Short-term microbiological effects of scaling and root planing and essential-oils mouthwash in Chinese adults

Short-term microbiological effects of scaling and root planing and essential-oils mouthwash in Chinese adults 1 first-author 2 2 3 3 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:huic66@hotmail.com †E-mail:huic66@hotmail.com huic66@hotmail.com Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Conservative Dentistry and Periodontics, Affiliated Hospital of Stomatology, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Stomatology, Hangzhou Aeromedicine Certification and Training Center of the PLA Air Force, Hangzhou 310013, China Department of Stomatology, Hangzhou Aeromedicine Certification and Training Center of the PLA Air Force, Hangzhou 310013, China Objective: 2 To assess the short-term effect of scaling and root planing (SRP) and essential-oils mouthwash on the levels of specific bacteria in Chinese adults. Methods: 2 Fifty Chinese adults with chronic periodontitis were randomly assigned to full-mouth SRP or a 7-d essential-oils mouthwash regimen. In addition, 22 periodontally healthy adults used essential-oils mouthwash for 7 d. Clinical examination and plaque/saliva sampling were performed at baseline and on Day 7. Quantitative real-time polymerase chain reaction (PCR) was used to measure Aggregatibacter actinomycetemcomitans ( Aa ), Fusobacterium nucleatum ( Fn ), Porphyromonas gingivalis ( Pg ), Prevotella intermedia ( Pi ), and total bacterial loads in saliva, supra- and sub-gingival plaque samples. Results: 2 The detection frequencies of four tested species remained unchanged after either treatment. However, the bacterial loads of Fn, Pg , and Pi were significantly reduced by SRP; the mean reduction of bacterial counts in saliva ranged from 52.2% to 62.5% ( p < 0.01), in supragingival plaque from 68.2% to 81.0% ( p < 0.05), and in subgingival plaque from 67.9% to 93.0% ( p < 0.01). Total bacterial loads were reduced after SRP in supra- and sub-gingival plaque ( p < 0.05). Essential-oils mouthwash reduced Fn levels in supragingival plaque by a mean of 53.2%, and reduced total bacterial loads in supra- and sub-gingival plaque ( p < 0.01). In subgingival plaque from periodontal patients, Pg and Pi reductions were high after SRP compared to essential-oils mouthwash (93.0% vs. 37.7% and 87.0% vs. 21.0%, p < 0.05). No significant bacterial reduction was observed in periodontally healthy subjects using essential-oils mouthwash. Conclusions: 2 SRP and essential-oils mouthwash both have an impact on saliva and gingival plaque flora in Chinese periodontitis patients in 7 d, with greater microbiological improvement by SRP. Oral microbiota Chronic periodontitis Scaling and root planing Essential-oils Quantitative detection Real-time PCR Introduction 1 Chronic periodontitis (CP), the most common periodontal disease, causes an inflammatory host response leading to bone loss and connective tissue destruction. The initiation and progression of CP are the result of oral bacteria and the host immune response. Approximately 700 different bacterial species can inhabit the human oral cavity (Paster et al., 2006 2006 ). Of these species, Aggregatibacter actinomycetemcomitans ( Aa ), Fusobacterium nucleatum ( Fn ), Porphyromonas gingivalis ( Pg ), and Prevotella intermedia ( Pi ) are closely associated with periodontal diseases (Armitage, 2010 2010 ). Periodontal therapy, which is aimed at controlling inflammatory processes and improving clinical parameters, has been mainly used to alter the periodontal environment by reducing or eliminating periodontopathogens in the dental plaque (Haffajee et al., 2006 2006 ). Anti-infective nonsurgical therapy includes both mechanical and chemotherapeutic approaches. Regular chemotherapeutic home-care by patients, such as applying chlorhexidine and essential-oils mouthwash, is generally very effective in controlling most inflammatory periodontal diseases, when used in conjunction with the professional removal of dental plaque (Drisko, 2001 2001 ). Chlorhexidine is a well-known antiseptic for antiplaque action with a long substantivity, but side-effects such as extensive tooth staining, taste alteration, and calculus deposition limit patient compliance for long-term use. Essential-oils mouthwash has been shown to help in reducing periodontopathogens and in controlling plaque and gingivitis (Fine et al., 1994 1994 ; Pizzo et al., 2008 2008 ) and may be a reliable alternative to chlorhexidine regarding gingival inflammation for long-term use (van Leeuwen et al., 2011 2011 ). The use of an essential-oils mouthwash has been recommended as adjunctive therapy in orthodontic patients with less than optimal oral hygiene, in medically compromised patients, and in patients at high risk of periodontal diseases (Barnett, 2006 2006 ). However, for CP patients, the clinical benefits of mouthwash alone are limited, and mechanical treatment is effective in reducing the bacterial loads resulting in clinical improvement (Slots et al., 1979 1979 ; Umeda et al., 2004 2004 ). Scaling and root planing (SRP) is the first-mode mechanical treatment recommended for most periodontal infections and remains an essential part of successful periodontal therapy (Cobb, 2002 2002 ). SRP alone or with antiseptic yields clinical benefits (Haffajee et al., 1997 1997 ; Heitz-Mayfield et al., 2002 2002 ; Faveri et al., 2006 2006 ). Although beneficial effects of SRP and essential-oils mouthwash on plaque control and clinical assessment have been reported, few studies have assessed their microbiological effect before and after treatment. Microbiological monitoring or evaluation is a useful supplement to clinical measurement to guide treatment strategies and achieve good treatment outcomes. After non-surgical periodontal treatment, oral niches in favor of periodontal bacteria can shift from a predominantly