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Efcacy of Amoxicillin and Metronidazole Combination for the Management of Generalized Aggressive Periodontitis
Emine Cifcibasi Yek,* Serdar Cintan,* Nursen Topcuoglu, Guven Kulekci, Halim _ Issever, and Alpdogan Kantarci
Background: The aim of this study is to evaluate the effects of metronidazoleamoxicillin combination on clinical and microbiologic parameters in patients with generalized aggressive periodontitis. Methods: Twenty-eight patients were randomly included. The test group (n = 12) received amoxicillinmetronidazole combination and scaling and root planing; the control group (n = 16) received scaling and root planing alone. In addition to the clinical examinations, subgingival plaque samples were analyzed for total cultivable bacteria and the presence of Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), Treponema denticola, Prevotella intermedia, Prevotella nigrescens, Prevotella pallens, and Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) using polymerase chain reaction. Results: All clinical parameters improved signicantly compared to baseline (P <0.05) in both groups. There was a statistically signicant reduction of pockets and clinical attachment gain in the combined group compared to the control group (P <0.05). Total counts of bacteria also decreased signicantly at 3 and 6 months in both groups (P <0.05). T. denticola and T. forsythia were the most prevalent bacteria throughout the study. T. denticola showed a continuous decrease over 6 months in the test group, whereas no change was seen in the control group beyond 3 months. P. gingivalis decreased signicantly at 3 months (P <0.05), whereas T. forsythia was the only pathogen decreased below detection limits by the combination therapy with a significant difference compared to the control group (P <0.05). Conclusions: The results from this study suggest that combined amoxicillin and metronidazole use as an adjunct to scaling and root planing leads to better clinical healing compared to mechanical treatment alone. The polypharmaceutical approach used results in a signicant and substantial decrease in T. forsythia and prevents its recolonization for 6 months, suggesting that T. forsythia may determine the long-term stability of periodontal treatment outcomes. J Periodontol 2010;81:964-974. KEY WORDS Aggressive periodontitis; amoxicillin; metronidazole; Tannerella forsythia.
* Department of Periodontology, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. Department of Oral Microbiology, Faculty of Dentistry, Istanbul University. Department of Biostatistics, Faculty of Medicine, Istanbul University. Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, Boston, MA.

eneralized aggressive periodontitis (GAgP) is a complex periodontal disease affecting the entire dentition with pronounced and rapid destruction of the periodontium and resulting in loss of teeth.1 Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), and Tannerella forsythia (previously T. forsythensis) have been strongly associated with GAgP.2-5 Recent studies with more sensitive detection methods suggest other species could also be associated with GAgP.6 In addition to its etiology, GAgP presents with a complex pathogenesis.7 Therefore, treatment of the disease and maintenance of therapeutic outcomes present major challenges for clinicians. As in other forms of periodontal diseases, the primary approach in the treatment of GAgP is through mechanical means using non-surgical and surgical techniques. One of the concerns regarding the use of mechanical techniques alone, however, is the lack of predictability of long-term success. This concern is based on the assumption that bacterial recolonization

