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Objective-Microbial biofilms are communities of sessile microorganisms formed by cells that are attached irreversibly to a substratum or interface or to each other and embedded in a hydrated matrix of extracellular polymeric substances. Microbial biofilms have been implicated in >80% of human infections such as periodontitis, urethritis, endocarditis, and device-associated infections. Thus far, intra-amniotic infection has been attributed to planktonic (free-floating) bacteria. A case is presented in which "amniotic fluid sludge" was found to contain microbial biofilms. This represents the first report of a microbial biofilm in the amniotic cavity.Study Design-"Amniotic fluid sludge" was detected by transvaginal sonography, and retrieved by transvaginal amniotomy. Bacteria were identified using scanning electron microscope and fluorescence in situ hybridization for conserved regions of the microbial genome; and the exopolymeric matrix was identified by histochemistry using the wheat germ agglutinin lectin method. The structure of the biofilm was imaged with confocal laser scanning microscopy.Results-"Amniotic fluid sludge" was imaged with scanning electron microscopy, which allowed identification of bacteria embedded in an amorphous material and inflammatory cells. Bacteria were demonstrated using fluorescent in situ hybredization using a eubacteria probe. Extracellular matrix was identified with the wheat germ agglutinin lectin stain. Confocal microscopy allowed threedimensional visualization of the microbial biofilm.Conclusion-Microbial biofilms have been identified in a case of intra-amniotic infection with "amniotic fluid sludge."

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Background Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)–biofilm colonizing titanium implants. Methods Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume. Results Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100%of biofilm-inoculated implants for up to 3 weeks and 25%for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks. Conclusions These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and antibiofilm therapy.

Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.Bacteria are usually found concentrated at solid-liquid interfaces, where they colonize natural and man-made surfaces in community-like structures termed bacterial biofilms (6). Bacteria in biofilms exhibit protein expression patterns different from those of planktonic bacteria (41). Within a biofilm, bacteria are typically embedded in an extracellular polymeric substance (EPS) matrix (7,17,43) that protects the microbial community members from desiccation, from phagocytosis, and, in the case of human pathogens, from the host immune system (8).While a variety of biofilms have been studied in depth by optical imaging approaches, only a few have been faithfully preserved and visualized at high resolution by transmission electron microscopy (3,22,25,26,45,48,49). A recent ultrastructural study of Pseudomonas aeruginosa biofilms shows that high-pressure freezing/freeze-substitution results in superior preservation (22). Unlike conventional sample preparation, millisecond cryofixation and low-temperature dehydration protocols minimize artifacts from chemical fixation, extraction, and aggregation (15,16,19,20,22).Conventional two-dimensional (2D) imaging provides x,y positional information but cannot resolve features along the z direction. Conventional 2D projection imaging of thin (ϳ70-to 100-nm) sections results in the superposition of ϳ5 to 20 layers of protein along the electron path, obscuring molecular details. True 3D visualization requires electron tomographic data acquisition and 3D reconstruction, upon which both intracellular and extracellular features are resolved. In this way, cellular organelles, molecular machines, and macromolecular complexes can be visualized in their native microenvironment. To date, electron tomographic 3D reconstruction of a bacterial biofilm has not been reported.Myxococcus xanthus is a well-studied gram-negative soil scavenger and predator (21), and it serves as a model system for biofilm formation. M. xanthus moves over surfaces either by adventurous (A) motility or social (S) motility (for a review, see reference 18)...

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A novel coaxial tubular device capable of generating a 2.5 cm long pencil-like plasma plume in ambient atmosphere has recently been developed to disinfect root canal systems during endodontic treatment. Powered with short (%100 ns), intense (6 kV) electric pulses at 1 kHz, the plasma dental probe is safe for operation, electromagnetic noisefree, with low power consumption (an average power of %1 W) and minimal heating of materials under treatment. It thus has the essential features required for oral and dental disinfection. In this communication, we present the design of the device and evidence that the plasma dental probe is effective for tooth surface disinfection. Scanning electron microscopy shows complete destruction of endodontic biofilms for a depth of 1 mm inside a root canal after plasma treatment for 5 min. Plasma emission spectroscopy identifies atomic oxygen as one of the likely active agents for the bactericidal effect.

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