In Situ Chlorhexidine Substantivity on Saliva and Plaque Like Biofilm: Influence of Circadian Rhythm

In Situ Chlorhexidine Substantivity on Saliva and Plaque Like Biofilm: Influence of Circadian Rhythm This document is a pre print version Article Published in Journal of Periodontology November, 2013 on You can find the original article at the following link: http://www.joponline.org/doi/abs/10.1902/jop.2013.120475?url_ver=z39.88 2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dpubmed&

TITLE: IN SITU CHLORHEXIDINE SUBSTANTIVITY ON SALIVA AND PLAQUE-LIKE BIOFILM: INFLUENCE OF CIRCADIAN RHYTHM RUNNING TITLE: CHX SUBSTANTIVITY AND CIRCADIAN RHYTHM AUTHORS: Tomás I, DDS, PhD*, García-Caballero L, DDS,PhD*, López-Alvar E, DDS*, Suárez M, DDS, PhD*, Seoane J, MD,DDS,PhD* *Department of Stomatology. School of Medicine and Dentistry. Santiago de Compostela University. Santiago de Compostela, Spain Summary sentence: Our results support the greater physiologic dynamics of the salivary flora and the possible reservoir function of de novo PL-biofilm. Source of funding This work was supported by project FIS 2011/PF004 (PI 11/01383) from Institute of Health Carlos III, Madrid, Spain. Word count= 4028; number of figures= 2; number of tables= 3 CORRESPONDING AUTHOR Inmaculada Tomás Carmona School of Medicine and Dentistry. Santiago de Compostela University C./ Entrerrios s/n 15872 Santiago de Compostela, Spain Tel: +34 981 563100 ext: 12344; Fax: +34 981 562226 e-mail: inmaculada.tomas@usc.es 1

ABSTRACT Background. To assess in situ substantivity of a single mouthrinse with 0.2% Clorhexidine (CHX) on saliva and on undisturbed de novo plaque-like biofilm (PL-biofilm), differentiating between 2 times of application: CHX mouthrinse in the morning and CHX mouthrinse at night. Methods. The study subjects were 10 healthy volunteers who wore an individualized splint with glass disks for 48 hours to boost the growth of PL-biofilm. Saliva samples were collected and 2 disks were removed from each volunteer s splint at 8, 10, and 12 hours after performing a mouthrinse with 0.2% CHX at 7 AM (M-0.2% CHX-diurnal) and 1 AM (M- 0.2% CHX-nocturnal). The saliva and plaque samples were analyzed by epifluorescence and confocal laser scanning microscopy, respectively, using SYTO 9/propidium iodide staining. Results. With M-0.2% CHX-diurnal, the frequency of vital bacteria in saliva was significantly higher than in the PL-biofilm at 8, 10, and 12 hours post-mouthrinse. After M- 0.2% CHX-nocturnal, the frequency of vital bacteria in saliva was significantly lower than in the PL-biofilm at 8 hours and higher than in the PL-biofilm at 12 hours post-mouthrinse. Conclusions. These results support the more active physiologic dynamics of the salivary flora and the possible reservoir function associated with the structure of undisturbed de novo PL-biofilm. Keywords: chlorhexidine; saliva; biofilm; fluorescence microscopy; confocal laser scanning microscopy; circadian rhythm 2

