The multispecies microbial cluster of Fusobacterium, Parvimonas, Bacteroides and Faecalibacterium as a precision biomarker for colorectal cancer diagnosis.

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker.

Characterization and description of Faecalibacterium butyricigenerans sp. nov. and F. longum sp. nov., isolated from human faeces.

Exploiting a pure culture strategy to investigate the composition of the human gut microbiota, two novel anaerobes, designated strains AF52-21T and CM04-06T, were isolated from faeces of two healthy Chinese donors and characterized using a polyphasic approach. The two strains were observed to be gram-negative, non-motile, and rod-shaped. Both strains grew optimally at 37 °C and pH 7.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains clustered with species of the genus Faecalibacterium and were most closely related to Faecalibacterium prausnitzii ATCC 27768T with sequence similarity of 97.18% and 96.87%, respectively. The two isolates shared a 16S rRNA gene sequence identity of 98.69%. Draft genome sequencing was performed for strains AF52-21T and CM04-06T, generating genome sizes of 2.85 Mbp and 3.01 Mbp. The calculated average nucleotide identity values between the genomes of the strains AF52-21T and CM04-06T compared to Faecalibacterium prausnitzii ATCC 27768T were 83.20% and 82.54%, respectively, and 90.09% when comparing AF52-21T and CM04-06T. Both values were below the previously proposed species threshold (95-96%), supporting their recognition as novel species in the genus Faecalibacterium. The genomic DNA G + C contents of strains AF52-21T and CM04-06T calculated from genome sequences were 57.77 mol% and 57.51 mol%, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, we conclude that both strains represent two new Faecalibacterium species, for which the names Faecalibacterium butyricigenerans sp. nov. (type strain AF52-21T = CGMCC 1.5206T = DSM 103434T) and Faecalibacterium longum sp. nov. (type strain CM04-06T = CGMCC 1.5208T = DSM 103432T) are proposed.

Detection of cell-free microbial DNA using a contaminant-controlled analysis framework.

BACKGROUND: The human microbiome plays an important role in cancer. Accumulating evidence indicates that commensal microbiome-derived DNA may be represented in minute quantities in the cell-free DNA of human blood and could possibly be harnessed as a new cancer biomarker. However, there has been limited use of rigorous experimental controls to account for contamination, which invariably affects low-biomass microbiome studies. RESULTS: We apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients. CONCLUSIONS: Together, these observations indicate that plasma can harbour a low yet detectable level of cfmDNA. The results highlight the importance of accounting for contamination and provide an analytical decontamination framework to allow the accurate detection of cfmDNA for future biomarker studies in cancer and other diseases.

The Gut Microbiome in Patients With Chronic Pancreatitis Is Characterized by Significant Dysbiosis and Overgrowth by Opportunistic Pathogens.

INTRODUCTION: Exocrine pancreatic function is a critical host factor in determining the intestinal microbiota composition. Diseases affecting the exocrine pancreas could therefore influence the gut microbiome. We investigated the changes in gut microbiota of patients with chronic pancreatitis (CP). METHODS: Patients with clinical and imaging evidence of CP (n = 51) were prospectively recruited and compared with twice the number of nonpancreatic disease controls matched for distribution in age, sex, body mass index, smoking, diabetes mellitus, and exocrine pancreatic function (stool elastase). From stool samples of these 153 subjects, DNA was extracted, and intestinal microbiota composition was determined by bacterial 16S ribosomal RNA gene sequencing. RESULTS: Patients with CP exhibited severely reduced microbial diversity (Shannon diversity index and Simpson diversity number, P < 0.001) with an increased abundance of facultative pathogenic organisms (P < 0.001) such as Enterococcus (q < 0.001), Streptococcus (q < 0.001), and Escherichia.Shigella (q = 0.002). The CP-associated changes were independent of exocrine pancreatic insufficiency. Short-chain fatty acid producers, considered protective for epithelia such as Faecalibacterium (q < 0.001), showed reduced abundance in patients with CP. Of 4 additional patients with CP previously treated with antibiotics (ceftriaxone and metronidazole), 3 patients were characterized by distinct Enterococcus overgrowth. DISCUSSION: CP is associated with marked gut microbiota dysbiosis, greatly reduced diversity, and increased abundance of opportunistic pathogens, specifically those previously isolated from infected pancreatic necrosis. Taxa with a potentially beneficial role in intestinal barrier function are depleted. These changes can increase the probability of complications from pancreatitis such as infected fluid collections or small intestinal bacterial overgrowth (see Graphical Abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A383).

