over ecological processes at high spatiotemporal resolution, and can serve as predictive platforms on which to prototype interventions. Thus, it is a concerted integration of these techniques that will enable rational targeted manipulations of the gut ecosystem.

Conducting research on diet-microbiome interactions: A review of current challenges, essential methodological principles, and recommendations for best practice in study design.

Diet is one of the strongest modulators of the gut microbiome. However, the complexity of the interactions between diet and the microbial community emphasises the need for a robust study design and continued methodological development. This review aims to summarise considerations for conducting high-quality diet-microbiome research, outline key challenges unique to the field, and provide advice for addressing these in a practical manner useful to dietitians, microbiologists, gastroenterologists and other diet-microbiome researchers. Searches of databases and references from relevant articles were conducted using the primary search terms 'diet', 'diet intervention', 'dietary analysis', 'microbiome' and 'microbiota', alone or in combination. Publications were considered relevant if they addressed methods for diet and/or microbiome research, or were a human study relevant to diet-microbiome interactions. Best-practice design in diet-microbiome research requires appropriate consideration of the study population and careful choice of trial design and data collection methodology. Ongoing challenges include the collection of dietary data that accurately reflects intake at a timescale relevant to microbial community structure and metabolism, measurement of nutrients in foods pertinent to microbes, improving ability to measure and understand microbial metabolic and functional properties, adequately powering studies, and the considered analysis of multivariate compositional datasets. Collaboration across the disciplines of nutrition science and microbiology is crucial for high-quality diet-microbiome research. Improvements in our understanding of the interaction between nutrient intake and microbial metabolism, as well as continued methodological innovation, will facilitate development of effective evidence-based personalised dietary treatments.

Duodenal bacterial load as determined by quantitative polymerase chain reaction in asymptomatic controls, functional gastrointestinal disorders and inflammatory bowel disease.

BACKGROUND: Small intestinal bacterial overgrowth may play a role in gastrointestinal and non-gastrointestinal diseases. AIMS: To use quantitative polymerase chain reaction (qPCR) to determine and compare bacterial loads of duodenal biopsies in asymptomatic controls, and patients with functional gastrointestinal disorders (FGIDs) and inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn's disease (CD). To define effects of gastric acid inhibition on bacterial load, explore links of bacterial load and gastrointestinal symptoms in response to a standardised nutrient challenge and compare bacterial load with glucose breath test results. METHODS: In 237 patients (63 controls, 84 FGID and 90 IBD), we collected mucosal samples under aseptic conditions during endoscopy extracted and total DNA. Bacterial load metric was calculated utilising qPCR measurements of the bacterial 16S rRNA gene, normalised to human beta-actin expression. Standard glucose breath test and nutrient challenge test were performed. RESULTS: The duodenal microbial load was higher in patients with FGID (0.22 ± 0.03) than controls (0.07 ± 0.05; P = 0.007) and patients with UC (0.01 ± 0.05) or CD (0.02 ± 0.09), (P = 0.0001). While patients treated with proton pump inhibitors (PPI) had significantly higher bacterial loads than non-users (P < 0.05), this did not explain differences between patient groups and controls. Bacterial load was significantly (r = 0.21, P < 0.016) associated with the symptom response to standardised nutrient challenge test. Methane, but not hydrogen values on glucose breath test were associated with bacterial load measured utilising qPCR. CONCLUSIONS: Utilising qPCR, a diagnosis of FGID and treatment with PPI were independently associated with increased bacterial loads. Increased bacterial loads are associated with an augmented symptom response to a standardised nutrient challenge.

Induction of Meal-related Symptoms as a Novel Mechanism of Action of the Duodenal-Jejunal Bypass Sleeve.