INTRODUCTION Study of the in situ antibacterial activity of an antiseptic involves analysis of its immediate effect and of its substantivity. Substantivity is defined as the prolonged adherence of the antiseptic to the oral surfaces (teeth and mucosas) and its slow release at effective doses that guarantee the persistence of its antibacterial activity 1. Many authors have demonstrated that, in situ, Chlorhexidine (CHX) has a greater immediate antibacterial effect and substantivity than other antiseptics used in the oral cavity 2-5. For this reason, the majority of the more recent microbiological studies have used 0.12% or 0.2% CHX as the positive control in the analysis of results obtained with other active substances on oral ecosystems 6-8. However, there still remain certain aspects of the antibacterial activity of CHX that have not been studied in sufficient depth. It has been shown that the retention of CHX in the oral cavity depends not only on the nature of the product, but also on intrinsic factors associated with the antiseptic (such as concentration, time, and temperature) and extrinsic factors (such as the presence or absence of teeth, dental prostheses, or organic material, the salivary ph, and the intake of food or drink) 9-11 that could affect its antibacterial activity. To date, few authors have studied the in situ antibacterial activity of a single mouthrinse with CHX on the oral ecosystem, analyzing the influence of factors intrinsic and extrinsic to the mouthrinse 12-14. Since the first results reported by Schiött et al in 1970 15, many published studies have used salivary bacterial counts to evaluate the substantivity of CHX on the salivary flora 2-5. Although some authors have questioned the reliability of these methods and have proposed alternatives, such as the use of fluorescent methods that use specific fluorochromes to mark live and dead bacteria 16,17, there are few studies published in the literature in which the effects of antibacterial agents, including CHX, on the salivary flora have been investigated using epifluorescence techniques 18-20. In vitro research has demonstrated that the plaque-like (PL-biofilm) presents greater resistance to antibacterial agents because of its slow rate of growth, the difficulty of penetrating its structure, and possible inactivation of the agent within the biofilm 21. At present, the scientific community considers that the methodological design based on the use of special removable appliances (including disks) to obtain biofilm samples, and analysis of the samples by confocal laser scanning microscopy (in combination with other microscopic and microbiological techniques), is the most suitable approach for studying the in situ architecture and physiology of undisturbed PL-biofilm that forms on the oral 3

surfaces, as well as the antibacterial effect of antibacterial agents on this microbial structure 21-23. However, there are few published studies that have investigated the effects of CHX on in situ undisturbed PL-biofilm using confocal laser scanning microscopy combined with techniques to determine bacterial vitality 24-27. The objective of the present study was to evaluate the in situ substantivity of a single mouthrinse with 0.2% CHX digluconate on the salivary flora and on non-destructured PLbiofilm, differentiating between 2 times of application (daytime mouthrinse versus nighttime mouthrinse). The methodology involved the use of epifluorescence microscopy techniques, confocal laser microscopy (CLSM), and SYTO 9/propidium iodide staining. MATERIAL AND METHODS This was a randomized, double-blind, crossover study of the antibacterial efficacy of CHX on an in situ model of PL-biofilm growth. Selection of the study group The study group was formed of 10 adult volunteers between 20 and 45 years of age and who presented a good oral health status: minimum of 24 permanent teeth with no evidence of gingivitis or periodontitis (Community Periodontal Index score=0) 28, and an absence of caries. The following exclusion criteria were applied: smoker, presence of dental prostheses or orthodontic devices, antibiotic treatment or routine use of oral antiseptics during the previous three months, and presence of any systemic disease that could alter the production or composition of the saliva. Scaling was performed on all volunteers before starting the study. This project was approved by the Ethics Committee of the Faculty of Medicine and Dentistry of the University of Santiago de Compostela. Written informed consent was obtained from all participants in the study. This study was conducted between October 2011 and March 2012 in the Faculty of Medicine and Dentistry of the University of Santiago de Compostela. Design of the disk-holding splint After considering a number of previously described in situ models 22,29, an individualized splint of the lower arch was created for each volunteer; the splints were able to hold 6 glass disks (6 mm diameter, 1 mm thickness) polished at 4000 grit. The splint was formed of two vinyl sheets, an internal sheet with a thickness of 1 mm that held and supported the 4

disks and an external sheet of 0.5 mm thickness that was fenestrated to permit contact of the vestibular surface of the disks with the saliva whilst protecting them from the action of the cheeks and tongue. Three disks were positioned on each hemiarch. The anterior disk (disk 1) was positioned between the distal part of the canine tooth and the mesial part of the first premolar; the middle disk (disk 2) was positioned between the distal part of the second premolar and the mesial part of the first molar; and the posterior disk (disk 3) was positioned between the distal part of the first molar and the mesial part of the second molar 30 (ES2380252B2) (Figure 1). The splint with the glass disks was worn by the volunteer for 48 hours to favor growth of the bacterial biofilm, withdrawing it from the oral cavity only during meals (it was stored in sterile saline solution) and to perform oral hygiene using only mechanical removal of bacterial plaque with water without the use of toothpaste or mouthrinse. Application of the chlorhexidine protocol Unstimulated saliva samples (1 ml) were collected from each volunteer and 2 of the glass disks were withdrawn from the splint (right and left; in a mesial-distal direction) at 8, 10, and 12 hours after performing one of the following mouthrinses under supervision: 1) A single, 30-second mouthrinse with 10 ml of 0.2% CHX * at 7 AM (M-0.2% CHXdiurnal). OR 2) A single, 30-second mouthrinse with 10 ml of 0.2% CHX * at 1 AM (M-0.2% CHXnocturnal). On the day of the experiment, the volunteers were not allowed to eat or drink during the course of the tests. Subjects were randomly assigned to either M-0.2% CHX-diurnal or M- 0.2% CHX-nocturnal. Following a washout period of 2 weeks, subjects repeated this procedure with the alternate mouthrinse. The unstimulated saliva samples were collected using a previously described method (the spitting method) 31. Preparation of the fluorescence solution The dual stain solution is formed of 2 fluorochromes, SYTO 9 and propidium iodide (PI), that, when applied simultaneously, enable bacteria with intact membranes (emitting green fluorescence) to be differentiated from bacteria with damaged membranes (emitting red fluorescence). The fluorescence solution was prepared in accordance with the manufacturer s instructions in 5 ml of sterile water, and filtered using a membrane filter, 5

