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

CLINICAL TRIAL published: 23 November 2017 doi: 10.3389/fmicb.2017.02162 Frontiers in Microbiology | www.frontiersin.org 1November 2017 | Volume 8 | Article 2162 Edited by: Sara María Soto, ISGlobal, Spain Reviewed by: Jens Kreth, Oregon Health and Science University, United States Thomas Thurnheer, University of Zurich, Switzerland *Correspondence: Inmaculada Tomás [email protected] Specialty section: This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology Received: 02 August 2017 Accepted: 20 October 2017 Published: 23 November 2017 Citation: Quintas V, Prada-López I, Carreira MJ, Suárez-Quintanilla D, Balsa-Castro C and Tomás I (2017) In Situ Antibacterial Activity of Essential Oils with and without Alcohol on Oral Biofilm: A Randomized Clinical Trial. Front. Microbiol. 8:2162. doi: 10.3389/fmicb.2017.02162 In Situ Antibacterial Activity of Essential Oils with and without Alcohol on Oral Biofilm: A Randomized Clinical Trial Victor Quintas1, Isabel Prada-López1, María J. Carreira2, David Suárez-Quintanilla1, Carlos Balsa-Castro1and Inmaculada Tomás1* 1Oral Sciences Research Group, Department of Surgery and Medical Surgical Specialties, School of Medicine and Dentistry, Health Research Institute of Santiago (IDIS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain, 2Centro Singular de Investigación en Tecnoloxías da Información, Health Research Institute of Santiago (IDIS), Universidade de Santiago de Compostela, Santiago de Compostela, Spain 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®BacLightTM 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. Keywords: anti-infective agents, local, biofilm, dental plaque, essential oils, microscopy, fluorescence This Clinical Trial was registered at clinicaltrials.gov with the number NCT03146390 URL: https://clinicaltrials. gov/ct2/show/NCT03146390. Quintas et al. Essential Oils Activity on Biofilm INTRODUCTION The accumulation of bacterial biofilms on tooth surfaces results in two of the most prevalent infectious diseases—caries and periodontitis. Although prevention and control of these diseases can be achieved by the daily mechanical removal of biofilms, many people are either unable or unwilling to practice these procedures as regularly or as efficiently as necessary. There is, therefore, great interest in the possibility of using chemicals to replace or augment, mechanical preventive, and therapeutic procedures (Marsh and Bradshaw, 1993; ten Cate and Marsh, 1994; Newman, 1996). The active ingredients present in the mouthwashes that are most commonly used in the oral cavity include: chlorhexidine, combinations of essential oils (EO), triclosan, cetylpyridinium chloride, and various metal salts such as zinc compounds and stannous fluoride. Of all of these, chlorhexidine mouthwashes are considered to be the gold standard, as they have thus far been the most effective in microbiological and clinical studies (McDonnell and Russell, 1999; Tomás et al., 2008; von Ohle et al., 2010). However, their well-known undesirable secondary effects, mainly after regular use (Van Strydonck et al., 2012), have led to the scientific community exploring the existence of effective alternatives, especially when continuous daily use is required. Accordingly, EO have been found to be as effective as chlorhexidine at controlling gingival inflammation after 6 months of use, although the latter performs better at reducing plaque levels (Van Leeuwen et al., 2011; Neely, 2012). EO are composed of a wide variety of products. As a consequence, their antimicrobial activity is related to their composition, configuration, amount, and possible interactions (Lis-Balchin et al., 1998). The traditional formulation containing EO (T-EO) (Listerine R MentolTM, Johnson & Johnson)1 are a complex mix of phenolic compounds combined with various EO: 0.092% eucalyptol, 0.064% thymol, 0.06% methylsalicylate, and 0.042% menthol. All of these are included in a hydroalcoholic vehicle containing between 21.6 and 26.9% alcohol (Fine, 1988). As a result, T-EO contains ethanol, which is a chemical compound used to dissolve and stabilize the numerous substances present in the rinse. The concentration of ethanol present in the T-EO rinses is more than 20%. Such amounts have been found to be sufficient to dissolve the EO, but insufficient when it comes to having a direct antibacterial effect (Sissons et al., 1996; Marchetti et al., 2009). In fact, the manufacturer presents the alcohol contained in the rinse (21.6%) as being, inter alia, an inactive ingredient in its formula. Over the years, the use of ethanol in mouthwashes, as well as their effects on the surfaces of composite restorations (Penugonda et al., 1994) and their possible role in the development of oropharyngeal cancer, have been discussed (Smigel, 1991; Llewelyn, 1994). A direct cause-and-effect correlation between the development 1Listerine Cool Mint Composition. Available online at: https://www.listerine.com/ mouthwash/antiseptic/listerine-cool-mint-mouthwash (Accessed on September 14, 2017). Listerine Cool Mint Zero alcohol composition. Available online at: https://www.listerine.com/products/listerine-zero/listerine-cool-mint-zeroalcohol#product-overview (Accessed on November 1, 2017). of oropharyngeal carcinoma and the use of alcohol-containing rinses has not been demonstrated (Moazzez et al., 2011; Bagán et al., 2012; Gandini et al., 2012), and probably never will be (at least by epidemiological studies; Lachenmeier, 2012). However, it is considered desirable to eliminate ethanol from daily mouthwashes, especially for pediatric populations and patients at higher risk for oral cancer (McCullough and Farah, 2008; La Vecchia, 2009). Furthermore, the fact that the alcohol is present has meant that some clinical practitioners do not prescribe the T-EO due to this controversy (Vlachojannis et al., 2013). All of this has led to the development of a new alcoholfree formulation of EO (Af-EO) (Listerine R ZeroTM, Johnson & Johnson)1. The composition of Af-EO is the same regarding their active ingredients (eucalyptol, thymol, methyl salicylate, and menthol), but sodium fluoride has been added to the mixture. Some differences are found in their inactive ingredients. These are based on the alcohol contained in the T-EO solution, which is not present in the Af-EO rinse, and the presence of propylene glycol, sodium lauryl sulfate and sucralose in the Af-EO solution, but not the T-EO rinse. Two different concepts should be taken into account to measure the efficacy of antiseptics against dental plaque: the substantivity and the antiplaque effect. The substantivity of an oral antiseptic is defined as the prolonged adherence to oral surfaces (teeth and mucosa) and its slow release at effective doses which guarantee the continuation of the antimicrobial activity (Manau Navarro and Guasch Serra, 2003). The more substantivity an oral antiseptic has, the better. To study this in situ, the most popular models are those that analyze the effect that a single mouthwash has on a biofilm of more than 24 h (García-Caballero et al., 2013; Quintas et al., 2015b). The second concept that should be studied, the antiplaque effect, is defined as the capacity that an agent has to prevent the formation of bacterial aggregates (plaque) on oral surfaces. To study this in situ effect, models start from a baseline sample with levels of plaque near to zero in order to assess the power of the antiseptic when it comes to reducing the formation of bacterial plaque (normally dental plaque) compared to the control. A 6-month clinical study using a determinate antiplaque agent is necessary to tag an antiseptic as effective (Council on Dental Therapeutics, 1986). However, in the literature, there is an established model of 4 days of plaque regrowth with which authors can assess the inhibitory activity that different agents have; furthermore, this determines the relative efficacy of the different formulations as being predictive of the antiplaque effect of an antiseptic (Singh et al., 2013; Quintas et al., 2015a). In addition, another important methodological aspect in the in situ study of an oral antiseptic is the need to conserve the oral biofilm intact at all stages, namely the formation, collection and analysis of the oral samples. The goal is to not interfere with the delicate three-dimensional structure of the oral biofilm, which has been proved to be essential in terms of the resistance to the effects of an antimicrobial agent (Wood et al., 2000; Beyth et al., 2010). For these reasons, the use of intraoral disks held in specially designed apparatus for biofilm formation combined with the application of confocal laser scanning microscopy has Frontiers in Microbiology | www.frontiersin.org 2November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm proved to be extremely valuable when it comes to analyzing the oral biofilm in its intact, hydrated natural state (Dong et al., 2010; Gosau et al., 2010; Gu et al., 2012; García-Caballero et al., 2013; Quintas et al., 2015a,b). As Af-EO solution has come to the market, it seems appropriate to compare their antibacterial effects to those of traditional mouthwashes. Although, there are some studies evaluating these effects of T-EO and Af-EO (Marchetti et al., 2009, 2011, 2017a,b; Charles et al., 2012; Pizzo et al., 2013; Ulkur et al., 2013), none of them have assessed and compared their substantivity and antiplaque impact in an in situ model of a nondestructured oral biofilm (PL-biofilm). For this reason, the aim of the present study was to compare the in situ antibacterial activity (immediate effect, substantivity and antiplaque effect) of EO with and without alcohol on the PL-biofilm. MATERIALS AND METHODS This research is a randomized, double-blind, crossover study of the antibacterial and antiplaque efficacy of two available EO solutions: a traditional formula of EO with alcohol (T-EO) and an alcohol-free formula of EO (Af-EO). The supporting CONSORT checklist is available as supporting information (Supplementary Table 1). The study received the approval of the Clinical Research Ethics Committee of Galicia (number 2014/008) and was registered at clinicaltrials.gov with the number NCT03146390. URL: https://clinicaltrials.gov/ct2/show/NCT03146390. The “a priori” sample size calculation was performed using the program G∗Power 