Clue Cells and Pseudo Clue Cells in Different Morphotypes of Bacterial Vaginosis.

Introduction: Clue cells (epithelial cells heavily covered with adherent bacteria) are an accepted clue to the diagnosis of bacterial vaginosis. However, the exact morphologic criteria of clue cells and bacterial adherence were never elaborated. Materials and Methods: We investigated adhesive and cohesive patterns of main microbiota groups in vaginal discharge using fluorescence in situ hybridization (FISH). Samples from 500 women diagnosed with bacterial vaginosis and positive for clue cells with classic microscopy were collected from 42 gynecologic practices in Berlin and reexamined in our FISH laboratory for the spatial distribution of Bifidobacteriaceae, Gardnerella, Fannyhessea vaginae (Atopobium); low G+C (guanine+cytosine) bacteria, lactobacilli, Lactobacillus iners; Lactobacillus crispatus, Gamma-Proteobacteria; and Enterobacteriaceae, Prevotella-Bacteroides, Veillonella, and Coriobacterium groups. Results: Bacterial taxa present in vaginal smears were not accidentally assembled according to their relative abundance but were built in group-specific distribution patterns, which can be well described by two features: cohesiveness to each other and adherence to epithelial cells. Accordingly, four patterns can be distinguished: dispersed (non-adherent bacteria), dispersed adherent bacteria, cohesive (non-adherent) bacteria, and cohesive adherent bacteria. Direct cohesive adherence to the epithelial cells representing true clue cells was unique for Gardnerella species and observed only in 56% of the investigated samples. In the remaining vaginal samples, the epithelial cells were mechanically entrapped in bacterial masses, and the composition was unrelated to the epithelial cell surface, building non-adherent pseudo clue cells. The proportion of women with true clue cells in their samples from different gynecologic practices varied from 19% to 80%. Discussion: Taxon indifferent imaging is inadequate for the exact analysis of the microbial layer adjacent to the vaginal epithelial cells. Morphologically seen bacterial vaginosis is a mix of at least two different conditions: biofilm vaginosis and bacterial excess vaginosis.

Antimicrobial Susceptibility of Microbiota in Bacterial Vaginosis Using Fluorescence In Situ Hybridization.

BACKGROUND: Testing of antibiotic resistance of intact vaginal microbiota in pure culture is not feasible. METHODS: Metronidazole, antiseptic octenisept®, antimycotic ciclopirox, bacterial probiotic Lactobacillus crispatus, yeast probiotic Saccharomyces boulardii, Gardnerella-phage-endolysin named phagolysin and phagolysin in combination with probiotics were tested for bacteriolytic activity. Included were vaginal swabs from 38 random women with Amsel-confirmed bacterial vaginosis (BV). Test aliquots were incubated by 37° for 2 and 24 h. Gardnerella, low G+C, Atopobium, lactobacilli, Lactobacillus iners and crispatus, Prevotella-Bacteroides, and Gammaproteobacteria microbial groups were quantified using fluorescence in situ hybridization (FISH). RESULTS: The probiotic strain Lactobacillus crispatus demonstrated the weakest bacteriolytical effects, followed by metronidazole. Both had no impact on Gardnerella species, instead lysing Prevotella-Bacteroides, Enterobacteriaceae (by L.crispatus) or LGC, Atopobium and Prevotella-Bacteroides (by metronidazole) groups of the microbiota. Cytolytic activity on Gardnerella was highly pronounced and increased from octenisept to ciclopirox, phagolysin, phagolysin with L.crispatus, being best in the combination of phagolysin with S.boulardii. Universally active ciclopirox and octenisept® suppressed nearly all microbial groups including those which are regarded as beneficial. Phagolysin had no effect on naturally occurring Lactobacillus crispatus. Conclusions: FISH susceptibility testing allows unique efficacy evaluation of individually adjusted topical therapy without microbial isolation facilitating optimal therapy choice.

Reduced Mass and Diversity of the Colonic Microbiome in Patients with Multiple Sclerosis and Their Improvement with Ketogenic Diet.

