In situ substrate-formed biofilms using IDODS mimic supragingival tooth-formed biofilms.

This study aimed to compare the bacterial viability and diversity of a substrate-formed biofilm (SF-biofilm) in situ to a supragingival tooth-formed biofilm (TF-biofilm) in the same group of individuals. The impact of the device/disc position and toothbrushing during the formation of SF-biofilm was also assessed. Two tests were run. In test 1, 15 volunteers wore two hemi-splints carrying six discs of human enamel, glass, and hydroxyapatite for 2 days, and were instructed to not perform any oral hygiene measure. Biofilm samples were collected from the substrates and the contralateral tooth and were analysed using CLSM. In five volunteers, half of the biofilm present on the discs and their contralateral teeth were scraped and analysed using 16S  pyrosequencing. In test 2, the microscopic analysis was repeated only on the SF-biofilm samples, and the volunteers were allowed to brush their teeth. Multivariate analyses revealed that the donors had a significant effect on the composition of the biofilm, confirming its subject-dependent character. The bacterial composition of the SF-biofilm was similar to the TF-biofilm, with significant differential abundance detected in very few taxa of low abundance. The toothbrushing during the formation of SF-biofilm was the only factor that conditioned the thickness or bacterial viability.

In Situ Antibacterial Activity of Essential Oils with and without Alcohol on Oral Biofilm: A Randomized Clinical Trial.

Currently, there is little evidence on the in situ antibacterial activity of essential oils (EO) without alcohol. This study aimed to evaluate in situ the substantivity and antiplaque effect on the plaque-like biofilm (PL-biofilm) of two solutions, a traditional formulation that contains EO with alcohol (T-EO) and an alcohol-free formulation of EO (Af-EO). Eighteen healthy adults performed a single mouthwash of: T-EO, Af-EO, and sterile water (WATER) after wearing an individualized disk-holding splint for 2 days. The bacterial viability (BV) and thickness of the PL-biofilm were quantified at baseline, 30 s, and 1, 3, 5, and 7 h post-rinsing (Test 1). Subsequently, each volunteer wore the splint for 4 days, applying two daily mouthwashes of: T-EO, Af-EO, and WATER. The BV, thickness, and covering grade (CG) of the PL-biofilm were quantified (Test 2). Samples were analyzed by confocal laser scanning microscopy after staining with the LIVE/DEAD® BacLight™ solution. To conduct the computations of the BV automatically, a Matlab toolbox called Dentius Biofilm was developed. In test 1, both EO antiseptics had a similar antibacterial effect, reducing BV after a single rinse compared to the WATER, and keeping it below baseline levels up to 7 h post-rinse (P < 0.001). The mean thickness of the PL-biofilm after rinsing was not affected by any of the EO formulations and ranged from 18.58 to 20.19 µm. After 4 days, the T-EO and Af-EO solutions were significantly more effective than the WATER, reducing the BV, thickness, and CG of the PL-biofilm (P < 0.001). Although, both EO antiseptics presented a similar bactericidal activity, the Af-EO rinses led to more significant reductions in the thickness and CG of the PL-biofilm than the T-EO rinses (thickness = 7.90 vs. 9.92 µm, P = 0.012; CG = 33.36 vs. 46.61%, P = 0.001). In conclusion, both essential oils antiseptics had very high immediate antibacterial activity and substantivity in situ on the 2-day PL-biofilm after a single mouthwash. In the 4-day PL-biofilm, both essential oils formulations demonstrated a very good antiplaque effect in situ, although the alcohol-free formula performed better at reducing the biofilm thickness and covering grade.

Devices for In situ Development of Non-disturbed Oral Biofilm. A Systematic Review.

OBJECTIVE: The aim of this review was to assess the types of devices used for in situ development of oral biofilm analyzed microbiologically. MATERIALS AND METHODS: A systematic search of the literature was conducted to identify all in situ studies of oral biofilm which used an oral device; the Ovid MEDLINE and EMBASE databases complemented with manual search were used. Specific devices used to microbiologically analyze oral biofilm in adults were included. After reading of the selected full texts, devices were identified and classified according to the oral cavity zone and manufacturing material. The "ideal" characteristics were analyzed in every group. RESULTS: The search provided 787 abstracts, of which 111 papers were included. The devices used in these studies were classified as palatal, lingual or buccal. The last group was sub-classified in six groups based on the material of the device. Considering the analyzed characteristics, the thermoplastic devices and the Intraoral Device of Overlaid Disk-holding Splints (IDODS) presented more advantages than limitations. CONCLUSIONS: Buccal devices were the most commonly used for the study of in situ biofilm. The majority of buccal devices seemed to slightly affect the volunteer's comfort, the IDODS being the closest to the "ideal" model. CLINICAL RELEVANCE: New devices for in situ oral biofilm microbiological studies should take into account the possible effect of their design on the volunteer's comfort and biofilm formation.

