Antiplaque Effect of Essential Oils and 0.2% Chlorhexidine on an In Situ Model of Oral Biofilm Growth: A Randomised Clinical Trial

RESEARCH ARTICLE Antiplaque Effect of Essential Oils and 0.2% Chlorhexidine on an In Situ Model of Oral Biofilm Growth: A Randomised Clinical Trial Víctor Quintas 1 , Isabel Prada-López 1 , Nikolaos Donos 2 , David Suárez-Quintanilla 1 , Inmaculada Tomás 1* 1Oral Sciences Research Group, School of Medicine and Dentistry, University of Santiago de Compostela, Santiago de Compostela, Spain, 2Periodontology Unit, UCL Eastman Dental Institute, London, United Kingdom *[email protected] Abstract 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 μmvs. 10.0 μm; p<0.05) and covering grade by the biofilm (20.0% vs. 54.3%; p<0.001). PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 1/18 OPEN ACCESS Citation: Quintas V, Prada-López I, Donos N, Suárez-Quintanilla D, Tomás I (2015) Antiplaque Effect of Essential Oils and 0.2% Chlorhexidine on an In Situ Model of Oral Biofilm Growth: A Randomised Clinical Trial. PLoS ONE 10(2): e0117177. doi:10.1371/journal.pone.0117177 Academic Editor: Kimon Divaris, UNC School of Dentistry, University of North Carolina-Chapel Hill, UNITED STATES Published: February 17, 2015 Copyright: © 2015 Quintas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by project PI11/ 01383 from Carlos III Institute of Health (General Division of Evaluation and Research Promotion, Madrid, Spain), which is integrated in National Plan of Research, Development and Innovation (PN I+D+I 2008-2011). This project was cofinanced by European Regional Development Fund (ERDF 2007- 2013). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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 Introduction The accumulation and maturation of oral biofilm in the gingival margin is widely recognised to be the primary aetiological factor in the development of chronic gingivitis [1,2]. Based on this association, the current treatment of gingivitis is focused on biofilm disruption, which will normally include mechanical processes, both professionally and at home. However, for patients, it is not easy to achieve a proper level of plaque control. The efficient plaque control techniques are very time consuming and require a special motivation and skills for their optimum use [3]. It was at this point where mouthwashes become important, due to the fact that they include diverse types of antimicrobial agents to complement the results of mechanical oral hygiene measures [4]. Chlorhexidine (CHX) is considered the “gold standard”of oral antiseptics; nevertheless it has not been recommended for long periods of time due to its well-known secondary effects [5]. All of these inconveniences have limited its acceptability among dental professionals and users; in contrast, however, are the exceptional antiseptic properties, promoting the interest of researchers in other alternative antiplaque agents [6]. Mouthwashes containing essential oils (EO) in their formulation have received a lot of attention. Their antiplaque activity has been demonstrated in numerous clinical studies, in which they were used in conjunction with mechanical oral hygiene measures [7,8]. Taking into account the fact that bacteria present in a biofilm can be from 10–1000 times more tolerant to antimicrobial agents than those in planktonic phase [9], a more predictive evaluation of the efficacy of an antiseptic agent present in a mouthwash could be done using biofilm models. Numerous studies have been carried out regarding the activity of EO and CHX on the oral biofilm both in vitro and in situ. The latter are more valuable when establishing the antiseptic efficacy of mouthwashes, due to the fact that their activity is tested under in vivo clinical conditions [10]. In order to achieve a better understanding of the clinical effects that these agents produce in the interior of the biofilm, it is necessary to apply a methodology in which the biofilm grows directly in the interior of the oral cavity but its three-dimensional structure is not distorted by manipulation [11,12]. Most in situ studies on “non-destructured”dental plaque do not use natural teeth; instead, they use disks made of different materials that are introduced in the mouth Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 2/18 Competing Interests: The authors have declared that no competing interests exist. for a variable period of time, during which they are exposed to the intraoral conditions of an individual. Therefore, it is not dental plaque itself, but a biofilm which is presumably very similar to dental plaque, which is generated in similar conditions and is set on a substrate which is generally artificial. Hereinafter, it will be denominated plaque-like biofilm (PL-Biofilm) [13,14]. The study of the antiplaque effect of an antimicrobial agent can be performed in long and short term clinical studies. Among the latter, 4-day models have particular importance. This model can be described as an established method for assessing the inhibitory activity against dental plaque that mouthwashes have, per se, and determines the relative effectiveness of the different formulations [15,16]. Thereby, they have been widely used by different research groups to study various antiplaque agents that are commonly used in the oral cavity [15–19]. Confocal laser scanning microscopy (CLSM), despite having poorer resolution than transmission electron microscopy (TEM) or scanning electron microscopy (SEM) [20], has eliminated or considerably reduced the distortion produced by preparation of the samples. The main advantage of CLSM is that it permits the analysis of biofilm in situ, without altering its delicate structure, keeping it hydrated with no need for fixation or drying [21,22]. CLSM also facilitates the observation of biofilms in situ and in “real”time, with all of the benefits of most sophisticated image analysis [23]. Systems based on the identification of bacterial vitality by fluorescence have become increasingly important, since they are accepted as a simple, accurate, reproducible and highly sensitive method for the quantitation of attached microorganisms [23]. Consequently, numerous authors have examined biofilms with the help of CLSM and fluorescence staining, incorporating both to analyse their structure [11,24], such as the spatial distribution of the vital and non-vital bacteria [13,25,26]. Various combinations of dyes have been used in the literature [10,11,27–29]. The combination of SYTO 9 and propidium iodide (PI) has been posed as a reliable alternative [30] to traditional blend of fluorescein diacetate with ethidium bromide, mainly because of the destructive properties of this combination and the toxicity and instability of the ethidium bromide [30]. This staining method allows for a visual demonstration of bactericidal activity as well as its quantification using computerized image analysis [31]. The aim of this study was to evaluate the in situ antiplaque effect of 2 antimicrobial agents in the short term with a posterior analysis on “non-destructured”biofilm with CLSM combined with fluorescence staining. Materials and Methods This was a randomised, observer-masked, crossover study of the antiplaque efficacy of 2 available formulas based on EO (Listerine Mentol Listerine Johnson & Johnson, Madrid, Spain) and 0.2% CHX (Oraldine Perio Johnson & Johnson, Madrid, Spain) on an in situ model of PL- Biofilm growth. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see S1 CONSORT Checklist and S1 Protocol. The trial was registered in ClinicalTrials.gov with the ID number NCT02124655. The authors confirm that all ongoing and related trials for this intervention are registered. URL of the registered trial: (http:// clinicaltrials.gov/ct2/show/NCT02124655?term = essential+oils+chlorhexidine +biofilm&rank=1). Selection of the study group To calculate “a priori”sample size, the following statistical criteria were established: an effect size of 0.35, an alpha error of 0.05 and a statistical power of 80%. Assuming these criteria and the possible application of repeated measures ANOVA test, a sample size of 15 subjects was required (Fig. 1). The sample size calculation was performed using the program GPower 3.1.5. Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 3/18 The participants were collected among dental students at the Faculty of Medicine and Dentistry of Santiago de Compostela where volunteer enrollment was asked by advertisements asking for the participation in a research study at the Faculty hall. All of these volunteers were revised by the same calibrated clinician to ensure they fulfilled all inclusion and exclusion criteria. The volunteers chosen were systemically healthy adult volunteers between 20 and 45 years old, who presented a good oral health status: a minimum of 24 permanent teeth with no evidence of gingivitis or periodontitis (Community Periodontal Index score = 0) [32] and an absence of untreated caries at the beginning of the study. The following exclusion criteria were applied: smoker or former smoker, presence of dental prostheses or orthodontic devices, antibiotic treatment or routine use of oral antiseptics in the previous 3 months, and presence of any systemic disease that could alter the production or composition of saliva. Before the start of each study, a full mouth scaling with ultrasonics and teeth polishing with rubber cup after dental disclosing was performed by the same calibrated clinician on all selected participants. This project was approved (number 2012/393) by the Clinical Research Ethics Committee of Galicia. Written informed consent was obtained from all participants in the study. Production of the Intraoral Device of Overlaid Disk-holding Splints (IDODS) After considering a number of previously described in situ models [10,24,25,33], an individualised splint of the lower arch was created for each volunteer, which was capable of holding 6 glass disks (6 mm in diameter, 1 mm thickness); these were polished at 800 grit. The characteristics of this splint have been previously described [34], although a new variety of partial splint has been already introduced [14]. Mouthwash protocol During the 96 hour (4 days) duration of each course of study, each volunteer wore the splints with the glass disks, withdrawing them from the oral cavity only during meals and to perform oral hygiene procedures, using only the mechanical removal of bacterial plaque with water, without the use of any toothpaste or mouthwash. They were advised to do just 3 meals per day avoiding eating or drinking in-between. The only drink permitted during meals was still water. While eating and brushing their teeth, the splints were stored in a provided opaque plastic box (the type used to store orthodontic removable devices). Volunteers were told to impregnate a sterile gauze with 5 ml of saline (the gauzes and the saline were provided as well), and extend it on the base of the plastic box, support the splints on it and close the box, leaving it at room temperature. The maximum allowed time that the volunteers had to eat and perform the oral hygiene measures were 20 minutes. Using the permitted mechanical oral hygiene measures (without the splints), the volunteers performed the following protocols based on the manufacturers’instructions, with the splints in the oral cavity, during the 4 days in the morning (8.30) after breakfast and at night (22.00) after dinner: A) 20 ml rinses for 30 seconds with essential oils/twice daily (4D-EO). -or- B) 10 ml rinses for 30 seconds with 0.2% chlorhexidine/twice daily (4D-0.2% CHX; positive control). -or- C) 20 ml rinses for 30 seconds with sterile water (4D-WATER; negative control). A number was assigned to each participant (closed envelope) by an investigator who was unaware of the study design. Using an internet-based balanced randomisation system [35] Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 4/18 introducing the numbers and the 3 test cycles (A, B and C), a random sequence indicating the mouthwash that each subject would use first, second and third was obtained. Although they were not told the type of mouthwash they were going to use, the obvious differences in taste between the 3 mouthwashes made allocation concealment to the volunteer impossible. The antiseptics/control were prepared