Background: Colonic microbiome is thought to be involved in auto-immune multiple sclerosis (MS). Interactions between diet and the colonic microbiome in MS are unknown. Methods: We compared the composition of the colonic microbiota quantitatively in 25 MS patients and 14 healthy controls.Fluorescence in situ hybridization (FISH) with 162 ribosomal RNA derived bacterial FISH probes was used. Ten of the MS patients received a ketogenic diet for 6 months. Changes in concentrations of 35 numerically substantial bacterial groups were monitored at baseline and at 2, 12, and 23/24 weeks. Results: No MS typical microbiome pattern was apparent.The total concentrations and diversity of substantial bacterial groups were reduced in MS patients (P < 0.001). Bacterial groups detected with EREC (mainly Roseburia), Bac303 (Bacteroides), and Fprau (Faecalibacterium prausnitzii) probes were diminished the most. The individual changes were multidirectional and inconsistent. The effects of a ketogenic diet were biphasic. In the short term, bacterial concentrations and diversity were further reduced. They started to recover at week 12 and exceeded significantly the baseline values after 23-24 weeks on the ketogenic diet. Conclusions: Colonic biofermentative function is markedly impaired in MS patients.The ketogenic diet normalized concentrations of the colonic microbiome after 6 months.

Impact of humic acids on the colonic microbiome in healthy volunteers.

AIM: To test the effects of humic acids on innate microbial communities of the colon. METHODS: We followed the effects of oral supplementation with humic acids (Activomin®) on concentrations and composition of colonic microbiome in 14 healthy volunteers for 45 d. 3 × 800 mg Activomin® were taken orally for 10 d followed by 3 × 400 mg for 35 d. Colonic microbiota were investigated using multicolor fluorescence in situ hybridization (FISH) of Carnoy fixated and paraffin embedded stool cylinders. Two stool samples were collected a week prior to therapy and one stool sample on days 10, 31 and 45. Forty-one FISH probes representing different bacterial groups were used. RESULTS: The sum concentration of colonic microbiota increased from 20% at day 10 to 30% by day 31 and remained stable until day 45 (32%) of humic acid supplementation (P < 0.001). The increase in the concentrations in each person was due to growth of preexisting groups. The individual microbial profile of the patients remained unchanged. Similarly, the bacterial diversity remained stable. Concentrations of 24 of the 35 substantial groups increased from 20% to 96%. Two bacterial groups detected with Bac303 (Bacteroides) and Myc657 (mycolic acid-containing Actinomycetes) FISH probes decreased (P > 0.05). The others remained unaffected. Bacterial groups with initially marginal concentrations (< 0.1 × 109/mL) demonstrated no response to humic acids. The concentrations of pioneer groups of Bifidobacteriaceae, Enterobacteriaceae and Clostridium difficile increased but the observed differences were statistically not significant. CONCLUSION: Humic acids have a profound effect on healthy colonic microbiome and may be potentially interesting substances for the development of drugs that control the innate colonic microbiome.

Functional anatomy of the colonic bioreactor: Impact of antibiotics and Saccharomyces boulardii on bacterial composition in human fecal cylinders.

Sections of fecal cylinders were analyzed using fluorescence in situ hybridization targeting 180 bacterial groups. Samples were collected from three groups of women (N=20 each) treated for bacterial vaginosis with ciprofloxacin+metronidazole. Group A only received the combined antibiotic regimen, whereas the A/Sb group received concomitant Saccharomyces boulardii CNCM I-745 treatment, and the A_Sb group received S. boulardii prophylaxis following the 14-day antibiotic course. The number of stool cylinders analyzed was 188 out of 228 in group A, 170 out of 228 in group A/Sb, and 172 out of 216 in group A_Sb. The colonic biomass was organized into a separate mucus layer with no bacteria, a 10-30µm broad unstirred transitional layer enriched with bacteria, and a patchy fermentative area that mixed digestive leftovers with bacteria. The antibiotics suppressed bacteria mainly in the fermentative area, whereas abundant bacterial clades retreated to the transitional mucus and survived. As a result, the total concentration of bacteria decreased only by one order. These effects were lasting, since the overall recovery of the microbial mass, bacterial diversity and concentrations were still below pre-antibiotic values 4 months after the end of antibiotic treatment. Sb-prophylaxis markedly reduced antibiotic effects and improved the recovery rates. Since the colon is a sophisticated bioreactor, the study indicated that the spatial anatomy of its biomass was crucial for its function.

Polymicrobial Gardnerella biofilm resists repeated intravaginal antiseptic treatment in a subset of women with bacterial vaginosis: a preliminary report.