achieving a 1:1 ratio of the 2 fluorochromes; the solution was stored at -20 C. The excitatory wavelengths (emission range) for SYTO 9 and PI are 488 nm (492-550 nm) and 561 nm (588-655 nm), respectively. Processing of the saliva samples The saliva samples were centrifuged at 2000 rpm for 6 minutes. The supernatant was discarded and the pellet thus obtained was resuspended in 100 µl of sterile water. After homogenizing the bacterial suspension by shaking, 100 µl of the fluorescence solution was added and the mixture was kept in darkness at room temperature for 15 minutes. Microscope examination was performed by 2 investigators who were blinded to the study design, using a microscope fitted with a filter set for fluorescein and Texas Red. The count of vital and dead bacteria was performed at high magnification (x100) on 20 fields (10 fields per slide) that presented a minimum of 100 bacteria (bacterial aggregates were excluded). The mean percentage of vital bacteria was calculated for each saliva sample (Figure 2). Processing of the samples of bacterial plaque The glass disks were withdrawn from the splint and were immediately submerged in 100 µl of fluorescence solution and were kept in darkness at room temperature for 15 minutes. Microscope observation was performed by a single investigator who was blinded to the study design, using a laser scanning spectral confocal microscope ǁ with an HCX APOL 63x/0.9 water-immersion lens. Four randomly selected fields or XYZ series in the central part of each disk were evaluated. Fluorescence emission was determined in series of XY images in which each image corresponded with each one of the Z positions (depth). The optical sections were scanned in 1 micron sections from the surface of the biofilm to its base, measuring the maximum thickness of the field and subsequently the maximum thickness of the biofilm of the corresponding sample. Quantification of bacterial vitality in the series of XY images was determined using cytofluorographic analysis. In this analysis, the images of each fluorochrome are defined as channels (SYTO 9 occupies the green channel and PI the red channel), obtaining values for the area (µm 2 ) occupied by each channel, the total area occupied by the biofilm, and the corresponding percentage vitality. Determination of the mean percentage bacterial vitality in each field required sections with a minimum area of biofilm of 250 µm 2, and the 6

mean percentage bacterial vitality of the biofilm was then calculated for the corresponding sample. Statistical analysis The results were analyzed using a statistical package #. The intraclass correlation coefficient (2-factor model, random effects) and the degree of homogeneity of the elements from the absolute agreement perspective were calculated for the intra-observer and inter-observer analysis of the epifluorescence microscopy technique. The values of the quantitative variables analyzed (percentage bacterial vitality) showed a normal distribution, evaluated using the Kolmogorov-Smirnov test. The ANOVA test (1- factor model and 2-factor model, both with repeated measures) was applied, and paired comparisons were performed (using the Bonferroni correction) of the intra-mouthrinse and inter-mouthrinse analysis of PL-biofilm thickness and bacterial vitality in the salivary flora and in the PL-biofilm, together with an analysis of vitality in the 2 oral ecosystems after each type of mouthrinse (M-0.2% CHX-diurnal and M-0.2% CHX-nocturnal). Significance was taken as a P value of less than 0.05. RESULTS The intraclass correlation coefficients for the intra-observer analysis of the epifluorescence microscopy technique were 0.937 for observer 1 (I.T.C.) and 0.928 for observer 2 (L.G.C) The correlation coefficient for the inter-observer analysis was 0.916. After application of the mouthrinses, the mean thickness of the biofilm detected at 8, 10, and 12 hours after the mouthrinse varied between 9 and 26 µm with the M-0.2% CHXdiurnal and between 6 and 26 µm with M-0.2% CHX-nocturnal. Table 1 shows the mean thickness of the PL-biofilm (in µm) at 8, 10, and 12 hours after the application of 0.2% CHX, as well as the intra-mouthrinse and inter-mouthrinse comparisons. The mean thickness of the PL-biofilm varied between 15.26 ± 2.82 µm and 18.40 ± 5.10 µm. No statistically significant differences were found in the thickness of the PL-biofilm in the intramouthrinse analysis (between the different sample collection times) or in the intermouthrinse analysis (between M-0.2% CHX-diurnal and M-0.2% CHX-nocturnal) Table 2 shows the percentage bacterial vitality in the salivary flora at 8, 10, and 12 hours after performing a single mouthrinse with 0.2% CHX, and the intra-mouthrinse and inter- 7