in opaque bottles labelled with an A, B, or C depending on the containing solution, with 10 ml more than the quantity needed for completing the whole series of mouthwashes. The day before the start of each experiment, after the full mouth dental scaling and polishing, participants were given by a masked investigator the corresponding previously made splints, the allocated opaque bottle, a plastic glass and a sterile 20 ml serum syringe with the objective of being the more precise possible with the quantity of solution used for the mouthwash. The day of the sample analysis, they were asked to bring back the bottles to measure the quantity of solution left in the bottle. All volunteers performed the 3 rinsing cycles, with a rest period of 2 weeks between each test (Fig. 2). Fig 1. Flow diagram of the study with enrollment, allocation, follow-up and analysis of participants. doi:10.1371/journal.pone.0117177.g001 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 5/18 Fig 2. Scheme of the sequence of protocols followed by the volunteers. doi:10.1371/journal.pone.0117177.g002 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 6/18 Study endpoints The primary endpoints with respect to the antiplaque efficacy of the 2 antiseptics on the PL- Biofilm were the bacterial vitality, in terms of detection of non-vital bacteria in the biofilm; the thickness and the covering grade, in terms of quantification of height and density of biofilm, respectively. Collection of the samples of PL-Biofilm Sample collection was done individually at the Unit of Confocal Microscopy of the University of Santiago de Compostela at 8 am in the morning, so that the samples of each volunteer were analysed on different days. It was determined that a minimum of 10 hours should have elapsed since the last mouthwash on the previous night. As the glass disks (in total, 6) were removed from the splint, they were immediately immersed in 100 μL of fluorescence solution LIVE/DEAD BacLigh and kept in a dark chamber at room temperature for 15 minutes. The characteristics of LIVE/DEAD BacLight fluorescence solution (Molecular Probes, Leiden, the Netherlands), as well as its preparation, have been described by the authors in a previous paper [36]. Microscopic observation was performed by a single investigator who was unaware of the study design, using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) with an HCX APOL 63x/0.9 water-immersion lens. Processing of the samples of PL-Biofilm Four selected fields or XYZ series in the central part of each disk were evaluated. These fields were considered representative of the whole sample after the observer’s general examination. Fluorescence emission was determined in a series of XY images in which each image corresponded to each of the Z positions (depth). The optical sections were scanned in 1 μm sections from the surface of the biofilm to its base, measuring the maximum thickness of the field and subsequently the mean thickness of the biofilm of the corresponding sample. The maximum thickness of biofilm field was defined as the distance between the substrate (in perpendicular) and the peaks of the highest cell clusters [37]. The maximum biofilm thickness of each field was divided into 3 zones or equivalent layers: outer layer (layer 1), middle layer (layer 2) and inner layer (layer 3) (Fig. 3). The capture of the data was done with the same settings in all cases. The spatial scan mode (XYZ) and the 1024x1024 pixels scan format resolution were used. The Argon-ion and DPSS laser were used at a 13% and 78% of maximum intensity, respectively. The values for the pinhole, zoom and scan speed were 121.58 microns, 1 and 400Hz, respectively. The only values that were different depending on the sample were the offset (range between-1% to 1%) and PMT gain which was different for channel red and green, being in general terms, higher for green than for red (test and positive control), due to the fact that there was more presence of red than green signal, being for the negative control the opposite. These values were always adjusted to get a good quality capture without