[Functional biostructure of colonic microbiota (central fermenting area, germinal stock area and separating mucus layer) in healthy subjects and patients with diarrhea treated with Saccharomyces boulardii]. / Biostructure fonctionnelle du microbiote colique(zone de fermentation centrale, zone de réservegerminale et couche de mucus séparatrice) chez lessujets sains et chez les patients atteints de diarrhéetraités par Saccharomyces boulardii.

The colonic content can be compared to a spatially structured high output bioreactor composed of three functionally different regions: a separating mucus layer, a germinal stock area, and a central fermenting area. The stool mirrors this structure and can be used for diagnosis in health and disease. In a first part, we introduce a novel method based on fluorescence in situ hybridization (FISH) of sections of punched-out stool cylinders, which allows quantitatively monitor microbiota in the mucus, the germinal stock and the central fermenting areas. in a second part, we demonstrate the practical implementation of this method, describing the biostructure of stool microbiota in healthy subjects and patients with chronic idiopathic diarrhea treated with Saccharomyces boulardii. Punched stool cylinders from 20 patients with chronic idiopathic diarrhea and 20 healthy controls were investigated using fluorescence in situ hybridization. Seventy-three bacterial groups were evaluated. Fluctuations in assembly of 11 constitutive bacterial groups were monitored weekly for 3 weeks prior to, 3 weeks during, and 3 weeks after oral Saccharomyces boulardii supplementation. Typical findings in healthy subjects were a 5-60 µm mucus separating layer; homogeneous distribution and fluorescence, high concentrations (>10 × 10(10) bacterial/mL) of the three habitual bacterial groups: Bacteroides, Roseburia and Faecalibacterium prausnitzii; and low concentrations of the occasional bacterial groups. The diarrhea could be described in terms of increased separating effort, purging, decontamination, bacterial substitution. Typical findings in diarrhea were: increased thickness of the protective mucus layer, its incorporation in the stool, absolute reduction in concentrations of the habitual bacterial groups, suppression of bacterial metabolism in the central fermenting area (hybridization silence), stratification of the stool structure by watery ingredients, and substitutive increase in the concentrations of occasional bacterial groups. The microbial and clinical symptoms of diarrhea were reversible with Saccharomyces boulardii therapy. The structure-functional analysis of stool microbiota allows to quantitatively monitor colonic malfunction and its response to therapy. Saccharomyces boulardii significantly improves the stool biostructure in patients with chronic idiopathic diarrhea and has no influence on the stool microbiota in healthy subjects.

Mucosal flora in Crohn's disease and ulcerative colitis - an overview.

The intestinal flora harbors varies pathogens. Clostridium perfringens (gas gangrene), Enterococci (endocarditis), Enterobacteriaceae (sepsis), Bacteroides (abscesses) are present in the large intestine of every healthy person in high concentrations. These bacteria are, however, separated from the colonic wall by an impenetrable mucus layer and are tolerated by the host. This separation is disturbed in patients with inflammatory bowel disease (IBD), where bacteria adhere to the mucosa and invade epithelial cells with concomitant inflammatory response. This chronic bowel inflammation can not subside as long as the mucus barrier remains defective. The inflammatory response interferes with the state of tolerance to the intestinal bacteria and leads to characteristic changes in the biostructure of the faecal microbiota. These changes in the biostructure of faecal microbiota are specific for active Crohn's disease and ulcerative colitis (UC) and can be longitudinally monitored. The reason for the defect of the mucus barrier in IBD patients is unclear. Epidemiologic studies indicate a negative role of western lifestyle and foods and document the rise in the incidence of IBD in the industrialized countries during the 20(th) century. In parallel to this, detergents were introduced in households and emulsifiers were increasingly added to food. The cleaning effect of these on the colonic mucus has to be investigated. The present contribution summarizes new data on the biostructure of the intestinal microbiota.

[Biomorphology of the Bacterial Invasion in Chronic pharyngotonsillitis ]. / Biomorphologie der Infektionsherde in rezidivierenden Mandelentzündungen.

