Antibiotics promote intestinal growth of carbapenem-resistant Enterobacteriaceae by enriching nutrients and depleting microbial metabolites.

The intestine is the primary colonisation site for carbapenem-resistant Enterobacteriaceae (CRE) and serves as a reservoir of CRE that cause invasive infections (e.g. bloodstream infections). Broad-spectrum antibiotics disrupt colonisation resistance mediated by the gut microbiota, promoting the expansion of CRE within the intestine. Here, we show that antibiotic-induced reduction of gut microbial populations leads to an enrichment of nutrients and depletion of inhibitory metabolites, which enhances CRE growth. Antibiotics decrease the abundance of gut commensals (including Bifidobacteriaceae and Bacteroidales) in ex vivo cultures of human faecal microbiota; this is accompanied by depletion of microbial metabolites and enrichment of nutrients. We measure the nutrient utilisation abilities, nutrient preferences, and metabolite inhibition susceptibilities of several CRE strains. We find that CRE can use the nutrients (enriched after antibiotic treatment) as carbon and nitrogen sources for growth. These nutrients also increase in faeces from antibiotic-treated mice and decrease following intestinal colonisation with carbapenem-resistant Escherichia coli. Furthermore, certain microbial metabolites (depleted upon antibiotic treatment) inhibit CRE growth. Our results show that killing gut commensals with antibiotics facilitates CRE colonisation by enriching nutrients and depleting inhibitory microbial metabolites.

Pathobionts in the tumour microbiota predict survival following resection for colorectal cancer.

BACKGROUND AND AIMS: The gut microbiota is implicated in the pathogenesis of colorectal cancer (CRC). We aimed to map the CRC mucosal microbiota and metabolome and define the influence of the tumoral microbiota on oncological outcomes. METHODS: A multicentre, prospective observational study was conducted of CRC patients undergoing primary surgical resection in the UK (n = 74) and Czech Republic (n = 61). Analysis was performed using metataxonomics, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), targeted bacterial qPCR and tumour exome sequencing. Hierarchical clustering accounting for clinical and oncological covariates was performed to identify clusters of bacteria and metabolites linked to CRC. Cox proportional hazards regression was used to ascertain clusters associated with disease-free survival over median follow-up of 50 months. RESULTS: Thirteen mucosal microbiota clusters were identified, of which five were significantly different between tumour and paired normal mucosa. Cluster 7, containing the pathobionts Fusobacterium nucleatum and Granulicatella adiacens, was strongly associated with CRC (PFDR = 0.0002). Additionally, tumoral dominance of cluster 7 independently predicted favourable disease-free survival (adjusted p = 0.031). Cluster 1, containing Faecalibacterium prausnitzii and Ruminococcus gnavus, was negatively associated with cancer (PFDR = 0.0009), and abundance was independently predictive of worse disease-free survival (adjusted p = 0.0009). UPLC-MS analysis revealed two major metabolic (Met) clusters. Met 1, composed of medium chain (MCFA), long-chain (LCFA) and very long-chain (VLCFA) fatty acid species, ceramides and lysophospholipids, was negatively associated with CRC (PFDR = 2.61 × 10-11); Met 2, composed of phosphatidylcholine species, nucleosides and amino acids, was strongly associated with CRC (PFDR = 1.30 × 10-12), but metabolite clusters were not associated with disease-free survival (p = 0.358). An association was identified between Met 1 and DNA mismatch-repair deficiency (p = 0.005). FBXW7 mutations were only found in cancers predominant in microbiota cluster 7. CONCLUSIONS: Networks of pathobionts in the tumour mucosal niche are associated with tumour mutation and metabolic subtypes and predict favourable outcome following CRC resection. Video Abstract.

Effects of bowel preparation on intestinal bacterial associated urine and faecal metabolites and the associated faecal microbiome.

BACKGROUND: Urinary and faecal metabolic profiling have been extensively studied in gastrointestinal diseases as potential diagnostic markers, and to enhance our understanding of the intestinal microbiome in the pathogenesis these conditions. The impact of bowel cleansing on the microbiome has been investigated in several studies, but limited to just one study on the faecal metabolome. AIM: To compare the effects of bowel cleansing on the composition of the faecal microbiome, and the urine and faecal metabolome. METHODS: Urine and faecal samples were obtained from eleven patients undergoing colonoscopy at baseline, and then at day 3 and week 6 after colonoscopy. 16S rRNA gene sequencing was used to analyse changes in the microbiome, and metabonomic analysis was performed using proton nuclear magnetic resonance (1H NMR) spectroscopy. RESULTS: Microbiomic analysis demonstrated a reduction in alpha diversity (Shannon index) between samples taken at baseline and three days following bowel cleansing (p = 0.002), and there was no significant difference between samples at baseline and six weeks post colonoscopy. Targeted and non-targeted analysis of urinary and faecal bacterial associated metabolites showed no significant impact following bowel cleansing. CONCLUSIONS: Bowel cleansing causes a temporary disturbance in bacterial alpha diversity measured in faeces, but no significant changes in the faecal and urine metabolic profiles, suggesting that overall the faecal microbiome and its associated metabolome is resistant to the effects of an induced osmotic diarrhoea.