background noise, avoiding excessive saturation of the brightest pixels of the image. As the technician was blinded to the experiment, they were advised to make the adjustments always consistent with what was seeing by the objective of the microscope, obtaining an image which was the closest as possible to reality. Quantification of bacterial vitality in the series of XY images was determined using cytofluorographic analysis (Leica Confocal Software). In this analysis, the images of each fluorochrome were defined as “channels”(SYTO 9 occupies the green channel and PI the red channel). Square capture masks were used to measure the area occupied (μm 2 ) by the pixels in each channel, determining the total area occupied by the biofilm and the corresponding Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 7/18 percentage of vitality. The intensity ranges that were considered as positive signal were between 100 and 255. Determination of the mean percentage of bacterial vitality in each field required sections with a minimum area of biofilm of 250 μm 2 , and the mean percentage of bacterial vitality of the biofilm was calculated for the corresponding sample and for each biofilm layer. For quantification of the percentage of surface substrate covered by the biofilm (covering grade), the cytofluorogram itself was used. From the maximum projection (superposition of all planes captured) of each of the analysed fields, the percentage of covering grade was obtained by calculating the sum of the bacterial mass (vital and non-vital) in regard to the total surface of the field (% positive within total area). Statistical analysis The results were analysed using the PASW Statistics Base 20 package for Windows (IBM, Madrid, Spain) by an investigator who was blinded to the type of interventions analysed. All values from the quantitative variables analysed (“biofilm thickness”and “bacterial vitality percentage”) presented a normal distribution, which was determined using the Kolmogorov-Smirnov test. One-way ANOVA with repeated measures was used for intra-mouthwash (differentiating between the 3 biofilm layers) comparisons using all of the PL-Biofilm samples. Two-way ANOVA with repeated measures was used for inter-mouthwash (differentiating between the 3 biofilm layers) comparisons using all of the PL-Biofilm samples. Pairwise comparisons (with the Bonferroni adjustment) were used for the analysis of intra- and inter-mouthwash results (including differentiating between the 3 biofilm layers). Statistical significance was taken as a pvalue less than 0.05. Results A total of 34 volunteers were evaluated for eligibility to achieve the calculated sample size (n = 15). When this number of participants meeting the inclusion and exclusion criteria was reached, the enrollment process was stopped. A total of 19 subjects were not selected as eligible for not fulfilling the inclusion criteria. In relation to demographic characteristics of the selected participants, 8 were female and 7 male with a mean age of 25.4 ± 2.3 years. No adverse or side Fig 3. Scheme of the divisions of the PL-Biofilm in layers. 1-Outer layer; 2-Middle layer; 3-Inner layer. *PL-Biofilm. **Glass disk (substrate). doi:10.1371/journal.pone.0117177.g003 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 8/18 effects were observed by investigators or reported by the volunteers after the completion of any of the rising cycles. PL-Biofilm: thickness, bacterial vitality and covering grade after 4-day- rinsing periods with essential oils and 0.2% chlorhexidine Both 4D-EO and 4D-0.2% CHX protocols were found to be significantly effective in regard to the 4D-WATER for reducing the thickness (9.99 ± 3.27 μm and 6.48 ± 1.82 μm respectively vs. 23.44 ± 4.78 μm) (Fig. 4)(S1,S2 and S3 Tables) and the bacterial vitality (14.67 ± 5.54% and 13.19 ± 18.09% respectively vs. 56.53 ± 14.40%) (Fig. 5)(S4 Table [see A], S5 Table [see A] and S6 Table [see A]). The covering grade was also significantly reduced by the 4D-EO and 4D- 0.2% CHX in regard to the 4D-WATER (54.32 ± 17.49% and 20.01 ± 16.52% respectively vs. 75.17 ± 16.51%) (Fig. 6)(S7,S8 and S9 Tables). Fig 4. On the left, lateral projections of the brightest point (X-Z) from images stacked in Y plane from the three rinsing cycles, presenting their respective mean thickness. On the right, maximum projections of the brightest point (X-Y) from images stacked in Z plane. 