Gram-negative to a Gram-positive subgingival microbiota. However, currently very little data are available in the literature on the microbiological outcome after non-surgical periodontal therapy in the Chinese population (Xu et al., 2004 2004 ; Cheng et al., 2008 2008 ). Moreover, while previous studies assessing the microbiological effects of periodontal therapy mainly used the techniques of culture, DNA probe or polymerase chain reaction (PCR), recent oral research has used quantitative real-time PCR (qRT-PCR), due to its accurate and rapid assessment of microbiological levels (Gomes et al., 2008 2008 ; Cionca et al., 2010 2010 ). The aim of this study was to quantify and evaluate the short-term microbiological effects following either SRP or essential-oils mouthwash in Chinese adults with CP. Furthermore, we aimed to examine the microbiological effect of essential-oils mouthwash in both periodontally healthy and diseased Chinese adults. We applied qRT-PCR based on 16S rRNA genes to assess the detection frequencies and bacterial loads of Aa , Fn , Pg , Pi , and total bacteria in saliva, supra- and sub-gingival plaque at baseline and after 7 d. Materials and methods 1 Participants and clinical assessment 2 Fifty CP patients, between the ages of 19 and 58 years who visited the Affiliated Hospital of Stomatology at Zhejiang University, were enrolled in this study on a rolling basis within a window of six months. An additional 22 periodontally healthy volunteers 20 to 46 years of age were recruited. Clinical measurements such as probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were made at six sites per tooth (buccal-mesial, mid-buccal, buccal-distal, lingual-mesial, mid-lingual, and lingual-distal). CP patients exhibited BOP, with at least one or more sites of CAL ≥4 mm, four sites of PD ≥4 mm, and radiographic alveolar bone loss in four or more teeth. Periodontally healthy subjects showed no clinical evidence of periodontal disease. Subjects were excluded if they (1) had used mouthwash in the previous year, (2) had used antibiotics within the last three months, (3) had received periodontal treatment in the previous six months, (4) were pregnant or had medical conditions such as heart disease and hypertension that may influence the progression of periodontal disease, or (5) had less than 20 teeth. This study was reviewed and approved by the Ethics Board of the Affiliated Hospital of Stomatology in Zhejiang University. All participants were informed about the study design and signed informed consent forms. Sample collection 2 Saliva, supra- and sub-gingival plaque samples were collected from each subject at the start of the treatment session. Subjects refrained from eating, drinking, and brushing for at least one hour prior to sample collection. Each subject was instructed to expectorate into an aseptic paper cup, and 500 μl saliva was transferred into a sterile 1.5 ml microcentrifuge tube. Supra- and sub-gingival plaque samples were collected from four teeth using a standardized method (Herrera et al., 2008 2008 ). The teeth to be sampled were isolated with sterile cotton rolls and gentle air drying. Supragingival plaque samples were collected from the mesial-buccal surface of the first molar (the second molar if the first molar was missing) in each quadrant. No participant in this study had both the first and second molars missing. Supragingival plaque samples were carefully scraped with sterile explorers from the gingival margin to prevent contamination with subgingival plaque, and the pooled supragingival sample from each subject was placed in a microcentrifuge tube containing 1.5 ml sterile reduced transfer fluid (RTF) buffer following Syed and Loesche ( 1972 1972 ). Subgingival plaque was sampled from the site of the deepest pocket of the tooth from which supragingival plaque was collected. A #30 sterile paper point was gently inserted into the periodontal pocket for 20 s and immediately removed to RTF buffer. Four paper points from each subject were pooled in the same microcentrifuge tube with RTF buffer. All samples were stored at −80 °C immediately after collection. Treatments 2 After verifying their eligibility and sampling their saliva and plaque, CP patients were randomly assigned to receive SRP ( N =25) or essential-oils mouthwash (Listerine, Johnson & Johnson, Bangkok, Thailand) ( N =25). Random allocation was carried out using a simple coin-tossing method. Periodontally healthy subjects ( N =22) received mouthwash treatment. For subjects assigned to receive SRP, single-visit one-stage full mouth dental debridement was performed using periodontal ultrasonic scaling (Satelec P5, Mérignac, France) without anesthesia. Subjects receiving essential-oils mouthwash were instructed to rinse with 20 ml mouthwash for 30 s twice a day for 7 d. All subjects maintained their own routine oral hygiene procedures during the 7-d study-period. BOP measurement was recorded for all patients returning to the hospital on the 7th day after treatment. In addition, saliva, supra- and sub-gingival plaque samples were collected using the same procedure as the initial visit. Microbiological examination: qRT-PCR 2 A total of 432 samples (72 each of saliva, supra- and sub-gingival plaque before and after treatment) were collected and qRT-PCR was used to assess the content of the four targeted species, Aa , Fn , Pg , Pi , and total bacteria as described by He et al. ( 2012 2012 ). Briefly, DNA from the samples and cultivated Aa ATCC 29523, Fn ATCC 25586, Pg ATCC 33277, and Pi ATCC 25611 were isolated using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The DNA concentration of cultivated bacterial strains was quantified by a Nanodrop 1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, USA) and the number of bacterial cell copies was quantified and calculated based on the molecular mass formula according