BACKGROUND: The study investigates whether relapses of chronicpharyngotonsillitis result from new infections caused by theoro-pharyngeal microbial flora or are reactivations of persistent bacterial infections of the tonsils. METHODS: 90 patients, who were surgically treated for chronicpharyngotonsillitis (age 13 months to 38 years, at least 5 episodes of disease and antibiotic treatment in the past) were included. The surgery was performed in the antibiotic- and symptom-free period (at least 6 weeks after the last exacerbation). Sections of tonsillar tissue were investigated for invasive bacteria using fluorescence in situ hybridization (FISH) with group and species-specific 15/23S RNA based probes. RESULTS: Abundant foci of invasive bacteria were found in 86% of the resected tonsils, despite previous treatment with antibiotica and absent symptoms of ongoing infection. The diffuse infiltration of the tonsils was most predominant in the youger children. Local invasive processes such as abscesses, fissures filled with pus and superficial infiltration of the tonsillar epithelium were more typical for adults. All of the foci were polymicrobial and contained up to 10 different species or groups of bacteria. The local concentrations of invasive bacteria were up to 1012 bacteria/ml. CONCLUSIONS: The chronic pharyngotonsillitis is the result of persistent invasive bacterial infections. The polymicrobial nature of the infectious foci enables them to resist the antibiotic treatment and to exacerbate afterwards. The surgical treatment is unavoidable as long as antibiotic treatment fails to clear the infection.

Spatial organisation of microbiota in quiescent adenoiditis and tonsillitis.

BACKGROUND: The reasons for recurrent adenotonsillitis are poorly understood. METHODS: The in situ composition of microbiota of nasal (5 children, 25 adults) and of hypertrophied adenoid and tonsillar tissue (50 children, 20 adults) was investigated using a broad range of fluorescent oligonucleotide probes targeted to bacterial rRNA. None of the patients had clinical signs of infection at the time of surgery. RESULTS: Multiple foci of ongoing purulent infections were found within hypertrophied adenoid and tonsillar tissue in 83% of patients, including islands and lawns of bacteria adherent to the epithelium, with concomitant marked inflammatory response, fissures filled with bacteria and pus, and diffuse infiltration of the tonsils by bacteria, microabscesses, and macrophages containing phagocytosed microorganisms. Haemophilusinfluenzae mainly diffusely infiltrated the tissue, Streptococcus and Bacteroides were typically found in fissures, and Fusobacteria,Pseudomonas and Burkholderia were exclusively located within adherent bacterial layers and infiltrates. The microbiota were always polymicrobial. CONCLUSIONS: Purulent processes persist during asymptomatic periods of adenotonsillitis. Most bacteria involved in this process are covered by a thick inflammatory infiltrate, are deeply invading, or are located within macrophages. The distribution of the bacteria within tonsils may be responsible for the failure of antibiotic treatment.

Bacterial biofilm within diseased pancreatic and biliary tracts.

BACKGROUND: Bacterial community structures in human pancreatic and biliary tracts were evaluated. METHODS: Gall bladder stones from 153 patients, 20 gall bladder walls, six common duct stones, 52 biliary stents, 21 duodenal biopsies, nine pancreatic duct biopsies, and five bile ducts were investigated using fluorescence in situ hybridisation (FISH) with ribosomal RNA targeted Cy3/Cy5 (carbocyanine) labelled oligonucleotide probes. RESULT: Duodenal, gall bladder, and bile duct walls were free of bacteria. A dense multispecies bacterial biofilm was present within the pancreatic duct of patients with calcific pancreatitis and within biliary stents, irrespective of diagnosis. The concentration, density, and amenability of the biofilm to FISH and DNA staining declined progressively with the grade of stent occlusion. The lowest detectable bacterial concentrations were found by FISH in completely occluded stents and brown/mixed gall stones. Bacteria were not detectable with FISH in cholesterol gall stones. CONCLUSIONS: A wide range of different branches and groups of bacteria participate in the development of biofilms on the surfaces of foreign bodies, such as biliary stents, mixed gall stones, or calcific pancreatic ducts, but not on the surface of pure cholesterol gall stones. Occlusion of stents leads to progressive extinction of the biofilm and mummification of its components. Deposition of cholesterol or other substances within the biofilm matrix may be a novel mechanism of host defence against bacteria present in these biofilms.