3.1.5 (Faul et al., 2007). The following statistical criteria were established: (1) an effect size of 0.7; (2) an alpha error of 0.05; and (3) a statistical power of 80%. A sample size of 19 subjects was required by these criteria and the application of the Wilcoxon signed-rank test to analyze the differences in the microscopic parameters between two rinsing protocols. The participants were selected among dental students at the School of Medicine and Dentistry of Santiago de Compostela (Universidade de Santiago de Compostela, Spain), where volunteer enrollment was sought by inviting responses to advertisements displayed in the faculty hall asking for participation in a research study. All these volunteers were assessed by the same trained clinician to ensure that they fulfilled all the inclusion and exclusion criteria that were applied in our group’s previous publications (García-Caballero et al., 2013; Prada-López et al., 2015a,b; Quintas et al., 2015a,b). The inclusion criteria were the following: systemically healthy adult volunteers aged between 20 and 45 with a good oral health status, namely a minimum of 24 permanent teeth with no evidence of gingivitis or periodontitis (Community Periodontal Index score =0; World Health Organization, 2013) and an absence of untreated caries at the start of the study. The following exclusion criteria were applied: smoker or former smoker, the presence of dental prostheses or orthodontic devices, antibiotic treatment, or routine use of oral antiseptics in the previous 3 months, and the presence of any systemic disease that could alter the production or composition of saliva. Before the start of each test or experiment, a full mouth scaling with ultrasonic instruments and teeth polishing with a rubber cup after dental disclosure were performed by the same trained clinician on all the selected participants (Figure 1). Written informed consent was obtained from all the volunteers. To achieve the aims of the study, all the participants performed two different tests. To test the antibacterial activity of the two EO solutions, an in situ model of PL-biofilm growth was used. An individualized thermoplastic splint called intraoral disk-holding splint (IDODS) (García-Caballero et al., 2013; Prada-López et al., 2013, 2015a,b,c, 2016; Quintas et al., 2015a,b) with a capacity to hold a total of six glass disks was made for each of the volunteers. Test 1 (Substantivity) The first experiment consisted of evaluating the immediate antibacterial effect and substantivity of the T-EO and Af-EO solutions. The volunteers wore an IDODS for 48 h to enable growth of the PL-biofilm. They were allowed to remove it from the oral cavity only during meals and to perform oral hygiene measures (when it had to be stored in a provided opaque container in humid conditions). In order to not disturb the formation of the PL-biofilm, the volunteers could not use any toothpaste or mouthwash as a complement to the mechanical removal of bacterial plaque. After 48 h, the glass disks were withdrawn one by one from the splint from each volunteer (from right to left in a distalmesial direction) at baseline, 30 s, and 1, 3, 5, 7h after performing the different mouthwashes. These mouthwashes were practiced under the investigator’s supervision with the IDODS present in the oral cavity, and they were: 1A) A single, 30-s mouthwash with 20 mL of sterile water (negative control) (M-WATER). -OR 1B) A single, 30-s mouthwash with 20 mL of a traditional EO formulation (Listerine R MentolTM, Listerine R , Johnson & Johnson, Madrid, Spain) (M-T-EO). -OR 1C) A single, 30-s mouthwash with 20 mL of an alcoholfree EO solution (Listerine R ZEROTM, Listerine R , Johnson & Johnson, Madrid, Spain) (M-Af-EO). On the day of the experiment, the volunteers were not allowed to eat or drink during the tests. Collection of the different PL-biofilm samples started at 11:50 a.m. (baseline sample) and finished at 7:00 p.m. (the final sample was obtained 7 h after using the mouthwash). Using an internet-based, balanced randomization system (Dallal)2, which indicated the mouthwash that each subject should use first, second and third, all the volunteers used the three mouthwashes, with a rest period of 2 weeks between each application (Figure 1). Test 2 (Antiplaque Effect) The second experiment consisted of evaluating the antiplaque effect of both EO formulas. During 96 h, each volunteer wore the splints with the glass disks, withdrawing them from their oral cavity only during meals (they were stored in an opaque 2Available online at: www.randomization.com (Accessed on September 03, 2017). Frontiers in Microbiology | www.frontiersin.org 3November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm FIGURE 1 | Protocol of the study. container in humid conditions) and to perform oral hygiene procedures involving only the mechanical removal of bacterial plaque with water, without the use of any toothpaste or mouthwash. Using the permitted mechanical oral hygiene measures (without the IDODS), the volunteers performed the different protocols based on the manufacturers’ instructions (with the IDODS in the oral cavity) over 4 days in the morning (8.30) Frontiers in Microbiology | www.frontiersin.org 4November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm after breakfast and at night (22.00) after dinner. These protocols were: 2A) A 30-s mouthwash with 20 mL of sterile water (negative control) (4D-WATER). -OR 2B) A 30-s mouthwash with 20 mL of a traditional EO formulation (Listerine R MentolTM, Listerine R , Johnson & Johnson, Madrid, Spain) (4D-T-EO). -OR 2C) A 30-s mouthwash with 20 mL of an alcohol-free EO solution (Listerine R ZEROTM, Listerine R , Johnson & Johnson, Madrid, Spain) (4D-Af-EO). The collection of the samples was carried out individually at 8 a.m. in the morning so that those of each volunteer were analyzed on different days. It was determined that a minimum of 10 h should have elapsed since the last use of the mouthwash the previous night. In this test, mouthwashes carried out by the volunteers were not supervised, but they were instructed to use a measured volume of the allocated solution. To assess the subject’s compliance with the rinsing protocol, the bottles containing the rinse were weighed before they were given to the volunteers. After the 4-day period, they were asked to bring the bottles back with the remaining mouthwash, and these were weighed again. Using an internet-based, balanced randomization system (Dallal), which indicated the rising protocol that each subject should use first, second and third, all the volunteers used the three regimes, with a rest period of 2 weeks between each protocol (Figure 1). Processing of the Samples of the PL-Biofilm As the glass disks were removed from the splint, they were immediately immersed in 100 µL of a fluorescence solution of LIVE/DEAD R BacLightTM and kept in a dark chamber at room temperature for 15 min. Microscope observations were 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. Four fields considered to be representative of the entirety of the samples were selected by an observer who was blind to the study’s conditions. 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 biofilm thickness of each field was divided into three zones or equivalent layers: the outer layer (layer 1), the middle layer (layer 2), and the inner layer (layer 3). The capture of the data was done with the same settings in all cases. The spatial scan mode (XYZ) and the 1,024 ×1,024 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 400 Hz, respectively. The only values that were different depending on the sample were the offset (range between −1 and 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. The quantification of bacterial viability (BV) in the series of XY images was determined using a cytofluorographic analysis (Leica confocal software). In this analysis, the images of each fluorochrome were defined as “channels” (SYTO 9 occupies the green channel and Propidium Iodide the red channel). To conduct the computations automatically, a Matlab toolbox called Dentius Biofilm was developed. The main program reads all the images from an experiment, organized in a folder tree, with the image folder at the top, the experiment folder below this and all the patient folders at the bottom. The program automatically computes the number of disks, fields and 1 µm sections from the images stored in each patient’s folder. The program considers the parameters fixed by experts: the BV is characterized by a high value in the green channel (over 100, with a range between 0 and 255) and a low value in the red channel (below 100). Bacteria are considered not to be viable if the values are high in the red channel (over 100) and low in the green channel (below 100). Values that are high in both channels (over 100) are visually orange and are considered nonviable bacteria. The program counts the number of pixels under these conditions to compute the BV percentage for each 1 µm section (viable bacteria/viable bacteria +non-viable bacteria × 100). Determination of the mean BV percentage in each field required sections with a minimum biofilm area of 250 µm2 (∼4,750 pixels). The program also considers the case where epithelial nuclei are present. These are characterized by compact red areas with a size that is greater than the bacteria, and these red points must not be counted as non-viable bacterial population. To eliminate these pixels, the program disregards epithelial cells, which are characterized by: having a high value in the red channel, an area >200 pixels, compact regions with a solidity >0.7, and a minimum value of the mean intensity of 180. These parameters were fixed using a training set. With this methodology, there could be some misdetections, but the effect on the BV was very low, as what was important was the elimination of large areas with a high intensity. All the results obtained for each section, field and disk from each patient were stored in a worksheet to be analyzed by the researchers. The BV percentage was also stored before and after eliminating the epithelial nuclei and their properties to localize them over the image. Frontiers in Microbiology | www.frontiersin.org 5November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm The mean BV percentage of the biofilm was calculated for the corresponding sample and for each biofilm