to the method of Novak et al. ( 2008 2008 ). Quantification of target species was achieved by using serial ten-fold dilutions from 10 2 to 10 7 cell copies of the quantified standard for each bacterium in the qRT-PCR analysis. Bacteria-specific primer pairs and a universal primer pair according to the literature based on the 16S rRNA gene were used to quantify each target bacterium and the total bacteria in samples (Baumgartner et al., 1999 1999 ; Sakamoto et al., 2001 2001 ; Nadkarni et al., 2002 2002 ; Maeda et al., 2003 2003 ; Suzuki et al., 2004 2004 ). All samples were run in duplicate in 96-well plates in a LightCycler® 480 Real-Time PCR system (Roche Diagnostics, Mannheim, Germany). Samples were assayed in a 10 μl reaction mixture containing 1 μl template DNA, 5 μl LightCycler® 480 SYBR Green I Master, 1 μl 2.5 μmol/L primer pair, and 3.5 μl ddH 2 O. The amplification cycling conditions were 95 °C for 10 min, 40 cycles of 10 s at 95 °C, 15 s at a bacterium-specific annealing temperature, and 40 s at 72 °C. Melting analysis was performed for each run to verify the specificity of the PCR products. Statistical analysis 2 The demographic and clinical characteristics of the two CP groups were compared using standard univariate statistics ( t -test and Fisher’s exact test). The BOP was assessed for the change from baseline to 7 d using the paired t -test. The main microbiological outcomes of interest were changes in detection frequencies and bacterial loads in saliva, supra- and sub-gingival plaque after 7 d of treatment. The absolute bacterial counts were log 10 transformed for analysis, and 1 was added to all the bacteria counts to circumvent issues of zero values with the log 10 transformation. Microbiological effectiveness of treatment was analyzed by applying the paired t -test to the detection frequencies and log 10 transformed bacterial counts before and after treatment. Furthermore, the percentage reduction was calculated by first subtracting the bacterial counts 7 d after treatment from the counts at baseline and then dividing by those at baseline. Wilcoxon rank-sum test was used to compare bacterial percentage reductions between SRP and essential-oils mouthwash in CP patients. Quantile regression was used to evaluate bacterial percentage reductions between the CP and periodontally healthy subjects receiving essential-oils mouthwash, adjusting for age and gender, as age and gender ratio were significantly different between the CP and periodontally healthy groups. We determined the number of subjects in the treatment regimen based on historical data, and the sample size (≥21 participants in each group) was calculated to provide 90% power to detect one standard difference of log 10 transformed bacterial counts between treatment groups. All tests were two-sided with a significance level of p < 0.05. All analyses were conducted with SAS V. 9.2. Results 1 The demographic and clinical characteristics of 72 participants at baseline are listed in Table 1 1 . No significant differences in age, gender, smoking history, BOP, PD, or CAL were observed between the SRP and essential-oils mouthwash groups of CP patients ( p > 0.12). The BOP improved in both the CP treatment groups on Day 7. CP patients who received SRP showed a reduced mean percentage of sites with BOP from 34.7% to 23.5% ( p =0.001) and those who received essential-oils mouthwash showed a reduction from 43.0% to 25.9% ( p < 0.001). None of the participants reported adverse experiences during the 7 d. Data are expressed as mean±standard deviation (SD) or number (percent). n male : number of males; n smoker : number of smokers; BOP: bleeding on probing; PD: probing depth; CAL: clinical attachment level. Mean PD (CAL) of a subject was calculated by averaging the PD (CAL) of all measured sites, with six sites per tooth, in the full mouth. PD s : PD of four sampling sites; CAL s : CAL of four sampling sites p < 0.05 between the CP and periodontally healthy groups Group Age (year) n male n smoker Site with BOP (%) PD (mm) CAL (mm) PD s (mm) CAL s (mm) Chronic periodontitis patients  SRP ( N =25) 31.0±6.8 21 (84%) 7 (28%) 34.7±16.3 2.5±0.6 1.8±1.4 3.8±0.6 3.6±1.4  Mouthwash ( N =25) 34.3±10.1 19 (76%) 10 (40%) 43.0±18.8 2.5±0.5 2.2±1.4 3.9±0.7 3.9±1.6 Healthy subjects  Mouthwash ( N =22) 26.2±6.1 * * 10 (45.5%) * * 3 (13.6%) 13.3±15.0 * * 1.6±0.4 * * 0.1±0.1 * * 2.4±0.4 * * 0.0±0.1 * * In the CP patients, neither treatment was followed by a significant reduction in the detection frequency ( p > 0.08, Table 2 2 ). Fn was detected in all the CP patients before and after treatment. Both Pg and Pi were still present, with detection frequencies of 92.0% (23/25) and 100% (25/25), respectively, in both the SRP and essential-oils mouthwash treatment groups. Aa was detected ≤50% at baseline and after treatment. Although they did not completely remove these four bacteria, to some extent both treatments reduced bacterial loads in CP patients (Fig. 1 1 ). The log 10 transformed bacterial counts before and after treatment on Day 7 for four species and total bacteria are listed in Table 3 3 . SRP reduced the Fn , Pg , and Pi in all samples of saliva, supra- and sub-gingival plaque; the mean reductions in bacterial counts in saliva were 52.2%, 62.5%, and 53.6%, respectively ( p < 0.01); the mean reductions in supragingival plaque were 68.2%, 81.0%, and 72.1% ( p < 0.05); and in subgingival plaque 67.9%, 93.0%, and 87.0% ( p < 0.01). After SRP, the total bacterial loads were reduced in supragingival plaque (mean 33.8%, p =0.02) and subgingival plaque (mean 58.1%, p =0.003). Essential-oils mouthwash reduced the Fn levels in supragingival plaque (mean 53.2%, p =0.004), and total bacteria in supragingival plaque (mean 57.2%, p =0.001) and subgingival plaque (mean 56.4%, p =0.003). Moreover, the percentage reductions of Pg and Pi in subgingival plaque in the SRP group