Ex vivo vs. in vivo antibacterial activity of two antiseptics on oral biofilm.

AIM: To compare the immediate antibacterial effect of two application methods (passive immersion and active mouthwash) of two antiseptic solutions on the in situ oral biofilm. MATERIAL AND METHODS: A randomized observer-masked crossover study was conducted. Fifteen healthy volunteers wore a specific intraoral device for 48 h to form a biofilm in three glass disks. One of these disks was used as a baseline; another one was immersed in a solution of 0.2% Chlorhexidine (0.2% CHX), remaining the third in the device, placed in the oral cavity, during the 0.2% CHX mouthwash application. After a 2-weeks washout period, the protocol was repeated using a solution of Essential Oils (EO). Samples were analyzed for bacterial viability with the confocal laser scanning microscope after previous staining with LIVE/DEAD® BacLight™. RESULTS: The EO showed a better antibacterial effect compared to the 0.2% CHX after the mouthwash application (% of bacterial viability = 1.16 ± 1.00% vs. 5.08 ± 5.79%, respectively), and was more effective in all layers (p < 0.05). In the immersion, both antiseptics were significantly less effective (% of bacterial viability = 26.93 ± 13.11%, EO vs. 15.17 ± 6.14%, 0.2% CHX); in the case of EO immersion, there were no significant changes in the bacterial viability of the deepest layer in comparison with the baseline. CONCLUSIONS: The method of application conditioned the antibacterial activity of the 0.2% CHX and EO solutions on the in situ oral biofilm. The in vivo active mouthwash was more effective than the ex vivo passive immersion in both antiseptic solutions. There was more penetration of the antiseptic inside the biofilm with an active mouthwash, especially with the EO. Trial registered in clinicaltrials.gov with the number NCT02267239. URL: https://clinicaltrials.gov/ct2/show/NCT02267239.

Post-tooth extraction bacteraemia: a randomized clinical trial on the efficacy of chlorhexidine prophylaxis.

OBJECTIVES: To investigate the development of post-extraction bacteraemia (PEB) after the prophylactic use of chlorhexidine (CHX). PATIENTS AND METHODS: A total of 201 patients who underwent a tooth extraction were randomly distributed into four groups: 52 received no prophylaxis (CONTROL), 50 did a mouthwash with 0.2% CHX before the tooth extraction (CHX-MW), 51 did a mouthwash with 0.2% CHX and a subgingival irrigation with 1% CHX (CHX-MW/SUB_IR) and 48 did a mouthwash with 0.2% CHX and a continuous supragingival irrigation with 1% CHX (CHX-MW/SUPRA_IR). Peripheral venous blood samples were collected at baseline, 30 seconds after performing the mouthwash and the subgingival or supragingival irrigation, and at 30 seconds and 15 minutes after completion of the tooth extraction. Blood samples were analysed applying conventional microbiological cultures under aerobic and anaerobic conditions performing bacterial identification of the isolates. RESULTS: The prevalences of PEB in the CONTROL, CHX-MW, CHX-MW/SUB_IR and CHX-MWSUPRA_IR groups were 52%, 50%, 55% and 50%, respectively, at 30 seconds and 23%, 4%, 10% and 27%, respectively, at 15 minutes. The prevalence of PEB at 15 minutes was significantly higher in the CONTROL group than in the CHX-MW group (23% versus 4%; p = 0.005). At the same time, no differences were found between CONTROL group and CHX-MW/SUB_IR or CHX-MW/SUPRA_IR groups. Streptococci (mostly viridans group streptococci) were the most frequently identified bacteria (69-79%). CONCLUSIONS: Performing a 0.2% CHX mouthwash significantly reduces the duration of PEB. Subgingival irrigation with 1% CHX didn't increase the efficacy of the mouthwash while supragingival irrigation even decreased this efficacy, probably due to the influence of these maneuvers on the onset of bacteraemia. CLINICAL RELEVANCE: These results confirm the suitability of performing a mouthwash with 0.2% CHX before tooth extractions in order to reduce the duration of PEB. This practice should perhaps be extended to all dental manipulations. TRIAL REGISTRATION: Clinicaltrials.gov NCT02150031.