4D-WATER = 4-day period during which rinses with 20 mL of sterile water are done twice a day; 4D-EO = 4-day period during which rinses with 20 mL of essential oils are done twice a day; 4D-0.2% CHX = 4-day period during which rinses with 10 mL of 0.2% chlorhexidine are done twice a day. doi:10.1371/journal.pone.0117177.g004 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 9/18 Acknowledgments The authors would like to express their gratitude to Ramiro Barreiro and Mercedes Rivas, staff of the Microscopy Unit of the Services to Support Research (University of Santiago de Compostela) for their technical advice on the CLSM techniques. Author Contributions Conceived and designed the experiments: IT ND DS-Q. Performed the experiments: VQ IP-L. Analyzed the data: IT. Contributed reagents/materials/analysis tools: IT. Wrote the paper: VQ IL-P IT. References 1. Loe H, Theilade E, Jensen SB (1965) Experimental Gingivitis in Man. J Periodontol 36: 177–187. PMID: 14296927 2. Tatakis DN, Trombelli L (2004) Modulation of clinical expression of plaque-induced gingivitis. I. Background review and rationale. J Clin Periodontol 31: 229–238. PMID: 15016250 3. Rodrigues JA, dos Santos PA, Garcia PP, Corona SA, Loffredo LC (2003) Evaluation of motivation methods used to obtain appropriate oral hygiene levels in schoolchildren. Int J Dent Hyg 1: 227–232. PMID: 16451505 4. Demke R (2012) Plaque inhibition: the science and application of oral rinses. Dent Today 31: 96–101; quiz 101, 195. PMID: 22970603 5. Van Strydonck DA, Slot DE, Van der Velden U, Van der Weijden F (2012) Effect of a chlorhexidine mouthrinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. J Clin Periodontol 39: 1042–1055. doi: 10.1111/j.1600-051X.2012.01883.x PMID: 22957711 6. Wu CD, Savitt ED (2002) Evaluation of the safety and efficacy of over-the-counter oral hygiene products for the reduction and control of plaque and gingivitis. Periodontol 2000 28: 91–105. 7. Charles CH, Mostler KM, Bartels LL, Mankodi SM (2004) Comparative antiplaque and antigingivitis effectiveness of a chlorhexidine and an essential oil mouthrinse: 6-month clinical trial. J Clin Periodontol 31: 878–884. PMID: 15367192 8. Sharma N, Charles CH, Lynch MC, Qaqish J, McGuire JA, et al. (2004) Adjunctive benefit of an essential oil-containing mouthrinse in reducing plaque and gingivitis in patients who brush and floss regularly: a six-month study. J Am Dent Assoc 135: 496–504. PMID: 15127875 9. Fine DH, Furgang D, Barnett ML (2001) Comparative antimicrobial activities of antiseptic mouthrinses against isogenic planktonic and biofilm forms of Actinobacillus actinomycetemcomitans. J Clin Periodontol 28: 697–700. PMID: 11422593 10. Auschill TM, Hein N, Hellwig E, Follo M, Sculean A, et al. (2005) Effect of two antimicrobial agents on early in situ biofilm formation. J Clin Periodontol 32: 147–152. PMID: 15691343 11. Wood SR, Kirkham J, Marsh PD, Shore RC, Nattress B, et al. (2000) Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy. J Dent Res 79: 21–27. PMID: 10690656 12. Beyth N, Yudovin-Farber I, Perez-Davidi M, Domb AJ, Weiss EI (2010) Polyethyleneimine nanoparticles incorporated into resin composite cause cell death and trigger biofilm stress in vivo. Proc Natl Acad Sci U S A 107: 22038–22043. doi: 10.1073/pnas.1010341107 PMID: 21131569 13. García-Caballero L, Quintas V, Prada-López I, Seoane J, Donos N, et al. (2013) Chlorhexidine Substantivity on Salivary Flora and Plaque-Like Biofilm: An In Situ Model. PLoS One 8: e83522. doi: 10. 1371/journal.pone.0083522 PMID: 24386220 14. Quintas V, Prada-López I, Prados-Frutos J, Tomas I (2014) In situ antimicrobial activity on oral biofilm: essential oils vs. 0.2% chlorhexidine. Clin Oral Investig. (In press). 