Fungal and Bacterial Loads: Noninvasive Inflammatory Bowel Disease Biomarkers for the Clinical Setting.

Microbiome sequence data have been used to characterize Crohn's disease (CD) and ulcerative colitis (UC). Based on these data, we have previously identified microbiomarkers at the genus level to predict CD and CD relapse. However, microbial load was underexplored as a potential biomarker in inflammatory bowel disease (IBD). Here, we sought to study the use of fungal and bacterial loads as biomarkers to detect both CD and UC and CD and UC relapse. We analyzed the fecal fungal and bacterial loads of 294 stool samples obtained from 206 participants using real-time PCR amplification of the ITS2 region and the 16S rRNA gene, respectively. We combined the microbial data with demographic and standard laboratory data to diagnose ileal or ileocolonic CD and UC and predict disease relapse using the random forest algorithm. Fungal and bacterial loads were significantly different between healthy relatives of IBD patients and nonrelated healthy controls, between CD and UC patients in endoscopic remission, and between UC patients in relapse and non-UC individuals. Microbial load data combined with demographic and standard laboratory data improved the performance of the random forest models by 18%, reaching an average area under the receiver operating characteristic curve (AUC) of 0.842 (95% confidence interval [CI], 0.65 to 0.98), for IBD diagnosis and enhanced CD and UC discrimination and CD and UC relapse prediction. Our findings show that fecal fungal and bacterial loads could provide physicians with a noninvasive tool to discriminate disease subtypes or to predict disease flare in the clinical setting.IMPORTANCE Next-generation sequence data analysis has allowed a better understanding of the pathophysiology of IBD, relating microbiome composition and functions to the disease. Microbiome composition profiling may provide efficient diagnosis and prognosis tools in IBD. However, the bacterial and fungal loads of the fecal microbiota are underexplored as potential biomarkers of IBD. Ulcerative colitis (UC) patients have higher fecal fungal and bacterial loads than patients with ileal or ileocolonic CD. CD patients who relapsed harbor more-unstable fungal and bacterial loads than those of relapsed UC patients. Fecal fungal and bacterial load data improved prediction performance by 18% for IBD diagnosis based solely on clinical data and enhanced CD and UC discrimination and prediction of CD and UC relapse. Combined with existing laboratory biomarkers such as fecal calprotectin and C-reactive protein (CRP), microbial loads may improve the diagnostic accuracy of IBD and of ileal CD and UC disease activity and prediction of UC and ileal CD clinical relapse.

Dietary supplementation with spray-dried porcine plasma has prebiotic effects on gut microbiota in mice.

In animal models of inflammation and in farm animals, dietary inclusion of spray-dried porcine plasma (SDP) reduces mucosal inflammation. Here, we study whether these effects could be mediated by changes in the intestinal microbiota and if these changes are similar to those induced by oral antibiotics. Weaned 21-day-old C57BL/6 mice were divided into 3 groups: the CTL group, fed the control diet; the COL group, administered low doses of neomycin and colistin; and the SDP group, supplemented with 8% SDP. After 14 days, analysis of the fecal microbiome showed that the microbiota profiles induced by SDP and the antibiotics were very different, thus, SDP has prebiotic rather than antibiotic effects. At the phylum level, SDP stimulated the presence of Firmicutes, considerably increasing the lactobacilli population. It also enhanced the growth of species involved in regulatory T-lymphocyte homeostasis and restoration of the mucosal barrier, as well as species negatively correlated with expression of pro-inflammatory cytokines. At the mucosal level, expression of toll-like receptors Tlr2, Tlr4 and Tlr9, and mucous-related genes Muc2 and Tff3 with regulatory and barrier stability functions, were increased. SDP also increased expression of Il-10 and Tgf-ß, as well as markers of macrophages and dendritic cells eventually promoting an immune-tolerant environment.

Metabolic adaptation of colonic microbiota to galactooligosaccharides: a proof-of-concept-study.

BACKGROUND: Prebiotics have been shown to reduce abdominal symptoms in patients with functional gut disorders, despite that they are fermented by colonic bacteria and may induce gas-related symptoms. AIM: To investigate changes in the metabolic activity of gut microbiota induced by a recognised prebiotic. METHODS: Healthy subjects (n = 20) were given a prebiotic (2.8 g/day HOST-G904, HOST Therabiomics, Jersey, Channel Islands) for 3 weeks. During 3-day periods immediately before, at the beginning and at the end of the administration subjects were put on a standard diet (low fibre diet supplemented with one portion of high fibre foods) and the following outcomes were measured: (i) number of daytime gas evacuations for 2 days by means of an event marker; (ii) volume of gas evacuated via a rectal tube during 4 h after a test meal; and (iii) microbiota composition by faecal Illumina MiSeq sequencing. RESULTS: At the beginning of administration, HOST-G904 significantly increased the number of daily anal gas evacuations (18 ± 2 vs. 12 ± 1 pre-administration; P < 0.001) and the volume of gas evacuated after the test meal (236 ± 23 mL vs. 160 ± 17 mL pre-administration; P = 0.006). However, after 3 weeks of administration, these effects diminished (11 ± 2 daily evacuations, 169 ± 23 mL gas evacuation). At day 21, relative abundance of butyrate producers (Lachnospiraceae) correlated inversely with the volume of gas evacuated (r = -0.52; P = 0.02). CONCLUSION: The availability of substrates induces an adaptation of the colonic microbiota activity in bacterial metabolism, which produces less gas and associated issues. Clinical trials.gov NCT02618239.

Colonisation by Faecalibacterium prausnitzii and maintenance of clinical remission in patients with ulcerative colitis.

