Gardnerella biofilm involves females and males and is transmitted sexually.

OBJECTIVE: To study the incidence and distribution of adherent Gardnerella vaginalis. METHODS: Bacteria adherent to desquamated epithelial cells in the urine were detected using fluorescence in situ hybridization (FISH). Urine from patients with bacterial vaginosis (BV, n = 20), their partners (n = 10) and different control populations (n = 344) including pregnant women and their partners, randomly selected populations of hospitalized man, women and children as also healthy controls was investigated. RESULTS: Gardnerella was found in two different forms: cohesive and dispersed. In the cohesive form, Gardnerella were attached to the epithelial cells in groups of highly concentrated bacteria. In the dispersed form, solitary Gardnerella were intermixed with other bacterial groups. Cohesive Gardnerella was present in all patients with proven BV and their partners, in 7% of men and 13% of women hospitalized for reasons other than BV, in 16% of pregnant women and 12% of their male partners, and in none of the healthy laboratory staff or children. In sexual partners, occurrence of cohesive Gardnerella was clearly linked. Dispersed Gardnerella were found in 10-18% of randomly selected females, 3-4% of males and 10% of children and not sexually linked. In daily longitudinal investigations over 4 weeks no transition between cohesive and dispersed Gardnerella and vice versa was observed. Transmission of a cohesive Gardnerella strain could be followed retrospectively over 15 years using molecular genetic methods. CONCLUSIONS: Cohesive Gardnerella biofilm is a distinct, clearly definable entity which involves both genders and is sexually transmitted. The correct name distinguishing it from symptom-defined conditions like BV should be gardnerellosis and for the bacterium Gardnerella genitalis.

Biostructure of fecal microbiota in healthy subjects and patients with chronic idiopathic diarrhea.

BACKGROUND & AIMS: Dysbiosis is a key component of intestinal disorders. Our aim was to quantitatively access the biostructure of fecal microbiota in healthy subjects and patients with chronic idiopathic diarrhea and evaluate the responses to Saccharomyces boulardii treatment. METHODS: We investigated punched fecal cylinders from 20 patients with chronic idiopathic diarrhea and 20 healthy controls using fluorescence in situ hybridization. Fluctuations in assembly of 11 bacterial groups were monitored weekly for 3 weeks before, during, and after oral S boulardii supplementation. RESULTS: The structural organization of fecal microbiota in healthy subjects was stable and unaffected by S boulardii. The assembly of fecal microbiota in idiopathic diarrhea was markedly different, characterized by mucus depositions within feces; mucus septa and striae; marked reduction in concentrations of habitual Eubacterium rectale, Bacteroides, and Faecalibacterium prausnitzii groups; suppression of bacterial fluorescence in the center of the feces; increased concentrations and spatial shift of mucotrop bacteria to the fecal core; and increased concentrations of occasional bacteria. Except for elevated concentrations of some occasional bacterial groups, all parameters typical for diarrhea improved significantly with S boulardii treatment and most changes persisted after cessation of therapy. The improvement of the fecal microbiota was accompanied by partial (40%) and complete normalization (30%) of the diarrheal symptoms. CONCLUSIONS: The fecal microbiota is highly structured. Fluorescence in situ hybridization analysis allowed us to quantitatively study the dysbiotic changes. S boulardii significantly improved the fecal biostructure in patients with diarrhea but had no influence on the feces in healthy subjects.

Active Crohn's disease and ulcerative colitis can be specifically diagnosed and monitored based on the biostructure of the fecal flora.

BACKGROUND: The intestinal microflora is important in the pathogenesis of inflammatory bowel disease (IBD). The impact of its spatial organization on health and disease is unknown. METHODS: We investigated sections of paraffin-embedded punched fecal cylinders. Fluctuations in spatial distribution of 11 bacterial groups were monitored in healthy subjects (n = 32), patients with IBD (n = 204), and other gastrointestinal diseases (n = 186) using fluorescence in situ hybridization (FISH). RESULTS: The microbial structure differed in patients with Crohn's disease (CD), ulcerative colitis (UC), and healthy and disease controls. The profiles of CD and UC were distinctly opposite in 6 of 11 FISH probes used. Most prominent were a depletion of Faecalibacterium prausnitzii (Fprau<1 x 10(9)/mL) with a normal leukocyte count in CD and a massive increase of leukocytes in the fecal-mucus transition zone (>30 leukocytes/10(4) microm(2)) with high Fprau in patients with UC. These 2 features alone enabled the recognition of active CD (Crohn's Disease Activity Index [CDAI] >150) or UC (Clinical Activity Index [CAI] >3) with 79%/80% sensitivity and 98%/100% specificity. The mismatch in the sensitivity was mainly due to overlap between single IBD entities, and the specificity was exclusively due to the similarity of Crohn's and celiac disease. When inflammatory bowel disease (IBD) patients were pooled the sensitivity was 100% for severe disease, 84% for moderate activity, 72% for IBD with < or =12 months remission, and 24% for IBD with >12 months remission. CONCLUSIONS: The fecal flora is highly structured and spatially organized. Diagnosing IBD and monitoring disease activity can be performed based on analysis of punched fecal cylinders independent from the patient's complaints.