doi: 10.1902/jop.2010.090522

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could not be prevented through mechanical treatment alone; thus, adjunctive use of antibiotics (e.g., penicillin, tetracycline, and metronidazole) will result in favorable clinical improvements.8-16 There is a consensus that the use of adjunctive antibiotics could provide a better outcome of attachment levels compared to mechanical therapy alone and comparable effects should be expected for most antibiotics.10 An emerging approach to stabilize the favorable effects of antibiotics suggests using a combination of multiple agents. This strategy assumes that multiple species can be simultaneously eliminated or suppressed during periodontal therapy, which leads to better stability of the microbiota and the host response and takes advantage of different specicities of the antibiotics and their spectra.10 As such, combined use of amoxicillin and metronidazole has been proposed as a useful regimen with increased bactericidal and spectral efcacy compared to monotherapy with each drug.17 In this combination, metronidazole, which metabolizes in liver where a highly effective hydroxymetabolite of the drug is produced, and amoxicillin synergistically act on A. actinomycetemcomitans, Treponema denticola, T. forsythia, and P. gingivalis.8,14,16,18-20 In addition to these species, Prevotella intermedia and Fusobacterium nucleatum have been shown to respond to metronidazoleamoxicillin combination.21,22 Systemically administered amoxicillin and metronidazole and professional removal of supragingival plaque weekly for 3 months in patients diagnosed with refractory periodontitis results in stably low levels of periodontopathogens for 2 years.23 These reports provide encouraging evidence for the polypharmaceutical use of adjunctive antibiotics in periodontal treatment. A limited number of studies suggest that there is indeed a strong potential for such a strategy in patients with aggressive periodontitis9,13,24,25 and treatment outcomes can be more predictable over a longer period because of increased effectiveness and wider spectra of activity.19 However, there is no clear consensus on the mechanism of action and efcacy of combined use of antibiotics during the treatment of GAgP. There is a considerable variation in study design, dosage, and duration. Very few studies have focused on the recolonization of periodontopathogens. Because recolonization of subgingival sites by the bacteria occurs shortly after mechanical treatment and determines the stability of the outcome, preventing or delaying recolonization is critical for long-term success. 26 This concern has been rarely addressed.9,27 To study these issues, we have designed our work with two aims: to analyze the effects of amoxicillinmetronidazole combination as an adjunct to scaling and root planning (SRP) on clinical and microbiologic parameters in patients with GAgP,

and to follow-up the outcomes and evaluate recolonization. MATERIALS AND METHODS Study Population, Design, and Clinical Procedures The study protocol has been approved by the ethical committee of Istanbul University, Istanbul, Turkey. All patients were informed about the nature of the study and their signed informed consent was obtained prior to study procedures. Thirty-two subjects with previously untreated GAgP were recruited from patients referred to the Department of Periodontology, Faculty of Dentistry, Istanbul University. Subjects were recruited and study procedures were completed between 2004 and 2006. The inclusion criteria for subject recruitment were as follows: 1) to have 20 teeth, 2) to exhibit 5 mm probing depth (PD) around 2 teeth in each quadrant, and 3) to have attachment and bone loss around 3 teeth other than incisors and rst molars. Subjects were excluded if they had restorations on teeth to be sampled; if they were pregnant, lactating, or allergic to the drugs used in the study; if they were smoking >10 cigarettes per day; if they had systemic conditions that might alter the host response or require antibiotic coverage during routine dental processes; if they had received antibiotic therapy during the last 6 months; or if they previously had periodontal treatment. The study included one test and one control group. Patients were randomly assigned to these groups by coin toss by one of the investigators (SC) who was not involved in the clinical procedures. After assigning the patients, the same investigator provided the prescriptions. All patients were treated by another investigator (EY) who was blinded to the designation of the groups and was not aware of which subjects were receiving the medications. This investigator provided the clinical treatment modalities, measurements, and sampling. The treatment codes of the study were not available to the treating investigator until the data were completely analyzed by the statistician (HI). Clinical measurements and plaque sampling were done at baseline and repeated after 3 and 6 months post-therapy. All patients were instructed for oral hygiene and monitored throughout the study period. Clinical parameters included plaque index (PI),28 gingival index (GI),29 PD, and clinical attachment level (CAL). Measurements were taken at six sites around all teeth excluding the third molars using a periodontal probe.i Subjects in the test group received SRP and adjunctive amoxicillin (500 mg, 3 1) and metronidazole# (500 mg, 3 1) combination, whereas the
i PCP-UNC15, Hu-Friedy, Chicago, IL. Mustafa Nevzat Pharmaceuticals, Istanbul, Turkey. # Eczacibasi Pharmaceuticals, Istanbul, Turkey.

