Phenalen-1-One-Mediated Antimicrobial Photodynamic Therapy and Chlorhexidine Applied to a Novel Caries Biofilm Model.

Antimicrobial photodynamic therapy (aPDT) may be useful as a supportive antimicrobial measure for caries-active subjects. In this study, the antimicrobial efficacy of aPDT with a phenalen-1-one photosensitizer was evaluated in a novel in vitro biofilm model comprising Actinomyces naeslundii, Actinomyces odontolyticus, and Streptococcus mutans and was compared to chlorhexidine. The proposed biofilm model allows high-throughput screening for antimicrobial efficacy while exhibiting a differentiated response to different antimicrobial approaches. While chlorhexidine 0.2% showed a reduction of ≈4 log10 for all species, aPDT led to a more pronounced reduction of S. mutans (2.8 log10) than of Actinomyces spp. (1.2 or 1.3 log10). A similar effect was also observed in monospecies biofilms. Therefore, aPDT may be more effective against S. mutans than against Actinomyces spp. when in biofilms, and this antimicrobial approach merits further investigations.

Influence of ultrasonic tip distance and orientation on biofilm removal.

OBJECTIVE: The aim of this study is to assess the effects of ultrasonic tip distance and orientation on the removal of a multispecies biofilm under standardized conditions in vitro. METHODS: Six-species biofilms were grown on hydroxyapatite discs for 64 h and treated with a magnetostrictive ultrasonic tip (Cavitron) placed either on contact or at 0.25- and 0.5-mm distance. The treatment was performed for 15 s with either the tip at right angle or sideways. Biofilm removal was evaluated by assessing the viable bacteria in each supernatant and compared to respective controls. In the latter, biofilms were mechanically removed and evaluated in supernatants to assess adhering and floating bacteria. Colony-forming units (CFU) were determined by cultivation on solid media. Any remaining biofilm on the treated discs was also visualized after staining with green-fluorescent SYTO® 9 stain using a confocal laser scanning microscope (CLSM). Mann-Whitney U tests and Bonferroni correction were used to analyze the results between the groups. RESULTS: Sideways application of the ultrasonic tip at distances of 0.25 and 0.5 mm removed as many bacteria as present on the control discs compared to the tip on contact (p < 0.05). All other application modes, especially the ultrasonic tip applied perpendicularly on contact, showed no statistical significance in removing biofilm. CONCLUSION: Overall, data indicated that bacterial detachment depended on tip orientation and distance, especially when the tip was applied sideways similar to the clinical setting. CLINICAL RELEVANCE: Biofilm removal by means of ultrasonic debridement remains a crucial aspect in the treatment of periodontal disease. To ensure sufficient biofilm removal, the tip does not necessarily require contact to the surface, but an application parallel to the surface on the side is recommended.

Incorporation of staphylococci into titanium-grown biofilms: an in vitro "submucosal" biofilm model for peri-implantitis.

OBJECTIVES: Staphylococcus spp. are postulated to play a role in peri-implantitis. This study aimed to develop a "submucosal" in vitro biofilm model, by integrating two staphylococci into its composition. MATERIALS AND METHODS: The standard "subgingival" biofilm contained Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Streptococcus anginosus, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, and was further supplemented with Staphyoccous aureus and/or Staphylococcus epidermidis. Biofilms were grown anaerobically on hydroxyapatite or titanium discs and harvested after 64 h for real-time polymerase chain reaction, to determine their composition. Confocal laser scanning microscopy and fluorescence in situ hybridization were used for identifying the two staphylococci within the biofilm. RESULTS: Both staphylococci established within the biofilms when added separately. However, when added together, only S. aureus grew in high numbers, whereas S. epidermidis was reduced almost to the detection limit. Compared to the standard subgingival biofilm, addition of the two staphylococci had no impact on the qualitative or quantitative composition of the biofilm. When grown individually in the biofilm, S. epidermidis and S. aureus formed small distinctive clusters and it was confirmed that S. epidermidis was not able to grow in presence of S. aureus. CONCLUSIONS: Staphyoccous aureus and S. epidermidis can be individually integrated into an oral biofilm grown on titanium, hence establishing a "submucosal" biofilm model for peri-implantitis. This model also revealed that S. aureus outcompetes S. epidermidis when grown together in the biofilm, which may explain the more frequent association of the former with peri-implantitis.