mouthrinse comparisons. In saliva, the percentage bacterial vitality varied between 22.22% ± 7.94% and 82.88% ± 6.48%. Statistically significant differences in bacterial vitality in the salivary flora were detected in the intra-mouthrinse analysis (between the different sample collection times) and in the inter-mouthrinse analysis (between M-0.2% CHX-diurnal and M-0.2% CHX-nocturnal) (P=0.025). In the paired intra-mouthrinse comparisons, the percentage bacterial vitality increased with the passage of time (8 hours versus 10 hours versus 12 hours) both with M-0.2% CHX-diurnal (the mean differences varied between -11.44 ± 8.20% and -23.22% ± 5.71%) and with M-0.2% CHX-nocturnal (the mean differences varied between -16.55% ± 6.54% and -35.00% ± 9.77%), achieving statistical significance in all comparisons (P<0.001 to P=0.026). In the paired intermouthrinse comparisons, the percentages of vital bacteria obtained with M-0.2% CHXdiurnal were significantly higher than those detected with M-0.2% CHX-nocturnal (the mean differences varied between 25.66% ± 9.56% and 37.44% ± 7.24%; P<0.001). Table 3 shows the percentage bacterial vitality in the biofilm at 8, 10, and 12 hours after performing a single mouthrinse with 0.2% CHX, as well as the intra-mouthrinse and intermouthrinse comparisons. No statistically significant differences were detected in bacterial vitality in the biofilm in the intra-mouthrinse analysis (between the different sample collection times) or in the inter-mouthrinse analysis (between M-0.2% CHX-diurnal and M- 0.2% CHX-nocturnal) (Figure 3). With the M-0.2% CHX-diurnal, the percentage of vital bacteria present in the salivary flora was significantly higher than that observed in the bacterial plaque at 8 hours (59.66% versus 39.76%; P<0.001), 10 hours (71.44% versus 35.85%; P<0.001), and 12 hours (82.88% versus 36.95%; P<0.001) after the mouthrinse. With the M-0.2% CHX-nocturnal, the percentage of vital bacteria present in the salivary flora was significantly lower than that observed in the bacterial plaque at 8 hours (22.22% versus 36.46%; P=0.014), was similar at 10 hours (40.60% versus 35.70%; P=0.107), and was significantly higher at 12 hours (57.22% versus 35.32%; P<0.001) after the mouthrinse. DISCUSSION Since the study published by Schiött et al in 1970 15, the scientific community has assumed that the substantivity of CHX persists for 12 to 14 hours after its application. However, there are few studies published in which the in situ substantivity of CHX on the salivary 8

