Microbiologic Observations After Four Treatment Strategies Among Patients With Periodontitis Maintaining a High Standard of Oral Hygiene: Secondary Analysis of a Randomized Controlled Clinical Trial.

Journal of Periodontology; Copyright 2015

DOI: 10.1902/jop.2015.140620

Microbiological Observations Following Four Treatment Strategies Among Periodontitis Patients Maintaining A High Standard of Oral Hygiene. A Secondary Analysis of a Randomized Controlled Clinical Trial Hans R Preus,* Gunnar Dahlen,† Per Gjermo,* Vibeke Baelum‡ *

Department of Periodontology, Institute of Clinical Odontology, Faculty of Dentistry, University of Oslo, Norway.

†

Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Sweden. ‡

Department of Dentistry, Health, Aarhus University, Denmark.

Background: The benefit of full-mouth disinfection (FDIS) over traditional scaling and root planing (SRP), with or without adjunctive metronidazole, when treating chronic, destructive periodontitis and the longterm association between clinical and microbiological outcomes after such strategies remains equivocal. Aim: To examine the relationship between clinical and microbiological outcomes of four different treatment strategies for chronic, destructive periodontitis among patients who maintain excellent oral hygiene and low gingival bleeding scores. Materials and Methods: 184 periodontitis patients capable of maintaining a high standard of oral hygiene were randomly allocated to one of four treatment groups 1) FDIS+metronidazole, 2) FDIS+placebo, 3) SRP+metronidazole, 4) SRP+placebo. Recordings of plaque, bleeding on probing, probing pocket depth and clinical attachment level were carried out in four sites per tooth at baseline, 3 and 12 months after treatment. Prior to treatment, pooled subgingival samples were obtained from the five deepest pockets, which were sampled again 3 and 12 months after treatment. Microbiological assessments of eight putative periodontopathogens were performed using the checkerboard DNA-DNA hybridization method. Results: The levels of the bacterial species were relatively low already at baseline. The only microbial factor statistically significantly associated with the clinical outcomes of treatment after 12 months was the association between the reductions of T. forsythia and being free from pockets ≥ 5 mm. Conclusion: In this clinical trial, the only microbial factor associated with the clinical outcomes after 12 months was a statistically significant association between the reductions of T. forsythia and being free from pockets ≥ 5 mm.

KEY WORDS: Oral hygiene, Dental Scaling, Root planing, Antibiotics, Periodontal Attachment Loss, Alveolar Bone Loss.

Changes in clinical attachment level (CAL) and probing pocket depth (PPD) are the key soft tissue outcome variables in clinical trials evaluating the effect of adjunctive antibiotics in periodontal therapy. As far as it has been possible to ascertain, the trial design usually involve baseline measurements of CAL and PPD obtained before any treatment procedures, including oral hygiene improvement, have commenced. Thereby, the treatment strategies tested in all these studies comprise a package composed of a personal oral hygiene part and a professional part, a major portion of which is SRP. Provided proper randomization and allocationconcealment has been used, this should not inflate the estimates of between-group differences in the observed CAL or PPD changes. However, the CAL and PPD changes observed following treatment are attributable to the total oral hygiene and treatment package, comprising supragingival plaque control (oral hygiene instruction, OHI), SRP as well as adjunct antibiotics. Unfortunately, in many of the adjunctive antibiotics trials the actual contents and thoroughness of the mechanical treatment remains unclear,1,2 and it is often not

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clear to what extent supragingival plaque control has in fact been part of the treatment strategies tested. It is well known that supragingival plaque control alone, i.e., treatment of the gingivitis component, leads to reduced CAL and PPD3 and has a marked effect on the subgingival microbiota of moderate to deep periodontal pockets,4 just as it reduces the subgingival species counts among treated patients.5 In a recently published longitudinal intervention study on chronic periodontitis,6 a unique design was used to reflect the effect of periodontitis treatment as distinct from the treatment of the gingivitis component. Hence, randomization as well as baseline clinical measurements and microbiological sampling were preceded by a three month hygiene phase that resulted in a mean baseline plaque and bleeding on probing (BOP) percent of 12-16% (depending on treatment group) and 10-13% respectively (i.e., “excellent oral hygiene”), while the baseline mean number of tooth surfaces with a PPD ≥ 5 mm remained high at 22-28 for the four treatment groups.6 Thus, The purpose of the present study was to report on a secondary analysis of the microbiological outcomes of the treatment modalities applied in that study,6 i.e., comparing adjunctive metronidazole therapy vs. placebo, and traditional scaling and root planing (SRP) vs. SRP within a single workday (FDIS), and to examine the relationship of the microbiological with the clinical outcomes of treatment.