Endodontic drug delivery for root surface disinfection: a laboratory feasibility evaluation.

OBJECTIVES: This study aims to assess the potential of a mixture of three antibiotics (TreVitaMix, TVM) as an intracanal dressing to disinfect the outer root surface by applying a new in vitro model. MATERIALS AND METHODS: Fifty freshly extracted bovine roots were endodontically treated. Forty samples were then thoroughly scaled, mounted to petri dishes, gas sterilized, and randomly allocated to four groups (n = 10/group) according to their intracanal medication: sterile saline (NaCl; control, A); the TVM carrier material alone, i.e., propylene glycol (PG; B); TVM (C); and calcium hydroxide (D). In an additional group (E), the cementum was not removed and TVM was placed. Petri dishes were filled with Fastidious Anaerobe Agar, inoculated with Fusobacterium nucleatum suspension and then anaerobically incubated during 48-h intervals at 37 °C up to 192 h. Inhibition zones around the roots were then measured after each incubation period (mm(2)). RESULTS: Only teeth inoculated with the TVM dressing showed inhibition at all time points, whereas the other treatments showed no peri-radicular growing inhibition. Presence of cementum had no negative effect on disinfection (p = 0.9320). CONCLUSION: TVM was able to penetrate through the dentine and inhibit the bacterial growth of F. nucleatum up to 192 h. CLINICAL RELEVANCE: TVM might have the potential to sustainably disinfect the outer root surface in perio-endo lesions and serve as an adjunctive antimicrobial agent.

Mechanical and hydrodynamic homecare devices to clean rough implant surfaces - an in vitro polyspecies biofilm study.

OBJECTIVES: To investigate the cleaning efficacy of a mechanical and a hydrodynamic homecare device on biofilm-coated titanium surfaces with and without chlorhexidine. MATERIAL AND METHODS: Six-species biofilms were grown on 108 SLA-titanium discs, which were cleaned as follows: sonic toothbrush alone (i) or in combination with either a 0.2% chlorhexidine (ii) or a placebo gel (iii) and oral irrigator (hydrodynamic action) with water (iv) or combined with 0.2% chlorhexidine solution (v). Untreated samples served as control (vi). Biofilms were then harvested either immediately after treatment (study part A) or after a regrowth phase of 24 h (study part B) and colony-forming units (CFU) were assessed. Results were analysed using Whitney U-tests between the treatment groups. After the Bonferroni correction, the significance level was set at α = 0.0033. RESULTS: The median CFU counts directly after instrumentation accounted - in ascending order (P-values in comparison with the control group A6 were <0.001 for all groups except for A3: P = 0.014) - 2.0E1 (A5), 1.1E5 (A4), 3.6E5 (A2), 3.3E5 (A1) and 6.8E6 (A3), respectively. The untreated control group showed the highest CFU counts: 1.8E7 (A6). After regrowth, the following CFU counts were measured in ascending order (all P-values <0.001 when compared to the control group B6 = 2.0E8): 1.6E2 (B5), 1.9E5 (B2), 1.4E7 (B4), 3.1E7 (B1) and 3.9E7 (B3). CONCLUSIONS: An oral irrigator combined with 0.2% chlorhexidine is effective in reducing biofilms attached to rough titanium surfaces immediately after cleaning. Following a regrowth phase of 24 h, micro-organisms could be equally effective removed with a sonic toothbrush combined with 0.2% chlorhexidine and an oral irrigator with 0.2% chlorhexidine.

Antibacterial Efficacy of a Propolis Toothpaste and Mouthrinse Against a Supragingival Multispecies Biofilm.