BACKGROUND: Treatment with a duodenal-jejunal bypass sleeve (DJBS) induces clinically significant weight loss, but little is known about the mechanisms of action of this device. AIM: The aim of this study was to characterize the mechanisms of action of the DJBS and determine the durability of weight loss and metabolic improvements. MATERIALS AND METHODS: We studied a cohort of 19 subjects with severe obesity and type 2 diabetes (baseline body mass index: 43.7±5.3 kg/m). Anthropometry, body composition, blood pressure, biochemical measures, and dietary intake were monitored for 48 weeks after DJBS implantation, and then for 1 year after device removal. Gastric emptying and triglyceride absorption were measured at baseline, 8 weeks after implant, and within 3 weeks of device explant. Visceral sensory function was assessed at baseline, 4 weeks after implant, and within 3 weeks after explant. RESULTS: Significant weight loss (P<0.01) occurred following DJBS placement, with a mean weight reduction of 17.0±6.5% at 48 weeks. The symptom burden following a standardized nutrient challenge was increased after DJBS implantation (P<0.05), returning to baseline after DJBS removal. Neither gastric emptying nor triglyceride absorption changed with the device in situ. A significant reduction in energy intake was observed [baseline: 7703±2978 kJ (1841±712 kcal), 24 weeks: 4824±2259 kJ (1153±540 kcal), and 48 weeks: 4474±1468 kJ (1069±351 kcal)]. After 1 year, anthropometry remained significantly improved, but there was no durable impact on metabolic outcomes. CONCLUSIONS: DJBS treatment resulted in substantial weight loss. Weight loss is related to reduced caloric intake, which seems linked to an augmented upper gastrointestinal symptom response, but not altered fat absorption.

Dysbiosis of the Duodenal Mucosal Microbiota Is Associated With Increased Small Intestinal Permeability in Chronic Liver Disease.

OBJECTIVES: Chronic liver disease (CLD) is associated with both alterations of the stool microbiota and increased small intestinal permeability. However, little is known about the role of the small intestinal mucosa-associated microbiota (MAM) in CLD. The aim of this study was to evaluate the relationship between the duodenal MAM and both small intestinal permeability and liver disease severity in CLD. METHODS: Subjects with CLD and a disease-free control group undergoing routine endoscopy underwent duodenal biopsy to assess duodenal MAM by 16S rRNA gene sequencing. Small intestinal permeability was assessed by a dual sugar (lactulose: rhamnose) assay. Other assessments included transient elastography, endotoxemia, serum markers of hepatic inflammation, dietary intake, and anthropometric measurements. RESULTS: Forty-six subjects (35 with CLD and 11 controls) were assessed. In subjects with CLD, the composition (P = 0.02) and diversity (P < 0.01) of the duodenal MAM differed to controls. Constrained multivariate analysis and linear discriminate effect size showed this was due to Streptococcus-affiliated lineages. Small intestinal permeability was significantly higher in CLD subjects compared to controls. In CLD, there were inverse correlations between microbial diversity and both increased small intestinal permeability (r = -0.41, P = 0.02) and serum alanine aminotransferase (r = -0.35, P = 0.04). Hepatic stiffness was not associated with the MAM. DISCUSSION: In CLD, there is dysbiosis of the duodenal MAM and an inverse correlation between microbial diversity and small intestinal permeability. TRANSLATIONAL IMPACT: Strategies to ameliorate duodenal MAM dysbiosis may ameliorate intestinal barrier dysfunction and liver injury in CLD.

Influence of cigarette smoking on the human duodenal mucosa-associated microbiota.

BACKGROUND: Cigarette smoking is a known risk factor in a number of gastrointestinal (GI) diseases in which the microbiota is implicated, including duodenal ulcer and Crohn's disease. Smoking has the potential to alter the microbiota; however, to date, the impact of smoking on the mucosa-associated microbiota (MAM), and particularly that of the upper GI tract, remains very poorly characterised. Thus, we investigated the impact of smoking on the upper small intestinal MAM. A total of 102 patients undergoing upper GI endoscopy for the assessment of GI symptoms, iron deficiency, or Crohn's disease, but without identifiable lesions in the duodenum, were recruited. Smoking status was determined during clinical assessment and patients classified as current (n = 21), previous smokers (n = 40), or having never smoked (n = 41). The duodenal (D2) MAM was profiled via 16S rRNA gene amplicon sequencing. RESULTS: Smoking, both current and previous, is associated with significantly reduced bacterial diversity in the upper small intestinal mucosa, as compared to patients who had never smoked. This was accompanied by higher relative abundance of Firmicutes, specifically Streptococcus and Veillonella spp. The relative abundance of the genus Rothia was also observed to be greater in current smokers; while in contrast, levels of Prevotella and Neisseria were lower. The MAM profiles and diversity of previous smokers were observed to be intermediate between current and never smokers. Smoking did not impact the total density of bacteria present on the mucosa. CONCLUSIONS: These data indicate the duodenal MAM of current smokers is characterised by reduced bacterial diversity, which is partially but not completely restored in previous smokers. While the precise mechanisms remain to be elucidated, these microbiota changes may in some part explain the adverse effects of smoking on mucosa-associated diseases of the GI tract. Smoking status requires consideration when interpreting MAM data.