were greater than those in CP patients who received essential-oils mouthwash (93.0% vs. 37.7%, p =0.004, and 87.0% vs. 21.0%, p =0.049, respectively). Aa : Aggregatibacter actinomycetemcomitans ; Fn : Fusobacterium nucleatum ; Pg : Porphyromonas gingivalis ; Pi : Prevotella intermedia ; SRP: scaling and root planing 3 Organism 4 Chronic periodontitis patients 2 Healthy subjects 2 SRP ( N =25) 2 Mouthwash ( N =25) 2 Mouthwash ( N =22) Baseline 7 d Baseline 7 d Baseline 7 d Aa  Saliva 9 7 14 12 7 5  Supragingival plaque 7 6 11 8 7 5  Subgingival plaque 3 2 8 6 2 2 Fn  Saliva 25 25 25 25 22 22  Supragingival plaque 25 25 25 25 22 22  Subgingival plaque 25 25 25 25 22 22 Pg  Saliva 23 23 23 23 10 10  Supragingival plaque 23 22 23 23 10 10  Subgingival plaque 23 22 23 23 9 7 Pi  Saliva 25 25 25 25 18 16  Supragingival plaque 25 25 25 24 15 14  Subgingival plaque 25 24 24 24 13 9 Aa : Aggregatibacter actinomycetemcomitans ; Fn : Fusobacterium nucleatum ; Pg : Porphyromonas gingivalis ; Pi : Prevotella intermedia ; SRP: scaling and root planing; SE: standard error 4 Organism 6 Chronic periodontitis patients 3 Healthy subjects 3 SRP ( N =25) 3 Mouthwash ( N =25) 3 Mouthwash ( N =22) 2 Mean (SE) 2 Reduction (%) 2 Mean (SE) 2 Reduction (%) 2 Mean (SE) 2 Reduction (%) Baseline 7 d Baseline 7 d Baseline 7 d Aa  Saliva 1.5 (2.2) 1.2 (2.1) 46.5 2.5 (2.4) 2.1 (2.4) 57.1 1.4 (2.2) 1.0 (2.0) 58.6  Supragingival plaque 0.9 (1.6) 0.9 (1.8) 4.0 1.6 (2.0) 1.5 (2.3) 23.7 1.0 (1.7) 0.8 (1.6) 40.2  Subgingival plaque 0.5 (1.5) 0.4 (1.4) 24.4 1.4 (2.2) 1.0 (2.0) 55.2 0.4 (1.2) 0.5 (1.5) −19.6 Fn  Saliva 6.6 (0.3) 6.3 (0.5) 52.2 6.4 (0.5) 6.4(0.5) 17.3 6.1 (0.6) 6.1 (0.6) 8.2  Supragingival plaque 7.3 (0.3) 6.8 (0.6) 68.2 7.3 (0.3) 7.0 (0.4) 53.2 6.8 (0.9) 6.6 (0.7) 30.2  Subgingival plaque 6.3 (0.6) 5.8 (0.7) 67.9 6.3 (0.5) 6.2 (0.5) 26.8 5.2 (0.8) 5.2 (1.0) 11.9 Pg  Saliva 6.6 (2.0) 6.1 (1.9) 62.5 6.5 (2.1) 6.3 (2.0) 38.1 2.8 (3.2) 2.8 (3.2) 2.6  Supragingival plaque 5.8 (1.9) 5.1 (2.2) 81.0 6.2 (2.1) 6.1 (2.0) 17.6 2.4 (2.8) 2.5 (2.9) −37.0  Subgingival plaque 5.9 (2.0) 4.7 (2.1) 93.0 5.9 (2.2) 5.7 (2.1) 37.7 1.9 (2.5) 1.7 (2.6) 37.6 Pi  Saliva 6.3 (0.5) 6.0 (0.6) 53.6 5.8 (0.7) 5.7 (0.7) 28.4 3.7 (2.1) 3.3 (2.2) 65.9  Supragingival plaque 6.1 (1.1) 5.5 (0.9) 72.1 5.9 (1.1) 5.6 (1.7) 52.9 2.9 (2.3) 3.0 (2.4) −7.4  Subgingival plaque 5.0 (1.1) 4.1 (1.3) 87.0 4.5 (1.5) 4.4 (1.6) 21.0 1.9 (1.8) 1.3 (1.7) 70.8 Total  Saliva 9.3 (0.2) 9.2 (0.3) 20.0 9.1 (0.5) 9.0 (0.6) 11.0 9.0 (0.5) 8.9 (0.5) 22.8  Supragingival plaque 9.1 (0.2) 8.9 (0.4) 33.8 9.1 (0.3) 8.8 (0.5) 57.2 8.8 (0.7) 8.7 (0.4) 20.7  Subgingival plaque 7.8 (0.4) 7.4 (0.6) 58.1 8.1 (0.4) 7.7 (0.4) 56.4 6.9 (0.6) 6.8 (0.9) 22.3 (a) SRP in CP patients; (b) Essential-oils mouthwash in CP patients; (c) Essential-oils mouthwash in periodontally healthy subjects. * p < 0.05, ** p < 0.01, *** p < 0.001 between baseline and 7 d post-treatment. Means with standard error bars are presented The periodontally healthy subjects who received essential-oils mouthwash showed no significant differences in the bacterial detection frequencies and bacterial loads during the 7-d follow-up period (Tables 2 2 and 3 3 ). Furthermore, there was no statistically significant difference in the percentage reduction between CP patients and periodontally healthy subjects receiving essential-oils mouthwash. Discussion 1 In the present study, the presence of four target bacteria was not influenced by either SRP or essential-oils mouthwash in 7 d. However, the bacterial loads on Day 7 were greatly reduced in CP patients treated with SRP. Essential-oils mouthwash showed some changes in the bacterial loads of CP patients but no change in periodontally healthy participants. That there was no difference in the presence of bacteria before and after SRP in this study is consistent with the report of Xu et al. ( 2004 2004 ), who used PCR and reverse hybridization assay and observed no difference in the presence of Aa , Pg , Pi , Tannerella forsythensis , or Treponema denticola in 20 CP patients in Hong Kong treated with SRP and adjunct hyaluronic acid gel. Darby et al. ( 2001 2001 ) also showed that the detection frequencies of these periodontal pathogens remained the same after SRP treatment using the PCR technique. In contrast, some groups reported significant reduction of the presence of periodontal pathogens after SRP using the checkerboard DNA-DNA hybridization or culture technique (Haffajee et al., 1997 1997 ; Bollen et al., 1998 1998 ; Colombo et al., 2005 2005 ). Discrepancies in the bacterial detection frequencies between different studies may be attributed to geographic differences, sampling procedures, or detection/enumeration methods. Teles et al. ( 2006 2006 ) explicitly discussed the impact of sampling strategies and detection techniques on the microbiological outcomes of previous studies of periodontal therapies and suspected that these factors largely account for the seeming discrepancies. In the paper cited above, Teles et al. ( 2006 2006 ) also pointed out that the most frequent microbiological outcome of periodontal therapy appears to be a significant reduction in the levels of periodontopathogens, although the most desirable effect would be to eradicate those species. As they suggested, the microbiological goal of periodontal therapy is to lower the periodontal bacterial loads to an extent that is both compatible with the host and can be maintained by clinicians and periodontal patients. In our study, although no significant difference in detection frequencies was found, SRP significantly reduced the counts of the periodontal pathogens Fn , Pg , and Pi , but not Aa , which is consistent with the work of others (Doungudomdacha et al., 2001 2001 ; Pawlowski et al., 2005 2005 ). Moreover, the fact that no difference in the amount of Aa was found after SRP in our study is also consistent with a