PURPOSE: Bacterial vaginosis is a recalcitrant polymicrobial biofilm infection that often resists standard antibiotic treatment. We therefore considered repeated treatment with octenidine, a local antiseptic that has previously been shown to be highly effective in several biofilm-associated infections. METHODS: Twenty-four patients with recurrent BV were treated with a 7-day course of octenidine (octenidine dihydrochloride spray application with the commercial product Octenisept). In case of treatment failure or relapse within 6 months, patients were re-treated with a 28-day course of octenidine. In case of recurrence within 6 months after the second treatment course, patients were treated again with a 28-day course followed by weekly applications for 2 months. Treatment effect was evaluated by assessment of the presence of the biofilm on voided vaginal epithelial cells through fluorescence in situ hybridisation. RESULTS: The initial cure rate following a 7-day course of octenidine was as high as 87.5%. The 6-month relapse rate was, however, as high as 66.6%. Repeated treatment for 28 days led to an overall cure rate of 75.0%; however, it was also associated with emergence of complete resistance to octenidine in a subset of women. The overall cure rate after three treatment courses with 1-year follow-up was 62.5 %, with 37.5 % of the patients showing complete resistance to octenidine. CONCLUSION: Our preliminary results showed that octenidine dihydrochloride was initially highly effective, but the efficacy of repeated and prolonged treatment dropped quickly as challenge with the antiseptic rapidly led to bacterial resistance in a considerable subset of women.

Increased Gardnerella vaginalis urogenital biofilm in inflammatory bowel disease.

INTRODUCTION: Inflammatory bowel disease (IBD) is a systemic inflammatory condition that affects the entire organism, not only the bowel. An impaired interaction with microbiota has been shown to be important. We looked for bacterial factors, which may contribute to the well-known higher incidence of poor reproductive outcome in IBD. METHODS: Urine specimen of patients with Crohn's disease (N=42), ulcerative colitis (N=46), and randomly selected patients attending the General Internal Medicine Outpatient Clinic of the Charité for non-IBD related medical conditions (N=49) was analyzed for bacteria adherent to desquamated epithelial cells and diffusely distributed bacteria in the urine using fluorescence in situ hybridization. RESULTS: The urine of IBD patients contained significantly more often Gardnerella vaginalis biofilms (CD 38%, UC 43%) than those of the control group (16%). There was no link between current disease activity, history of and present fistula and G. vaginalis biofilms, but the samples of patients with steroid refractory/dependent disease were significantly more often G. vaginalis biofilm positive. No significant differences in number of epithelial cells and leukocytes, and total bacterial counts were present. CONCLUSIONS: There is a significant link between IBD and G. vaginalis biofilm. This observation suggests an epithelial barrier dysfunction of the genital tract. Since G. vaginalis is believed to be one of the reasons responsible for bacterial vaginosis, it may be an important factor in the well-known higher incidence of poor reproductive outcome in IBD. Excessive G. vaginalis biofilms in steroid refractory/dependent disease suggests a need to avoid long-term steroid therapy.

Infection through structured polymicrobial Gardnerella biofilms (StPM-GB).

BACKGROUND: We analysed data on bacterial vaginosis (BV) contradicting the paradigm of mono-infection. METHODOLOGY: Tissues and epithelial cells of vagina, uterus, fallopian tubes and perianal region were investigated using fluorescence in situ hybridization (FISH) in women with BV and controls. RESULTS: Healthy vagina was free of biofilms. Prolific structured polymicrobial (StPM) Gardnerella-dominated biofilm characterised BV. The intact StPM-Gardnerella-biofilm enveloped desquamated vaginal/prepuce epithelial cells and was secreted with urine and sperma. The disease involved both genders and occurred in pairs. Children born to women with BV were negative. Monotherapy with metronidazole, moxifloxacin or local antiseptics suppressed but often did not eradicate StPM-Gardnerella-biofilms. There was no BV without Gardnerella, but Gardnerella was not BV. Outside of StPM-biofilm, Gardnerella was also found in a subset of children and healthy adults, but was dispersed, temporal and did not transform into StPM-Gardnerella-biofilm. CONCLUSIONS: StPM-Gardnerella-biofilm is an infectious subject. The assembly of single players to StPM-Gardnerella-biofilm is a not trivial every day process, but probably an evolutionary event with a long history of growth, propagation and selection for viability and ability to reshape the environment. The evolutionary memory is cemented in the structural differentiation of StPM-Gardnerella-biofilms and imparts them to resist previous and emerging challenges.

Presence of a polymicrobial endometrial biofilm in patients with bacterial vaginosis.

OBJECTIVE: To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract. STUDY DESIGN: Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria. RESULTS: A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0-76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0-341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6-205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis. CONCLUSION: Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.

Positive effects of local therapy with a vaginal lactic acid gel on dysuria and E.coli bacteriuria question our current views on recurrent cystitis.