PURPOSE: To determine in vitro the antibacterial properties of propolis toothpaste and mouthrinse against an in vitro multispecies biofilm model. MATERIALS AND METHODS: Six-species biofilms grown anaerobically on pellicle-coated hydroxyapatite disks were fed with glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in 37°C saliva between feedings for up to 64.5 h. At each interval, biofilms were exposed to six different slurries and solutions, including: 1) toothpaste without propolis, 2) toothpaste with propolis, 3) toothpaste with chlorhexidine, 4) mouthrinse with propolis, 5) mouthrinse with chlorhexidine, 6) saline solution (control). Afterwards, biofilms were harvested and the number of colony forming units were determined (CFU). The results were analysed using ANOVA, followed by the Bonferroni test at a 5% significance level. RESULTS: The strongest CFU reduction was shown after treatment with 0.12% chlorhexidine (p<0.0004). When comparing the different toothpastes, there was no statistically significant difference (p<0.05) in CFU reduction. However, they all showed a significant reduction in CFU of more than one log-step vs the saline control group. Nevertheless, the propolis-containing mouthrinse showed no significant reduction in CFU. CONCLUSION: All toothpastes under investigation displayed some growth inhibition in this supragingival biofilm model, which accounted for an approximately 80%-88% linear reduction. However, the propolis mouthwash had no effect.

Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation.

BACKGROUND: Periodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 "subgingival" bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition. RESULTS: Following 64 h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola, in the presence of P. gingivalis Arg-gingipain mutant. CONCLUSIONS: The gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola.

Static biofilm removal around ultrasonic tips in vitro.

OBJECTIVES: This study aims to investigate the biofilm removal capacity of two ultrasonic tips under standardized conditions using a multi-species biofilm model. METHODS: Six-species biofilms were grown on hydroxyapatite discs for 64.5 h and were treated for 15 s with a standardized load of 40 g with a piezoelectric or magnetostrictive device. Tips were applied either with the tip end or with the side facing downwards. Detached bacteria were determined in the supernatant and colony-forming units (CFUs) counted after 72 h of incubation. Untreated specimens served as controls. Moreover, the biofilms remaining on the hydroxyapatite surface after treatment were stained using the Live/Dead stain, and the pattern of their detachment was assessed by confocal laser scanning microscopy (CLSM). RESULTS: As compared to the untreated control, it was found that only a side application of the magnetostrictive device was able to remove efficiently the biofilm. In contrast, its tip application as well as both applications of the piezoelectric device removed significantly less bacteria from the biofilm structure. These findings were corroborated by CLSM observation. CONCLUSION: Both ultrasonic tips under investigations led to bacterial detachment, but the action mode as well as the tip configuration and adaptation appeared to be influenced by the biofilm removal effectiveness. CLINICAL RELEVANCE: Biofilm removal remains a main goal of ultrasonic debridement. This should be reflected in respective laboratory investigations. The presented combination of methods applied on a multi-species biofilm model in vitro allows the evaluation of the effectiveness of different ultrasonic scaler applications.

Metatranscriptome and Resistome of the Endodontic Microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

Dynamic interactions between Candida albicans and different streptococcal species in a multispecies oral biofilm.

The oral cavity is colonized by a plethora of bacteria, fungi, and archaea, including streptococci of the mitis group (MSG) and the yeast Candida albicans. This study aims to investigate the role of streptococcal species in the development of oral biofilm and the cross-kingdom interactions between some of the members of the commensal MSG and the pathogen yeast C. albicans using a multispecies supragingival biofilm model. A total of nine different in vitro biofilms were grown, quantified with culture analyses, and visually examined with confocal laser scanning microscopy (CLSM). A four-species biofilm without any streptococcal species was used as a basic biofilm. In each subsequent inoculum, one species of MSG was added and afterward combined with Streptococcus mutans. The eight-species biofilm contained all eight strains used in this study. Culture analyses showed that the presence of S. mutans in a four-species biofilm with Streptococcus oralis or S. oralis subsp. tigurinus did not differ significantly in C. albicans colony-forming unit (CFU) counts compared to biofilms without S. mutans. However, compared to other mitis species, Streptococcus gordonii combined with S. mutans resulted in the lowest CFUs of C. albicans. Visual observation by CLSM showed that biofilms containing both S. mutans and one species of MSG seemed to induce the formation of filamentous form of C. albicans. However, when several species of MSG were combined with S. mutans, C. albicans was again found in its yeast form.