layer. In Test 2, apart from the thickness and BV, the covering grade (CG) was also assessed. This parameter is the percentage of the surface substrate covered by the biofilm. The cytofluorogram itself was used for this purpose. From the maximum projection (superposition of all captured planes) of each of the analyzed fields, the CG percentage was obtained by calculating the sum of the bacterial mass (viable and non-viable) with regard to the total surface of the field (% positive within the total area). Statistical Analysis The statistical analyses were performed using the R software (R Core Team, 2016). The Shapiro–Wilk test was performed to analyze the distribution of the quantitative variables associated with the PLbiofilm (thickness, BV and CG), showing mostly these microscopic parameters a non-normal distribution in both tests. In Test 1 (substantivity) and Test 2 (antiplaque effect), the Friedman test was used for intra-mouthwash and intermouthwash comparisons using all the PL-biofilm samples (including differentiating between the three biofilm layers). In both tests, the Wilcoxon signed-rank test was used for pairwise comparisons (with Bonferroni adjustment) of the intraand inter-mouthwash results (including differentiating between the three biofilm layers). The significance level established was a P<0.05. In the Test 1, the Bonferroni-corrected P-values applied were <0.003 and <0.016, and in the Test 2, this value was <0.016. RESULTS A total of 30 volunteers were evaluated to obtain the calculated sample size (n=19). When this number of participants meeting the inclusion and exclusion criteria was achieved, the enrollment process was ended. A total of 11 subjects were ineligible as they did not meet all of the inclusion criteria. All the participants performed both of the tests, although a subject was excluded after performing the Test 1 for an unexpected event (Supplementary Table 2, CONSORT flow diagram). No adverse effects were reported by them at any stage of the experiment. Eighteen subjects completed the rinsing protocols satisfactorily in both tests. In Test 2, the returns of each product suggested good compliance with the instructions. Test 1 (Substantivity) Influence of a Single Mouthwash of T-EO and Af-EO on the Thickness of the PL-Biofilm Neither the T-EO nor the Af-EO antiseptics could reduce the thickness of the PL-biofilm of 48 h after a single application. Their baseline thicknesses were 21.81 ±5.28 µm and 20.71 ± 4.13 µm, respectively. After a single mouthwash, the thicknesses were slightly reduced (20.19 ±3.62 µm and 18.58 ±3.14 µm, respectively), but did not achieve statistical significance. Influence of a Single Mouthwash of T-EO and Af-EO on the Bacterial Viability of the PL-Biofilm The mean BV at baseline ranged between 63.99 ±19.7% and 79.54 ±5.31% for all the three rising protocols, with no statistical differences between them. Both EO formulations achieved similar results at all the time points measured. In fact, no differences were found between them from the immediate sample (30 s) to the 7-h sample (Figure 2). The EO formulations were FIGURE 2 | Percentages of bacterial viability of the PL-biofilm in baseline conditions, at 30 s, 1, 3, 5, and 7 h after a single mouthwash with sterile water (M-WATER), with essential oils with alcohol (M-T-EO), and with essential oils without alcohol (M-Af-EO). *Statistically significant differences in regard to the 30-s sample (P<0.003). +Statistically significant differences in regard to the baseline (P<0.003). Frontiers in Microbiology | www.frontiersin.org 6November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm effective at reducing the BV after a single mouthwash with respect to their respective baseline levels (BV at 30 s for M-T-EO and M-Af-EO =6.53 ±7.60% and 4.13 ±3.89%, respectively; P< 0.001). These results were statistically lower than those from the M-WATER (62.39 ±8.17%; P<0.001). Both solutions were able to keep the BV under baseline levels for 7 h (BV at 7 h for M-T-EO and M-Af-EO =18.20 ±9.38% and 20.10 ±10.27%, respectively; P<0.001). Again, these findings were statistically lower than those from the M-WATER (BV at 7 h =76.78 ±4.40%; P<0.001; Figures 2,3). In terms of BV recovery concerning the 30-s sample, significant recovery was not achieved until 7 h after the use of an M-T-EO rinse (BV at 30 s vs. at 7 h for M-EO =6.53 ±7.60% vs. 18.20 ±9.38%; P<0.001). However, for the M-Af-EO solution, FIGURE 3 | Representative images of the obtained bacterial viabilities at 30 s, 3 and 7 h after a single mouthwash with sterile water (M-WATER), traditional essential oils (M-T-EO), and alcohol-free essential oils (M-Af-EO). Frontiers in Microbiology | www.frontiersin.org 7November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm significant recovery was identified in the 3-h sample (BV at 30 s vs. at 3 h for M-Af-EO =4.13 ±3.89% vs. 12.35 ±8.86%; P< 0.001; Figure 2). When it comes to differentiating between the three biofilm layers, the two EO antiseptics had lower BV levels in all the layers (Table 1). No significant differences were found in BV for the same biofilm layer between the EO formulations, with the outer layers being generally more viable