A Diversified Dietary Pattern Is Associated With a Balanced Gut Microbial Composition of Faecalibacterium and Escherichia/Shigella in Patients With Crohn's Disease in Remission.

BACKGROUND AND AIMS: Crohn's disease [CD] is associated with alterations in gut microbial composition and function. The present controlled-intervention study investigated the relationship between patterns of dietary intake and baseline gut microbiota in CD patients in remission and examined the effects of a dietary intervention in patients consuming a non-diversified diet [NDD]. METHODS: Forty outpatients with quiescent CD were recruited in Calgary, Alberta, Canada. Based on 3-day food records, patients consuming a lower plant-based and higher red and processed meat-based diet were assigned to the NDD group [n = 15] and received a 12-week structured dietary intervention; all other patients were assigned to the diversified diet [DD] control group [n = 25] and received conventional management. Faecal microbiota composition, short chain fatty acids [SCFAs] and calprotectin were measured. RESULTS: At baseline the NDD and DD groups had a different faecal microbial beta-diversity [p = 0.003, permutational multivariate analysis of variance]. The NDD group had lower Faecalibacterium and higher Escherichia/Shigella relative abundances compared to the DD group [3.3 ±â€…5.4% vs. 8.5 ±â€…10.6%; 6.9 ±â€…12.2% vs. 1.6 ±â€…4.4%; p ≤ 0.03, analysis of covariance]. These two genera showed a strong negative correlation [rs = -0.60, q = 0.0002]. Faecal butyrate showed a positive correlation with Faecalibacterium [rs = 0.52, q = 0.002], and an inhibitory relationship with Escherichia/Shigella abundance [four-parameter sigmoidal model, R = -0.83; rs = -0.44, q = 0.01], respectively. After the 12 weeks of dietary intervention, no difference in microbial beta-diversity between the two groups was observed [p = 0.43]. The NDD group demonstrated an increase in Faecalibacterium [p < 0.05, generalized estimated equation model], and resembled the DD group at the end of the intervention [p = 0.84, t-test with permutation]. We did not find an association of diet with faecal SCFAs or calprotectin. CONCLUSIONS: Dietary patterns are associated with specific gut microbial compositions in CD patients in remission. A diet intervention in patients consuming a NDD modifies gut microbial composition to resemble that seen in patients consuming a DD. These results show that diet is important in shaping the microbial dysbiosis signature in CD towards a balanced community.

Gut microbiota in Mexican patients with common variable immunodeficiency

Introduction: Common variable immunodeficiency (CVID) is the main symptomatic primary immunodeficiency and is associated with complex immune disorders. Gut microbiota interacts closely with the immune system, and intestinal dysbiosis is related to multiple diseases. Objective: To describe for the first time the composition of gut microbiota in Mexican patients with CVID. Methods: Fecal samples from five patients with CVID were collected and massive sequencing of the V3-V4 region of 16S rRNA gene was carried out using illumina technology. Results: Bacterial relative abundance was observed at all taxonomic levels. Firmicutes, Actinobacteria and Verrucomicrobia were the predominant phyla. The Clostridia class and the Clostridial order were the most common in their respective taxon; the Ruminococcaceae family predominated. A total of 166 genera were reported, with the most abundant being Faecalibacterium. Five species were identified, but only Bifidobacterium longum was present in all patients. Conclusions: Unlike healthy subjects' gut microbiota, where Firmicutes and Bacteroidetes predominate, the microbiota of the patients with CVID considered in this study was abundant in Firmicutes, Actinobacteria and Verrucomicrobia. The low presence of Bacteroidetes and high abundance of Firmicutes might indicate the existence of intestinal dysbiosis in these patients.

Alterations in gut bacterial and fungal microbiomes are associated with bacterial Keratitis, an inflammatory disease of the human eye.

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n = 21) and bacterial Keratitis patients (BK, n = 19). An increase in abundance of several antiinflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut-eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.

Associations Between Race, Perceived Psychological Stress, and the Gut Microbiota in a Sample of Generally Healthy Black and White Women: A Pilot Study on the Role of Race and Perceived Psychological Stress.