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control group received SRP alone. Whole-mouth SRP was performed in two sequential visits. In the test group, systemic antibiotics were administered during the rst session and continued for 7 days. During the study period, all subjects were recalled monthly for oral hygiene instruction and supragingival scaling. Microbiologic Sampling and Culture of Subgingival Microbiota Subgingival plaque samples were taken from the three preselected deepest pockets of at least 5 mm depth using sterile paper points (#40).** Teeth were chosen from different quadrants to represent a prole of the whole mouth. The samples were pooled in 1 ml of VMGIII transport medium30 and transported to the laboratory. After vortexing for 1 minute, 10-fold serial dilutions were prepared. Aliquots of 0.1 ml were plated onto sheep blood agar plates, which were supplemented with hemin (5 mg/l) and vitamin K1 (1 mg/l) for isolation and growth of obligate anaerobic bacteria. The plates were incubated in anaerobic jars at 37C for 7 days. Total number of colonies and black-pigmented colonies were counted and presented as colony forming units. Polymerase Chain Reaction (PCR) Bacterial genomic DNA was extracted from pooled samples by modication of a technique previously reported.31 Briey, after adding 10 ml of lysis buffer (10 mM Tris/HCl, 10 mM EDTA, 10% non-ionic surfactant pH = 8), 90 ml suspension of each isolate was vortexed for 5 minutes, boiled for 5 minutes in a water bath, and immediately placed on ice for 10 minutes. Cell debris was removed by centrifugation and the supernatant was stored at -80C for PCR. Table 1 lists the species-specic PCR primers chosen based on previously published sequences.27,32 The PCR was performed according to a previously described protocol.33 PCR mix contained 1 mM of each primer, 1.5 mM MgCl2, 200 mM of deoxyribonucleotide triphosphate, 1 Taq polymerase buffer,ii 1.25 units of Taq DNA polymerase, and 10 ml of template DNA in 50-ml reaction volume. PCR reactions were carried out in a DNA thermal cycler. The cycle included a total of 36 cycles with an initial denaturation phase at 94C for 3 minutes followed by 30 seconds at 95C, 1 minute at 60C, and 2 minutes at 72C. Specic bacterial template DNA and sterile water were used as positive or negative controls, respectively. Electrophoresis was performed with ethidium bromide labeled 2% agarose gel by using 10 ml of the PCR product. A DNA ladder mix## was run as a molecular size marker in the gel. Data Analyses Prior to the initiation of the study, sample size and power were calculated. Type-1 error was assumed
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at 0.05, type-2 error was assumed at 0.02, and power was assumed at 80% predicting a 30% reduction between baseline and 3 months. The minimum sample size was calculated to consist of 12 subjects for each group. To compensate for potential dropouts, 16 patients were initially recruited per treatment group. For the clinical measurements, average wholemouth recordings were accepted as a unit. Repeated measures of analysis of variance and generalized linear model were used to detect intragroup and intergroup differences in PI, GI, PD, and CAL and the microbiologic data. PI and GI measurements were further analyzed as percentage of sites with 1 or 2 in each index. PD was further analyzed as percentage of sites with <4 mm, between 4 and 7 mm, or 7 mm at baseline and during the study. Clinical attachment gain was also analyzed based on the degree of change in study sites. These detailed investigations were analyzed using the Kruskal-Wallis and x2 tests. For the PCR analysis, when the bacteria were not detected, the value was accepted as zero. The McNemar test was used to detect intragroup differences. The Fisher exact x2 test was used for studying the intergroup differences for individual bacteria. Statistical signicance was accepted as P <0.05. All results were evaluated at 95% condence interval. Statistical software was used for analysis.*** RESULTS Demographic and Clinical Findings The demographics of study participants are shown in Table 2. Of 16 subjects in the test group, two individuals were excluded because of lack of compliance and two individuals became unavailable after moving to another city. In the control group, all 16 patients completed the study. Compliance was determined by counting the pills. This was performed at the end of the second treatment session at which mechanical therapy was completed at 7 days. There were no major drug-associated side effects in the test group with the exception of one patient reporting mild intestinal problems and one complaining of vertigo. Figure 1 presents the clinical ndings in this study as averages for all patients. There was a statistically signicant improvement in plaque scores between baseline and 3 months and between baseline and 6 months in both groups (P <0.05) with no signicant differences between 3 and 6 months (P >0.05; Fig.
** ii ## *** DiaDent, Burnaby, Canada. Gas Generation Kit, Oxoid, Basingstone, Hampshire, UK. Triton X-100, Sigma-Aldrich, St. Louis, MO. dNTP Set, Fermentas, VAB, Vilnius, Lithuania. Boehringer, Mannheim, Germany. Eppendorf PCR System, Mastercycler personal, Eppendorf, Germany. Gene Ruler, MBI Fermantas, Vilnius, Lithuania. Pass 2008/NCSS 2007, NCSS, Kaysville, UT.