than the inner ones in all the samples. There were no significant differences between the three layers for the M-T-EO rinse (BV at 30 s by layers =6.67 ±6.80% vs. 5.63 ±8.15% vs. 7.29 ±9.17%, respectively; P>0.016) or between the deeper layers for the M-Af-EO rinse (BV at 30 s by layers 2 and 3 =3.22 ±3.24% vs. 3.51 ±4.94%, respectively; P> 0.016; Table 1). Test 2 (Antiplaque Effect) Influence of a 4-Day Protocol of T-EO and Af-EO Mouthwashes on the Thickness and Covering Grade of the PL-Biofilm The Af-EO rinses were more effective than the T-EO formulation at reducing the thickness of the oral biofilm after 4 days of use (thickness for 4D-T-EO vs. 4D-Af-EO =9.92 ±2.87 µm vs. 7.90 ±2.91 µm; P=0.012), but both solutions were more powerful than the negative control (thickness for 4D-WATER =22.76 ± 6.21 µm; P<0.001; Table 2 and Figure 4). The Af-EO rinses were more effective than the T-EO solution at reducing the CG of the oral biofilm after 4 days of use (CG for 4D-T-EO vs. 4D-Af-EO =46.61 ±19.12% vs. 33.36 ±12.01%, respectively; P=0.001). The two EO solutions were significantly more effective than the negative control at reducing the CG (CG for 4D-WATER =73.92 ±17.49%; P<0.001; Table 2 and Figure 5). Influence of a 4-Day Protocol of T-EO and Af-EO Mouthwashes on the Bacterial Viability of the PL-Biofilm The T-EO and Af-EO formulations after 4 days of use showed similar effectiveness in maintaining the BV at significantly lower levels than the negative control (BV for 4D-T-EO and 4D-Af-EO vs. 4D-WATER =26.27 ±14.61% and 31.08 ±16.52% vs. 51.35 ±5.38%, respectively; P<0.001; Table 2). In terms of the BV by layers, the outer layers had significantly more BV than the inner ones in all the experiments. The T-EO and Af-EO formulations were significantly more effective at reducing the BV of layers 1 and 2 than the negative control, with layer 3 unaltered (BV for layer 1 =40.10 ±17.31% and 39.81 ±19.09% vs. 82.47 ±7.58%, respectively; BV for layer 2 =24.32 ±16.16% and 30.73 ±17.06% vs. 51.76 ±13.53%, respectively; P<0.001 for all comparisons; Table 3). DISCUSSION Methodology Approach This is the first study to compare the antibacterial activity in vivo that T-EO and Af-EO solutions have on the non-destructured oral biofilm. From a methodological perspective, to perform all the BV computations automatically through a Matlab toolbox called Dentius Biofilm ensures that the experiment’s findings are accurate, quick to obtain, reliable, and repeatable, which is very important when it comes to comparing results and extracting robust conclusions. In the substantivity study (test 1), mean BV data of each subject were obtained from analyzing ∼160 sections of 1 µm (around 40 sections per field X four fields X one disk) for each evaluated moment (six-time moments: baseline, 30 s, 1, 3, 5, and 7 h). In the antiplaque study (test 2), when the negative control was applied, the mean BV values of each subject were obtained from evaluating ∼1,200 sections (around 50 sections per field X four fields X six disks); when antiseptic solutions were used, the mean BV results of each subject were derived from the analysis of ∼360 sections (around 15 sections per field X four fields X six disks). In the present series, the BV was assessed by the Live/Dead R  BacLightTM fluorescence assay. This solution stains the bacteria in red or green depending on the permeability of their membrane (propidium iodide which stains the cell in red only if the membrane permeability is altered). Given that the tested antiseptics act mostly at this cellular element, this viability method may be suitable for this type of study. Although, firstly conceived as a technique only valid for BV assessment of single species model (Invitrogen communication), its simplicity and good results also in in situ studies containing multiple bacterial species (Boulos et al., 1999; Ihalin et al., 2003; Tomás et al., 2009; Beyth et al., 2010; Dong et al., 2010; Gosau et al., 2010; GarcíaCaballero et al., 2013; Tawakoli et al., 2013; Prada-López et al., 2015a,b; Quintas et al., 2015a,b) has produced the manufacturer to make a recommendation also for multiple species models (Live/Dead R BacLightTM User Manual)3. Despite this, there has been some discussion about the reliability of this technique (Hannig et al., 2010; Tawakoli et al., 2013; Netuschil et al., 2014), mainly due to the fact that not only red and green bacteria appear in the analysis, but also orange regions appear with “unknown” interpretation (Berney et al., 2007). Hannig et al. (2010) considered that live/dead staining methods were reliable when evaluating antimicrobial agents activity. However, they continued to ask the question about “how dead is dead?” due to several stages of vitality which have been discussed and described in the literature (viable and culturable, viable but non-culturable, dormant, non-viable and pre-lytic, and avital dead bacteria). The exact differentiation of these stages is still one of the greatest challenges in modern microbiology (Decker, 2001). Several attempts have been made to compare the Live/Dead R  BacLightTM with the gold standard -traditional plaque cultures - (Boulos et al., 1999; Ihalin et al., 2003; Tomás