Bacterial biofilm suppression with antibiotics for ulcerative and indeterminate colitis: consequences of aggressive treatment.

BACKGROUND: Antibiotics are commonly used in inflammatory bowel disease (IBD). Little is known about their effect on the mucosal flora. METHODS: The mucosal flora was investigated in colonoscopic biopsies from six groups of 20 IBD patients each. Patients were selected with regard to duration of/interval to combined metronidazole and ciprofloxacin therapy: group I patients with 1 day and group II with 7-14 days of antibiotic therapy, group III-V patients evaluated 1-4 weeks, 2-18 weeks, 26-36 weeks after cessation of antibiotic therapy, respectively. The control group VI included patients without antibiotic therapy. Thirty different fluorescent in situ hybridization (FISH) probes representative of the diversity of the human intestinal flora were applied to all specimens. RESULTS: Bacteria adherent to mucosa could be seen exclusively in DAPI stain and were practically nonamenable to FISH probes in patients on antibiotics (0.001-3+/-0.001-5)x10(10)/mL. Occurrence and concentrations were significantly reduced in groups I and II as compared to untreated controls. The mucosal bacteria were significantly augmented after cessation of antibiotic therapy in group III (13.2+/-4.3) and group IV (5.8+/-2) but not in group V (1.1+/-0.8) as compared to group VI (0.5+/-0.4)x10(10)/mL. Neither Bacteroides nor Enterobacteriaceae groups were permanently suppressed by metronidazole-ciprofloxacin therapy. CONCLUSIONS: The suppressing effects of antibiotics on the mucosal flora are accompanied by massive rebound effects. The concentrations of mucosal bacteria are dramatically increased as soon as 1 week after cessation of antibiotic therapy, remaining at a level that is at least one power higher over a period of 5 months as compared to the group without antibiotic treatment.

Viscosity gradient within the mucus layer determines the mucosal barrier function and the spatial organization of the intestinal microbiota.

BACKGROUND: Migration is an important virulence factor for intestinal bacteria. However, the role of bacterial mobility in the penetration of viscous mucus and their spatial organization within the colon is relatively unknown. METHODS: Movements of fecal bacteria were assessed in gels of varying agarose concentrations and were compared with patterns of bacterial distribution observed in colons from conventional and Enterobacter cloacae-monoassociated mice. Bacteria were visualized using fluorescence in situ hybridization. RESULTS: Long curly bacteria moved best in moderate viscosity gels, short rods and cocci preferred a low viscous environment, whereas high viscosity immobilized all bacterial groups. The spatial distribution of bacteria in the murine colon was also shape- and not taxonomy-dependent, indicating the existence of vertical (surface to lumen) and longitudinal (proximal to distal colon) viscosity gradients within the mucus layer. Our results suggest that mucus viscosity is low in goblet cells, at the crypt basis and close to the intestinal lumen, whereas sites adjacent to the columnar epithelium have a high mucus viscosity. The mucus viscosity increased progressively toward the distal colon, separating bacteria selectively in the proximal colon and completely in the distal colon. CONCLUSIONS: The site-specific regulation of mucus secretion and dehydration make the mucus layer firm and impenetrable for bacteria in regions close to the intestinal mucosa but loose and lubricating in regions adjacent to the luminal contents. Selective control of mucus secretion and dehydration may prove to be a key factor in the management of chronic diseases in which intestinal pathogens are involved.

Seladelpar (MBX-8025), a selective PPAR-δ agonist, in patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid: a double-blind, randomised, placebo-controlled, phase 2, proof-of-concept study.