The intraoral device of overlaid disk-holding splints as a new in situ oral biofilm model.

OBJECTIVES: To design a device that allows the formation of in situ oral biofilm with similar characteristics to those from the dental plaque, overcoming the limitations of previous devices. STUDY DESIGN: The Intraoral Device of Overlaid Disk-holding Splints (IDODS) was designed and manufactured. To test its validity, five healthy adult volunteers wore them for two and four days allowing the biofilm to grow without any type of distortion. After each period, the thickness, vitality and structure of the formed biofilm were measured with a Confocal Laser Scanning Microscope (CLSM) in combination with a dual fluorescence solution. All volunteers filled out a Likert-type questionnaire to evaluate the device. RESULTS: Mean bacterial vitality in the 2- and 4-day biofilms was 71% and 63%, respectively. Mean thicknesses were 21 µm and 28 µm, respectively. There was predominance in the open and heterogeneous structure whose complexity was ascending as the biofilm matured. The results obtained from the questionnaire were 2/5 in the influence in aesthetics, 3.4/5 in comfort, and 5/5 in ease of maintaining oral hygiene and withdrawal from the oral cavity. CONCLUSIONS: A biofilm with optimum characteristics was obtained by IDODS. Its use is associated with good aesthetic and comfort results and is absent of functional limitations, allowing optimal oral hygiene without altering the structure of the in situ oral biofilm. Key words:Confocal Laser Scanning Microscope, fluorochromes, in situ, intraoral device, oral biofilm.

Characteristics of in situ oral biofilm after 2 and 4 days of evolution.

OBJECTIVE: To analyze the evolution of the thickness, bacterial vitality, covering grade, and the structure after 2 and 4 days of aging in "non-disturbed" plaque-like biofilm (PL-biofilm). METHOD AND MATERIALS: Twenty healthy volunteers wore a specific appliance. After 2 days half of the samples were removed from the appliance. Posteriorly, after bacterial vital staining, samples were analyzed using a confocal laser scanning microscope. In the first volunteer, one of the disks was analyzed using a scanning electronic microscope. The same process was realized on the remaining disks after 4 days. RESULTS: The thicknesses of the PL-biofilm after 2 and 4 days were not significantly different. The bacterial vitality changed significantly from 72.50 ± 15.50% to 57.54 ± 15.66% over time, which was in contrast to the covering grade (53.08 ± 18.03% and 70.74 ± 19.11%). The structure changed from an irregular surface and compact deepest layer with a high predominance of the coccus shape to a complex structure with voids in the deepest layer and a great proportion of bacillus-shaped bacteria. CONCLUSION: The PL-biofilm thickness remained practically constant, decreasing the bacterial vitality and increasing the covering grade over time. Regarding the structure, differences were principally bacterial disposition in the surface and bacterial shape. Clinically, the findings show that new control strategies for combating the oral biofilm should be focused on inhibiting bacterial adhesion to tooth surfaces, which would reduce biofilm formation.

Antiplaque effect of essential oils and 0.2% chlorhexidine on an in situ model of oral biofilm growth: a randomised clinical trial.