Fecal Microbiota Transplant Mitigates Adverse Outcomes Seen in Patients Colonized With Multidrug-Resistant Organisms Undergoing Allogeneic Hematopoietic Cell Transplantation.

The gut microbiome can be adversely affected by chemotherapy and antibiotics prior to hematopoietic cell transplantation (HCT). This affects graft success and increases susceptibility to multidrug-resistant organism (MDRO) colonization and infection. We performed an initial retrospective analysis of our use of fecal microbiota transplantation (FMT) from healthy donors as therapy for MDRO-colonized patients with hematological malignancy. FMT was performed on eight MDRO-colonized patients pre-HCT (FMT-MDRO group), and outcomes compared with 11 MDRO colonized HCT patients from the same period. At 12 months, survival was significantly higher in the FMT-MDRO group (70% versus 36% p = 0.044). Post-HCT, fewer FMT-MDRO patients required intensive care (0% versus 46%, P = 0.045) or experienced fever (0.29 versus 0.11 days, P = 0.027). Intestinal MDRO decolonization occurred in 25% of FMT-MDRO patients versus 11% non-FMT MDRO patients. Despite the significant differences and statistically comparable patient/transplant characteristics, as the sample size was small, a matched-pair analysis between both groups to non-MDRO colonized control cohorts (2:1 matching) was performed. At 12 months, the MDRO group who did not have an FMT had significantly lower survival (36.4% versus 61.9% respectively, p=0.012), and higher non relapse mortality (NRM; 60.2% versus 16.7% respectively, p=0.009) than their paired non-MDRO-colonized cohort. Conversely, there was no difference in survival (70% versus 43.4%, p=0.14) or NRM (12.5% versus 31.2% respectively, p=0.24) between the FMT-MDRO group and their paired non-MDRO cohort. Collectively, these data suggest that negative clinical outcomes, including mortality associated with MDRO colonization, may be ameliorated by pre-HCT FMT, even in the absence of intestinal MDRO decolonization. Further work is needed to explore this observed benefit.

Multiomics Profiling Reveals Signatures of Dysmetabolism in Urban Populations in Central India.

BACKGROUND: Non-communicable diseases (NCDs) have become a major cause of morbidity and mortality in India. Perturbation of host-microbiome interactions may be a key mechanism by which lifestyle-related risk factors such as tobacco use, alcohol consumption, and physical inactivity may influence metabolic health. There is an urgent need to identify relevant dysmetabolic traits for predicting risk of metabolic disorders, such as diabetes, among susceptible Asian Indians where NCDs are a growing epidemic. METHODS: Here, we report the first in-depth phenotypic study in which we prospectively enrolled 218 adults from urban and rural areas of Central India and used multiomic profiling to identify relationships between microbial taxa and circulating biomarkers of cardiometabolic risk. Assays included fecal microbiota analysis by 16S ribosomal RNA gene amplicon sequencing, quantification of serum short chain fatty acids by gas chromatography-mass spectrometry, and multiplex assaying of serum diabetic proteins, cytokines, chemokines, and multi-isotype antibodies. Sera was also analysed for N-glycans and immunoglobulin G Fc N-glycopeptides. RESULTS: Multiple hallmarks of dysmetabolism were identified in urbanites and young overweight adults, the majority of whom did not have a known diagnosis of diabetes. Association analyses revealed several host-microbe and metabolic associations. CONCLUSIONS: Host-microbe and metabolic interactions are differentially shaped by body weight and geographic status in Central Indians. Further exploration of these links may help create a molecular-level map for estimating risk of developing metabolic disorders and designing early interventions.

Disease Prevention Not Decolonization: A Model for Fecal Microbiota Transplantation in Patients Colonized With Multidrug-resistant Organisms.

Fecal microbiota transplantation (FMT) yields variable intestinal decolonization results for multidrug-resistant organisms (MDROs). This study showed significant reductions in antibiotic duration, bacteremia, and length of stay in 20 patients colonized/infected with MDRO receiving FMT (compared with pre-FMT history, and a matched group not receiving FMT), despite modest decolonization rates.

Inflammatory Bowel Disease Outcomes Following Fecal Microbiota Transplantation for Recurrent C. difficile Infection.