study by Fujise et al. ( 2002 2002 ), which found that SRP did not significantly change the level of Aa using the qRT-PCR technique. The observed difference in bacterial loads but not presence after periodontal treatment in this study supports the idea that the levels of bacterial species, rather than their presence, are important in evaluating periodontal therapeutic effects (Haffajee et al., 2006 2006 ). The anti-plaque and anti-gingivitis effectiveness of essential-oils mouthwash on dental plaque control has previously been demonstrated under different clinical conditions (Charles et al., 2001 2001 ; Seymour, 2003 2003 ; Sharma et al., 2004 2004 ). Consistent with the work of others, we found that essential-oils mouthwash reduced the levels of total bacteria and Fn in 7 d, without significantly reducing the detection frequencies. The percentage reduction of Fn observed in supragingival plaque in CP patients is consistent with the findings of a previous randomized clinical trial investigating the effect of essential-oils mouthwash on the levels of plaque flora (Fine et al., 2007 2007 ). It is widely accepted that various periodontal pathogens are associated with periodontal disease status (Socransky and Haffajee, 2005 2005 ). We hypothesized that essential-oils mouthwash may have different effects on microbiological outcomes between CP patients and periodontally healthy subjects. Indeed, we found no significant change of bacterial loads in any of the four target bacterial species and total bacterial loads in periodontally healthy participants after a 7-d course of essential-oils mouthwash, while some changes in bacterial loads occurred in the CP patients after using this mouthwash. The stable bacterial loads of periodontally healthy subjects before and after using essential-oils mouthwash may be explained by the presence of a beneficial bacterial etiology in the healthy subjects, and the targeting on certain microorganisms in the oral cavity (Fine et al., 2000 2000 ; Stoeken et al., 2007 2007 ). Periodontopathogenic bacteria can colonize multiple ecological sites with varied distributions (Mager et al., 2003 2003 ). Bacteria in supragingival plaque or saliva can have an impact on the subgingival re-colonization of pockets after periodontal therapy, and the intraoral translocation of periodontal pathogens may jeopardize periodontal therapy (Quirynen et al., 1996 1996 ; 2001 2001 ). We assessed four target bacterial species in saliva, supra- and sub-gingival plaque before and after SRP and found that in all these locations Fn , Pg , and Pi were reduced, indicating that subgingival plaque control is important and affects the overall distribution of periodontal pathogens in the oral niches. Essential-oils mouthwash was approved by the American Dental Association for supragingival plaque control and anti-gingivitis in 1987, and it has become available over-the-counter in China in recent years. Even though the clinical and microbiological benefits of essential-oils mouthwash alone or as an adjunct to periodontal therapies on Europeans and North Americans have been reported, to the best of our knowledge, no published study to date has evaluated the effect of essential-oils mouthwash in the Chinese population using qRT-PCR. In this study, essential-oils mouthwash was assigned to participants for using in conjunction with unsupervised daily oral hygiene in an attempt to simulate the normal daily situation. Besides treating a group of periodontally healthy subjects with essential-oils mouthwash, a group of CP patients was also treated with this mouthwash for the following reasons. First, essential-oils mouthwash alone has been shown to improve clinical outcomes such as inflammation and reduce periodontopathogens in patients with periodontitis (Fine et al., 2007 2007 ; Haffajee et al., 2009 2009 ). Second, using essential-oils mouthwash may be a good home-care practice for Chinese adults to improve their oral hygiene, particularly for people whose oral hygiene is compromised because of temporary disability or lack of awareness. Third, this study was intended to compare the microbiological effect of essential-oils mouthwash between CP patients and periodontally healthy subjects. In conclusion, SRP significantly reduced the bacterial loads of Fn , Pg , Pi , and total bacteria in saliva, supra- and sub-gingival plaque in patients with CP during a short 7-d follow-up. Essential-oils mouthwash significantly reduced the levels of Fn and total bacteria in the CP patients, and this reduction may explain its effectiveness in reducing supragingival plaque and gingivitis. No significant changes in bacteria were observed in periodontally healthy participants. This was a short-term 7-d preliminary study; therefore, the successful reduction of microbes may be transitory. Longer follow-up studies are essential to assess whether, and to what extent, this initial beneficial microbiological effect of SRP and essential-oils mouthwash is sustained. Overall, the results show that SRP and essential-oils mouthwash have beneficial effects in reducing bacterial levels. Our understanding of the microbiological effects of SRP and essential-oils mouthwash sheds light on oral health and provides a scientific basis for recommending professional dental treatment and the use of the mouthwash in addition to daily self-performed oral hygiene in Chinese adults with periodontitis. References GC Armitage . Comparison of the microbiological features of chronic and aggressive periodontitis . Periodontol 2000 , 2010 . 53 ( 1 ): 70 - 88 . 20403106 DOI: 10.1111/j.1600-0757.2010.00357.x http://doi.org/10.1111/j.1600-0757.2010.00357.x . Comparison of the microbiological features of chronic and aggressive periodontitis ML Barnett . The rationale for the daily use of an antimicrobial mouthrinse . J Am Dent Assoc , 2006 . 137 16 - 21 . 