flora or bacterial plaque has been determined more than 7 hours after the application of the antiseptic, and the majority of those studies analyzed samples collected at 24 hours after the mouthrinse 32-34. In the present study we have determined the in situ substantivity of 0.2% CHX on 2 ecosystems (salivary flora and PL-biofilm) at 8, 10, and 12 hours after performing a single mouthrinse with this antiseptic, in order to provide results on its substantivity during this period. The SYTO 9/PI fluorescence solution detects bacterial vitality based on the integrity of the cytoplasmic membrane; it has therefore been considered particularly useful for analysis of the antibacterial activity of CHX 35,36. To date, the SYTO 9/PI solution has only been used to study the antibacterial action of CHX on the biofilm 8,35-37. Recently, however, our research group demonstrated that epifluorescence microscopy using the SYTO 9/PI dualstain solution is an effective technique for quantifying the antibacterial activity of CHX on the salivary flora in real time 20. As this is considered to be an observer-dependent technique 20, it is important to calculate the intra-observer and inter-observer correlations; in the present series, these correlations were higher than 0.9. Although some authors have used epifluorescence techniques for analysis of the salivary flora 18,19,38, we have found no studies (with the exception of those published by our research group 13,14,20,39) in which epifluorescence microscopy with the SYTO 9/PI solution has been used to evaluate the in situ antibacterial activity of CHX on the salivary flora. Comparisons of our results with those obtained by other authors using plate-culture techniques should therefore be interpreted with caution 20,39. Some authors studied the correlation between the 2 techniques (plate culture vs epifluorescence microscopy with the SYTO-9/PI dual staining) for the quantification of different bacterial populations and detected that the plate counting and SYTO-9/PI solution counting provided conflicting information on bacterial viability 16,40. In our opinion, the immediate antibacterial effect could be similarly interpreted with both microbiological techniques 20. However, in accordance with previous authors 16,18, we observed that the plate culture technique could overestimate the in vivo CHX substantivity, since a significant and progressive increase of the bacterial vitability in the different post-mouthrinse saliva samples was detected applying the epifluorescence microscopy with the SYTO-9/PI dual staining 20. The absence of correlation between fluorescence and plate bacterial count data is likely to be associated to the different characteristics described for each microbiological technique 20. Using epifluorescence microscopy with a solution of fluorescein diacetate and ethidium 9

bromide, Weiger et al 19 found that approximately 85% of the salivary flora is vital under basal conditions. Although a sample of saliva was not collected at baseline in the present study, previous studies performed by our research group using epifluorescence microscopy with the SYTO 9/PI solution have found that the baseline percentage bacterial vitality in the salivary flora is approximately 90% 20,39. Results obtained in a number of in situ studies based on plate-culture techniques have shown that the application of a mouthrinse with 0.2% CHX (10 ml/1 min) is associated with an immediate antibacterial effect with a reduction of 90% ( 1 log 10 CFU/mL) in the salivary bacterial concentration compared with baseline values 2,41-44, and that its substantivity persists for at least 7 hours after the mouthrinse, with a reduction 90% ( 1 log 10 CFU/mL) 2,41,42,44. Previous results obtained by our group using epifluorescence microscopy and the SYTO 9/PI solution found that a mouthrinse with 0.2% CHX gave rise to an immediate fall in the percentage of vital bacteria in the saliva ( 90%) and that antibacterial activity was still detectable at 7 hours after the mouthrinse 20,39. In the present study, 0.2% CHX showed greater antibacterial activity as, on comparison with the previously described baseline values 20,39, this activity persisted for up to 12 hours after the mouthrinse, with a reduction in vitality of 19% with M-0.2% CHX-diurnal and of 33% with M-0.2% CHX-nocturnal. The disk-holding splints that the participants wore probably affected these results, since they may have favored the retention of CHX, as has been demonstrated in patients with removable prostheses 10 ; as a result, the splints could have potentiated the antibacterial effect. In contrast to the results obtained in previous studies based on plate culture techniques 2,41,42,44, and in agreement with previous results obtained by our group 20,39, we found that bacterial vitality in the saliva samples showed a progressive recovery over time after the CHX mouthrinse (8 hours versus 10 hours versus 12 hours), and this recovery was statistically significant in all comparisons. It has been shown that CHX retention in the oral cavity is affected by extrinsic factors not associated with the mouthrinse 10. However, we found no studies of the influence of the time of application of the mouthrinse (day versus night) on the in situ substantivity of CHX on the salivary flora. In the present study, M-0.2% CHX-diurnal was associated with a significantly shorter duration of antibacterial activity on the salivary flora than was observed with M-0.2% CHX-nocturnal (the differences in the percentage bacterial vitality varied from 37% at 8 hours after the mouthrinse to 26% at 12 hours after the mouthrinse). In our opinion, the greater substantivity of M-0.2% CHX-nocturnal on the salivary flora could be attributed to the circadian rhythm of saliva secretion; it has been demonstrated 10