OBJECTIVE: Racial health disparities persist among black and white women for colorectal cancer. Understanding racial differences in the gut microbiota and related covariates (e.g., stress) may yield new insight into unexplained colorectal cancer disparities. METHODS: Healthy non-Hispanic black or white women (age ≥19 years) provided survey data, anthropometrics, and stool samples. Fecal DNA was collected and isolated from a wipe. Polymerase chain reaction was used to amplify the V4 region of the 16SrRNA gene and 250 bases were sequenced using the MiSeq platform. Microbiome data were analyzed using QIIME. Operational taxonomic unit data were log transformed and normalized. Analyses were conducted using linear models in R Package "limma." RESULTS: Fecal samples were analyzed for 80 women (M (SD) age = 39.9 (14.0) years, 47 black, 33 white). Blacks had greater average body mass index (33.3 versus 27.5 kg/m, p < .01) and waist circumference (98.3 versus 86.6 cm, p = .003) than whites. Whites reported more stressful life events (p = .026) and greater distress (p = .052) than blacks. Final models accounted for these differences. There were no significant differences in dietary variables. Unadjusted comparisons revealed no racial differences in alpha diversity. Racial differences were observed in beta diversity and abundance of top 10 operational taxonomic units. Blacks had higher abundances than whites of Faecalibacterium (p = .034) and Bacteroides (p = .038). Stress was associated with abundances of Bifidobacterium. The association between race and Bacteroides (logFC = 1.72, 0 = 0.020) persisted in fully adjusted models. CONCLUSIONS: Racial differences in the gut microbiota were observed including higher Bacteroides among blacks. Efforts to cultivate an "ideal" gut microbiota may help reduce colorectal cancer risk.

Characteristics of Faecal Microbiota in Paediatric Crohn's Disease and Their Dynamic Changes During Infliximab Therapy.

BACKGROUND: Crohn's disease [CD] is known to be associated with gut microbial dysbiosis. Infliximab [IFX] is increasingly used to treat paediatric CD; however, it is not clear how the gut microbiota is modified during IFX treatment. The aim of this study was to characterise the faecal microbiota community composition in paediatric CD patients and to assess its dynamic changes during IFX therapy. METHODS: A 16S rRNA sequencing approach was applied to determine the compositions of microbial communities in faecal samples. The composition and function of the faecal microbiota were compared between CD patients and healthy controls. RESULTS: Characteristics of faecal microbiome composition in paediatric CD patients before IFX treatment were represented by a lower biodiversity, a gain in Enterococcus, and a significant loss in multiple short-chain fatty acid [SCFA]-producing bacteria, including Anaerostipes, Blautia, Coprococcus, Faecalibacterium, Lachnospira, Odoribacter, Roseburia, Ruminococcus, and Sutterella. Additionally, alterations were observed in metabolic functions of the gut microbial community in CD. IFX treatment increased the biodiversity of gut microbiota and shifted its composition as well as its functional capabilities in the paediatric CD patients toward a healthy status. However, multiple SCFA-producing taxa were not significantly expanded. The sustained response of paediatric CD patients to IFX was associated with abundance of SCFA-producing bacteria. CONCLUSIONS: A lower biodiversity with alterations in the composition and function of faecal microbial community, characterising gut microbial dysbiosis, was observed in Chinese paediatric CD patients. IFX diminished the CD-associated gut microbial dysbiosis but was deficient in increasing certain SCFA-producing taxa.

The association between the gut microbiota and the inflammatory bowel disease activity: a systematic review and meta-analysis.

BACKGROUND: The pathogenesis of inflammatory bowel diseases (IBD) involves complex interactions between the microbiome and the immune system. We evaluated the association between the gut microbiota and disease activity in IBD patients. METHODS: Systematic review of clinical studies based on a published protocol. Included patients had ulcerative colitis (UC) or Crohn's disease (CD) classified as active or in remission. We selected bacteria assessed in at least three studies identified through electronic and manual searches (November 2015). Bias control was evaluated with the Newcastle Ottawa scale (NOS). Results of random-effects meta-analyses were presented as mean differences (MD). RESULTS: Three prospective and seven cross-sectional studies (NOS score 6-8) were included. Five studies included patients with CD (231 patients) and eight included patients with UC (392 patients). Compared to patients in remission, patients with active IBD had lower abundance of Clostridium coccoides (MD = -0.49, 95% CI: -0.79 to -0.19), Clostridium leptum (MD = -0.44, 95% CI: -0.74 to -0.14), Faecalibacterium prausnitzii (MD = -0.81, 95% CI: -1.23 to -0.39) and Bifidobacterium (MD = -0.37, 95% CI: -0.56 to -0.17). Subgroup analyses showed a difference in all four bacteria between patients with UC classified as active or in remission. Patients with active CD had fewer C. leptum, F. prausnitzii and Bifidobacterium, but not C. coccoides. CONCLUSION: This systematic review suggests that dysbiosis may be involved in the activity of IBD and that there may be differences between patients with CD and UC.