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cally signicant and steady improvement in clinical inSpecies-Specic PCR Primers Used in this Study ammation in both groups with no signicant differSpecies Primer Pairs (59-39) Amplicon Length (bp) ence between study groups. Both treatment modaliA. actinomycetemcomitans AAA CCC ATC TCT GAG TTC TTC TTC 478 to 1,034 (557) ties were found to be ATG CCA ACT TGA CGT TTAAT successful on PD measureT. forsythia GCG TAT GTA ACC TGC CCG CA 120 to 760 (641) ments and CALs over 3 and TGC TTC AGT GTC AGT TAT ACC T 6 months compared to the P. gingivalis AGG CAG CTT GCC ATA CTG CG 729 to 1,132 (404) baseline (P <0.05; Figs. ACT GTT AGC AAC TAC CGA TGT 1C and 1D, respectively). No signicant differences P. intermedia TTT GTT GGG GAG TAA AGC GGG 458 to 1,032 (575) were observed between TCA ACA TCT CTG TAT CCT GCG T the groups when the values P. nigrescens ATG AAA CAA AGG TTT TCC GGT AAG 219 to 1,022 (804) for PD and CAL were CCC ACG TCT CTG TGG GCT GCG A averaged per patient (P > 432 to 1,003 (562) P. pallens TGT GCG TTA TTG CAT GTA TCG TAT 0.05). When the percentCCC CGA AGG GCA TAT TTA TCT C age of sites with varying depths of periodontal T. denticola TAA TAC CGA ATG TGC TCA TTT ACA T 193 to 508 (316) pockets were evaluated beTCA AAG AAG CAT TCC CTC TTC TTC TTA tween groups at baseline, bp = base pair. 3 onths, and 6 months, both treatment modalities Table 2. resulted in statistically signicant reduction in PD Demographic Features and Clinical Characteristics of Study compared to baseline (TaPatients at Baseline ble 4). There was a statistically increased reduction Characteristics Control (n = 16) Test (n = 12) in the test group in the percentage of periodontal Age pockets with depths 7 SD 28.9 7.4 33.7 6.9 Range 15 to 45 25 to 45 mm compared to the control group at 3 and 6 Gender (females/males) 9:7 10:2 months (P <0.05). Table 5 Smokers (10 cigarettes per day)* 3 4 shows CAL gains. At 3 and 6 months, the test * There were no subjects in any group who smoked >10 cigarettes per day; P >0.05 for each parameter; analysis of 2 variance and x tests. group showed signicantly more sites with gain com1A). There was no signicant difference between the pared to the control group. The difference was signiftest and the control groups in PI (P >0.05), suggesting icant for both <4 mm and 4 mm of attachment gain a successful maintenance and effective oral hygiene (P <0.05). practice by the patients. When sampled sites were evaluated as areas with PI 1 or PI 2, both groups Microbiologic Findings showed similar changes over the study period (Table The mean total bacterial counts in test and control 3). There was a statistically signicant and steady imgroups at baseline, 3 months, and 6 months are shown provement in detectable plaque accumulation in both in Table 6. Comparison of initial PD of the sampling groups with no signicant difference between study sites between the test and the control groups showed groups. that there was no signicant difference between Similar to the PI scores, GI decreased signicantly groups (data not shown; P >0.05). There were signifin both groups at the end of 3 and 6 months compared icantly more bacteria at baseline in the test group to the baseline (P <0.05; Fig. 1B) with no signicant compared to the control group (P <0.05). The total difference between test and control groups (P >0.05). bacteria was signicantly reduced compared to baseWhen sampled sites were evaluated as areas with line after 3 months in the test group (P <0.05), GI 1 or GI 2, both groups showed similar changes whereas it returned to baseline levels at the end of 6 over the study period (Table 3). There was a statistimonths. The decrease at 3 months in the control Table 1.
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Figure 1.
Clinical ndings in study groups. A) Plaque index scores at baseline, 3 months, and 6 months in study groups. B) Gingival index scores at baseline, 3 months, and 6 months in study groups. C) Probing depth measurements at baseline, 3 months, and 6 months in study groups. D) Clinical attachment levels at baseline, 3 months, and 6 months in study groups (*P <0.05 compared to baseline). Repeated measures of analysis of variance and generalized linear model were used to detect intragroup and intergroup differences in PI, GI, PD, and CAL.