MATERIALS AND METHODS The present study thus represents a secondary analysis of data originating in a randomized double masked, four-arm, placebo-controlled clinical intervention trial carried out among 184 patients with severe chronic destructive periodontitis. Participants were recruited among patients referred from local dentists to a periodontal specialist clinic in Telemark County in the southern part of Norway. Recruitment and inclusion lasted from December 2007 to April 2009, treatment from March 2008, and the twelve months follow-up was completed September 22nd, 2010. The study rationale, design as well as the one-year clinical results has previously been described in detail.6 Briefly, participants were recruited among patients referred to a periodontal specialist clinic for treatment. Candidate participants (age between 35 and 75 years; no prior systematic periodontal treatment experience; no systemic diseases or continuous medication known to affect the severity or progression of periodontitis) underwent a three month pre-study hygiene phase resulting in an excellent oral hygiene (defined above), subsequent to which eligibility for the trial was assessed on the basis at least five sites remaining with a pocket depth ≥ 5 mm; absence of bacterial species with known low- or insensitivity to metronidazole (e.g. Aggregatibacter actinomycetemcomitans, Pseudomonas, Echerichia coli, Serratia, Shigella, or Acinetobacter); and no known allergies or adverse reactions from this antibiotic.6 Eligible participants were assigned to one of four treatment arms using a random allocation table.7 A codebook manager (PG) kept all patient and allocation data throughout the study - thereby securing blinding of the clinical research staff.6 The four treatment arms were: FDIS+Metronidazole (MET) (Group 1); FDIS+placebo (Group 2); SRP+MET (Group 3); and SRP+placebo (Group 4). Groups 1 & 2 received full mouth scaling and root planing (SRP) completed within a single workday (FDIS) using two sessions of 65 minutes each, two hours apart. In Groups 3 & 4 the SRP was completed using two 65-minute sessions, 21 days apart. Subsequent to all mechanical treatment sessions, in all groups, the patients rinsed for one minute with 10 ml 0.2 % CHX,* and following mechanical instrumentation all sulci and pockets were filled with CHX gel.† In addition, patients in Groups 1 and 3 received MET,‡ 400 mg x 3 for ten days, starting the day before the two mechanical treatment sessions in Group 1 and the day before the second SRP session (day 20) in Group 3. Patients in Groups 2 and 4 received pharmacy-packed placebo tablets according to the same scheme as for Groups 1 & 3, respectively. Four patients left the study during the