BACKGROUND: Many patients with primary biliary cholangitis have an inadequate response to first-line therapy with ursodeoxycholic acid. Seladelpar is a potent, selective agonist for the peroxisome proliferator-activated receptor-delta (PPAR-δ), which is implicated in bile acid homoeostasis. This first-in-class study evaluated the anti-cholestatic effects and safety of seladelpar in patients with an inadequate response to ursodeoxycholic acid. METHODS: The study was a 12-week, double-blind, placebo-controlled, phase 2 trial of patients with alkaline phosphatase of at least 1·67 times the upper limit of normal (ULN) despite treatment with ursodeoxycholic acid. Patients, recruited at 29 sites in North America and Europe, were randomly assigned to placebo, seladelpar 50 mg/day, or seladelpar 200 mg/day while ursodeoxycholic acid was continued. Randomisation was done centrally (1:1:1) by a computerised system using an interactive voice-web response system with a block size of three. Randomisation was stratified by region (North America and Europe). The primary outcome was the percentage change from baseline in alkaline phosphatase over 12 weeks, analysed in the modified intention-to-treat (ITT) population (any randomised patient who received at least one dose of medication and had at least one post-baseline alkaline phosphatase evaluation). This study is registered with ClinicalTrials.gov (NCT02609048) and the EU Clinical Trials Registry (EudraCT2015-002698-39). FINDINGS: Between Nov 4, 2015, and May 26, 2016, 70 patients were screened at 29 sites in North America and Europe. During recruitment, three patients treated with seladelpar developed fully reversible, asymptomatic grade 3 alanine aminotransferase increases (one on 50 mg, two on 200 mg), ranging from just over five to 20 times the ULN; as a result, the study was terminated after 41 patients were randomly assigned. The modified ITT population consisted of 12 patients in the placebo group, 13 in the seladelpar 50 mg group, and 10 in the seladelpar 200 mg group. Mean changes from baseline in alkaline phosphatase were -2% (SD 16) in the placebo group, -53% (14) in the seladelpar 50 mg group, and -63% (8) in the seladelpar 200 mg group. Changes in both seladelpar groups versus placebo were significant (p<0·0001 for both groups vs placebo), with no significant difference between the two seladelpar groups (p=0·1729). All five patients who received seladelpar for 12 weeks had normal alkaline phosphatase values at the end of treatment, based on a central laboratory ULN for alkaline phosphatase of 116 U/L. The most frequently reported adverse events were pruritus (16%; one patient on placebo, four on seladelpar 50 mg, and one on seladelpar 200 mg), nausea (13%; one patient on placebo, three on seladelpar 50 mg, and one on seladelpar 200 mg), diarrhoea (10%; two patients on placebo, one on seladelpar 50 mg, and one on seladelpar 200 mg), dyspepsia (8%; two patients on seladelpar 50 mg and one on seladelpar 200 mg), muscle spasms (8%; three patients on seladelpar 200 mg), myalgia (8%; one patient on placebo and two on seladelpar 200 mg), and dizziness (8%; one patient on placebo and two on seladelpar 50 mg). INTERPRETATION: Seladelpar normalised alkaline phosphatase levels in patients who completed 12 weeks of treatment. However, treatment was associated with grade 3 increases in aminotransferases and the study was stopped early. The effects of seladelpar should be explored at lower doses. FUNDING: CymaBay Therapeutics.

Bacterial overgrowth and inflammation of small intestine after carboxymethylcellulose ingestion in genetically susceptible mice.

BACKGROUND: Detergents and emulsifiers added to food may destroy the mucus barrier, which normally isolates bacteria from the intestinal wall, and lead to chronic bowel inflammation in susceptible persons. We investigated the influence of 2% carboxymethylcellulose (CMC) on the biostructure of the intestinal microbiota in IL-10 gene-deficient mice. METHODS: Twenty to 27-week-old IL-10 gene-deficient mice received either 2% CMC solution (n = 7) or water (n = 6) orally for 3 weeks. Intestinal bacteria were investigated using fluorescence in situ hybridization in paraffin-fixed sections of the intestine. RESULTS: CMC-treated IL-10 gene-deficient mice demonstrated a massive bacterial overgrowth, distention of spaces between villi, with bacteria filling these spaces, adherence of bacteria to the mucosa, and migration of bacteria to the bottom of the crypts of Lieberkuehn. Leukocytes migrated into the intestinal lumen in 4 of the 7 CMC mice. The changes were similar to those observed in Crohn's disease in humans and were absent in control animals. CONCLUSIONS: CMC induces bacterial overgrowth and small bowel inflammation in susceptible animals. Because of its ubiquity in products and its unrestricted use in food of the industrial world, CMC is an ideal suspect to account for the rise of IBD in the 20th century.