To identify which species of bacteria are found Percent Distribution of Study Sites According to Plaque Index in the study groups, we and Gingival Index Scores performed PCR analyses on pooled samples. FigPlaque Index Gingival Index ure 2 presents the species distribution at baseline in Study Group Score: 0 to 1 Score: 2 to 3 Score: 0 to 1 Score: 2 to 3 all subjects and during Control the course of the study in Baseline 11.7 88.3 42.2 57.8 control and test groups. T. 3 months 69.6* 30.4* 82.7* 17.3* denticola and T. forsythia 6 months 79.8* 20.2* 73.1* 26.9* were the most frequent bacteria in subgingiTest Baseline 19.9 80.1 44.9 55.1 val samples at baseline, 3 months 70.7* 29.3* 81.6* 18.4* whereas A. actinomyce6 months 78.1* 21.9* 76.5* 23.5* temcomitans and Prevo* Change in treatment groups are statistically signicant compared to baseline (P <0.01); Kruskal-Wallis and x2 tella nigrescens had the tests. lowest detection frequencies (4.6%). As a result of the treatment, there was a substantial group was not signicant compared to the baseline (P decrease in T. forsythia in the control group, whereas T. >0.05). The change in the test group at 3 months comforsythia decreased below the detection limits in pared to the control group was statistically signicant the test group suggesting the additional effect of com(P <0.05). Table 6 also demonstrates the mean counts bination therapy over SRP alone on this species. The of black-pigmented bacteria during the study. Both decrease in T. forsythia was maintained over the 6 treatment strategies resulted in statistically signicant months of study period. In both control and test reductions in the counts of black-pigmented bacteria groups, the difference at 3 and 6 months was statistiafter 3 months compared to the baseline (P <0.05) cally signicant compared to baseline levels of T. forand the reduction was sustained over 6 months (P sythia (P <0.05). P. gingivalis was detected in 33.3% <0.05). There was no statistically signicant difference between groups at any sampling time (P >0.05). of control subjects and 27.3% of test subjects at Table 3.
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baseline. In response to adjunctive antibiotics and SRP, P. gingivalis decreased signicantly to 11.1% at 3 months (P <0.05) in the test group, whereas no change was observed in the control group. In both