15. Riep BG, Bernimoulin JP, Barnett ML (1999) Comparative antiplaque effectiveness of an essential oil and an amine fluoride/stannous fluoride mouthrinse. J Clin Periodontol 26: 164–168. PMID: 10100042 16. Arweiler NB, Boehnke N, Sculean A, Hellwig E, Auschill TM (2006) Differences in efficacy of two commercial 0.2% chlorhexidine mouthrinse solutions: a 4-day plaque re-growth study. J Clin Periodontol 33: 334–339. PMID: 16634954 17. Rosin M, Welk A, Kocher T, Majic-Todt A, Kramer A, et al. (2002) The effect of a polyhexamethylene biguanide mouthrinse compared to an essential oil rinse and a chlorhexidine rinse on bacterial counts and 4-day plaque regrowth. J Clin Periodontol 29: 392–399. PMID: 12060421 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 16 / 18 18. Pizzo G, La Cara M, Licata ME, Pizzo I, D’Angelo M (2008) The effects of an essential oil and an amine fluoride/stannous fluoride mouthrinse on supragingival plaque regrowth. J Periodontol 79: 1177–1183. doi: 10.1902/jop.2008.070583 PMID: 18597599 19. Singh A, Daing A, Dixit J (2013) The effect of herbal, essential oil and chlorhexidine mouthrinse on de novo plaque formation. Int J Dent Hyg 11: 48–52. doi: 10.1111/j.1601-5037.2012.00556.x PMID: 22583681 20. Dige I, Nilsson H, Kilian M, Nyvad B (2007) In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization. Eur J Oral Sci 115: 459–467. PMID: 18028053 21. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, et al. (1994) Biofilms, the customized microniche. J Bacteriol 176: 2137–2142. PMID: 8157581 22. Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15: 167–193. PMID: 11932229 23. Hahnel S, Rosentritt M, Burgers R, Handel G (2008) Surface properties and in vitro Streptococcus mutans adhesion to dental resin polymers. J Mater Sci Mater Med 19: 2619–2627. doi: 10.1007/ s10856-007-3352-7 PMID: 18197372 24. Netuschil L, Reich E, Unteregger G, Sculean A, Brecx M (1998) A pilot study of confocal laser scanning microscopy for the assessment of undisturbed dental plaque vitality and topography. Arch Oral Biol 43: 277–285. PMID: 9839703 25. Auschill TM, Arweiler NB, Netuschil L, Brecx M, Reich E, et al. (2001) Spatial distribution of vital and dead microorganisms in dental biofilms. Arch Oral Biol 46: 471–476. PMID: 11286812 26. Hope CK, Wilson M (2006) Biofilm structure and cell vitality in a laboratory model of subgingival plaque. J Microbiol Methods 66: 390–398. PMID: 16487610 27. Korber DR, Lawrence JR, Hendry MJ, Caldwell DE (1993) Analysis of spatial variability within mot+ and mot- Pseudomonas fluorescens biofilms using representative elements. Biofouling 7: 339–358. 28. Arweiler NB, Lenz R, Sculean A, Al-Ahmad A, Hellwig E, et al. (2008) Effect of food preservatives on in situ biofilm formation. Clin Oral Investig 12: 203–208. doi: 10.1007/s00784-008-0188-6 PMID: 18357475 29. Gu H, Fan D, Gao J, Zou W, Peng Z, et al. (2012) Effect of ZnCl2 on plaque growth and biofilm vitality. Arch Oral Biol 57: 369–375. doi: 10.1016/j.archoralbio.2011.10.001 PMID: 22071420 30. Tawakoli PN, Al-Ahmad A, Hoth-Hannig W, Hannig M, Hannig C (2013) Comparison of different live/ dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clin Oral Investig 17: 841–850. doi: 10.1007/s00784-012-0792-3 PMID: 22821430 31. Pan P, Barnett ML, Coelho J, Brogdon C, Finnegan MB (2000) Determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method. J Clin Periodontol 27: 256–261. PMID: 10783839 32. WHO (1997) Oral health surveys, Basic methods. In: Organization WH, editor. 4th ed. Geneva. 33. Arweiler NB, Hellwig E, Sculean A, Hein N, Auschill TM (2004) Individual vitality pattern of in situ dental biofilms at different locations in the oral cavity. Caries Res 38: 442–447. PMID: 15316188 34. Tomás I, Henderson B, Diz Dios P, Donos N (2010) In vivo oral biofilm analysis by confocal laser scanning microscopy: methodological approaches. In: A M-V, editor. Microscopy Science, technology, applications and education. Badajoz (Spain): Formatex. pp. 597–606. 35. Dallal GE. Available: www.randomization.com. Accessed 2014 February 5. doi: 10.1016/j.humpath. 