Dietary fiber intervention on gut microbiota composition in healthy adults: a systematic review and meta-analysis.

Background: Dysfunction of the gut microbiota is frequently reported as a manifestation of chronic diseases, and therefore presents as a modifiable risk factor in their development. Diet is a major regulator of the gut microbiota, and certain types of dietary fiber may modify bacterial numbers and metabolism, including short-chain fatty acid (SCFA) generation. Objective: A systematic review and meta-analysis were undertaken to assess the effect of dietary fiber interventions on gut microbiota composition in healthy adults. Design: A systematic search was conducted across MEDLINE, EMBASE, CENTRAL, and CINAHL for randomized controlled trials using culture and/or molecular microbiological techniques evaluating the effect of fiber intervention on gut microbiota composition in healthy adults. Meta-analyses via a random-effects model were performed on alpha diversity, prespecified bacterial abundances including Bifidobacterium and Lactobacillus spp., and fecal SCFA concentrations comparing dietary fiber interventions with placebo/low-fiber comparators. Results: A total of 64 studies involving 2099 participants were included. Dietary fiber intervention resulted in higher abundance of Bifidobacterium spp. (standardized mean difference (SMD): 0.64; 95% CI: 0.42, 0.86; P < 0.00001) and Lactobacillus spp. (SMD: 0.22; 0.03, 0.41; P = 0.02) as well as fecal butyrate concentration (SMD: 0.24; 0.00, 0.47; P = 0.05) compared with placebo/low-fiber comparators. Subgroup analysis revealed that fructans and galacto-oligosaccharides led to significantly greater abundance of both Bifidobacterium spp. and Lactobacillus spp. compared with comparators (P < 0.00001 and P = 0.002, respectively). No differences in effect were found between fiber intervention and comparators for α-diversity, abundances of other prespecified bacteria, or other SCFA concentrations. Conclusions: Dietary fiber intervention, particularly involving fructans and galacto-oligosaccharides, leads to higher fecal abundance of Bifidobacterium and Lactobacillus spp. but does not affect α-diversity. Further research is required to better understand the role of individual fiber types on the growth of microbes and the overall gut microbial community. This review was registered at PROSPERO as CRD42016053101.

Isolation of Genetically Tractable Most-Wanted Bacteria by Metaparental Mating.

Metagenomics has rapidly advanced our inventory and appreciation of the genetic potential inherent to the gut microbiome. However it is widely accepted that two key constraints to further genetic dissection of the gut microbiota and host-microbe interactions have been our inability to recover new isolates from the human gut, and the paucity of genetically tractable gut microbes. To address this challenge we developed a modular RP4 mobilisable recombinant vector system and an approach termed metaparental mating to support the rapid and directed isolation of genetically tractable fastidious gut bacteria. Using this approach we isolated transconjugants affiliated with Clostridium cluster IV (Faecalibacterium and Oscillibacter spp.), Clostridium cluster XI (Anaerococcus) and Clostridium XIVa (Blautia spp.) and group 2 ruminococci amongst others, and demonstrated that the recombinant vectors were stably maintained in their recipient hosts. By a similar approach we constructed fluorescently labelled bacterial transconjugants affiliated with Clostridium cluster IV (including Flavonifractor and Pseudoflavonifractor spp.), Clostridium XIVa (Blautia spp.) and Clostridium cluster XVIII (Clostridium ramosum) that expressed a flavin mononucleotide-based reporter gene (evoglow-C-Bs2). Our approach will advance the integration of bacterial genetics with metagenomics and realize new directions to support a more mechanistic dissection of host-microbe associations relevant to human health and disease.