study groups, 63.6% of the subject population was infected with T. denticola at baseline. T. denticola showed a steady decrease in the test group over 6 months, whereas no further change was seen in the control group after 3 months. A similar numTable 4. ber of subjects was positive for P. intermedia in the Percent Distribution of Study Sites According to Probing test and control groups at Depth Measurements baseline and there was no signicant change in test Sites or control groups in reStudy Group <4 mm (%) 4 to 6 mm (%) 7 mm (%) Total (%) sponse to treatment. The prevalence of P. nigrescens Control and Prevotella pallens did Baseline 54.8 35.1 10 100 not show statistically sig4.9 100 3 months 83.3* 11.8 nicant changes during 6 months 85.6* 12.1 2.2 100 the course of treatment in Test any groups. Overall, all Baseline 47 28.5 24.5 100 members of the P. interme 3 months 77.7* 20.2* 2.2 100 dia group showed either no 6 months 82.3* 15.9 1.8 100 change or some increase Change in probing depth measurements in treatment groups are statistically signicant compared to baseline. after the antibiotic therapy. * Signicantly different from baseline (P <0.05). Signicantly different from baseline (P <0.01). The red complex, Signicantly different from baseline (P <0.001). 2 comprising P. gingivalis, Signicantly different from the change in control group (P <0.05); Kruskal-Wallis and x tests. T. forsythia, and T. denticola, has become recognized as a diseaseTable 5. related complex.34 Table 7 presents the prevalence Percent Distribution of Study Sites According to the Gain in of these pathogens. At Clinical Attachment baseline, 16.7% of control and 18.2% of test subjects Sites were infected with all of the red complex pathogens. Study Group 0 mm (%) 1 to 3 mm (%) 4 mm (%) Total (%) At 3 and 6 months, there Control were no subjects in any of 0 to 3 months 52.6 40.2 7.1 100 the groups infected with 0 to 6 months 54.8 40 5.2 100 the entire red comTest plex members, suggesting 0 to 3 months 38 48.6* 13.5* 100 that the cumulative effect 0 to 6 months 37.8 51* 11.2* 100 of red complex bacteria 2 * Signicantly different from control group (P <0.05); x test. was successfully reduced Table 6.

Total Viable Bacterial Counts and Black-Pigmented Bacterial Counts in Study Populations
Total Viable Counts (106 CFU/ml) Study Group Control (n = 16) Test (n = 12) Baseline 362.64 99.83 493.69 154.72* 3 months 324.84 115.05 270.67 102.49 6 months 412.89 92.40 456.46 119.81 Black-Pigmented Bacterial Counts (106 CFU/ml) Baseline 26.98 15.10 80.77 22.31 3 months 0.88 0.46 1.95 1.23 6 months 0.38 0.18 4.07 1.63

* Signicantly different from control group (P <0.05). Signicantly different from baseline (P <0.05). Signicantly different from the change in control group over time (P <0.05).

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Figure 2.
Proportions of seven bacterial species at baseline in all patients and distribution in control and test groups at baseline, 3 months, and 6 months. The McNemar test was used to detect intragroup differences. The Fisher exact x2 test was used for studying the intergroup differences for individual bacteria.

Table 7.

Prevalence of Red Complex Pathogens in Study Groups

P. gingivalis + T. forsythia or P. gingivalis + T. denticola or T. denticola + T. forsythia (%) Study Group Control (n = 16) Test (n = 12) Baseline 41.7 36.4
2

P. gingivalis + T. forsythia + T. denticola (%) 6 months 22.2 25 Baseline 16.7 18.2 3 months 0 0 6 months 0 0

3 months 16.7* 11.1*

* P <0.05 compared to baseline; Fisher exact x test.

