Sonja Swidsinski

Adherent Biofilms in Bacterial Vaginosis

OBJECTIVE: Bacterial vaginosis is a common infectious disorder. Although known since ancient times, little progress has occurred in identifying causal factors. Our aims were to study the bacterial community structure and the spatial organization of microbiota on the epithelial surfaces of vaginal biopsy specimens. METHODS: We investigated the composition and spatial organization of bacteria associated with the vaginal epithelium in biopsy specimens from 20 patients with bacterial vaginosis and 40 normal premenopausal and postmenopausal controls using a broad range of fluorescent bacterial group-specific rRNA-targeted oligonucleotide probes. RESULTS: Bacterial vaginosis was associated with greater occurrence and higher concentrations of a variety of bacterial groups. However, only Gardnerella vaginalis developed a characteristic adherent biofilm that was specific for bacterial vaginosis. CONCLUSION: A biofilm comprised of confluent G vaginalis with other bacterial groups incorporated in the adherent layer is a prominent feature of bacterial vaginosis. LEVEL OF EVIDENCE: II-2

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.

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.

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.

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.

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.

Su1233 Changes of Mouth Microbiota Under the Influence of Oral Lavage With Saccharomyces Boulardii in Gastroenterologic Patients With Oral Discomfort and Halitosis

(3000-5000/μl) and concentration of 6-thioguanine (235-400pmol/8×108RBCs). Patients were classified into two groups according to the time of initiating thiopurine treatment after diagnosis of CD as follows: early induction group (,1.5 years, n=27); late induction group (.1.5 years, n=21). Cumulative remission-maintenance rate were determined and we assessed patients characteristics that were associated with long-term remission as well as the effect of early induction of thiopurines on long-term clinical remission. Clinical remission was defined as a Crohns Disease Activity Index (CDAI) less than 150 points maintained for at least 6 months. Results: Based on Kaplan-Meier analysis, overall cumulative remissionmaintenance rate of 48 CD patients was 65.7% at 85.0 months. A greater cumulative remission-maintenance rate was found in CD patients without a) stenosis or b) a history of surgery compared patients with either factors, respectively (a) 79.9% at 85.0 months vs. 47.1% at 77.6 months; p,0.05: b) 74.3% at 85.0 months vs.47.1% at 77.6 months; p= 0.05). No other patients factors were identified that correlated with an increased rate of remission. Cumulative remission-maintenance rate of early induction group was significantly higher than that of late induction group (72.1% at 85.0 months vs. 56.1% at 77.6 months; p,0.05). Moreover, there was no significant difference of cumulative remission-maintenance rates in CD patients without stenosis between early and late induction group (82.2% at 85.0 months vs. 71.4% at 74.0 months; p=0.41). Conclusion: Overall, early treatment with thiopurines resulted in a greater rate of remission in CD patients. Our results suggested that early induction of thiopurines prior to development of intestinal stricture contributes to long-term clinical remission in patients with CD. Additional studies including prospective studies are needed to confirm our findings.

Pertussis-PCR Neuer Standard in der Keuchhustendiagnostik Auswertung eines ersten multizentrischen Ringversuchs in Deutschland und der Schweiz

ZusammenfassungFragestellung: Der kulturelle Nachweis von Bordetella pertussis ist für Kinderärzte wenig hilfreich, da die Erregeranzüchtung nur bei 30–60% der Keuchhustenpatienten gelingt und das Ergebnis frühestens nach 3–5 Tagen vorliegt. Zur Verbesserung der Keuchhustendiagnostik sind verschiedene Polymerasekettenreaktionsprotokolle (PCR-Protokolle) entwickelt worden. Methode: Das Nationale Referenzzentrum Pertussis hat einen Ringversuch initiiert mit dem Ziel, die Leistungsfähigkeit der verschiedenen PCR-Protokolle zu vergleichen. Ergebnisse: Von 15 teilnehmenden Laboratorien haben 14 die Anforderungen des Ringversuchs erfüllt. Die Nachweisgrenze der Pertussis-PCR lag bei 10–100 koloniebildenden Einheiten/Tupfer. Die PCR war damit um mindestens 1 Zehnerpotenz empfindlicher als der kulturelle Nachweis. Die Rate der falsch-positiven Befunde betrug 4%. Schlußfolgerungen: Die Pertussis-PCR ist eine hochempfindliche Methode, die für die klinische Routinediagnostik des Keuchhustens empfohlen werden kann. Wegen der Möglichkeit falsch-positiver Befunde durch Kreuzreaktionen oder Kontaminationen gehört diese Methode, wie alle PCR-Verfahren, zwingend in die Hände erfahrener und qualifizierter Laborspezialisten. Zudem sollten alle PCR-Laboratorien, die Routinediagnostik betreiben, verpflichtet werden, an Ringversuchen teilzunehmen.SummaryObjective: Cultural detection of Bordetella pertussis is not very helpful in the management of patients with whooping cough, since culture takes three to five days until results are available, and only about 30–60% of patients may be detected by this method. In order to improve the laboratory diagnosis of whooping cough, different protocols for the polymerase chain reaction (PCR) were developed. Methods: The German National Reference Laboratory for pertussis has initiated a multi-center study in order to compare the sensitivity and specificity of different protocols for pertussis-PCR. Results: Fourteen of fifteen participating laboratories have reported acceptable results. The detection limit was shown 10–100 colony-forming-units (CFU)/swab and it was, thus, 10 times more sensitive than cultural detection. Four percent of false positive PCR-results were reported. Conclusions: The polymerase chain reaction is an improved tool to diagnose whooping cough. False positive test results are due to cross reaction or contamination. Therefore PCR should only be applied by highly experienced molecular biological laboratories, who participate in an intensive program of external quality control.