16456992 . The rationale for the daily use of an antimicrobial mouthrinse JC Baumgartner , BJ Watkins , KS Bae , T Xia . Association of black-pigmented bacteria with endodontic infections . J Endod , 1999 . 25 ( 6 ): 413 - 415 . 10530240 DOI: 10.1016/S0099-2399(99)80268-4 http://doi.org/10.1016/S0099-2399(99)80268-4 . Association of black-pigmented bacteria with endodontic infections CM Bollen , C Mongardini , W Papaioannou , D van Steenberghe , M Quirynen . The effect of a one-stage full-mouth disinfection on different intra-oral niches. Clinical and microbiological observations . J Clin Periodontol , 1998 . 25 ( 1 ): 56 - 66 . 9477021 DOI: 10.1111/j.1600-051X.1998.tb02364.x http://doi.org/10.1111/j.1600-051X.1998.tb02364.x . The effect of a one-stage full-mouth disinfection on different intra-oral niches. Clinical and microbiological observations CH Charles , NC Sharma , HJ Galustians , J Qaqish , JA McGuire , JW Vincent . Comparative efficacy of an antiseptic mouthrinse and an antiplaque/antigingivitis dentifrice: a six-month clinical trial . J Am Dent Assoc , 2001 . 132 ( 5 ): 670 - 675 . 11367972 . Comparative efficacy of an antiseptic mouthrinse and an antiplaque/antigingivitis dentifrice: a six-month clinical trial RH Cheng , WK Leung , EF Corbet . Non-surgical periodontal therapy with adjunctive chlorhexidine use in adults with down syndrome: a prospective case series . J Periodontol , 2008 . 79 ( 2 ): 379 - 385 . 18251655 DOI: 10.1902/jop.2008.070247 http://doi.org/10.1902/jop.2008.070247 . Non-surgical periodontal therapy with adjunctive chlorhexidine use in adults with down syndrome: a prospective case series N Cionca , C Giannopoulou , G Ugolotti , A Mombelli . Microbiologic testing and outcomes of full-mouth scaling and root planing with or without amoxicillin/metronidazole in chronic periodontitis . J Periodontol , 2010 . 81 ( 1 ): 15 - 23 . 20059413 DOI: 10.1902/jop.2009.090390 http://doi.org/10.1902/jop.2009.090390 . Microbiologic testing and outcomes of full-mouth scaling and root planing with or without amoxicillin/metronidazole in chronic periodontitis CM Cobb . Clinical significance of non-surgical periodontal therapy: an evidence-based perspective of scaling and root planing . J Clin Periodontol , 2002 . 29 ( s2 ): 6 - 16 . 12010523 DOI: 10.1034/j.1600-051X.29.s2.4.x http://doi.org/10.1034/j.1600-051X.29.s2.4.x . Clinical significance of non-surgical periodontal therapy: an evidence-based perspective of scaling and root planing AP Colombo , RP Teles , MC Torres , W Rosalem , MC Mendes , RM Souto , M Uzeda . Effects of non-surgical mechanical therapy on the subgingival microbiota of brazilians with untreated chronic periodontitis: 9-month results . J Periodontol , 2005 . 76 ( 5 ): 778 - 784 . 15898939 DOI: 10.1902/jop.2005.76.5.778 http://doi.org/10.1902/jop.2005.76.5.778 . Effects of non-surgical mechanical therapy on the subgingival microbiota of brazilians with untreated chronic periodontitis: 9-month results IB Darby , J Mooney , DF Kinane . Changes in subgingival microflora and humoral immune response following periodontal therapy . J Clin Periodontol , 2001 . 28 ( 8 ): 796 - 805 . 11442741 DOI: 10.1034/j.1600-051X.2001.280812.x http://doi.org/10.1034/j.1600-051X.2001.280812.x . Changes in subgingival microflora and humoral immune response following periodontal therapy S Doungudomdacha , A Rawlinson , TF Walsh , CW Douglas . Effect of non-surgical periodontal treatment on clinical parameters and the numbers of Porphyromonas gingivalis , Prevotella intermedia and Actinobacillus actinomycetemcomitans at adult periodontitis sites . J Clin Periodontol , 2001 . 28 ( 5 ): 437 - 445 . 11350507 DOI: 10.1034/j.1600-051x.2001.028005437.x http://doi.org/10.1034/j.1600-051x.2001.028005437.x . Effect of non-surgical periodontal treatment on clinical parameters and the numbers of Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans at adult periodontitis sites CH Drisko . Nonsurgical periodontal therapy . Periodontol 2000 , 2001 . 25 ( 1 ): 77 - 88 . 11155183 DOI: 10.1034/j.1600-0757.2001.22250106.x http://doi.org/10.1034/j.1600-0757.2001.22250106.x . Nonsurgical periodontal therapy M Faveri , LC Gursky , M Feres , JA Shibli , SL Salvador , LC de Figueiredo . Scaling and root planing and chlorhexidine mouthrinses in the treatment of chronic periodontitis: a randomized, placebo-controlled clinical trial . J Clin Periodontol , 2006 . 33 ( 11 ): 819 - 828 . 16965522 DOI: 10.1111/j.1600-051X.2006.00994.x http://doi.org/10.1111/j.1600-051X.2006.00994.x . Scaling and root planing and chlorhexidine mouthrinses in the treatment of chronic periodontitis: a randomized, placebo-controlled clinical trial DH Fine , D Furgang , ML Barnett , C Drew , L Steinberg , CH Charles , JW Vincent . Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels . J Clin Periodontol , 2000 . 27 ( 3 ): 157 - 161 . 10743861 DOI: 10.1034/j.1600-051x.2000.027003157.x http://doi.org/10.1034/j.1600-051x.2000.027003157.x . Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels DH Fine , K Markowitz , D Furgang , D Goldsmith , CH Charles , TA Lisante , MC Lynch . Effect of an essential oil-containing antimicrobial mouthrinse on specific plaque bacteria in vivo . J Clin Periodontol , 2007 . 34 ( 8 ): 652 - 657 . 17535287 DOI: 10.1111/j.1600-051X.2007.01096.x http://doi.org/10.1111/j.1600-051X.2007.01096.x . Effect of an essential oil-containing antimicrobial mouthrinse on specific plaque bacteria in vivo JB Fine , DS Harper , JM Gordon , CA Hovliaras , CH Charles . Short-term microbiological and clinical effects of subgingival irrigation with an antimicrobial mouthrinse . J Periodontol , 1994 . 65 ( 1 ): 30 - 36 . 8133413 DOI: 10.1902/jop.1994.65.1.30 http://doi.org/10.1902/jop.1994.65.1.30 . Short-term microbiological and clinical effects of subgingival irrigation with an antimicrobial mouthrinse O Fujise , T Hamachi , K Inoue , M Miura , K Maeda . Microbiological markers for prediction and assessment of treatment outcome following non-surgical periodontal therapy . J Periodontol , 2002 . 