below limits of detection by both methods of treatment. When two of the red complex bacteria were evaluated, a similar trend was observed. There were no statistically signicant differences between groups regarding red complex pathogens sampled in triple or double alliances (P >0.05). DISCUSSION The aim of this randomized clinical trial is to evaluate the clinical and microbiologic effects of systemic metronidazoleamoxicillin therapy as an adjunct to nonsurgical therapy in patients with GAgP. A panel of seven periodontopathogen bacteria was tested and
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clinical measurements were taken to assess plaque accumulation, gingival inammation, PD, and CAL. The outcomes of the treatment were followed over a period of 6 months. The ndings indicate that both treatment modalities resulted in signicant and stable improvements in clinical parameters with reduced clinical inammation and PD, and a gain in CAL. Consistent with other studies administering amoxicillin and metronidazole in GAgP,15,34-36 test subjects in the present study showed a greater reduction in PDs and clinical attachment gains. Parallel to the clinical improvements, total viable counts signicantly decreased in the test group in response to the adjunctive amoxicillin and metronidazole use after 3 months

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compared to the control group. At the end of 6 months, both groups rebounded to baseline levels, and this rebound was consistent with other studies.37-44 When individual species were analyzed, the most signicant difference between the addition of the antibiotic regimen to the mechanical treatment and the mechanical treatment alone was the pronounced effect of the antibiotic combination on T. forsythia. This impact was stable over 6 months, suggesting the important role that T. forsythia may be playing in the long-term stability of periodontal treatment outcome. A similar impact was observed on T. denticola. Although P. gingivalis was also reduced during the rst 3 months after the treatment, the addition of the systemic antibiotics did not result in longterm stability of this reduction, most likely as a result of recolonization from extracrevicular oral sources.43,45 Overall, the results of the present study show that mechanical treatment with or without polypharmaceutical antibiotic support result in predictable clinical healing over 6 months, whereas the most substantial reduction of the bacterial species during this period can be observed in T. forsythia. GAgP presents a therapeutic challenge for clinicians. The clinical treatment protocol is to use SRP followed by the surgical approach to eliminate or regenerate the infrabony defects. Although the surgical approach is useful for direct access to the infrabony involvement and correction of defects, in advanced cases the predictability of the outcome and the stability of the surgical success are questionable.35,46 Nonsurgical technique involving SRP provides a less invasive and more conservative approach, however, where the clinical status can be maintained for an extended period of time.39,40,47 The maximum benet from SRP is generally expected to occur within 2 to 3 months after therapy.8,17,42 It has also been reported that the microbial dental plaque and disease parameters tend to return to baseline levels after 2 to 3 months, demonstrating the need for maintenance therapy every 3 months,41 whereas studies beyond 3 months are very limited. Our results supported the clinical reports that with repeated oral hygiene every 3 months the clinical response could be maintained. This response was more favorable and stable when adjunctive antibiotics were used. Overall, successful suppression of supragingival plaque build-up and maintenance contributes to clinical outcomes,43,44 whereas GAgP patients respond favorably to the suppression of T. forsythia. Studies have proposed that unsteady subgingival microora, differences in host response, and environmental and geographic differences could account for variations of treatment efcacy in otherwise systemically healthy subjects diagnosed with GAgP.48-50 Although several papers have suggested varying spe-

