Brazilian red propolis reduces orange-complex periodontopathogens growing in multispecies biofilms

This study investigated the antimicrobial effects of the ethanolic extract of Brazilian red propolis (BRP) on multispecies biofilms. A seven-day-old subgingival biofilm with 32 species was grown in a Calgary device. Biofilms were treated with BRP (1,600, 800, 400 and 200?μg ml^?1) twice a day for 1?min, starting from day 3. Chlorhexidine (0.12%) and dilution-vehicle were used as positive and negative controls, respectively. On day 7, metabolic activity and the microbial composition of the biofilms by DNA-DNA hybridization were determined. The viability data were analyzed by one-way ANOVA followed by Tukey’s post hoc, whereas the microbial composition data were transformed via BOX-COX and analyzed using Dunnett’s post hoc. BRP (1,600?μg ml^?1) decreased biofilm metabolic activity by 45%, with no significant difference from chlorhexidine-treated samples. BRP (1,600?μg ml^?1) and chlorhexidine significantly reduced levels of 14 bacterial species compared to the vehicle control. Taken together, BRP showed promising antimicrobial properties which may be useful in periodontal disease control.     

A Biofilm Pocket Model to Evaluate Different Non-Surgical Periodontal Treatment Modalities in Terms of Biofilm Removal and Reformation,Surface Alterations and Attachment of Periodontal Ligament Fibroblasts

Background and Aim

There is a lack of suitable in vitro models to evaluate various treatment modalities intending to remove subgingival bacterial biofilm. Consequently, the aims of this in vitro-study were: a) to establish a pocket model enabling mechanical removal of biofilm and b) to evaluate repeated non-surgical periodontal treatment with respect to biofilm removal and reformation, surface alterations, tooth hard-substance-loss, and attachment of periodontal ligament (PDL) fibroblasts.

Material and Methods

Standardized human dentin specimens were colonized by multi-species biofilms for 3.5 days and subsequently placed into artificially created pockets. Non-surgical periodontal treatment was performed as follows: a) hand-instrumentation with curettes (CUR), b) ultrasonication (US), c) subgingival air-polishing using erythritol (EAP) and d) subgingival air-polishing using erythritol combined with chlorhexidine digluconate (EAP-CHX). The reduction and recolonization of bacterial counts, surface roughness (Ra and Rz), the caused tooth substance-loss (thickness) as well as the attachment of PDL fibroblasts were evaluated and statistically analyzed by means of ANOVA with Post-Hoc LSD.

Results

After 5 treatments, bacterial reduction in biofilms was highest when applying EAP-CHX (4 log10). The lowest reduction was found after CUR (2 log10). Additionally, substance-loss was the highest when using CUR (128±40 µm) in comparison with US (14±12 µm), EAP (6±7 µm) and EAP-CHX (11±10) µm). Surface was roughened when using CUR and US. Surfaces exposed to US and to EAP attracted the highest numbers of PDL fibroblasts.

Conclusion

The established biofilm model simulating a periodontal pocket combined with interchangeable placements of test specimens with multi-species biofilms enables the evaluation of different non-surgical treatment modalities on biofilm removal and surface alterations. Compared to hand instrumentation the application of ultrasonication and of air-polishing with erythritol prevents from substance-loss and results in a smooth surface with nearly no residual biofilm that promotes the reattachment of PDL fibroblasts.     

<Emphasis Type="Italic">Filifactor alocis</Emphasis> - involvement in periodontal biofilms

Background  

Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms.     

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.     

Interleukin-8 Responses of Multi-Layer Gingival Epithelia to Subgingival Biofilms: Role of the “Red Complex” Species

Periodontitis is an infectious inflammatory disease that results in the destruction of the tooth-supporting (periodontal) tissues. The Gram-negative anaerobic species Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, (also known as the “red complex” species) are highly associated with subgingival biofilms at periodontitis-affected sites. A major chemokine produced by the gingival epithelium in response to biofilm challenge, is interleukin (IL)-8. The aim of this in vitro study was to investigate the relative effect of the “red complex” species as constituents of subgingival biofilms, on the regulation of IL-8 by gingival epithelia. Multi-layered organotypic human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its 7-species variant, excluding the “red complex”. IL-8 gene expression and secretion analyses were performed by qPCR and ELISA, respectively. After 3 h, both biofilms up-regulated IL-8 gene expression, but the presence of the “red complex” resulted in 3-fold greater response. IL-8 secretion was also up-regulated by both biofilms, with no differences between them. After 24 h, the 10-species biofilm reduced IL-8 secretion to 50% of the control, but this was not affected when the “red complex” was absent. In conclusion, as part of biofilms, “red complex” species differentially regulate IL-8 in gingival epithelia, potentially affecting the chemotactic responses of the tissue.     

Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Population diversity in model potable water biofilms receiving chlorine or chloramine residual

Abstract

Most water utilities use chlorine or chloramine to produce potable water. These disinfecting agents react with water to produce residual oxidants within a water distribution system (WDS) to control bacterial growth. While monochloramine is considered more stable than chlorine, little is known about the effect it has on WDS biofilms. Community structure of 10-week old WDS biofilms exposed to disinfectants was assessed after developing model biofilms from unamended distribution water. Four biofilm types were developed on polycarbonate slides within annular reactors while receiving chlorine, chloramine, or inactivated disinfectant residual. Eubacteria were identified through 16S rDNA sequence analysis. The model WDS biofilm exposed to chloramine mainly contained Mycobacterium and Dechloromonas sequences, while a variety of alpha- and additional beta-proteobacteria dominated the 16S rDNA clone libraries in the other three biofilms. Additionally, bacterial clones distantly related to Legionella were found in one of the biofilms receiving water with inactivated chlorine residual. The biofilm reactor receiving chloraminated water required increasing amounts of disinfectant after 2 weeks to maintain chlorine residual. In contrast, free chlorine residual remained steady in the reactor that received chlorinated water. The differences in bacterial populations of potable water biofilms suggest that disinfecting agents can influence biofilm development. These results also suggest that biofilm communities in distribution systems are capable of changing in response to disinfection practices.     

On the release of fluoride from biofilm reservoirs during a cariogenic challenge: an in situ study

Abstract

Biofilm fluoride reservoirs may be a source of fluoride to the fluid phase during a sugar challenge reducing tooth mineral loss. However, the evidence for that is conflicting and has not been studied in biofilms containing different fluoride levels. In order to test fluoride release from biofilms with distinct fluoride concentrations, biofilms were grown in situ exposed to a combination of placebo, calcium and fluoride rinses forming biofilms with no (fluoride-free rinses), low (fluoride-only rinses) or high (calcium followed by fluoride rinses) fluoride concentrations, and collected before and 5?min after a sucrose challenge. Rinsing with fluoride increased fluoride concentration in the biofilm (p?<?0.05), mainly when a calcium pre-rinse was used before the fluoride (p?<?0.05). However, after a sugar challenge, no significant increase in the biofilm fluid fluoride concentration was observed, even in the fluoride-rich biofilms (p?>?0.05). Fluoride-rich biofilms do not release fluoride to the fluid phase during a sugar challenge.     

Molecular Assessment of Inoculated and Indigenous Bacteria in Biofilms from a Pilot-Scale Perchlorate-Reducing Bioreactor

Bioremediation of perchlorate-contaminated groundwater can occur via bacterial reduction of perchlorate to chloride. Although perchlorate reduction has been demonstrated in bacterial pure cultures, little is known about the efficacy of using perchlorate-reducing bacteria as inoculants for bioremediation in the field. A pilot-scale, fixed-bed bioreactor containing plastic support medium was used to treat perchlorate-contaminated groundwater at a site in Southern California. The bioreactor was inoculated with a field-grown suspension of the perchlorate-respiring bacterium Dechlorosoma sp. strain KJ and fed groundwater containing indigenous bacteria and a carbon source amendment. Because the reactor was flushed weekly to remove accumulated biomass, only bacteria capable of growing in biofilms in the reactor were expected to survive. After 26 days of operation, perchlorate was not detected in bioreactor effluent. Perchlorate remained undetected by ion chromatography (detection limit 4 μg L⁻¹) during 6 months of operation, after which the reactor was drained. Plastic medium was subsampled from top, middle, and bottom locations of the reactor for shipment on blue ice and storage at −80°C prior to analysis. Microbial community DNA was extracted from successive washes of thawed biofilm material for PCR-based community profiling by 16S-23S ribosomal intergenic spacer analysis (RISA). No DNA sequences characteristic of strain KJ were recovered from any RISA bands. The most intense bands yielded DNA sequences with high similarities to Dechloromonas spp., a closely related but different genus of perchlorate-respiring bacteria. Additional sequences from RISA profiles indicated presence of representatives of the low G+C gram-positive bacteria and the Cytophaga–Flavobacterium–Bacteroides group. Confocal scanning laser microscopy and fluorescence in situ hybridization (FISH) were also used to examine biofilms using genus-specific 16S ribosomal RNA probes. FISH was more sensitive than RISA profiling in detecting possible survivors from the initial inoculum. FISH revealed that bacteria hybridizing to Dechlorosoma probes constituted <1% of all cells in the biofilms examined, except in the deepest portions where they represented 3–5%. Numbers of bacteria hybridizing to Dechloromonas probes decreased as biofilm depth increased, and they were most abundant at the biofilm surface (23% of all cells). These spatial distribution differences suggested persistence of low numbers of the inoculated strain Dechlorosoma sp. KJ in parts of the biofilm nearest to the plastic medium, concomitant with active colonization or growth by indigenous Dechloromonas spp. in the biofilm exterior. This study demonstrated the feasibility of post hoc analysis of frozen biofilms following completion of field remediation studies.     