et al., 2009; Tawakoli et al., 2013). One of these was performed several years ago by the authors (Tomás et al., 2009) in a study on the in situ substantivity of the chlorhexidine on the salivary bacteria. The mean BV obtained was compared with plaque cultures. A good correlation was observed between both techniques in the baseline and immediate samples. However, this correlation 3Live/Dead R BacLightTM User Manual. Available online at: https://tools. thermofisher.com/content/sfs/manuals/mp07007.pdf (Accessed on September 30, 2017). Frontiers in Microbiology | www.frontiersin.org 8November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm TABLE 1 | Bacterial viability in 2-day PL-biofilm under basal conditions and in the samples collected at 30 s and 1, 3, 5, and 7 h after a single mouthrinse with: sterile water, traditional essential oils solution, and alcohol-free essential oils solution. BASAL 30 S 1 H 3 H 5 H 7 H BACTERIAL VIABILITY (%) OF THE 2-DAY PL-BIOFILM; MEAN ±STANDARD DEVIATION, MEDIAN (INTERQUARTILE RANGE) M-WATER Layer 1 (outer layer) 86.82 ±3.57 87.51 (2.18) 81.86 ±6.63 80.48 (8.15) 85.46 ±5.78 83.31 (6.89) 89.11 ±6.13 91.90 (8.74) 86.26 ±3.76 87.36 (6.29) 90.75 ±3.45 89.54 (4.58) Layer 2 (middle layer) 82.06 ±5.02 (80.48 (3.87) 69.21 ±7.83 68.53 (8.15) 73.94 ±9.79 75.54 (6.02) 82.75 ±5.98 80.91 (8.06) 77.20 ±8.07 78.34 (11.84) 83.07 ±4.25 84.73 (5.48) Layer 3 (inner layer) 69.74 ±16.21 73.69 (22.91) 36.08 ±20.97 31.09 (26.52) 44.18 ±14.80 44.34 (30.87) 50.12 ±13.22 56.27 (15.80) 48.95 ±17.78 52.45 (32.48) 56.53 ±12.59 60.34 (9.10) M-T-EO Layer 1 (outer layer) 75.54 ±17.28 75.53 (19.71) 6.67 ±6.80 4.44 (6.06) 9.90 ±17.56 4.16 (4.80) 18.49 ±14.37 16.36 (20.45) 22.35 ±14.73 15.56 (28.93) 35.42 ±19.00 35.55 (31.05) Layer 2 (middle layer) 67.86 ±21.95 75.91 (32.06) 5.63 ±8.15 2.77 (3.53) 7.97 ±18.16 1.69 (4.65) 8.04 ±10.79 3.68 (7.61) 8.58 ±7.78 5.45 (6.14) 13.26 ±9.09 9.91 (11.96) Layer 3 (inner layer) 48.56 ±28.37 51.87 (32.67) 7.29 ±9.17 1.72 (12.59) 9.02 ±17.95 1.35 (6.06) 5.81 ±9.26 0.52 (7.15) 4.76 ±7.62 1.37 (4.29) 5.92 ±9.21 2.56 (5.05) M-Af-EO Layer 1 (outer layer) 84.74 ±15.33 90.03 (9.74) 5.66 ±5.72 3.88 (6.75) 10.01 ±8.30 8.82 (10.24) 20.74 ±14.73 18.56 (12.87) 27.69 ±18.15 21.85 (21.27) 41.99 ±18.96 46.53 (27.42) Layer 2 (middle layer) 76.37 ±19.38 81.93 (27.11) 3.22 ±3.24 1.76 (5.33) 4.04 ±4.25 2.46 (4.84) 9.70 ±8.52 7.62 (10.00) 12.67 ±10.22 8.32 (14.72) 14.28 ±11.27 11.35 (14.96) Layer 3 (inner layer) 51.95 ±31.30 59.06 (56.86) 3.51 ±4.94 2.01 (3.13) 1.23 ±1.18 0.98 (1.30) 6.63 ±8.39 4.23 (7.16) 6.53 ±7.06 3.38 (8.52) 4.02 ±6.50 1.50 (4.27) BACTERIAL VIABILITY, INTRA-MOUTHWASH ANALYSIS; STATISTICAL SIGNIFICANCE M-WATER Layer 1 vs. Layer 2 P<0.016 P<0.016 P<0.016 — P<0.016 P<0.016 Layer 1 vs. Layer 3 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 Layer 2 vs. Layer 3 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 M-T-EO Layer 1 vs. Layer 2 P<0.016 — P<0.016 P<0.016 P<0.016 P<0.016 Layer 1 vs. Layer 3 P<0.016 — — P<0.016 P<0.016 P<0.016 Layer 2 vs. Layer 3 P<0.016 — — P<0.016 — P<0.016 M-Af-EO Layer 1 vs. Layer 2 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 Layer 1 vs. Layer 3 P<0.016 — P<0.016 P<0.016 P<0.016 P<0.016 Layer 2 vs. Layer 3 P<0.016 — P<0.016 P<0.016 P<0.016 P<0.016 BACTERIAL VIABILITY, INTER-MOUTHWASH ANALYSIS; STATISTICAL SIGNIFICANCE M-WATER vs. M-T-EO Layer 1 vs. Layer 1 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 Layer 2 vs. Layer 2 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 Layer 3 vs. Layer 3 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 M-WATER vs. M-Af-EO Layer 1 vs. Layer 1 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 Layer 2 vs. Layer 2 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 Layer 3 vs. Layer 3 — P<0.016 P<0.016 P<0.016 P<0.016 P<0.016 M-T-EO vs. M-Af-EO Layer 1 vs. Layer 1 — — — — — — Layer 2 vs. Layer 2 — — — — — — Layer 3 vs. Layer 3 — — — — — — Differences between the three biofilm layers, as well as intra-mouthrinse and inter-mouthrinse comparisons. M-WATER, A single, 30-s mouthwash with 20 mL of sterile water; M-T-EO, A single, 30-s mouthwash with 20 mL of a traditional essential oils solution; M-Af-EO, A single, 30-s mouthwash with 20 mL of an alcohol-free essential oils solution. —, no statistical significance. Frontiers in Microbiology | www.frontiersin.org 9November 2017 | Volume 8 | Article 2162 Quintas et al. Essential Oils Activity on Biofilm Pizzo, G., Compilato, D., Di Liberto, B., Pizzo, I., and 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. Pizzo, G., La Cara, M., Licata, M. E., Pizzo, I., and 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 Prada-López, I., Quintas, V., Casares-De-Cal, M. A., Suárez-Quintanilla, J. A., Suárez-Quintanilla, D., and Tomás, I. (2015a). Ex vivo vs. in vivo antibacterial activity of two antiseptics on oral biofilm. Front. Microbiol. 