Dysbiosis Across Multiple Body Sites in Critically Ill Adult Surgical Patients.

The microbiota of critically ill patients likely undergoes dramatic changes but has not been rigorously studied with a culture-independent high-throughput approach. The aim of this study was to characterize spatial and temporal variation in the microbiota of critically ill patients. Trauma and acute surgery patients admitted to the intensive care unit (ICU) were sampled at five body sites (stool, tongue, skin, trachea, urine) every 3 to 4 days. A mean of 10.8 samples was collected from 32 patients with a mean sampling period of 8.8 days. Bacterial 16S rRNA sequences were amplified and sequenced for microbiota analyses. Results were compared to data from unhospitalized adult participants in the American Gut and Human Microbiome Projects. Relative to healthy adults, alpha diversity was decreased in ICU gut and skin samples at all time points. Diversity in tongue swabs decreased over time. Beta diversity measures indicated differences in community membership between critically ill and healthy adults at each body site. Taxonomic alterations in the ICU included depletion of important commensal bacteria such as Faecalibacterium in GI samples and Corynebacterium in skin swabs and enrichment with pathogens such as Enterococcus, Mycoplasma, and Staphylococcus. A high proportion of ICU sample sets contained pathogens present simultaneously at three body sites indicating widespread colonization. In several cases, clinically relevant airway infections were preceded by the appearance of the causative pathogen in tracheal microbiome profiles. These results demonstrate that the microbiome of critically ill patients undergoes a loss of diversity, loss of site specificity, and a shift toward dominant pathogens. These changes may provide opportunities to precisely modulate the microbiome and thereby improve patient outcomes.

Campylobacter concisus utilizes blood but not short chain fatty acids despite showing associations with Firmicutes taxa.

Campylobacter concisus is a member of the oral microbiota that has been associated with the development of inflammatory bowel diseases. However, the role of the bacterium in disease aetiology remains poorly understood. Here, we examine optimal conditions for the growth of C. concisus, and the pathogenic potential of this bacterium in human gastrointestinal cells from the upper tract. Further, the presence of C. concisus in the lower tract of Crohn's disease (CD) patients undergoing therapy is observed, and the associations of C. concisus with the abundance of other microbial taxa and compounds they produce are evaluated. C. concisus strains had the ability to tolerate moderate levels of acidity, adhere to and invade esophageal and gastric cells; however, these properties did not correlate with their pathogenic potential in intestinal cells. The presence of the bacterium in the lower gut of CD patients was associated with an increased relative abundance of Faecalibacterium and Lachnospiraceae incertae sedis. Short chain fatty acids that can be produced by these microbial species did not appear to be responsible for this association. However, we identified genetic similarity between C. concisus and Firmicutes, specifically within aspartate and glutamate racemases. The potential pathogenesis of C. concisus in the upper gastrointestinal tract, and the responsiveness of the bacterium to therapy in a subset of CD patients warrant further investigation into whether this bacterium has a causal role in disease or its presence is incidental.

Microbiological profiles of sputum and gastric juice aspirates in Cystic Fibrosis patients.

Gastro-Oesophageal Reflux (GOR) is a key problem in Cystic Fibrosis (CF), but the relationship between lung and gastric microbiomes is not well understood. We hypothesised that CF gastric and lung microbiomes are related. Gastric and sputum cultures were obtained from fifteen CF patients receiving percutaneous endoscopic gastrostomy feeding. Non-CF gastric juice data was obtained through endoscopy from 14 patients without lung disease. Bacterial and fungal isolates were identified by culture. Molecular bacterial profiling used next generation sequencing (NGS) of the 16S rRNA gene. Cultures grew bacteria and/or fungi in all CF gastric juice and sputa and in 9/14 non-CF gastric juices. Pseudomonas aeruginosa(Pa) was present in CF sputum in 11 patients, 4 had identical Pa strains in the stomach. NGS data from non-CF gastric juice samples were significantly more diverse compared to CF samples. NGS showed CF gastric juice had markedly lower abundance of normal gut bacteria; Bacteroides and Faecalibacterium, but increased Pseudomonas compared with non-CF. Multivariate partial least squares discriminant analysis demonstrated similar bacterial profiles of CF sputum and gastric juice samples, which were distinct from non-CF gastric juice. We provide novel evidence suggesting the existence of an aerodigestive microbiome in CF, which may have clinical relevance.