The role of bacteria in gallstone pathogenesis.

Bacteria are often found in high concentrations in brown pigment and less so in cholesterol gallstones. Although it is intriguing to hypothesize that cholesterol stone formation is non-bacterial in nature and principally different from the pathogenesis of "infectious" brown pigment gallstones, it is more likely that significant overlap exists between the two processes. Most gallstones are composite in nature. Using molecular-genetic methods, bacteria can be found in most pure cholesterol gallstones (i.e. those whose structure consists of more than 90% cholesterol). The natural history of the gallstones development is unknown. It is likely that brown pigment stones can evolve in their chemical composition after the termination of the infectious process that initiate their formation, and may further develop into either mixed or nearly pure cholesterol stones. In a similar fashion, cholesterol-poor or black pigment gallstones may act as foreign bodies to enhance the propensity of bacterial colonization in the presence of pre-existing gallstones or cholangitis, thereby activating pathways of bacterial lithogenesis and resulting in the encasement of cholesterol nuclei with pigment shells and/or in the internal remodeling of extant stones. It is often difficult, if not impossible, to ascertain whether bacterial infection of bile arose before stone formation or vice-versa. The development of gallstones (nucleation, assembly of microcalculi, growth, remodeling) includes the interaction of both bacterial and non-bacterial mechanisms, working discontinuously over years and decades and shaping the structural individuality of each stone. At cholecystectomy, the gallstone removed from the patient represents the end product of a long pathologic process. Although our understanding of the exact temporal contribution of bacteria in lithogenesis is incomplete, it is important for the clinician to realize that most gallstones are colonized by a bacterial biofilm, even though the bile may be culture-negative.

Association between intraepithelial Escherichia coli and colorectal cancer.

BACKGROUND & AIMS: Although multiple studies have focused on Helicobacter pylori, little is known about the mucosa-associated flora of the colon. The aim of this study was to detect bacteria directly in colonic mucosa from patients screened for colorectal cancer. METHODS: Bacteria were quantified with the polymerase chain reaction and identified by comparative sequence analysis in colonoscopic biopsy specimens from 31 asymptomatic and 34 symptomatic controls with normal colonoscopic findings, 29 patients with colonic adenoma, and 31 patients with colorectal carcinoma. In 41 patients, intra- and extracellular location of bacteria was confirmed with the gentamicin protection assay. RESULTS: No bacteria were detected in biopsy specimens from 97% of asymptomatic and 69% of symptomatic controls. In contrast, bacterial concentrations of 10(3)-10(5) colony-forming units per microliter were detected in biopsy specimens from both malignant and macroscopically normal tissue in 90% and 93% of patients with adenoma and carcinoma, respectively. E. coli and coli-like bacteria were shown to colonize the colonic mucosa in 82% of these patients. The gentamicin protection assay indicated that E. coli was partially intracellular in 87% of patients with adenoma and carcinoma and in none of the controls. CONCLUSIONS: The colonic mucosa of patients with colorectal carcinoma but not normal colonic mucosa is colonized by intracellular E. coli.

Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstones.