that during sleep there is a significant reduction in the flow of saliva and a change in its composition 45,46. As a result, there is a reduction in the protective effects of the saliva on the oral cavity and, simultaneously, there is a fall in nutrient supply; both alterations have a direct effect on bacterial growth 46,47. On this subject, Dige et al 48 observed that the in situ formation of initial dental biofilm decreases during the night, which may reflect differences in the availability of salivary nutrients. The methodology based on the use of CLSM in combination with fluorescence solutions requires the biofilm to be evaluated to be grown on certain substrates incorporated into removable appliances of various designs 21,22,49,50. In published studies, a number of substrates of different characteristics have been used, including human enamel 21,51,52, bovine enamel 24,53, bovine dentine 27, and glass 22,25,29,51. The time the appliance remains in the oral cavity has varied between 6 hours 27 and 168 hours 50, depending on the type of biofilm to be analyzed. Auschill et al 29 demonstrated that the mean thickness of the biofilm at 48 hours was not affected by the position of the removable appliance within the oral cavity (maxillary versus mandibular) or by the position of the disk (distal versus mesial; right versus left). Although the roughness of the surface and its free energy are considered important factors for the in situ growth of the biofilm 29, Netuschil et al 51 did not find any major differences in the thickness of the 48-hour biofilm on using enamel or glass disks; however, some authors recommend using glass to avoid any optical disturbance due to the known autofluorescence of enamel 51,54. In addition, Arweiler et al 22 observed that the pattern of bacterial vitality was not affected by the site of the disk in the oral cavity. In the present study, the removable appliances were placed on the inferior dental arch for 48 hours; the substrate for the biofilm was provided by glass disks and an analysis was performed of 2 disks (right and left; extracted in a mesial-distal direction) at each collection time. Focusing on the characteristics of the 48-hour biofilms, Auschill et al 29 and Arweiler et al 22 described the marked interindividual variability in the values of maximum thickness (between 14 and 150 µm) and bacterial vitality (between 64% and 77%). A number of authors agree that this type of biofilm has an open architecture model, characterized by the presence of a complex system of channels 22,27,29. Although these parameters were not evaluated in the present series, the results obtained previously by our research group using an in situ model of the 48-hour biofilm revealed a maximum thickness of 20 to 51 µm and a mean percentage bacterial vitality of 80% (data not published). Despite the in-depth knowledge about the antibacterial activity of CHX on in vitro 11

biofilm 36,55, there are few studies published in the literature in which the effects of CHX on in situ PL-biofilm at predetermined time points have been investigated using both CLSM and bacterial vitality techniques 24-27. None of the previous studies on the antibacterial effects of CHX on PL-biofilm after a single application evaluated CHX substantivity on the PL-biofilm and the influence on its thickness 26,27. Recently, our group used CLSM and the SYTO 9/PI solution to evaluate in situ the immediate effect and substantivity up to 7 hours after the application of a single mouthrinse with 0.2% CHX (10 ml/30 seconds) on undisturbed 48-hour PL-biofilm (data not published). In contrast to the baseline vitality (77%), the frequencies detected in measurements made after the mouthrinse were significantly lower (5% at 30 seconds, 16% at 1 hour, 36% at 3 hours, 25% at 5 hours, and 32% at 7 hours). In agreement with those results, a vitality of around 30% was detected at 8 hours after the mouthrinse in the present study, with persistence of the antibacterial effect up to 12 hours after the application of the CHX. In contrast to what occurs with the salivary flora, no progressive recovery of the ecosystem was detected in the biofilm, and there were no differences in vitality between the 2 times the CHX mouthrinses were applied (M-0.2% CHX-diurnal versus M-0.2%CHX-nocturnal). Comparison of the mean thickness of the 48-hour biofilm with the mean thickness previously reported by our group (data not published), showed that the biofilm in the present study was not as thick (6.00 µm to 26.00 µm versus 11.75 µm to 33.00 µm), which could suggest a certain antiplaque effect after performing a single mouthrinse with CHX. Numerous authors have demonstrated that bacteria growing in in vitro structured communities can be 10 to 1000 times more resistant to antibacterial treatment than those grown in planktonic phase 56,57. Although it has been stated that the antibacterial activity of CHX on the salivary flora cannot be extrapolated to its action on bacteria present in the bacterial plaque 34, saliva is the medium through which oral microorganisms disseminate from one ecological niche to another 58. Sekino et al 59 demonstrated that the concentration of bacteria in the saliva after the application of different CHX protocols could affect the levels of bacterial plaque during the initial phases of its formation. Despite the interrelationship between the 2 ecosystems and the importance of their study, very few authors have simultaneously investigated the antibacterial activity of a single application of 0.2% CHX on the saliva and on oral biofilms 60. In agreement with results from a previous study by our group using CLSM together with SYTO 9/PI solution (data not published), the percentages of bacterial vitality detected in the present study at 8, 10, and 12 hours after M-0.2%CHX-diurnal and M-0.2% CHX-nocturnal were significantly higher in the salivary 12