BACKGROUND: Although incrimination of the intestinal microbiota in the pathogenesis of IBD is widely accepted, few data are available about the role of specific bacteria. Potentially, Faecalibacterium prausnitzii, bacteria with anti-inflammatory properties, might be deficient in ulcerative colitis (UC). AIM: To quantify F. prausnitzii in the faecal microbiota of UC patients in remission and determine its relationship with relapse. METHODS: A cross-sectional study included 116 UC patients in remission, 29 first-degree relatives and 31 healthy controls. A subset of eighteen patients, recruited during the first month of remission, underwent a 1-year follow-up. Total bacteria and F. prausnitzii were measured by quantitative Real Time PCR (qPCR, copies/g). Calprotectin was determined as inflammatory index (µg/g). RESULTS: We found that F. prausnitzii was reduced in patients (median, IQR: 1.4 × 108 , 5.1 × 107-4.5 × 108) and relatives (1.7 × 108, 9.3 × 107-5.1 × 108) vs. controls (6.5 × 108, 3.7 × 108-1.6 × 109, P < 0.0001). Moreover, low counts of F. prausnitzii were associated with less than 12 months of remission (8.0 × 107, 2.0 × 107-3.5 × 108 vs. 2.1 × 108, 1.0 × 108-7.9 × 108, P < 0.001) and more than 1 relapse/year (8.0 × 107, 3.2 × 107-3.8 × 108 vs. 1.9 × 108, 6.8 × 107-6.0 × 108, P < 0.01). When patients were followed up, F. prausnitzii increased steadily until reaching similar levels to those of controls if remission persisted (2.9 × 108, 9.3 × 106-1.2 × 109; calprotectin: 76, 19-212), whereas it remained low if patients relapsed (2.2 × 108, 1.4 × 106-3.3 × 108; calprotectin: 1760, 844-3662 P < 0.05 vs. controls). CONCLUSIONS: Defective gut colonisation by F. prausnitzii occurred in UC patients during remission and in their unaffected relatives. The recovery of the F. prausnitzii population after relapse is associated with maintenance of clinical remission.

Review article: the role of bacteria in onset and perpetuation of inflammatory bowel disease.

We review the evidence that strongly suggests a role of the intestinal microbiota in the onset and perpetuation of inflammatory bowel disease (IBD). Experimental studies consisted of suppressing micro-organisms from the microbiota (using germ-free or gnotoxenic animals or antibiotics), introducing new micro-organisms or microbial components (e.g. probiotics, CpG-DNA) or selectively increasing some endogenous bacteria (e.g. using prebiotics). Intervention studies were performed in patients or animal models of spontaneous or chemically-induced colitis. Information was also obtained from observational studies that described the composition of the faecal and mucosal microbiota at various stages of the disease process and in controls. Many have used culture-independent techniques that identify bacteria based on the nucleic acid sequence of ribosomal RNA molecules. Microbiota in patients with IBD seem to be characterized by high concentrations of bacteria in contact with the mucosa, instability, the presence of high numbers of unusual bacteria and sometimes a reduction in the biodiversity. Studies searching for a generalized or localized dysbiosis in IBD are discussed, as well as those trying to identify bacterial molecules and receptors, which may be implicated in triggering the inflammatory process.

Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach.

BACKGROUND AND AIM: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn's disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. METHODS: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25,000 clones, for the 16S rRNA gene by DNA hybridisation. RESULTS: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n=12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). CONCLUSION: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.

Genomic choice of codons in 16 microbial species.

We study the codon usage over whole set of ORFs of 16 unicellular microbial species: eight archaebacteria, seven eubacteria, and one eukarya. We first try to define, for each species, the neutral expected codon usage to better approach subsequently the influence of selection. Overlapping triplets counted from the complete DNA genomic sequence and mean amino acid composition of ORFs allow us to build satisfying expected codon usage for each species. Within species deviation from this neutral model is then studied through Correspondence Analysis and characterization with bias index, N(C)' (effective number of codons reported to neutral model). Our results are compared to previously published ones for three species and let appear good agreement in spite of very different methods. We thus propose set of codons probably preferred by selection for nine other species. In the four last species, no clear preference can be evidenced. Finally, we characterize variation of codon usage over functional categories. We propose that the high degree of bias of proteins involved in translation, ribosomal structure and biogenesis has a positive influence on overexpression of the corresponding genes under optimum growth conditions and is a negative regulator of the same genes when amino acids become limited resources.

Susceptibility of human herpesvirus 6 to antiviral compounds by flow cytometry analysis.

BACKGROUND: The emergence of human herpesvirus 6 (HHV-6) as a human pathogen led to the possibility of specific therapy against HHV-6 and the development of standardized susceptibility assays of HHV-6 to antivirals. METHODS: We have developed a flow cytometry method to analyze the multiplication of the HST strain of human herpesvirus 6 (HHV-6) variant B in vitro using monoclonal antibodies specific to virus proteins. This method was subsequently used to determine the sensitivity of HST multiplication in MT4 cells to four antiviral compounds of three different classes: acyclovir (ACV) and ganciclovir (GCV), two acyclic guanosine analogs; cedofovir (CDV), an acyclic nucleoside phosphonate; and phosphonoformic acid (PFA), a pyrophosphate analog. RESULTS: The 50% inhibitory concentrations (IC(50)) of ACV, GCV, CDV, and PFA determined by flow cytometry assay were 25.3, 6.4, 0.95, and 6.0 microM, respectively (5.7, 1.6, 0.3, and 1.8 microg/ml, respectively). These data together with the results of cytotoxicity assays confirmed the high efficiency and selectivity of CDV and PFA against HHV-6 B in vitro, suggested by previous results. CONCLUSIONS: Our flow cytometric assay appeared as a reproducible specific method to characterize HHV-6 susceptibility to antiviral compounds. It can be considered as a convenient alternative to the other immunologic and DNA hybridization assays used for that purpose.