BACKGROUND: Recurrent Clostridioides difficile infection (CDI) in patients with inflammatory bowel disease (IBD) is a clinical challenge. Fecal microbiota transplantation (FMT) has emerged as a recurrent CDI therapy. Anecdotal concerns exist regarding worsening of IBD activity; however, prospective data among IBD patients are limited. METHODS: Secondary analysis from an open-label, prospective, multicenter cohort study among IBD patients with 2 or more CDI episodes was performed. Participants underwent a single FMT by colonoscopy (250 mL, healthy universal donor). Secondary IBD-related outcomes included rate of de novo IBD flares, worsening IBD, and IBD improvement-all based on Mayo or Harvey-Bradshaw index (HBI) scores. Stool samples were collected for microbiome and targeted metabolomic profiling. RESULTS: Fifty patients enrolled in the study, among which 15 had Crohn's disease (mean HBI, 5.8 ± 3.4) and 35 had ulcerative colitis (mean partial Mayo score, 4.2 ± 2.1). Overall, 49 patients received treatment. Among the Crohn's disease cohort, 73.3% (11 of 15) had IBD improvement, and 4 (26.6%) had no disease activity change. Among the ulcerative colitis cohort, 62% (22 of 34) had IBD improvement, 29.4% (11 of 34) had no change, and 4% (1 of 34) experienced a de novo flare. Alpha diversity significantly increased post-FMT, and ulcerative colitis patients became more similar to the donor than Crohn's disease patients (P = 0.04). CONCLUSION: This prospective trial assessing FMT in IBD-CDI patients suggests IBD outcomes are better than reported in retrospective studies.

A Subset of Roux-en-Y Gastric Bypass Bacterial Consortium Colonizes the Gut of Nonsurgical Rats without Inducing Host-Microbe Metabolic Changes.

Roux-en-Y gastric bypass (RYGB) is an effective weight loss surgery, resulting in a characteristic increase of fecal Gammaproteobacteria The contribution of this compositional change to metabolic benefits of RYGB is currently debatable. Therefore, this study employed 16S rRNA gene sequencing and metabolic profiling to monitor the dynamic colonization of the RYGB microbial consortium and their metabolic impact on the host. Eleven Wistar rats received vancomycin and enrofloxacin, followed by fecal microbiota transplantation (FMT) of cecal slurry obtained from either RYGB- or sham-operated rats. Urine and feces from the microbiota recipients (RYGB microbiota recipients [RYGBr], n = 6; sham microbiota recipients [SHAMr], n = 5) were collected pre- and post-antibiotics and 1, 3, 6, 9, and 16 days post-FMT. No significant differences in body weight and food intake were observed between RYGBr and SHAMr. While neither group reached the community richness of that of their donors, by day 6, both groups reached the richness and diversity of that prior to antibiotic treatment. However, the typical signature of RYGB microbiome-increased Enterobacteriaceae-was not replicated in these recipients after two consecutive FMT, suggesting that the environmental changes induced by the anatomical rearrangements of RYGB could be key for sustaining such a consortium. The transplanted bacteria did not induce the same metabolic signature of urine and feces as those previously reported in RYGB-operated rats. Future work is required to explore environmental factors that shape the RYGB microbiota in order to further investigate the metabolic functions of the RYGB microbiota, thereby teasing out the mechanisms of the RYGB surgery.IMPORTANCE Roux-en-Y gastric bypass (RYGB) surgery results in a long-term gut bacterial shift toward Gammaproteobacteria in both patients and rodents. The contribution of this compositional shift, or the RYGB bacterial consortium, to the metabolic benefit of the surgery remains debatable. It is unclear how well these bacteria colonize in an anatomically normal gut. This is a fundamental question in both defining the function of the RYGB microbiota and evaluating its potential as a nonsurgical treatment for obesity. We monitored the dynamic colonization of the RYGB bacterial consortium and observed that while approximately one-third of the bacterial taxa from the RYGB donor colonized in the gut of the nonoperated recipients, Gammaproteobacteria were unable to colonize for longer than 3 days. The study highlighted that a successful long-term colonization of Gammaproteobacteria-rich RYGB microbiota in nonsurgical animals requires key environmental factors that may be dictated by the intestinal anatomical modification by the surgery itself.

Multiomic features associated with mucosal healing and inflammation in paediatric Crohn's disease.