OBJECTIVE: We tested the effect of vaginally applied lactic acid gel on symptoms and bacteriuria in acutely exacerbated recurrent Eschericia coli cystitis. METHODS: Carnoy fixed samples of the morning urine from 20 women with a history of recurrent E.coli cystitis were prospectively investigated for bacteriuria using fluorescence in situ hybridization (FISH). RESULTS: In 11/20 women with acute cystitis, the symptoms and bacteriuria were regressive with lactic acid gel treatment, without the need for antibiotic treatment. The complete regression of symptoms took between 1 week (7 women) and 4 weeks (4 women). In parallel with this regression, the microscopic shape of E.coli bacteria in these women changed from short rods to long curly filaments starting within the first days of therapy. The filamentous transformation affected 100% of the E.coli population in six women and at least 50% of E.coli population in five women and was not observed in urine samples from untreated women or in women without clinical response to lactic acid gel. This could not happen if the bladder was the origin of the infection. CONCLUSIONS: A number of recurrent and probably acute cystitis is a local vagino-urethritis caused by an adhesive invasive E.coli biofilm of the vaginal surface.

Mucosal invasion by fusobacteria is a common feature of acute appendicitis in Germany, Russia, and China.

BACKGROUND/AIM: To investigate the geographic occurrence of mucosa-invading Fusobacteria in acute appendicitis. PATIENTS AND METHODS: Carnoy- and formalin-fixated appendices from Germany, Russia, and China were comparatively investigated. Bacteria were detected using fluorescent in situ hybridization. Cecal biopsies from patients with inflammatory bowel disease and other conditions were used as disease controls. RESULTS: Fusobacteria represented mainly by Fusobacterium nucleatum were the major invasive component in bacterial infiltrates in acute appendicitis but were completely absent in controls. The occurrence of invasive Fusobacteria in Germany, Russia, and China was the same. The detection rate in Carnoy-fixated material was 70-71% and in formalin-fixated material was 30-36%. CONCLUSIONS: Acute appendicitis is a polymicrobial infectious disease in which F. nucleatum and other Fusobacteria play a key role.

Common biostructure of the colonic microbiota in neuroendocrine tumors and Crohn's disease and the effect of therapy.

BACKGROUND: The aims were to comparatively investigate the biostructure of colonic microbiota in patients with neuroendocrine tumors and Crohn's disease (CD) and to study the response of the microbiota to therapy. METHODS: Sections of fecal cylinders from 66 patients with neuroendocrine tumors (NET; 25 foregut, 30 midgut, 11 hindgut), 50 patients with CD (Crohn's Disease Activity Index [CDAI] ≥150), and 30 patients with chronic idiopathic diarrhea seen at the Charité Hospital and 25 healthy controls were investigated using fluorescence in situ hybridization with probes specific for five bacterial groups: Faecalibacterium prausnitzii, Clostridium group XIVa / Roseburia group, Bacteroides, Enterobacteriaceae, and Bifidobacteriaceae. RESULTS: We found a striking F. prausnitzii (Fprau) depletion in the stool of patients with NET of the midgut and patients with CD. The changes of the microbiota in the two other NET groups were uncharacteristic and similar to those observed in patients with chronic idiopathic diarrhea. Fprau depletion was reversible with chemotherapy and with interferon alpha-2b treatment in patients with midgut NET. Somatostatin analogs had no influence on Fprau concentrations. CONCLUSIONS: Patients with NET and CD show similarities in their abnormalities of the fecal biostructure. Interferon alpha and systemic chemotherapy significantly improved the fecal biostructure in patients with midgut NET.

Response of Gardnerella vaginalis biofilm to 5 days of moxifloxacin treatment.

Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.

Acute appendicitis is characterised by local invasion with Fusobacterium nucleatum/necrophorum.

BACKGROUND: Acute appendicitis is a local intestinal inflammation with unclear origin. The aim was to test whether bacteria in appendicitis differ in composition to bacteria found in caecal biopsies from healthy and disease controls. METHODS AND PATIENTS: We investigated sections of 70 appendices using rRNA-based fluorescence in situ hybridisation. Four hundred caecal biopsies and 400 faecal samples from patients with inflammatory bowel disease and other conditions were used as controls. A set of 73 group-specific bacterial probes was applied for the study. RESULTS: The mucosal surface in catarrhal appendicitis showed characteristic lesions of single epithelial cells filled with a mixed bacterial population ('pinned cells') without ulceration of the surroundings. Bacteria deeply infiltrated the tissue in suppurative appendicitis. Fusobacteria (mainly Fusobacterium nucleatum and necrophorum) were a specific component of these epithelial and submucosal infiltrates in 62% of patients with proven appendicitis. The presence of Fusobacteria in mucosal lesions correlated positively with the severity of the appendicitis and was completely absent in caecal biopsies from healthy and disease controls. Main faecal microbiota represented by Bacteroides, Eubacterium rectale (Clostridium group XIVa), Faecalibacterium prausnitzii groups and Akkermansia muciniphila were inversely related to the severity of the disease. The occurrence of other bacterial groups within mucosal lesions of acute appendicitis was not related to the severity of the appendicitis. No Fusobacteria were found in rectal swabs of patients with acute appendicitis. CONCLUSIONS: Local infection with Fusobacterium nucleatum/necrophorum is responsible for the majority of cases of acute appendicitis.