flora than in the PL-biofilm. This situation could be related to a slower growth rate in the PL- biofilm 61 or to the presence of an open architecture with channels and spaces in the in situ PL-biofilm that could presumably provide direct communication between the oral environment and the enamel surface 21, contributing a possible reservoir function for antibacterial agents 8. CONCLUSIONS The application of a single mouthrinse with 0.2% CHX had a lower substantivity on the salivary flora between 8 and 12 hours after its application than was found with undisturbed 48-hour PL-biofilm. In the saliva, the bacterial vitality recovered progressively and was affected by the time of application of the mouthrinse (day or night); it was more effective when performed at night. In the PL-biofilm, bacterial vitality remained stable for up to 12 hours after the mouthrinse and was not affected by the time of day the mouthrinse was performed. These results support the more active physiologic dynamics of the salivary flora and the possible reservoir function associated with the structure of undisturbed de novo PL-biofilm. 13

FOOTNOTES * Oraldine Perio, Johnson and Johnson, Madrid, Spain. LIVE/DEAD BacLight TM fluorescence solution, Molecular Probes, Leiden, The Netherlands. Millipore membrane filter, Millipore Ibérica S.A., Madrid, Spain. Olympus BX51 microscope, Olympus, Tokyo, Japan. ǁ Leica TCS SP2 laser scanning spectral confocal microscope, Leica Microsystems Heidelberg GmbH, Mannheim, Alemania. Leica Confocal Software, Leica Microsystems Heidelberg GmbH, Mannheim, Alemania. # PASW Statistics Base 18 package for Windows, IBM, Madrid, Spain. 14

ACKNOWLEDGEMENTS Our gratitude to Mercedes Rivas, a member of the Microscopy Unit of the Services to Support Research (Santiago de Compostela University) for her technical advice on the CLSM technique. 15

CONFLICT OF INTEREST The authors declare that they have no conflict of interests 16

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Table 1. Mean thickness (µm) of the PL-biofilm at 8, 10, 12 hours after a single morning or nighttime mouthrinse with 0.2% chlorhexidine, and the intra-mouthrinse and intermouthrinse comparisons. Mean ± Standard Deviation (µm) 8 h after the CHX application 10 h after the CHX application 12 h after the CHX application M-0.2% CHX-diurnal 17.22 ± 3.58 18.40 ± 5.10 15.51 ± 3.54 M-0.2% CHX-nocturnal 15.48 ± 4.16 15.91 ± 1.55 15.26 ± 2.82 INTRA-MOUTHRINSE ANALYSIS Mean Difference ± Standard Deviation, P value M-0.2% CHX-diurnal: 8 h vs 10 h M-0.2% CHX-diurnal: 8 h vs 12 h M-0.2% CHX-diurnal: 10 h vs 12 h M-0.2% CHX-nocturnal: 8 h vs 10 h M-0.2% CHX-nocturnal: 8 h vs 12 h M-0.2% CHX-nocturnal: 10 h vs 12 h -1.17 ± 4.13 1.71 ± 3.88 2.88 ± 3.02-0.43 ± 3.54 0.22 ± 3.77 0.65 ± 3.28 1.000 0.670 0.063 1.000 1.000 1.000 INTER-MOUTHRINSE ANALYSIS Mean Difference ± Standard Deviation, P value 8 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 10 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 12 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 1.73 ± 5.29 2.48 ± 5.87 0.25 ± 3.19 0.354 0.240 0.820 M-0.2% CHX-diurnal= A single, 30-second mouthrinse with 10 ml of 0.2% CHX performed at 7 AM M-0.2% CHX-nocturnal= A single, 30-second mouthrinse with 10 ml of 0.2% CHX performed at 1 AM 22