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active treatment phase; one died; two were diagnosed with cancer, and one with diabetes mellitus, leaving 180 patients still in trial at the 12-month follow-up (Figure 1). Additional details regarding patient recruitment, eligibility assessment, in- and exclusion criteria, randomization procedures, patient flow and clinical assessments have been published elsewhere.6 The project protocol was approved by the Privacy Ombudsman for the Norwegian Universities (#15768) and Regional Committee for Medical Research Ethics, (Oslo, Norway) (REC South East 2.2006.3573/S-06458b) and all patients provided written informed consent prior to participation. U.S. National Institutes of Health Clinical Trials Registry number http://www.clinicaltrials.gov – is NCT01318928. Microbiological Sampling: The sampling was performed by the project leader (HRP) as follows: At baseline, after identifying the sites showing periodontal breakdown, a pooled sample was collected from the five deepest pockets of each patient. Following therapy, pooled samples from the same five sites were obtained at 3 and 12 months. Sampling was done using a curette and paper point,§ and the harvest was transferred to 1 ml PBS in a sterile tubes.|| One paper point was inserted for 10 sec for the planktonic state in each site, and subsequently used to wipe off any remaining substance on the curette used to sample that site. After closing the lid, and vigorous shaking, one half of the harvest was stored frozen at - 20oC for checkerboard diagnosis.8 Checkerboard DNA-DNA Hybridization. The checkerboard (CKB) method was used according to the description and evaluation given in a previous publication.8 Briefly, following frozen storage and thawing, 100 µl of the samples were transferred to 100µl TE buffer¶ and 100 µl 0.5 M NaOH were added and the suspensions were boiled for 5 min. After boiling 800 µl 5M ammonium acetate were added to each tube and were kept deep frozen until the samples were processed with the checkerboard methodology according to standardized protocols.9,10 The membranes were loaded so that the Baseline (BL), 3 and 12-month samples were on the same membrane to minimize between membrane variations for each patient (Figure 1). Specific whole genomic DNA probes and standard preparations of the same species were constructed for the following periodontitis associated bacterial species/strains: Porphyromonas gingivalis,# Prevotella intermedia,** Campylobacter rectus,†† Tannerella forsythia,‡‡ Aggregatibacter actinomycetemcomitans,§§ Fusobacterium nucleatum,|||| Treponema denticola,¶¶ Haemophilus parainfluenzae.## Even though all patients were found to be negative for Aggregatibacter actinomycetemcomitans at baseline, this bacterium was included to assess the possibility of regrowth of this microorganism. Four microorganisms, known to cause classical opportunistic infections, were also included (Enterococcus faecalis,*** Staphylococcus aureus,††† Enterobacter cloacea‡‡‡ and Candida albicans,§§§ but as these species were infrequently detected.8 and in any case not considered putative periodontal pathogens, they are not further dealt with here. Digoxigenin-labeled, whole genomic probes were prepared by random priming using a labelling kit‖‖‖ from the 12 microbial strains of the panel, described previously.8 The DNAprobes were applied using every second lane perpendicularly to the applied sample preparations. The hybrids formed between the bacterial DNA and the probes were detected by application of an anti-digoxigenin antibody conjugated with alkaline phosphatase and incubation with a chemiluminescent substitute.¶¶¶ The intensity of the chemiluminesence signals ### was assessed in biomedical light units (BLU). For most samples there was

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sufficient sample material to run the checkerboard analysis twice,8 and the microbiological data analysed are therefore based on duplicate assessments for the vast majority of samples. Data Analysis and Statistical Evaluation: The BLU signals for each sample were expressed as a percentage of the BLU signal intensity of pooled standard samples containing 106 (high-standard) of each of the eight bacterial species tested for. This BLU percentage was log transformed (log10), in such a way that a value of 6 would correspond to a sample bacterial 106, a value of 5 to a bacterial count of 105, etc. Whenever possible the log10(counts) used in the analyses were obtained as the average of the two duplicate assessments of the samples. For each bacterial species the change in the log10(count) was calculated thus obtaining values from baseline to 3 months, and from baseline to 12 months. These change values were linearly regressed on FDIS (yes=FDIS; no=SRP) and metronidazole (yes=metronidazole; no=placebo) to assess if the treatment approaches influenced the bacterial counts. In these regression analyses, allowance was made for the possible interaction between mechanical treatment mode (FDIS/SRP) and antibiotic regimen (metronidazole/placebo). Intention to treat analysis was used, and linear or logistic regression analysis were applied as appropriate to assess the extent to which the 12 month clinical outcomes in terms of the mean pocket depth reduction or the absence of pockets ≥ 5 mm was related to the changes in the microbiological parameters..

RESULTS Table 1 shows that the BLU signals observed at baseline corresponded to mean log10(count) values in the order of magnitude of 4 to 5 for the eight periodontitis associated species tested, i.e., P. gingivalis, P. intermedia, C. rectus, T. forsythia, F. nucleatum, T. denticola, A. actinomycetemcomitans, and H. parainfluenzae. Only rarely were log10(counts) of 6 or more observed, even at baseline (Figure 2). Periodontal treatment resulted in reductions in the log10(counts) for six species, i.e., P. gingivalis, P. intermedia, C. rectus, F. nucleatum, T. denticola and T. forsythia, whereas no overall changes were observed for the remaining two species, A. actinomycetemcomitans, and H. parainfluenzae (Figure 2). The reductions in the log10(counts) for the six species tended to be greater at 3 months than at 12 months for three of the four treatment groups; the exception being the FDIS-only group where reductions were either greater or similar at 12 months compared with the 3 months results (Table 2). Except for T. forsythia where log10(count) reductions up to 0.85 were observed in the FDIS+MET group, most reductions were modest (Table 2). Regression analyses showed limited, and mostly statistically insignificant, influence of the treatment modality (FDIS vs. SRP, or metronidazole vs. placebo) on the reductions in the bacterial log10(counts), whether at 3 months or at 12 months after therapy (Table 3). The exceptions were a statistically significantly greater reduction in the log10(counts) of C. rectus and T. forsythia after metronidazole treatment at 3 months, and, for T. forsythia, also at 12 months; and a statistically significantly greater reduction in the log10(counts) of these bacteria following FDIS at 12 months (Table 3). For T. forsythia and C. rectus there was a statistically significant interaction between the mechanical (FDIS vs. SRP) and the antibiotic (MET vs. placebo) treatment regimen, such that FDIS and metronidazole treatment potentiated each other. Regression analyses showed that the only microbial factor associated with the clinical outcomes of treatment after 12 months (Table 4) was a statistically significant (p=0.04) association between the reduction in the log10(count) for T. forsythia and being free from pockets ≥ 5 mm at 12 months.