cies dominating the microora of GAgP patients, there is a consensus that the microbial composition of the biolm in GAgP is dominated by specic bacteria.7 Suspected pathogens of aggressive periodontitis (P. gingivalis, T. forsythia, and A. actinomycetemcomitans) and spirochetes are susceptible to antibiotics; still, the complexity of the periodontal biolm could prevent the complete elimination of bacteria during the periodontal treatment.15,51 Patients with high levels of P. intermedia and P. nigrescens, which are also among possible pathogens of GAgP, could present a decreased response to periodontal therapy. The bacterial composition of the plaque, however, showed variations between mechanical treatment and adjunctive use of antibiotics. The microbiologic sampling method, using the three deepest pockets to represent the whole periodontal microbiota in a particular patient group, could account for these variations. We have used this strategy to represent each individual with a microbiota that would populate the deepest pockets in the oral cavity; however, this also presents limitations, such as not being able to detect bacterial species in unsampled pockets or less shallow pockets not being probed. For example, this could also explain the lack of detection of A. actinomycetemcomitans in the control group at baseline where these patients could very well harbor A. actinomycetemcomitans in other periodontal pockets. Nevertheless, the strategy taken here has demonstrated that certain bacteria do respond to periodontal treatment with or without adjunctive antibiotics. Most species in the SRP group returned to baseline levels after 6 months with the exception of the decline in T. forsythia and increase in A. actinomycetemcomitans. In the test group, not only was the reduction in T. forsythia maintained, but the tendency of increase in A. actinomyctemecomitans was also prevented. The decrease in T. denticola was also more profound in the test group, whereas the difference was not signicant compared to the control group. Overall, these ndings suggest that the polypharmaceutical approach in periodontal treatment could help the stabilization of the outcomes of mechanical techniques. However, the observation that the eradication was not complete and some species proportionally increased in the test group implies that more studies are needed to optimize the antibiotic protocols, possibly at the individual patient level. This view conrms the complexity of choice of ideal antibiotics or their combinations in periodontal treatment10,52 and warrants further studies of longer duration and testing of more clinical treatment modalities. In a similar and recent study where GAgP patients were treated with full-mouth root planing, systemic antibiotics, and chlorhexidine rinses, the extent of
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invasion of epithelial cells by A. actinomycetemcomitans, P. intermedia, P. gingivalis, T. forsythia, and T. denticola was analyzed.45 The results demonstrate that clinical measurements improved signicantly following treatment, all bacterial species except P. gingivalis were signicantly reduced in plaque between baseline and 3 months, and all species showed a trend to repopulate between 3 and 6 months. Most importantly, all species were detected intracellularly and the percentage of cells infected intracellularly was not affected by therapy. The authors of this study argued that because the presence of the tested species within epithelial cells was not altered after treatment, recolonization may originate from the oral mucosa, and extracrevicular reservoirs of bacteria exist. Our results support these observations. The inadequacy of antibiotic or antiseptic-based elimination of bacterial species53 could also be overcome by the use of host-modulating agents, which could provide a less favorable environment for the recolonization of the pathogenic microbiota.54,55 CONCLUSIONS The results from this study suggest that combined amoxicillin and metronidazole use as an adjunct to SRP leads to a better clinical healing compared to mechanical treatment alone. The polypharmaceutical approach resulted in a signicant and substantial decrease in T. forsythia and prevented its recolonization for 6 months, suggesting that T. forsythia may determine the long-term stability of periodontal treatment outcomes. ACKNOWLEDGMENTS The authors acknowledge the kind technical assistance of Drs. Fahriye Keskin and Sevgi Ciftci from the Department of Oral Microbiology, Faculty of Dentistry, Istanbul University, Istanbul, Turkey. This study was supported by the Research Fund of Istanbul University (project no. T657/170305). The authors report no conicts of interest related to this study. REFERENCES
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52. Beikler T, Abdeen G, Schnitzer S, et al. Microbiological shifts in intra- and extraoral habitats following mechanical periodontal therapy. J Clin Periodontol 2004; 31:777-783. 53. Ehmke B, Moter A, Beikler T, Milian E, Flemmig TF. Adjunctive antimicrobial therapy of periodontitis: long-term effects on disease progression and oral colonization. J Periodontol 2005;76:749-759. 54. Kantarci A, Hasturk H, Van Dyke TE. Host-mediated resolution of inammation in periodontal diseases. Periodontol 2000 2006;40:144-163.

55. Salvi GE, Lang NP. Host response modulation in the management of periodontal diseases. J Clin Periodontol 2005;32:108-129. Correspondence: Dr. Serdar Cintan, Department of Periodontology, Faculty of Dentistry, Istanbul University, Capa 34390, Istanbul, Turkey. Fax: 0212-5340807; e-mail: scintan@istanbul.edu.tr. Submitted September 5, 2009; accepted for publication February 27, 2010.

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