Development of a high throughput and low cost model for the study of semi-dry biofilms

Abstract

The persistence of microorganisms as biofilms on dry surfaces resistant to the usual terminal cleaning methods may pose an additional risk of transmission of infections. In this study, the Centre for Disease Control (CDC) dry biofilm model (DBM) was adapted into a microtiter plate format (Model 1) and replicated to create a novel in vitro model that replicates conditions commonly encountered in the healthcare environment (Model 2). Biofilms of Staphylococcus aureus grown in the two models were comparable to the biofilms of the CDC DBM in terms of recovered log₁₀ CFU well^?1. Assessment of the antimicrobial tolerance of biofilms grown in the two models showed Model 2 a better model for biofilm formation. Confirmation of the biofilms’ phenotype with an extracellular matrix deficient S. aureus suggested stress tolerance through a non-matrix defined mechanism in microorganisms. This study highlights the importance of conditions maintained in bacterial growth as they affect biofilm phenotype and behaviour.     

Effect of heterogeneous structure in mechanically unstressed biofilms on overall growth

In contrast to their name, biofilms are not always flat and homogeneous but instead often exhibit complex structural heterogeneity. It has been suggested that nonhomogeneous geometry is selected in order to increase biofilm growth rate. A previous study (Dockery and Klapper (2002) SIAM J. Appl. Math., 62, 853–869) of a model biofilm system in a static bulk fluid demonstrated that under some circumstances a flat biofilm-bulk fluid interface is linearly unstable to perturbation due to growth induced forces. Computations indicated that subsequent nonlinear evolution results in fingers and mushrooms of biofilm similar to structures observed in actual biofilms. However, the important complementary issue of biological functionality was not considered. Here a weakly nonlinear analysis of the simple growing biofilm layer model in Dockery and Klapper (2002, SIAM J. Appl. Math., 62, 853–869) is presented. It is argued that, at least in the case of biofilms free of external mechanical stress, overall growth is in fact generally inhibited by the presence of growing perturbations in the linear stage. Hence a more complex explanation of function is necessary.     

Characterization of polymicrobial biofilms obtained from saliva or carious lesions in dentin

Abstract

This study aimed to compare the formation of polymicrobial biofilms using carious dentin or saliva as inoculum for application in in vitro microbiological studies on caries research. For biofilm growth, combined samples of infected dentin or saliva from three donors were used. The biofilms were grown on glass coverslips, under a regimen of intermittent exposure (6?h day^?1) to 1% sucrose for 4?days. Total bacterial loads, as well as specific aciduric bacteria and mutans streptococci loads were quantified and correlated with biofilm acidogenicity and susceptibility to chlorhexidine. The data were evaluated using the Student’s-t, Mann Whitney and Kruskal-Wallis tests. The two biofilms showed similar microbial loads (total bacteria, aciduric bacteria and mutans streptococci) on day 4, and high acidogenicity after 48?h and were susceptible to chlorhexidine at different time intervals. In conclusion, both dentin and saliva can be used as an inoculum in in vitro studies of processes related to biofilm formation.     

Continuous flow system for biofilm formation using controlled concentrations of Pseudomonas putida from chicken carcass and coupled to electrochemical impedance detection

Abstract

Most studies dealing with monitoring the dynamics of biofilm formation use microbial suspensions at high concentrations. These conditions do not always represent food or water distribution systems. A continuous flow system capable of controlling the concentration of the microbial suspension stream from 10⁴ to 10⁶ CFU ml^?1 is reported. Pseudomonas putida biofilms formed using 100-fold, 1,000-fold or 10,000-fold diluted bacterial suspensions were monitored in-line by electrochemical impedance spectroscopy (EIS) and total plate counts. Randles equivalent circuit model and a modified Randles model with biofilm elements were used to fit the EIS data. In Randles equivalent circuit, the charge transfer resistance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the charge transfer resistance. In the biofilm model, the biofilm resistance and the double layer capacitance decreased as the biofilm formed. The log colony counts of the biofilm correlated to the biofilm resistance.     