The role of bacteria in gallstone formation could not be conclusively evaluated until bacterial presence or absence in a stone was consistently shown. Cultural bacteriologic investigations at the time of cholecystectomy, however, led to the assumption that cholesterol gallstones were free of bacteria. In this study, we used a culture independent, molecular genetic approach to detect, quantify, and identify bacteria in cholesterol gallstones from 100 patients at the time of cholecystectomy and 6 months following. Bacterial growth was recorded in the culture in 9 of 100 gallstones; bacterial DNA, however, was detected in 82 of 91 sterile gallstones. High concentrations corresponding to between 10(6) to 10(7) bacteria/g were detected in 11 stones and low concentrations of 10(5) bacteria/g were detected in 71 sterile stones. The infection in stones with a positive bacterial culture was characterized by the predominance of single bacterial sequence(s) of the bacteria cultured. A similar predominance, indicating a recent infection, was found in sterile gallstones with low DNA concentrations. A high diversity of non-repeating bacterial sequences, possibly arising from previous overlapping infections, was found in sterile gallstones with high concentrations of bacterial DNA. After 6 months concentrations of bacterial DNA fell significantly in all groups of gallstones. As bacterial DNA is quickly destroyed upon storage, but is nevertheless readily found in most gallstones at the time of cholecystectomy, there must be a mechanism by which it is replenished. One such mechanism is the frequently reoccurring, possibly self-terminating infection and another one is the permanent colonization of the gallstone with bacteria at low concentrations. Both can promote cholecystolithiasis.

Molecular genetic evidence of bacterial colonization of cholesterol gallstones.

BACKGROUND/AIMS: Cholesterol gallstone formation is believed to be unrelated to the presence of bacteria because attempts to culture potentially causative bacteria from surgically removed cholesterol stones have failed. However, the formation of gallbladder gallstones takes years. Embedded bacteria may be damaged or killed. The aim of this study was to search for bacterial DNA sequences in cholesterol stones with negative bacterial culture. METHODS: Bacterial gene fragments were amplified in vitro from DNA extracted from cholesterol gallbladder stones. Comparative 16S ribosomal RNA sequence analysis was used for identification. RESULTS: Gallstones with cholesterol content between 70% to 90% harbored bacterial DNA (16 of 17 patients). No bacterial DNA was found in the gallstones with cholesterol content of > 90% (3 patients). Three bacterial groups typical for gallstone colonization were identified. Propionibacteria-related DNA was found in the stones of 9 patients (45%). Enterobacterial type sequences were obtained in 5 patients (25%). A more heterogenous sequence collection was retrieved from 7 patients (35%) and could be assigned to the major bacterial line of gram-positive bacteria with a low DNA guanine and cytosine content. CONCLUSIONS: Most cholesterol gallstones harbor bacterial DNA. It is important to determine the actual role of these microorganisms in gallstone formation.

[Fc IgG carrying T lymphocytes in healthy adults and patients following kidney transplantation]. / Fc-IgG-Rezeptor-tragende T-Lymphozyten bei gesunden Erwachsenen und Patienten nach Nierentransplantation).

In 28 healthy adults aged 19 to 52 years, normal values of the TG lymphocytes amount to 17.8 +/- 2.3% and 253.1 +/- 96.1 per microliters respectively and the non TG/TG ratio to 4.72 +/- 0.82. When compared with normal controls, patients on dialysis had a disturbed non TG/TG ratio (4.72 +/- 0.82 vs. 6.00 +/- 1.94, p less than 0.05). During the first posttransplant month the total TG cell count was significantly reduced, but the relative TG cell count did not significantly differ from the normal as well as praeoperative TG cell count. In connection with rejection crises we could not observe any changes in the TG subset. Beside this general dynamics we observed two different kinds of changes of the non TG/TG ratio in the individual posttransplant course. Either the non TG/TG ratio were lower than praetransplant or higher. In 9 out of 9 cases the lowering of the non TG/TG ratio (that means a relative or absolute increase of Fc-IgG receptor bearing T cells) were connected with a good graft function. In 4 out of 6 cases a postoperative increase of the non TG/TG ratio were connected with an early graft failure. A change of T subsets for the benefit of TG cells which includes suppressor cells seems to be favourable with regard to the graft survival. Therefore, we think the determination of the prae- and posttransplant non TG/TG ratio is of some prognostic value.