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DISCUSSION The primary analysis of the clinical data from this trial demonstrated that MET treatment resulted in statistically significant additional CAL gains and PPD reductions, and higher odds of being free from pockets ≥ 5 mm, after one year as compared to placebo treatment.6 In contrast, the mechanical treatment strategy (FDIS or standard SRP) had no influence over the clinical outcomes after treatment. The results showed that the clinical benefits of adjunct MET treatment were accompanied by modest, and mostly statistically insignificant, reductions in the counts of the eight putative periodontal pathogens studied. The notable exceptions from this rule were the statistically significant post-therapy reductions in T. forsythia and C. rectus, which for T. forsythia persisted at 12 months. Moreover, the T. forsythia reduction was the only bacterial change that was statistically significantly associated with the clinical outcome; absence of pockets ≥ 5 mm after 12 months. Relatively few studies have reported on the effect on clinical and microbiological outcomes of mechanical treatment with or without adjunctive MET.1,11 From such studies,6,12,13-16 it is evident that there is considerable heterogeneity in both the mechanical treatment approaches as well as in the antibiotic regimens. Hence the duration of antibiotic treatment ranged from 7 13,15 to 14 days;12,14,16 and the daily dose ranged from 600 mg/day 13 to 1200 mg/day.6,12,14,16 This variability resulted in total antibiotic load ranging from 4,200 mg13 to 16,800 mg.12,14,16 Moreover, with the exception of Preus et al.,6 none of the studies12,13-16 applied microbiological diagnosis prior to the antibiotic therapy, and this might mean that some of the patients have been treated using an antibiotic not expected to target all the periodontal pathogens present, i.e. species that show low- or no sensitivity to metronidazole. There were also variations in the commencement of the antibiotic regimens, and with the exception of one study, 6 the antibiotic treatment was never started prior to the mechanical intervention. These are all factors that may compromise the validity of betweenstudy comparisons.2 Taking these methodological differences into account, the findings from this study are in concert with those of Haffajee and coworkers17 who found significant reduction of T. forsythia in both SRP+MET and SRP-only treatment groups after 12 months. They also found a significant reduction of P. gingivalis in their SRP+MET group.17 In comparative studies,12,13,17,18 it was found that the mean proportions of the members of the red complex19 were reduced significantly 12 months after therapy in MET+SRP groups as compared to SRP only groups. In most of these study populations,6,12,13-18 as well as in the present one, adjunctive MET was found to provide a variable, but generally better clinical outcome than SRP alone. In contrast to the above mentioned articles,12,13,17,18 the present study did not find any effect of adjunctive MET on the presence of P. gingivalis and any clinical endpoint studied (mean CAL, mean PPD, absence of pockets >5mm). A possible explanation for this may be the long and stringent oral hygiene phase imposed on the present population, and that the baseline of this study was after this hygiene phase (see discussion below). The observation that Enterococcus faecalis, Staphylococcus aureus, Enterobacter cloacea and Candida albicans were infrequently detected8 indicates that use of adjunctive MET did not cause a selective pressure for the opportunistic growth of these organisms. The reason for finding A.actinomycetemcomitans in a limited number of samples, despite that a prerequisite for inclusion was the absence of this bacterium in prestudy and baseline bacterial samples, was probably related to the method of diagnosis at baseline, which was culture and not CKB. The detection limit for A.actinomycetemcomitans upon culture is 102 103cells/ml in the seeded sample.20,21 Moreover, regarding post-therapy samples, it is possible that all four treatment strategies reduced the presence of other biofilm bacteria to such an