In vitro versus in situ biofilms for evaluating the antimicrobial effectiveness of herbal mouthrinses.

For centuries, diverse mouthrinses have been applied for medicinal purposes in the oral cavity. In view of the growing resistance of oral microorganisms against conventional antimicrobial agents e.g. chlorhexidine, the implementation of alternative treatments inspired by nature has lately gained increasing interest. The aim of the present study was to compare in vitro biofilm models with in situ biofilms in order to evaluate the antimicrobial potential of different natural mouthrinses. For the in vitro study a six-species supragingival biofilm model containing A. oris, V. dispar, C. albicans, F. nucleatum, S. mutans and S. oralis was used. Biofilms were grown anaerobically on hydroxyapatite discs and treated with natural mouthrinses Ratanhia, Trybol and Tebodont. 0.9% NaCl and 10% ethanol served as negative controls, while 0.2% CHX served as positive control. After 64h hours, biofilms were harvested and quantified by cultural analysis CFU. For the in situ study, individual test splints were manufactured for the participants. After 2h and 72h the biofilm-covered samples were removed and treated with the mouthrinses and controls mentioned above. The biofilms were quantified by CFU and stained for vitality under the confocal laser scanning microscope. In the in vitro study, 0.2% CHX yielded the highest antimicrobial effect. Among all mouthrinses, Tebodont (4.708 ± 1.294 log10 CFU, median 5.279, p<0.0001) compared with 0.9% NaCl showed the highest antimicrobial potential. After 72h there was no significant reduction in CFU after 0.2% CHX treatment. Only Trybol showed a statistically significant reduction of aerobic growth of microorganisms in situ (5.331 ± 0.7350 log10 CFU, median 5.579, p<0.0209). After treatment with the positive control 0.2% CHX, a significant percentage of non-vital bacteria (42.006 ± 12.173 log10 CFU, median 42.150) was detected. To sum up, a less pronounced effect of all mouthrinses was shown for the in situ biofilms compared to the in vitro biofilms.

Antibiotic Resistance among Fusobacterium, Capnocytophaga, and Leptotrichia Species of the Oral Cavity.

PURPOSE: Antibiotics play an important role in treating periodontal diseases. Due to the effectiveness of antibiotic therapies, their usage in dentistry has significantly increased. The aim of this study focused on the in-vitro susceptibility of different gram-negative oral bacteria species - which are associated with periodontal diseases (Fusobacterium spp., Capnocytophaga spp. and Leptotrichia buccalis) and have different geographical origins (Asia and Europe) - against antimicrobials that are clinically relevant in dental therapy. MATERIALS AND METHODS: A total of 45 strains were tested (29 Fusobacterium spp., 13 Capnocytophaga spp. and 3 L. buccalis) that were either isolated from Chinese patients or were obtained from different strain collections. Their antimicrobial susceptibility to the antimicrobial agents benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, clindamycin, doxycycline, tetracycline and metronidazole was tested using the E-Test. Strains with particular resistance to penicillin, clindamycin and metronidazole were further analysed for resistance genes. RESULTS: All tested bacterial isolates were sensitive to amoxicillin, amoxicillin-clavulanic acid, doxycycline and tetracycline, but showed variable sensitivity towards other antibiotics such as benzylpenicillin, ciprofloxacin, moxifloxacin, clindamycin and metronidazole. CONCLUSION: The results of the present study suggest that certain periodontal disease-related bacterial strains can be resistant towards antimicrobial agents commonly used in adjuvant periodontal therapy.