TLR2-targeted secreted proteins from Mycobacterium tuberculosis are protective as powdered pulmonary vaccines.

Despite considerable research efforts towards effective treatments, tuberculosis (TB) remains a staggering burden on global health. Suitably formulated sub-unit vaccines offer potential as safe and effective generators of protective immunity. The Mycobacterium tuberculosis antigens, cutinase-like proteins (Culp) 1 and 6 and MPT83, were conjugated directly to the novel adjuvant Lipokel (Lipotek Pty Ltd), a TLR2 ligand that delivers antigen to immune cells in a self-adjuvanting context. Protein-Lipokel complexes were formulated as dry powders for pulmonary delivery directly to the lungs of mice by intra-tracheal insufflation, leading to recruitment of neutrophils and antigen presenting cell populations to the lungs at 72 h, that persisted at 7 days post immunisation. Significant increases in the frequency of activated dendritic cells were observed in the mediastinal lymph node (MLN) at 1 and 4 weeks after homologous boosting with protein-Lipokel vaccine. This was associated with the increased recruitment of effector CD4(+) and CD8(+) T-lymphocytes to the MLN and systemic antigen-specific, IFN-γ producing T-lymphocyte and IgG responses. Notably, pulmonary immunisation with either Culp1-6-Lipokel or MPT83-Lipokel powder vaccines generated protective responses in the lungs against aerosol M. tuberculosis challenge. The successful combination of TLR2-targeting and dry powder vaccine formulation, together with important practical benefits, offers potential for pulmonary vaccination against M. tuberculosis.

Host cell-induced components of the sulfate assimilation pathway are major protective antigens of Mycobacterium tuberculosis.

New therapies to control tuberculosis are urgently required because of the inability of the only available vaccine, BCG, to adequately protect against tuberculosis. Here we demonstrate that proteins of the Mycobacterium tuberculosis sulfate-assimilation pathway (SAP) represent major immunogenic targets of the bacillus, as defined by strong T-cell recognition by both mice and humans infected with M. tuberculosis. SAP proteins displayed increased expression when M. tuberculosis was resident within host cells, which may account in part for their ability to stimulate anti-M. tuberculosis host immunity. Vaccination with the first enzyme in this pathway, adenosine-5'-triphosphate sulfurylase, conferred significant protection against murine tuberculosis and boosted BCG-induced protective immunity in the lung. Therefore, we have identified SAP components as a new family of M. tuberculosis antigens, and we have demonstrated that these components are promising candidate for inclusion in new vaccines to control tuberculosis in humans.

Cutinase-like protein-6 of Mycobacterium tuberculosis is recognised in tuberculosis patients and protects mice against pulmonary infection as a single and fusion protein vaccine.

Infection with Mycobacterium tuberculosis continues to be a leading cause of death in many regions of the world, and control of this disease is hampered by the lack of a safe and effective vaccine. Secreted proteins of M. tuberculosis are an important group of antigens for subunit vaccines which target this infection. We have tested three secreted members of the cutinase-like protein (CULP) family of M. tuberculosis for their potential as protein vaccine antigens. Culp6 elicited a strong T lymphocyte response in M. tuberculosis infected mice, and importantly, in tuberculosis (TB) patients tested. Culp1, Culp2 and Culp6 when delivered as protein vaccines to mice, induced potent IFN-gamma responses which in turn translated into a significant level of protection against aerosol M. tuberculosis infection. A Culp1-6 fusion protein provided an increased level of protection against infection compared to Culp1 or Culp6 alone. The data presented here may indicate that the cell wall-associated, putatively essential protein Culp6, shown here for the first time to be recognised in TB patients, is an attractive candidate for inclusion in future subunit vaccines.