2014.04.012 PMID: 24908143 36. Tomás I, García-Caballero L, Cousido MC, Limeres J, Álvarez M, et al. (2009) Evaluation of chlorhexidine substantivity on salivary flora by epifluorescence microscopy. Oral Dis 15: 428–433. doi: 10.1111/ j.1601-0825.2009.01570.x PMID: 19413678 37. Roberts SK, Bass C, Brading M, Lappin-Scott H, Stoodley P (1999) Biofilm formation and structure; What’s new? In: Newman HN WM, editor. Dental plaque revisited-oral biofilms in health and disease. Cardiff: BioLine. pp. 1–36. PMID: 11323939 38. Moran J, Addy M, Kohut B, Hovliaras CA, Newcombe RG (1994) Efficacy of mouthrinses in inhibiting the development of supragingival plaque over a 4-day period of no oral hygiene. J Periodontol 65: 904–907. PMID: 7823270 39. Jentsch H, Hombach A, Beetke E, Jonas L (2002) Quantitative transmission electron microscopic study of dental plaque—an in vivo study with different mouthrinses. Ultrastruct Pathol 26: 309–313. PMID: 12396241 40. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43: 5721–5732. PMID: 16272510 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 17 / 18 41. Hannig C, Follo M, Hellwig E, Al-Ahmad A (2010) Visualization of adherent micro-organisms using different techniques. J Med Microbiol 59: 1–7. doi: 10.1099/jmm.0.015420-0 PMID: 19815663 42. Decker EM (2001) The ability of direct fluorescence-based, two-colour assays to detect different physiological states of oral streptococci. Lett Appl Microbiol 33: 188–192. PMID: 11555201 43. Pizzo G, Compilato D, Di Liberto B, Pizzo I, Campisi G (2013) Effects of two essential oil mouthrinses on 4-day supragingival plaque regrowth: a randomized cross-over study. Am J Dent 26: 156–160. PMID: 23986963 44. Tomás I, García-Caballero L, López-Álvar E, Suárez M, Diz P, et al. (2013) In Situ Chlorhexidine Substantivity on Saliva and Plaque-Like Biofilm: Influence of Circadian Rhythm. J Periodontol 84: 1662–1672. doi: 10.1902/jop.2013.120475 PMID: 23327586 45. Jentsch H, Mozaffari E, Jonas L (2013) Scanning electron microscopy of growing dental plaque: a quantitative study with different mouth rinses. Ultrastruct Pathol 37: 233–240. doi: 10.3109/01913123. 2013.763882 PMID: 23758106 46. Koo H, Nino de Guzman P, Schobel BD, Vacca Smith AV, Bowen WH (2006) Influence of cranberry juice on glucan-mediated processes involved in Streptococcus mutans biofilm development. Caries Res 40: 20–27. PMID: 16352876 47. Pratten J, Barnett P, Wilson M (1998) Composition and susceptibility to chlorhexidine of multispecies biofilms of oral bacteria. Appl Environ Microbiol 64: 3515–3519. PMID: 9726908 48. Al-Ahmad A, Wiedmann-Al-Ahmad M, Carvalho C, Lang M, Follo M, et al. (2008) Bacterial and Candida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacement materials. J Biomed Mat Res A 87: 933–943. doi: 10.1002/jbm.a.31832 PMID: 18228269 49. Al-Ahmad A, Wiedmann-Al-Ahmad M, Auschill TM, Follo M, Braun G, et al. (2008) Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro. Arch Oral Biol 53: 765–772. doi: 10.1016/j.archoralbio.2008.02.014 PMID: 18395697 50. Ulkur F, Arun T, Ozdemir F (2013) The effects of three different mouth rinses in a 4-day supragingival plaque regrowth study. Eur J Dent 7: 352–358. doi: 10.4103/1305-7456.115420 PMID: 24926216 51. Stoeken JE, Paraskevas S, van der Weijden GA (2007) The long-term effect of a mouthrinse containing essential oils on dental plaque and gingivitis: a systematic review. J Periodontol 78: 1218–1228. PMID: 17608576 52. Van Leeuwen MP, Slot DE, Van der Weijden GA (2011) Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review. J Periodontol 82: 174–194. doi: 10.1902/jop.2010.100266 PMID: 21043801 53. Neely AL (2012) Essential oil mouthwash (EOMW) may be equivalent to chlorhexidine (CHX) for longterm control of gingival inflammation but CHX appears to perform better than EOMW in plaque control. J Evid Based Dent Pract 12: 69–72. doi: 10.1016/S1532-3382(12)70017-9 PMID: 23253835 54. Gandini S, Negri E, Boffetta P, La Vecchia C, Boyle P (2012) Mouthwash and oral cancer risk quantitative meta-analysis of epidemiologic studies. Ann Agric Environ Med 19: 173–180. PMID: 22742785 Essential Oils and Chlorhexidine Antiplaque Effect on Oral Biofilm PLOS ONE | DOI:10.1371/journal.pone.0117177 February 17, 2015 18 / 18