Multispecies biofilm behavior and host interaction support the association of Tannerella serpentiformis with periodontal health.

The recently identified bacterium Tannerella serpentiformis is the closest phylogenetic relative of Tannerella forsythia, whose presence in oral biofilms is associated with periodontitis. Conversely, T. serpentiformis is considered health-associated. This discrepancy was investigated in a comparative study of the two Tannerella species. The biofilm behavior was analyzed upon their addition and of Porphyromonas gingivalis-each bacterium separately or in combinations-to an in vitro five-species oral model biofilm. Biofilm composition and architecture was analyzed quantitatively using real-time PCR and qualitatively by fluorescence in situ hybridization/confocal laser scanning microscopy, and by scanning electron microscopy. The presence of T. serpentiformis led to a decrease of the total cell number of biofilm bacteria, while P. gingivalis was growth-promoting. This effect was mitigated by T. serpentiformis when added to the biofilm together with P. gingivalis. Notably, T. serpentiformis outcompeted T. forsythia numbers when the two species were simultaneously added to the biofilm compared to biofilms containing T. forsythia alone. Tannerella serpentiformis appeared evenly distributed throughout the multispecies biofilm, while T. forsythia was surface-located. Adhesion and invasion assays revealed that T. serpentiformis was significantly less effective in invading human gingival epithelial cells than T. forsythia. Furthermore, compared to T. forsythia, a higher immunostimulatory potential of human gingival fibroblasts and macrophages was revealed for T. serpentiformis, based on mRNA expression levels of the inflammatory mediators interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein-1 and tumor necrosis factor α, and production of the corresponding proteins. Collectively, these data support the potential of T. serpentiformis to interfere with biological processes relevant to the establishment of periodontitis.

Advancement of the 10-species subgingival Zurich biofilm model by examining different nutritional conditions and defining the structure of the in vitro biofilms.

BACKGROUND: Periodontitis is caused by a highly complex consortium of bacteria that establishes as biofilms in subgingival pockets. It is a disease that occurs worldwide and its consequences are a major health concern. Investigations in situ are not possible and the bacterial community varies greatly between patients and even within different loci. Due to the high complexity of the consortium and the availability of samples, a clear definition of the pathogenic bacteria and their mechanisms of pathogenicity are still not available. In the current study we addressed the need of a defined model system by advancing our previously described subgingival biofilm model towards a bacterial composition that reflects the one observed in diseased sites of patients and analysed the structure of these biofilms. RESULTS: We further developed the growth media by systematic variation of key components resulting in improved stability and the firm establishment of spirochetes in the 10-species subgingival Zurich biofilm model. A high concentration of heat-inactivated human serum allowed the best proliferation of the used species. Therefore we further investigated these biofilms by analysing their structure by confocal laser scanning microscopy following fluorescence in situ hybridisation. The species showed mutual interactions as expected from other studies. The abundances of all organisms present in this model were determined by microscopic counting following species-specific identification by both fluorescence in situ hybridisation and immunofluorescence. The newly integrated treponemes were the most abundant organisms. CONCLUSIONS: The use of 50% of heat-inactivated human serum used in the improved growth medium resulted in significantly thicker and more stable biofilms, and the quantitative representation of the used species represents the in vivo community of periodontitis patients much closer than in biofilms grown in the two media with less or no human serum. The appearance of T. denticola, P. gingivalis, and T. forsythia in the top layer of the biofilms, and the high abundance of T. denticola, reflects well the microbial situation observed at diseased sites. The improved model biofilms will allow further investigations of interactions between individual species and of the effects of atmospheric or nutritional changes, as well as interactions with tissue cells.

Microbial approaches for the assessment of toothpaste efficacy against oral species: A method comparison.