6:655. doi: 10.3389/fmicb.2015.00655 Prada-López, I., Quintas, V., Donos, N., Álvarez-Fernández, M., and Tomás, I. (2013). “In situ substantivity of the essential oils in the oral cavity,” in Microbial Pathogens and Strategies for Combating Them: Science, Technology and Education, ed A. Méndez-Vilas (Badajoz: Formatex Research Center), 1112–1122. Prada-López, I., Quintas, V., Donos, N., Suárez-Quintanilla, D., and Tomás, I. (2015b). Characteristics of in situ oral biofilm after 2 and 4 days of evolution. Quintessence Int. 46, 287–298. doi: 10.3290/j.qi.a33402 Prada-López, I., Quintas, V., and Tomás, I. (2015c). The intraoral device of overlaid disk-holding splints as a new in situ oral biofilm model. J. Clin. Exp. Dent. 7, e126–e132. doi: 10.4317/jced.52093 Prada-López, I., Quintas, V., Vilaboa, C., Suárez-Quintanilla, D., and Tomás, I. (2016). Devices for in situ development of non-disturbed oral biofilm. A systematic review. Front. Microbiol. 7:1055. doi: 10.3389/fmicb.2016. 01055 Pratten, J., Barnett, P., and Wilson, M. (1998). Composition and susceptibility to chlorhexidine of multispecies biofilms of oral bacteria. Appl. Environ. Microbiol. 64, 3515–3519. Quintas, V., Prada-López, I., Donos, N., Suarez-Quintanilla, D., and Tomás, I. (2015a). 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:e0117177. doi: 10.1371/journal.pone.0117177 Quintas, V., Prada-López, I., Prados-Frutos, J. C., and Tomás, I. (2015b). In situ antimicrobial activity on oral biofilm: essential oils vs. 0.2 % chlorhexidine. Clin Oral Investig 19, 97–107. doi: 10.1007/s00784-0141224-3 R Core Team (2016). R: A Language and Environment for Statistical Computing. Vienna: R foundation for statistical computing. Available online at: http://www. R-project.org (Accessed on July 30, 2017). Robinson, C., Strafford, S., Rees, G., Brookes, S. J., Kirkham, J., Shore, R. C., et al. (2006). Plaque biofilms: the effect of chemical environment on natural human plaque biofilm architecture. Arch. Oral Biol. 51, 1006–1014. doi: 10.1016/j.archoralbio.2006.04.010 Rosin, M., Welk, A., Kocher, T., Majic-Todt, A., Kramer, A., and Pitten, F. A. (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. doi: 10.1034/j.1600-051X.2002.290503.x Singh, A., Daing, A., and 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 Sissons, C. H., Wong, L., and Cutress, T. W. (1996). Inhibition by ethanol of the growth of biofilm and dispersed microcosm dental plaques. Arch. Oral Biol. 41, 27–34. doi: 10.1016/0003-9969(95) 00103-4 Smigel, K. (1991). High-alcohol mouthwashes are under scrutiny. J. Natl. Cancer Inst. 83:751. doi: 10.1093/jnci/83.11.751 Tawakoli, P. N., Al-Ahmad, A., Hoth-Hannig, W., Hannig, M., and 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 ten Cate, J. M., and Marsh, P. D. (1994). Procedures for establishing efficacy of antimicrobial agents for chemotherapeutic caries prevention. J. Dent. Res. 73, 695–703. doi: 10.1177/00220345940730031601 Tomás, I., Cousido, M. C., Tomás, M., Limeres, J., García-Caballero, L., and Diz, P. (2008). In vivo bactericidal effect of 0.2% chlorhexidine but not 0.12% on salivary obligate anaerobes. Arch. Oral Biol. 53, 1186–1191. doi: 10.1016/j.archoralbio.2008.07.009 Tomás, I., García-Caballero, L., Cousido, M. C., Limeres, J., Álvarez, M., and Diz, P. (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 Ulkur, F., Arun, T., and 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 Van Leeuwen, M. P., Slot, D. E., and Van der Weijden, G. A. (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 Van Strydonck, D. A., Slot, D. E., Van der Velden, U., and 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 Vlachojannis, C., Chrubasik-Hausmann, S., Hellwig, E., and Al-Ahmad, A. (2015). A preliminary investigation on the antimicrobial activity of Listerine(R), its components, and of mixtures thereof. Phytother. Res. 29, 1590–1594. doi: 10.1002/ptr.5399 Vlachojannis, C., Winsauer, H., and Chrubasik, S. (2013). Effectiveness and safety of a mouthwash containing essential oil ingredients. Phytother. Res. 27, 685–691. doi: 10.1002/ptr.4762 von Ohle, C., Gieseke, A., Nistico, L., Decker, E. M., DeBeer, D., and Stoodley, P. (2010). Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine. Appl. Environ. Microbiol. 76, 2326–2334. doi: 10.1128/AEM.02090-09 Waaler, S. M., Rolla, G., Skjorland, K. K., and Ogaard, B. (1993). Effects of oral rinsing with triclosan and sodium lauryl sulfate on dental plaque formation: a pilot study. Scand. J. Dent. Res. 101, 192–195. doi: 10.1111/j.1600-0722.1993.tb01103.x Wood, S. R., Kirkham, J., Marsh, P. D., Shore, R. C., Nattress, B., and Robinson, C. (2000). Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy. J. Dent. Res. 79, 21–27. doi: 10.1177/00220345000790010201 Wood, S. R., Kirkham, J., Shore, R. C., Brookes, S. J., and Robinson, C. (2002). Changes in the structure and density of oral plaque biofilms with increasing plaque age. FEMS Microbiol. Ecol. 39, 239–244. doi: 10.1111/j.1574-6941.2002.tb00926.x World Health Organization (2013). Oral Health Surveys: Basic Methods. World Health Organization. Zaura-Arite, E., van Marle, J., and ten Cate, J. M. (2001). Conofocal microscopy study of undisturbed and chlorhexidine-treated dental biofilm. J. Dent. Res. 80, 1436–1440. doi: 10.1177/00220345010800051001 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2017 Quintas, Prada-López, Carreira, Suárez-Quintanilla, BalsaCastro and Tomás. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 16 November 2017 | Volume 8 | Article 2162