Table 2. Mean percentages of bacterial vitality in salivary flora at 8, 10, 12 hours after a single morning or nighttime mouthrinse with 0.2% chlorhexidine, and the intra-mouthrinse and inter-mouthrinse comparisons. SALIVARY FLORA Mean ± Standard Deviation (%) M-0.2% CHX-diurnal M-0.2% CHX-nocturnal 8 h after the CHX application 10 h after the CHX application 12 h after the CHX application 59.66 ± 8.73 71.44 ± 8.77 82.88 ± 6.48 22.22 ± 7.94 40.66 ± 9.00 57.22 ± 8.77 INTRA-MOUTHRINSE ANALYSIS Mean Difference ± Standard Deviation (%), P value M-0.2% CHX-diurnal: 8 h vs 10 h M-0.2% CHX-diurnal: 8 h vs 12 h M-0.2% CHX-diurnal: 10 h vs 12 h M-0.2% CHX-nocturnal: 8 h vs 10 h M-0.2% CHX-nocturnal: 8 h vs 12 h M-0.2% CHX-nocturnal: 10 h vs 12 h -11.77 ± 10.25-23.22 ± 5.71-11.44 ± 8.20-18.44 ± 8.26-35.00 ± 9.77-16.55 ± 6.54 0.026 <0.001 0.009 <0.001 <0.001 <0.001 INTER-MOUTHRINSE ANALYSIS Mean Difference ± Standard Deviation (%), P value 8 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 10 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 12 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 37.44 ± 7.24 30.77 ± 11.91 25.66 ± 9.56 <0.001 <0.001 <0.001 M-0.2% CHX-diurnal= A single, 30-second mouthrinse with 10 ml of 0.2% CHX performed at 7 AM M-0.2% CHX-nocturnal= A single, 30-second mouthrinse with 10 ml of 0.2% CHX performed at 1 AM 23

Table 3. Mean percentages of bacterial vitality in PL-biofilm at 8, 10, 12 hours after a single morning or nighttime mouthrinse with 0.2% chlorhexidine, and the intra-mouthrinse and inter-mouthrinse comparisons. PL-BIOFILM Mean ± Standard Deviation (%) 8 h after the CHX application 10 h after the CHX application 12 h after the CHX application M-0.2% CHX-diurnal 39.76 ± 13.32 35.85 ± 10.80 36.95 ± 12.85 M-0.2% CHX-nocturnal 36.46 ± 12.03 35.78 ± 10.56 35.32 ± 10.93 INTRA-MOUTHRINSE ANALYSIS Mean Difference ± Standard Deviation (%), P value M-0.2% CHX-diurnal: 8 h vs 10 h M-0.2% CHX-diurnal: 8 h vs 12 h M-0.2% CHX-diurnal: 10 h vs 12 h M-0.2% CHX-nocturnal: 8 h vs 10 h M-0.2% CHX-nocturnal: 8 h vs 12 h M-0.2% CHX-nocturnal: 10 h vs 12 h 3.90 ± 12.63 2.80 ± 13.47-1.09 ± 12.91 0.67 ± 10.24 1.14 ± 9.15 0.46 ± 11.36 1.000 1.000 1.000 1.000 1.000 1.000 INTER-MOUTHRINSE ANALYSIS Mean Difference ± Standard Deviation (%), P value 8 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 10 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 12 h: M-0.2% CHX-diurnal vs M-0.2% CHX-nocturnal 3.29 ± 9.91 0.07 ± 16.44 1.63 ± 12.01 0.348 0.990 0.694 M-0.2% CHX-diurnal= A single, 30-second mouthrinse with 10 ml of 0.2% CHX performed at 7 AM M-0.2% CHX-nocturnal= A single, 30-second mouthrinse with 10 ml of 0.2% CHX performed at 1 AM 24

Figure 1. A) Individualized splint of the lower arch: 1. internal vinyl sheet; 2. polished glass discs; 3. fenestrated external vinyl sheet. B) Clinical view of the individualized splint with the glass discs inserted (arrows). Figure 2. Salivary bacteria with intact membranes (emitting green fluorescence) and with damaged membranes (emitting red fluorescence) on epithelial cells (100x objective). Figure 3. Images representing the changes in bacterial vitality in the de novo plaque-like biofilm: a) Sample collected at 8 hours after performing a M-0.2% CHX-diurnal, b) Sample collected at 10 hours after performing a M-0.2% CHX-diurnal, c) Sample collected at 10 hours after performing a M-0.2% CHX-diurnal. M-0.2% CHX-diurnal= A single, 30-second mouthrinse with 10 ml of 0.2% chlorhexidine performed at 7 AM. 25

Figure 1. 26

Figure 2. 27

Figure 3. a b c 28