Antibacterial properties of toothpastes enable chemical plaque control in limited-access tooth regions that are mechanically not sufficiently reached by toothbrushes. Therefore, this study aimed to compare different microbial methods to assess antimicrobial toothpaste properties and evaluate different toothpastes in terms of their antibacterial efficacy against different oral microorganisms in an in vitro setting. Six toothpaste suspensions with varying antibacterial supplements were applied to a multispecies biofilm model (Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, and Streptococcus mutans) as well as to each microorganism. A culture method was used to assess the anti-biofilm effects and two different agar diffusion assays were performed for testing the antimicrobial effect on each microorganism. The measurements of the culture and diffusion analyses were statistically normalized and compared and toothpastes were ranked according to their antimicrobial efficacy. The results of both agar diffusion assays showed a high correlation across all tested species (Spearman correlation coefficients ρs > 0.95). The results of the multispecies biofilm model, however, substantially differed in its assessment of antibacterial properties (ρs ranging from 0.22 to 0.87) compared to the results of both diffusion assays. Toothpastes with amine fluoride (with and without stannous fluoride), and toothpastes with triclosan resulted in the highest antimicrobial efficacy. Activated carbon supplements in toothpastes were comparable in their antimicrobial action to the negative control NaCl. The appropriate selection of a broad range of oral microorganisms seems crucial when testing the chemical impact of toothpaste and toothpaste supplements.

Antibiofilm Activity of LL-37 Peptide and D-Amino Acids Associated with Antibiotics Used in Regenerative Endodontics on an Ex Vivo Multispecies Biofilm Model.

The antimicrobial peptide LL-37 and D-amino acids (D-AAs) have been proposed as antibiofilm agents. Therefore, this study aimed to test the antimicrobial effect of antibiofilm agents associated with antibiotics used in regenerative endodontic procedures (the triple antibiotic paste­TAP: ciprofloxacin + metronidazole + minocycline). An endodontic-like biofilm model grown on bovine dentin discs was used in this study. After 21-day growth, the biofilms were treated with 1 mg/mL TAP, 10 µM LL-37, an association of LL-37 + TAP, 40 mM D-AAs solution, an association of D-AAs + TAP, and phosphate-buffered saline (negative control). Colony forming unit (CFU) data were analyzed by two-way ANOVA and Tukey's multiple comparison test (p < 0.05). LL-37 + TAP showed the best antibacterial activity (7-log10 CFU/mL ± 0.5), reaching a 1 log reduction of cells in relation to the negative control (8-log10 CFU/mL ± 0.7) (p < 0.05). In turn, no significant reduction in bacterial cells was observed with TAP, LL-37, D-AAs, and D-AAs + TAP compared to the negative control. In conclusion, the combination of antibiotics and LL-37 peptide showed mild antibacterial activity, while the combination of antibiotics and D-AAs showed no activity against complex biofilms.

Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms.

BACKGROUND: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. RESULTS: As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. CONCLUSIONS: Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of them detect non-oral species and phylogenetic groups of importance in a variety of medical conditions and the food industry.

Biofilm Models to Study the Etiology and Pathogenesis of Oral Diseases.

More than 700 microbial species inhabit the complex environment of the oral cavity. For years microorganisms have been studied in pure cultures, a highly artificial situation because microorganisms in natural habitats grow as complex ecologies, termed biofilms. These resemble multicellular organisms and are characterized by their overall metabolic activity upon multiple cellular interactions. Microorganisms in biofilms express different genes than their planktonic counterparts, resulting in higher resistance to antimicrobials, different nutritional requirements, or creation of a low redox potential allowing the growth of strictly anaerobic bacteria in the presence of oxygen. Multiple in vitro biofilm models have been described in the literature so far. The main emphasis here will be on multispecies biofilm batch culture models developed in Zurich. The standard 6-species supragingival biofilm model has been used to study basic aspects of oral biofilms such as structure, social behavior, and spatial distribution of microorganisms, or diffusion properties. Numerous parameters related to the inhibition of dental plaque were tested illustrating the high reliability of the model to predict the in vivo efficiency of antimicrobials. Modifications and advancements led to a 10-species subgingival model often combined with human gingival epithelial cells, as an integral part of the oral innate immune system, eliciting various cell responses ranging from cytokine production to apoptosis. In conclusion, biofilm models enable a multitude of questions to be addressed that cannot be studied with planktonic monocultures. The Zurich in vitro biofilm models are reproducible and reliable and may be used for basic studies, but also for application-oriented questions that could not be addressed using culture techniques. Oral biofilm research will certainly lead to a more realistic assessment of the role of microorganisms in the oral cavity in health and disease. In this respect, substantial progress has been made, but there is still more to explore.