OBJECTIVE: To evaluate the in situ antiplaque effect after 4 days of using of 2 commercial antimicrobial agents in short term on undisturbed plaque-like biofilm. TRIAL DESIGN AND PARTICIPANTS: An observer-masked, crossover randomised clinical trial on 15 oral and systemically healthy volunteers between 20-30 years who were randomly and sequentially allocated in the same group which performed 3 interventions in different randomised sequences. INTERVENTION: The participants wore an appliance in 3 different rinsing periods doing mouthwashes twice a day (1/0/1) with essential oils, 0.2% chlorhexidine or sterile water (negative control). At the end of each 4-day mouthwash period, samples were removed from the appliance. Posteriorly, after bacterial vital staining, samples were analysed using a Confocal Laser Scanning Microscope. MAIN OUTCOME MEASURES: Bacterial vitality, thickness and covering grade by the biofilm after 4 days of applying each of the mouthwashes. RESULTS: The essential oils and the 0.2% chlorhexidine were significantly more effective than the sterile water at reducing bacterial vitality, thickness and covering grade by the biofilm. No significant differences were found between the 0.2% chlorhexidine and the essential oils at reducing the bacterial vitality (13.2% vs. 14.7%). However, the 0.2% chlorhexidine showed more reduction than the essential oils in thickness (6.5 µm vs. 10.0 µm; p<0.05) and covering grade by the biofilm (20.0% vs. 54.3%; p<0.001). CONCLUSION: The essential oils and 0.2% chlorhexidine showed a high antiplaque effect. Although the 0.2% chlorhexidine showed better results with regard to reducing the thickness and covering grade by the biofilm, both antiseptics showed a high and similar antibacterial activity. CLINICAL RELEVANCE: Daily essential oils or 0.2% chlorhexidine mouthwashes are effective when reducing dental plaque formation in the short term. Although 0.2% chlorhexidine continues to be the "gold standard" in terms of antiplaque effect, essential oils could be considered a reliable alternative. TRIAL REGISTRATION: ClinicalTrials.gov NCT02124655.

In situ antimicrobial activity on oral biofilm: essential oils vs. 0.2 % chlorhexidine.

OBJECTIVES: This study aims to evaluate the in situ antibacterial activity of a mouthwash containing essential oils (M-EO) on undisturbed de novo plaque-like biofilm (PL-biofilm) up to 7 h after its application. PATIENTS AND METHODS: An appliance was designed to hold six glass disks on the buccal sides of the lower teeth, allowing PL-biofilm growth. Fifteen healthy volunteers wore the appliance for 48 h and then performed a M-EO. Disks were removed after 30 s and at 1, 3, 5, and 7 h later. After a washout period, the same procedure was repeated with a M-WATER and a M-0.2 % chlorhexidine. After PL-biofilm vital staining, samples were analyzed using a confocal laser scanning microscope. RESULTS: At 30 s after M-EO, the levels of bacterial vitality were 1.18 %, significantly lower than that of the basal sample (p < 0.001). After 7 h, the antibacterial effect of essential oils was still patent with a 47.86 % difference in bacterial vitality compared to the basal sample (p < 0.001). CONCLUSION: A single M-EO presents high antibacterial immediate activity and penetration capacity in situ and a substantivity which lasts for at least 7 h after its application over de novo biofilm. These results were better than those observed with 0.2 % chlorhexidine under the same conditions. CLINICAL RELEVANCE: A single M-EO is an effective measure against the de novo biofilm, presenting a good alternative to clorhexidine such as a preoperative rinse, in periodontal procedures or post-treatment applications.

Chlorhexidine substantivity on salivary flora and plaque-like biofilm: an in situ model.

OBJECTIVE: To evaluate the in situ antibacterial activity of a mouthrinse with 0.2% Chlorhexidine (M-0.2% CHX) on undisturbed de novo plaque-like biofilm (PL-biofilm) and on salivary flora up to 7 hours after its application. METHODS: A special acrylic appliance was designed, with 3 inserted glass disks on each buccal side, allowing for PL-biofilm growth. Fifteen healthy volunteers wore the appliance for 48 hours and then performed an M-0.2% CHX; disks were removed at 30 seconds and 1, 3, 5 and 7 hours after the mouth-rinsing. Applying a washout period, saliva samples were collected from each volunteer at 30 seconds and 1, 3, 5 and 7 hours after performing an M-0.2% CHX. The PL-biofilm and saliva samples were analysed by confocal laser scanning and epifluorescence microscopes, respectively. RESULTS: At 30 seconds after M-0.2% CHX, the levels of viable bacteria detected in saliva were significantly lower than those observed in PL-biofilm. The difference in the percentage of live bacteria detected in saliva was significantly higher than that observed in PL-biofilm at 5 and 7 hours after M-0.2% CHX. CONCLUSION: After a single mouthrinse of the 0.2% CHX formulation tested in the present study, the 2-day PL-biofilm presented a significantly higher resistance to this antiseptic in situ than that observed in salivary flora. However, this 0.2% CHX formulation showed a higher substantivity on PL-biofilm than on salivary flora at 5 and 7 hours after mouth-rinsing, which could be related to the slower growth rate of PL-biofilm and the possible reservoir function for antimicrobial agents associated with the undisturbed de novo PL-biofilm.