73 ( 11 ): 1253 - 1259 . 12479627 DOI: 10.1902/jop.2002.73.11.1253 http://doi.org/10.1902/jop.2002.73.11.1253 . Microbiological markers for prediction and assessment of treatment outcome following non-surgical periodontal therapy SC Gomes , C Nonnenmacher , C Susin , RV Oppermann , R Mutters , RAC Marcantonio . The effect of a supragingival plaque-control regimen on the subgingival microbiota in smokers and never-smokers: evaluation by real-time polymerase chain reaction . J Periodontol , 2008 . 79 ( 12 ): 2297 - 2304 . 19053920 DOI: 10.1902/jop.2008.070558 http://doi.org/10.1902/jop.2008.070558 . The effect of a supragingival plaque-control regimen on the subgingival microbiota in smokers and never-smokers: evaluation by real-time polymerase chain reaction AD Haffajee , MA Cugini , S Dibart , C Smith , Jr RL Kent , SS Socransky . The effect of SRP on the clinical and microbiological parameters of periodontal diseases . J Clin Periodontol , 1997 . 24 ( 5 ): 324 - 334 . 9178112 DOI: 10.1111/j.1600-051X.1997.tb00765.x http://doi.org/10.1111/j.1600-051X.1997.tb00765.x . The effect of SRP on the clinical and microbiological parameters of periodontal diseases AD Haffajee , RP Teles , SS Socransky . The effect of periodontal therapy on the composition of the subgingival microbiota . Periodontol 2000 , 2006 . 42 ( 1 ): 219 - 258 . 16930312 DOI: 10.1111/j.1600-0757.2006.00191.x http://doi.org/10.1111/j.1600-0757.2006.00191.x . The effect of periodontal therapy on the composition of the subgingival microbiota AD Haffajee , C Roberts , L Murray , N Veiga , L Martin , RP Teles , M Letteri , SS Socransky . Effect of herbal, essential oil, and chlorhexidine mouthrinses on the composition of the subgingival microbiota and clinical periodontal parameters . J Clin Dent , 2009 . 20 ( 7 ): 211 - 217 . 20128317 . Effect of herbal, essential oil, and chlorhexidine mouthrinses on the composition of the subgingival microbiota and clinical periodontal parameters J He , W Huang , Z Pan , H Cui , G Qi , X Zhou , H Chen . Quantitative analysis of microbiota in saliva, supragingival, and subgingival plaque of Chinese adults with chronic periodontitis . Clin Oral Investig , 2012 . 16 ( 6 ): 1579 - 1588 . 22169888 DOI: 10.1007/s00784-011-0654-4 http://doi.org/10.1007/s00784-011-0654-4 . Quantitative analysis of microbiota in saliva, supragingival, and subgingival plaque of Chinese adults with chronic periodontitis LJ Heitz-Mayfield , L Trombelli , F Heitz , I Needleman , D Moles . A systematic review of the effect of surgical debridement vs. non-surgical debridement for the treatment of chronic periodontitis . J Clin Periodontol , 2002 . 29 ( s3 ): 92 - 102 . 12787211 DOI: 10.1034/j.1600-051X.29.s3.5.x http://doi.org/10.1034/j.1600-051X.29.s3.5.x . A systematic review of the effect of surgical debridement vs. non-surgical debridement for the treatment of chronic periodontitis D Herrera , A Contreras , J Gamonal , A Oteo , A Jaramillo , N Silva , M Sanz , JE Botero , R Leon . Subgingival microbial profiles in chronic periodontitis patients from chile, colombia and spain . J Clin Periodontol , 2008 . 35 ( 2 ): 106 - 113 . 18081862 DOI: 10.1111/j.1600-051X.2007.01170.x http://doi.org/10.1111/j.1600-051X.2007.01170.x . Subgingival microbial profiles in chronic periodontitis patients from chile, colombia and spain H Maeda , C Fujimoto , Y Haruki , T Maeda , S Kokeguchi , M Petelin , H Arai , I Tanimoto , F Nishimura , S Takashiba . Quantitative real-time PCR using TaqMan and SYBR green for Actinobacillus actinomycetemcomitans , Porphyromonas gingivalis , Prevotella intermedia , tetQ gene and total bacteria . FEMS Immunol Med Microbiol , 2003 . 39 ( 1 ): 81 - 86 . 14557000 DOI: 10.1016/S0928-8244(03)00224-4 http://doi.org/10.1016/S0928-8244(03)00224-4 . Quantitative real-time PCR using TaqMan and SYBR green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria DL Mager , LA Ximenez-Fyvie , AD Haffajee , SS Socransky . Distribution of selected bacterial species on intraoral surfaces . J Clin Periodontol , 2003 . 30 ( 7 ): 644 - 654 . 12834503 DOI: 10.1034/j.1600-051X.2003.00376.x http://doi.org/10.1034/j.1600-051X.2003.00376.x . Distribution of selected bacterial species on intraoral surfaces MA Nadkarni , FE Martin , NA Jacques , N Hunter . Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set . Microbiology , 2002 . 148 257 - 266 . 11782518 . Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set MJ Novak , KF Novak , JS Hodges , S Kirakodu , M Govindaswami , A Diangelis , W Buchanan , PN Papapanou , BS Michalowicz . Periodontal bacterial profiles in pregnant women: response to treatment and associations with birth outcomes in the obstetrics and periodontal therapy (OPT) study . J Periodontol , 2008 . 79 ( 10 ): 1870 - 1879 . 18834241 DOI: 10.1902/jop.2008.070554 http://doi.org/10.1902/jop.2008.070554 . Periodontal bacterial profiles in pregnant women: response to treatment and associations with birth outcomes in the obstetrics and periodontal therapy (OPT) study BJ Paster , I Olsen , JA Aas , FE Dewhirst . The breadth of bacterial diversity in the human periodontal pocket and other oral sites . Periodontol 2000 , 2006 . 42 ( 1 ): 80 - 87 . 16930307 DOI: 10.1111/j.1600-0757.2006.00174.x http://doi.org/10.1111/j.1600-0757.2006.00174.x . The breadth of bacterial diversity in the human periodontal pocket and other oral sites AP Pawlowski , A Chen , BM Hacker , LA Mancl , RC Page , FA Roberts . Clinical effects of scaling and root planing on untreated teeth . J Clin Periodontol , 2005 . 32 ( 1 ): 21 - 28 . 15642054 DOI: 10.1111/j.1600-051X.2004.00626.x http://doi.org/10.1111/j.1600-051X.2004.00626.x . Clinical effects of scaling and root planing on untreated teeth G Pizzo , M la Cara , ME Licata , I Pizzo , M D′Angelo . The effects of an essential oil and an amine fluoride/stannous fluoride mouthrinse on supragingival plaque regrowth . J Periodontol , 2008 . 