Antibacterial Effect of High-Purity Nisin Alone and in Combination with D-Amino Acids or Chlorhexidine in an Endodontic-Like Biofilm Model.

New strategies to eradicate endodontic biofilms are needed. Therefore, we evaluated the effect of high-purity nisin alone and in combination with D-amino acids (D-AAs) or chlorhexidine (CHX) against an "endodontic-like" biofilm model. Biofilms were grown on hydroxyapatite discs for 64 h and treated with nisin, eight D-AAs mixture, nisin + eight D-AAs, 2% CHX, and nisin + 2% CHX. After the 5 min and 24 h treatments, biofilm cells were harvested and total colony-forming units were counted. Differences between groups were tested by two-way ANOVA followed by Tukey's multiple comparisons test (p < 0.05). Nisin and D-AAs, alone or in combination, were not effective in reducing bacteria after short or long exposure times. After 5 min, treatment with 2% CHX and nisin + 2% CHX resulted in 2 and 2.4-log cell reduction, respectively, compared with the no treatment control (p < 0.001). After 24 h, 2% CHX and nisin + 2% CHX drastically reduced bacterial counts. In conclusion, high-purity nisin alone or in combination with D-AAs did not show antibacterial activity against multispecies biofilms. Moreover, combined treatment using nisin and CHX showed similar antibiofilm activity compared with the use of CHX alone.

Low Concentrations of Chlorhexidine Inhibit the Formation and Structural Integrity of Enzyme-Treated Multispecies Oral Biofilms.

The self-produced matrix of biofilms, consisting of extracellular polymeric substances, plays an important role in biofilm adhesion to surfaces and the structural integrity of biofilms. In dentistry, biofilms cause multiple diseases such as caries, periodontitis, and pulpitis. Disruption of these biofilms adhering to dental hard tissues may pose a major challenge since biofilms show higher tolerance to antimicrobials and antibiotics than planktonic cells. In this study, the effect of low concentrations of chlorhexidine (CHX) on enzyme-treated multispecies oral biofilm was investigated in an in vitro model. Six-species biofilms were enzymatically treated by anaerobic growth in a medium containing DNase I and proteinase K. Biofilms were exposed to a low concentration of CHX at defined time points. After 64h, biofilms were either harvested and quantified by cultural analyses or stained for confocal laser scanning microscopy (CLSM) analyses using either Live/Dead kit or different fluorescent dyes. A mixture of YoPro1 and SYTOX™ Green, Fluorescent Brightener 28 (Calcofluor), and SYPRO™ Ruby Protein Gel Stain was used to stain total DNA, exopolysaccharides, and extracellular proteins, respectively. Extracellular DNA (eDNA) was visualized via an indirect immunofluorescence assay (Mouse anti-DNA IgG, Goat anti-Mouse IgG, Streptavidin-Cy3). Overall, the total colony-forming units significantly decreased after combined treatment with a low concentration of CHX and enzymes compared to the group treated with CHX alone (p<0.001). These findings also apply to five species individually (Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Veillonella dispar, and Candida albicans) occurring in the biofilms, with Fusobacterium nucleatum being the only exception. Furthermore, CLSM images showed less dense biofilms and a reduction in cell numbers after combined treatment compared to the group without enzymes. The combination of enzymes capable of disturbing the matrix integrity with antimicrobial agents thus appears to be a promising approach for biofilm disruption and killing.