Biofilms consist of microbial communities embedded in a 3D extracellular matrix. The matrix is composed of a complex array of extracellular polymeric substances (EPS) that contribute to the unique ...attributes of biofilm lifestyle and virulence. This ensemble of chemically and functionally diverse biomolecules is termed the ‘matrixome’. The composition and mechanisms of EPS matrix formation, and its role in biofilm biology, function, and microenvironment are being revealed. This perspective article highlights recent advances about the multifaceted role of the ‘matrixome’ in the development, physical–chemical properties, and virulence of biofilms. We emphasize that targeting biofilm-specific conditions such as the matrixome could lead to precise and effective antibiofilm approaches. We also discuss the limited knowledge in the context of polymicrobial biofilms, and the need for more in-depth analyses of the EPS matrix in mixed communities that are associated with many human infectious diseases.
The 'matrixome' is the inventory of currently known biomolecules (polysaccharides, nucleic acids, proteins, lipids, and lipoproteins) and their molecular, structural, and functional diversity associated with biofilm assembly, and its physicochemical and virulence attributes.The structural and biochemical properties of the matrixome provide the emergent properties of biofilms, including surface adhesion, spatial and chemical heterogeneities, synergistic/competitive polymicrobial interactions, antimicrobial recalcitrance, and biofilm virulence.Combinatorial treatment strategies are crucial to eradicate biofilms by targeting the functionally and structurally complex extracellular polymeric matrix and embedded microbial cells.Due to limited knowledge of the polymicrobial EPS matrix there is an urgent need for more experimental polymicrobial biofilm and in vivo mechanistic studies.
Summary
Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining ...this balance. Using the six‐species Zürich “supragingival” biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony‐forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries‐favoring dysbiotic state.
Summary
The development of dental caries and periodontal diseases result from distinct shifts in the microbiota of the tooth‐associated biofilm. This in vitro study aimed to investigate changes in ...biofilm composition and structure, during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile. Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar were aerobically grown in saliva‐containing medium on hydroxyapatite disks. After 64 h, Campylobacter rectus, Prevotella intermedia and Streptococcus anginosus were further added along with human serum, while culture conditions were shifted to microaerophilic. After 96 h, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were finally added and the biofilm was grown anaerobically for another 64 h. At the end of each phase, biofilms were harvested for species‐specific quantification and localization. Apart from C. albicans, all other species gradually increased during aerobic and microaerophilic conditions, but remained steady during anaerobic conditions. Biofilm thickness was doubled during the microaerophilic phase, but remained steady throughout the anaerobic phase. Extracellular polysaccharide presence was gradually reduced throughout the growth period. Biofilm viability was reduced during the microaerophilic conversion, but was recovered during the anaerobic phase. This in vitro study has characterized the dynamic structural shifts occurring in an oral biofilm model during the switch from aerobic to anaerobic conditions, potentially modeling the conversion of supragingival to subgingival biofilms. Within the limitations of this experimental model, the findings may provide novel insights into the ecology of oral biofilms.
In addition to occasional opportunistic colonization of the oral mucosa, Candida albicans is frequently found in carious dentin. The yeast’s potential to induce dental caries as a consequence of its ...pronounced ability to produce and tolerate acids was investigated. Eighty caries-active Osborne-Mendel rats were raised on an ampicillin-supplemented diet and exposed to C. albicans and/or Streptococcus mutans, except for controls. Throughout the 28-day test period, the animals were offered the modified cariogenic diet 2000a, containing 40% various sugars. Subsequently, maxillary molars were scored for plaque extent. After dissection, the mandibular molars were evaluated for smooth surface and fissure caries. Test animals exposed to C. albicans displayed considerably more advanced fissure lesions (p < 0.001) than non-exposed controls. While S. mutans yielded similar results, a combined association of C. albicans and S. mutans had no effect on occlusal caries incidence. Substituting dietary sucrose by glucose did not modify caries induction by C. albicans. However, animals fed a diet containing 20% of both sugars showed no differences to non-infected controls. Smooth surface caries was not generated by the yeast. This study provides experimental evidence that C. albicans is capable of causing occlusal caries in rats at a high rate.
Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this ...study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species
Actinomyces naeslundii,
Candida albicans,
Fusobacterium nucleatum,
Streptococcus oralis,
Streptococcus sobrinus, and
Veillonella dispar were grown anaerobically for 64.5 h at 37 °C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of
A. naeslundii), EUK116 (
C. albicans), FUS664 (
F. nucleatum), MIT447 and MIT588 (
S. oralis), SOB174 (
S. sobrinus), and VEI217 (
V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.
Background and Objective
Subgingival biofilms are the prime etiological factor of periodontal disease. Owing to their complex polymicrobial nature, quantification of individual bacterial species ...within the biofilm for research and diagnostic purposes can be methodologically challenging. The aims of this study were to establish a quantitative real‐time PCR (qPCR) assay to quantify the bacteria used in our 10‐species in vitro ‘subgingival’ biofilm model and to compare the quantitative outcome with fluorescence microscopy and colony‐forming unit (CFU) counts on selective agar plates.