Antimicrobial effects of a pulsed electromagnetic field: an in vitro polymicrobial periodontal subgingival biofilm model

Abstract

The objective was to test the influence of a pulsed electromagnetic field (PEMF) on bacterial biofilm colonization around implants incorporated with healing abutments. Healing abutments with (test group) and without (control group) active PEMF devices were placed in a multispecies biofilm consisting of 31 different bacterial species. The biofilm composition and total bacterial counts (x10⁵) were analyzed by checkerboard DNA-DNA hybridization. After 96?h, the mean level of 7 out of the 31 bacterial species differed significantly between groups, namely Eubacterium nodatum, Fusobacterium nucleatum ssp. nucleatum, Streptococcus intermedius, Streptococcus anginosus, Streptococcus mutans, Fusobacterium nucleatum ssp. Vicentii and Capnocytophaga ochracea were elevated in the control group (p?<?0.05). The mean total bacterial counts were lower in the Test group vs the control group (p?<?0.05). An electromagnetic healing cap had antimicrobial effects on the bacterial species and can be used to control bacterial colonization around dental implants. Further clinical studies should be conducted to confirm these findings.     

Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface

Summary Formation and activity of bacterial nitrifying biofilms play an important role in the closed seawater systems for shrimp cultivation. The structure of microbial biofilm on empty oyster shells, used as a biofilm carrier in biofiltration of aquacultural water, was studied using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. FISH was performed with specific oligonucleotide probes for Bacteria and ammonia-oxidizing Nitrosomonas spp. The bacterial cells were arranged within the biofilm as a layer of vertically elongated aggregates. Aggregates of ammonia-oxidizing bacteria were embedded within the matrix formed by other bacteria. Vertically elongated cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio than that of laminated biofilms.     

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ～80% was in the range of ng?cm^?2 of colonized area (mean biofilm density 2.33?×?10⁹?cells?cm^?2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.     

Microbial Exopolymers Link Predator and Prey in a Model Yeast Biofilm System

Protistan grazing on biofilms is potentially an important conduit enabling energy flow between microbial trophic levels. Contrary to the widely held assumption that protistan feeding primarily involves ingestion of biofilm cells, with negative consequences for the biofilm, this study demonstrated preferential grazing on the noncellular biofilm matrix by a ciliate, with selective ingestion of yeast and bacterial cells of planktonic origin over attached and biofilm-derived planktonic cells. Introducing a ciliate to two biofilm-forming Cryptococcus species, as well as two bacterial species in a model biofilm system, fluorescent probes were applied to determine ingestion of cellular and noncellular biofilm fractions. Fluoromicroscopy, as well as photometric quantification, confirmed that protistan grazing enhanced yeast biofilm metabolism, and an increase in biofilm biomass and viability. We propose that the extracellular polymeric matrix of biofilms may act as an interface regulating interaction between predator and prey, while serving as source of nutrients and energy for protists.     

Induction of prostaglandin E(2) and interleukin-6 in gingival fibroblasts by oral biofilms

Polymicrobial oral biofilms attaching on tooth surfaces can trigger inflammatory responses by the neighbouring tooth-supporting periodontal tissues. An excessive inflammatory response can cause destruction of the periodontal tissues, including the alveolar bone, thus resulting in periodontitis. Mediators of inflammation, such as prostaglandin E(2) (PGE(2) ) and interleukin-6, are primary regulators of alveolar bone destruction in periodontitis. The present study aimed to comparatively investigate the effects of in vitro supragingival and subgingival biofilms, on the regulation of PGE(2) and interleukin-6 in human gingival fibroblasts. The cells were challenged with culture supernatants of the two biofilms for 6?h. Cyclo-oxygenase (COX)-2, an enzyme responsible for the conversion of PGE(2) , and interleukin-6 gene expression were analysed by quantitative real-time PCR. The production of PGE(2) and interleukin-6 by the cells was analysed by ELISA. While the supragingival biofilm did not induce significant changes, the subgingival biofilm caused an 8.6- and 2.9-fold enhancement of COX-2 and interleukin-6 gene expression, respectively, and a 72.5- and 1.5-fold enhancement of PGE(2) and interleukin-6 production, respectively. In conclusion, subgingival biofilms are potent inducers of PGE(2) in gingival fibroblasts, providing further mechanistic insights into the association of subgingival biofilms with bone resorption periodontitis.