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.

Dissimilarity in the occurrence of Bifidobacteriaceae in vaginal and perianal microbiota in women with bacterial vaginosis.

Recent data point at the similarity between the perianal and vaginal microflora in terms of Lactobacillus species involved. Bacterial vaginosis, the most common perturbation of the vaginal microflora involving primarily overgrowth of Gardnerella vaginalis, has also been suggested to involve a recto-vaginal pathway. We addressed this issue with regard to bacteria of the Bifidobacteriaceae family. In particular, we investigated the putative concordance of the presence of G. vaginalis and a series of Bifidobacteria between the perianal and vaginal microflora in 10 patients with bacterial vaginosis through multicolor fluorescence in situ hybridization analysis of desquamated epithelial cells. G. vaginalis was found in a biofilm mode of growth at the perianal and vaginal sites. In most women at least one of the following species was detected perianally: Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium breves, Bifidobacterium bifidum and Bifidobacterium catenulatum. At the vaginal site, none of these Bifidobacteria was found. We conclude that bacterial vaginosis does not occur as a result of simple growth per continuum of perianal bacteria. Only some species originating from the intestinal tract do display pronounced vaginotropism, like G. vaginalis, whereas many other species do not.

Desquamated epithelial cells covered with a polymicrobial biofilm typical for bacterial vaginosis are present in randomly selected cryopreserved donor semen.

We tested whether the bacterial biofilm typical for bacterial vaginosis (BV) can be found on desquamated epithelial cells in cryopreserved donor semen. Bacteria were detected with FISH. Bacterial biofilm, covering the epithelial layer in vaginal biopsies of 20 women with BV, was evaluated on desquamated epithelial cells found in the urine of these same women and their male partners (N=20) and compared with the bacterial biofilm found on desquamated epithelial cells in randomly selected cryopreserved semen samples (N=20). Urine from 20 healthy women of laboratory and clinic personnel and urine from their partners were used as controls. Desquamated epithelial cells covered with a polymicrobial Gardnerella biofilm were identified in urine samples from all women with BV and 13 of their male partners and in none of the female controls and their partners. Gardnerella biofilm, typical for BV, was found in the semen of three of the 20 donors. Donor semen might be a vector for BV.

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.

Altered bile acid metabolism in childhood functional constipation: inactivation of secretory bile acids by sulfation in a subset of patients.

OBJECTIVE: An elevated concentration in the colon of the primary bile acid chenodeoxycholic acid (CDCA) or the secondary bile acid deoxycholic acid (DCA) is known to induce water secretion, causing diarrhea. We hypothesized that of the many fecal bile acids, only CDCA and DCA function as endogenous laxatives; therefore, a decrease in their proportion may be a cause of childhood functional constipation. To test this possibility, fecal bile acid composition was determined in children with functional constipation and in nonconstipated control children. PATIENTS AND METHODS: Fecal samples were obtained from 207 children, 103 with functional constipation and 104 with normal bowel habits. Bile acid classes were determined by use of electrospray ionization-single ion monitoring-mass spectrometry (ESI-SIM-MS), and individual bile acids were measured by gas chromatography (GC)-MS (GC-MS). The structure of individual sulfated bile acids was obtained by use of liquid chromatography (LC)-MS (LC-MS). RESULTS: By ESI-SIM-MS, the proportions of DCA did not differ in constipated children (n = 73) from that in control children (n = 92), but monosulfated dihydroxy bile acids were greater (P < 0.05). The difference was attributable to 6 patients in the constipated group whose major fecal bile acid by LC-MS was the 3-sulfate of CDCA. Sulfation of CDCA is known to abolish its secretory activity. By GC-MS, the bile acid profile was identical in the 2 groups. CONCLUSIONS: In most children with functional constipation, the fecal bile acid profile seems to be normal. There is a small subset of children, however, whose dominant fecal bile acid is the 3-sulfate of CDCA, indicating a novel disturbance in bile acid metabolism. Such sulfation abolishes the secretory activity of CDCA and may contribute to constipation.