BACKGROUND: The gastrointestinal microbiota has an important role in mucosal immune homoeostasis and may contribute to maintaining mucosal healing in Crohn's disease (CD). AIM: To identify changes in the microbiota, metabolome and protease activity associated with mucosal healing in established paediatric CD METHODS: Twenty-five participants aged 3-18 years with CD, disease duration of over 6 months, and maintenance treatment with biological therapy were recruited. They were divided into a low calprotectin group (faecal calprotectin <100 µg/g, "mucosal healing," n = 11), and a high calprotectin group (faecal calprotectin >100 µg/g, "mucosal inflammation," n = 11). 16S gene-based metataxonomics, 1 H-NMR spectroscopy-based metabolic profiling and protease activity assays were performed on stool samples. RESULTS: Relative abundance of Dialister species was six-times greater in the low calprotectin group (q = 0.00999). Alpha and beta diversity, total protease activity and inferred metagenomic profiles did not differ between groups. Pentanoate (valerate) and lysine were principal discriminators in a machine-learning model which differentiated high and low calprotectin samples using NMR spectra (R2 0.87, Q2 0.41). Mean relative concentration of pentanoate was 1.35-times greater in the low calprotectin group (95% CI 1.03-1.68, P = 0.036) and was positively correlated with Dialister. Mean relative concentration of lysine was 1.54-times greater in the high calprotectin group (95% CI 1.05-2.03, P = 0.028). CONCLUSIONS: This multiomic study identified an increase in Dialister species and pentanoate, and a decrease in lysine, in patients with "mucosal healing." It supports further investigation of these as potential novel therapeutic targets in CD.

A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague-Dawley Rats.

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague-Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.

In vitro models of the human microbiota and microbiome.

Gut microbiome studies have been gaining popularity over the years, especially with the development of new technologies (e.g. metataxonomics, metagenomics, metatranscriptomics, and metabonomics) that makes it easier for researchers to characterize the composition and functionality of these complex microbial communities. The goal of these studies is to identify a microorganism, group of microbes, or microbial metabolite which correlates with a disease state (e.g. inflammatory bowel disease, colorectal cancer, and obesity). Many of these are cross-sectional studies, where fecal samples from a group of diseased individuals are compared with those from a group of healthy individuals at a single time point. However, there are a wide range of variables that can affect the gut microbiota of humans which make mechanistic studies challenging. Longitudinal studies are required for research to more reliably correlate interventions or disease status to microbiota composition and functionality. However, longitudinal studies in humans and animals are difficult, expensive, and time-consuming. This review will discuss in vitro gut fermentation models and how they can be used to perform longitudinal studies that complement in vivo microbiome studies. Gut fermentation models support the growth of stable, reproducible, and diverse microbial communities in a tightly controlled environment set to mimic the conditions microbes encounter in the gastrointestinal tract. Gut fermentation models will make it easier for researchers to perform mechanistic studies and aid in the development of novel treatments that are both targeted and maintained over time.

Administration of defined microbiota is protective in a murine Salmonella infection model.

Salmonella typhimurium is a major cause of diarrhea and causes significant morbidity and mortality worldwide, and perturbations of the gut microbiota are known to increase susceptibility to enteric infections. The purpose of this study was to investigate whether a Microbial Ecosystem Therapeutic (MET-1) consisting of 33 bacterial strains, isolated from human stool and previously used to cure patients with recurrent Clostridium difficile infection, could also protect against S. typhimurium disease. C57BL/6 mice were pretreated with streptomycin prior to receiving MET-1 or control, then gavaged with S. typhimurium. Weight loss, serum cytokine levels, and S. typhimurium splenic translocation were measured. NF-κB nuclear staining, neutrophil accumulation, and localization of tight junction proteins (claudin-1, ZO-1) were visualized by immunofluorescence. Infected mice receiving MET-1 lost less weight, had reduced serum cytokines, reduced NF-κB nuclear staining, and decreased neutrophil infiltration in the cecum. MET-1 also preserved cecum tight junction protein expression, and reduced S. typhimurium translocation to the spleen. Notably, MET-1 did not decrease CFUs of Salmonella in the intestine. MET-1 may attenuate systemic infection by preserving tight junctions, thereby inhibiting S. typhimurium from gaining access to the systemic circulation. We conclude that MET-1 may be protective against enteric infections besides C. difficile infection.

Differential expression of 26S proteasome subunits and functional activity during neonatal development.

Proteasomes regulate many essential cellular processes by degrading intracellular proteins. While aging is known to be associated with dysfunction of the proteasome, there are few reports detailing activity and function of proteasomes in the early stages of life. To elucidate the function and development of mammalian proteasomes, 26S proteasomes were affinity-purified from rat intestine, spleen and liver. The developmental expression of core, regulatory and immunoproteasome subunits was analyzed by immunoblotting and reverse-transcriptase PCR of mRNA subunits, and proteasome catalytic function was determined by fluorogenic enzymatic assays. The expression of core (ß2, ß5, α7 and ß1) and regulatory (Rpt5) subunits was found to be present at low levels at birth and increased over time particularly at weaning. In contrast, while gradual developmental progression of proteasome structure was also seen with the immunoproteasome subunits (ß1i, ß5i, and ß2i), these were not present at birth. Our studies demonstrate a developmental pattern to 26S proteasome activity and subunit expression, with low levels of core proteasome components and absence of immunoproteasomes at birth followed by increases at later developmental stages. This correlates with findings from other studies of a developmental hyporesponsiveness of the adaptive immune system to allow establishment of microbial colonization immediately after birth.