Material and Methods
The 10 species included in the in vitro biofilm were Streptococcus oralis, Streptococcus anginosus, Veillonella dispar, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Actinomyces oris, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia. The numbers of each species were quantified at two time points using qPCR, microscopy counting following fluorescence in‐situ hybridization (FISH) or immunofluorescence staining, and counting of CFUs after growth on selective agar plates.
Results
All 10 species were successfully quantified using qPCR and FISH or immunofluorescence, and the eight species culturable on selective agar plates were also quantified by counting the numbers of CFUs after growth on selective agar. In early biofilm cultures, all methods showed a significant correlation, although the absolute numbers differed between methods. In late biofilm cultures, measurements obtained using qPCR and FISH or immunofluorescence, but not by CFU counts, maintained significant correlation. CFU counts yielded lower values than did measurements made using the other two methods.
Conclusion
Quantitative PCR and epifluorescence microscopy can be easily combined with each other to determine species‐specific bacterial numbers within biofilms. However, conventional bacterial cultures cannot be as efficiently combined using these molecular detection methods. This may be crucial in designing and selecting appropriate clinical diagnostic methods for subgingival biofilm samples.
Summary
Periodontitis is the chronic inflammatory destruction of periodontal tissues as a result of bacterial biofilm formation on the tooth surface. Proteins secreted by the gingival epithelium ...challenged by subgingival biofilms represent an important initial response for periodontal inflammation. The aim of this in vitro study was to characterize the whole secreted proteome of gingival epithelial tissue challenged by subgingival biofilms, and to evaluate the differential effects of the presence of the red‐complex species in the biofilm. Multi‐layered human gingival epithelial cultures were challenged with a 10‐species in vitro biofilm model or its seven‐species variant excluding the red complex. Liquid chromatography‐tandem mass spectrometry for label‐free quantitative proteomics was applied to identify and quantify the secreted epithelial proteins in the culture supernatant. A total of 192 proteins were identified and quantified. The biofilm challenge resulted in more secreted proteins being downregulated than upregulated. Even so, presence of the red complex in the biofilm was responsible for much of this downregulatory effect. Over 24 h, the upregulated biological processes were associated with inflammation and apoptosis, whereas the downregulated processes were associated with the disruption of epithelial tissue integrity and impairment of tissue turnover. Over 48 h, negative regulation of several metabolic processes and degradation of various molecular complexes was further intensified. Again, many of these biological regulations were attributed to the presence of the red complex. In conclusion, the present study provides the secreted proteome profile of gingival epithelial tissue to subgingival biofilms, and identifies a significant role for the red‐complex species in the observed effects.
Experiments were carried out to investigate the performance of different fuels used in a internal combustion engine: gasoline, methane and fuel blends containing methane with 5%, 10% and 15% hydrogen ...by volume, respectively. A two-litre naturally aspirated bi-fuel engine with port fuel injection was used. The engine was operated stoichiometrically. For each fuel the spark advance for best efficiency was determined. Experiments were conducted at 2000
rpm and 2
bar brake mean effective pressure. A heat release analysis and a loss analysis were performed for all fuels. The main findings are that increasing the hydrogen fraction of the methane hydrogen fuel blend decreases the overall burn duration. This decrease is predominantly achieved by a shortened duration of the fist stage of combustion (ignition to 5% mass fraction burned). The faster combustion comes along with an increase in fuel conversion efficiency. The different losses for gasoline and pure methane operation interact such that equal fuel conversion efficiencies result. However, care has to be taken when comparing fuel conversion efficiencies among the different fuels as the relative error in fuel conversion efficiency for the gaseous fuels is 0.2% at most, whereas it is about 1% for gasoline.
Experiments were carried out to investigate different injection strategies on a heavy-duty diesel engine. In particular, pilot/main, post/main and single main injection strategies were considered. ...Engine speed, load, exhaust gas recirculation rate and relative air/fuel ratio were held constant, whereas pilot and post injection fuel masses and injection timings were varied. For each measurement, heat release and detailed loss analyses were carried out to compare the influence of different injection strategies on the fuel conversion process and its associated efficiencies. As it is known, fuel conversion efficiency depends strongly on the injection timing, in particular on the main injection timing. However, the detailed loss analysis reveals that fuel conversion efficiency is largely determined by the interaction between real combustion and wall heat losses, respectively. To complete the analysis, emissions of nitric oxides, soot mass and particle number of the different injection settings are compared.
Internal combustion engines deliver work using an intermittent thermodynamic process. For control and diagnosis purposes, it is useful to detect key events relative to the crank angle position. A new ...method to detect the intake valve closing (IVC), start of combustion (SOC), end of combustion (EOC) or exhaust valve opening (EVO), using the measured cylinder pressure as input, is described. The method is based on the observation that the compression and expansion processes are of polytropic nature. It is shown that the events can be detected by detecting the points where the real and the polytropic volume diverge.
► A novel approach to detect valve and combustion key events in internal combustion engines is presented. ► The measured in-cylinder pressure is used as an input. ► The method is based on the polytropic change of state during compression and expansion. ► A polytropic volume is calculated which can easily and intuitively be compared with the known real cylinder volume. ► Events are detected using wavelet transformation.
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