79 ( 7 ): 1177 - 1183 . 18597599 DOI: 10.1902/jop.2008.070583 http://doi.org/10.1902/jop.2008.070583 . The effects of an essential oil and an amine fluoride/stannous fluoride mouthrinse on supragingival plaque regrowth M Quirynen , W Papaioannou , D van Steenberghe . Intraoral transmission and the colonization of oral hard surfaces . J Periodontol , 1996 . 67 ( 10 ): 986 - 993 . 8910838 DOI: 10.1902/jop.1996.67.10.986 http://doi.org/10.1902/jop.1996.67.10.986 . Intraoral transmission and the colonization of oral hard surfaces M Quirynen , M de Soete , K Dierickx , D van Steenberghe . The intra-oral translocation of periodontopathogens jeopardises the outcome of periodontal therapy: a review of the literature . J Clin Periodontol , 2001 . 28 ( 6 ): 499 - 507 . 11350516 DOI: 10.1034/j.1600-051x.2001.028006499.x http://doi.org/10.1034/j.1600-051x.2001.028006499.x . The intra-oral translocation of periodontopathogens jeopardises the outcome of periodontal therapy: a review of the literature M Sakamoto , Y Takeuchi , M Umeda , I Ishikawa , Y Benno . Rapid detection and quantification of five periodontopathic bacteria by real-time PCR . Microbiol Immunol , 2001 . 45 ( 1 ): 39 - 44 . 11270605 . Rapid detection and quantification of five periodontopathic bacteria by real-time PCR R Seymour . Additional properties and uses of essential oils . J Clin Periodontol , 2003 . 30 ( s5 ): 19 - 21 . 12787199 DOI: 10.1034/j.1600-051X.30.s5.7.x http://doi.org/10.1034/j.1600-051X.30.s5.7.x . Additional properties and uses of essential oils N Sharma , CH Charles , MC Lynch , J Qaqish , JA McGuire , JG Galustians , LD Kumar . 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 , 2004 . 135 ( 4 ): 496 - 504 . 15127875 . 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 Slots , P Mashimo , MJ Levine , RJ Genco . Periodontal therapy in humans. I. Microbiological and clinical effects of a single course of periodontal scaling and root planing, and of adjunctive tetracycline therapy . J Periodontol , 1979 . 50 ( 10 ): 495 - 509 . 385821 DOI: 10.1902/jop.1979.50.10.495 http://doi.org/10.1902/jop.1979.50.10.495 . Periodontal therapy in humans. I. Microbiological and clinical effects of a single course of periodontal scaling and root planing, and of adjunctive tetracycline therapy SS Socransky , AD Haffajee . Periodontal microbial ecology . Periodontol 2000 , 2005 . 38 ( 1 ): 135 - 187 . 15853940 DOI: 10.1111/j.1600-0757.2005.00107.x http://doi.org/10.1111/j.1600-0757.2005.00107.x . Periodontal microbial ecology JE Stoeken , S Paraskevas , GA van der Weijden . The long-term effect of a mouthrinse containing essential oils on dental plaque and gingivitis: a systematic review . J Periodontol , 2007 . 78 ( 7 ): 1218 - 1228 . 17608576 DOI: 10.1902/jop.2007.060269 http://doi.org/10.1902/jop.2007.060269 . The long-term effect of a mouthrinse containing essential oils on dental plaque and gingivitis: a systematic review N Suzuki , A Yoshida , T Saito , M Kawada , Y Nakano . Quantitative microbiological study of subgingival plaque by real-time PCR shows correlation between levels of Tannerella forsythensis and Fusobacterium spp . J Clin Microbiol , 2004 . 42 ( 5 ): 2255 - 2257 . 15131205 DOI: 10.1128/JCM.42.5.2255-2257.2004 http://doi.org/10.1128/JCM.42.5.2255-2257.2004 . Quantitative microbiological study of subgingival plaque by real-time PCR shows correlation between levels of Tannerella forsythensis and Fusobacterium spp SA Syed , WJ Loesche . Survival of human dental plaque flora in various transport media . Appl Microbiol , 1972 . 24 ( 4 ): 638 - 644 . 4628799 . Survival of human dental plaque flora in various transport media RP Teles , AD Haffajee , SS Socransky . Microbiological goals of periodontal therapy . Periodontol 2000 , 2006 . 42 ( 1 ): 180 - 218 . 16930311 DOI: 10.1111/j.1600-0757.2006.00192.x http://doi.org/10.1111/j.1600-0757.2006.00192.x . Microbiological goals of periodontal therapy M Umeda , Y Takeuchi , K Noguchi , Y Huang , G Koshy , I Ishikawa . Effects of nonsurgical periodontal therapy on the microbiota . Periodontol 2000 , 2004 . 36 ( 1 ): 98 - 120 . 15330945 DOI: 10.1111/j.1600-0757.2004.03675.x http://doi.org/10.1111/j.1600-0757.2004.03675.x . Effects of nonsurgical periodontal therapy on the microbiota MP van Leeuwen , DE Slot , GA van der Weijden . Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review . J Periodontol , 2011 . 82 ( 2 ): 174 - 194 . 21043801 DOI: 10.1902/jop.2010.100266 http://doi.org/10.1902/jop.2010.100266 . Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review Y Xu , K Hofling , R Fimmers , M Frentzen , PM Jervoe-Storm . Clinical and microbiological effects of topical subgingival application of hyaluronic acid gel adjunctive to scaling and root planing in the treatment of chronic periodontitis . J Periodontol , 2004 . 75 ( 8 ): 1114 - 1118 . 15455740 DOI: 10.1902/jop.2004.75.8.1114 http://doi.org/10.1902/jop.2004.75.8.1114 . Clinical and microbiological effects of topical subgingival application of hyaluronic acid gel adjunctive to scaling and root planing in the treatment of chronic periodontitis Compliance with ethics guidelines Jia-yan HE, Gang-gang QI, Wu-jing HUANG, Xu-dong SUN, Yu TONG, Chun-mei PENG, Xue-ping ZHOU, and Hui CHEN declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000(5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1200350 Project supported by the Zhejiang Provincial Natural Science Foundation of China (No. Y2080253), the Education Department of Zhejiang Province (No. Y200909376), the Science and Technology Department of Zhejiang Province (No. 2007C24010), and 2011 China State Key Clinical Department Grants 2012-12-18 2013-04-15 2013-04-01 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 14