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extent that undetected A.actinomycetemcomitans had the opportunity to grow into detectable levels. The detection frequency of A. actinomycetemcomitans was not significantly different among the groups at any time. In this study, intention to treat analysis was used, i.e. participants were analysed in the group to which they had been randomized, whether they complied with the antibiotic regime or not. Missing data were a minor problem. All 184 patients contributed clinical baseline data, and 180 patients contributed clinical follow-up data. A total of 178 microbiological samples were available for analysis at baseline, whereas 174 samples were analysed at 3 month too little material for analysis in a few samples), and 179 samples were analysed at 12 months. As previously emphasized, the present study has introduced an uncommon practice of enrolling participants only after a stringent and long oral hygiene phase. This clinical strategy was deliberately chosen to explore the real ‘adjunct’ effects of mechanical and antibiotic treatment over and beyond the improvements that can be brought about by proper oral hygiene alone. The pocket depth (PD) is a variable sum of increased gingival volume due to edema (gingivitis) and apical migration of periodontal attachment (periodontitis). More importantly, the gingival edema may jeopardize the measuring procedure in as much as the bottom of the pocket becomes softer and more penetrable to instruments (periodontal probe and scalers).22 Both of these factors contribute to erroneous diagnoses resulting in an untoward selection of participants that is biased towards patients with more gingivitis than periodontitis, just as the observed clinical treatment effects (changes from baseline) inevitably will be inflated. The pre-study hygiene phase employed in the present study served to reduce the inflammatory component of the gingiva to a minimum; just as it reduced the PD caused by gingivitis, and tightened the fibers coronal to the top of the periodontal membrane by reducing the edema, and possibly increasing collagen contents. This was desirable to reduce the suggested risk for inflated PD measurements at baseline,22 but also to avoid anticipated damage inflicted by the scaling procedures whereby an iatrogenic post-treatment CAL loss might be induced.23 While it could be argued that periodontitis does not exist in the absence of gingivitis, it remains a fact that the initial oral hygiene phase alone will significantly influence the clinical parameters of periodontitis, just as the microbiological diagnosis is changed.24-26 Certainly, the low baseline bacterial counts observed in the present study might indicate that a key microbiological effect of a pre-study oral hygiene phase is a reduction of the bacterial load. Only infrequently were baseline bacterial levels observed in the order of magnitude of 106, which is otherwise commonly observed in trials reporting on the treatment-wise ‘pristine’ microbiological profile.27,28 This reduced baseline load might also explain why relatively small and insignificant bacterial reductions were observed as a result of the subsequent mechanical and antibiotic treatment. It is thus clear that the lower the starting point, the smaller will a reduction necessarily be, just as low baseline levels may mean that the signalto-noise ratio becomes unfavorable for the detection of changes owing to the methodological errors inherent in microbiological analyses.8 Unfortunately, a checkerboard analysis of the microbiological samples taken before patients were subject to the hygiene phase was not performed, as the samples taken at that time were used to provide a microbiological diagnosis of the sensitivity to metronidazole.6 Presently, evidence for the interpretation of the effect of the pre-study hygiene phase on the bacterial levels cannot be presented, other than by reporting a substantial reduction of sites with plaque from pre-study baseline (screening) average value above 60% (Unpublished observations) to the study baseline values of 12-16%.6 However, a study is in the process to investigate the microbiological effects of meticulous oral hygiene prior to mechanical SRP in a new group of untreated periodontitis patients.

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It may be calculated that the logistic regression coefficient of -0.43 for the association between a one unit reduction in the log10(count) of T. forsythia and the persistence of pockets ≥ 5 mm at 12 months (Table 4) corresponds to a 35% reduced risk of pocket persistence at 12 months in participants with a 12-month T. forsythia reduction to 10% of the baseline level. Even so, the results of the present study indicate that the statistically significant adjunctive effect on metronidazole treatment on the clinical parameters CAL, PD and absence of pockets ≥ 5 mm at 12 months is accompanied by only modest and mostly statistically insignificant microbiological changes.6 In fact, the only statistically significant association observed between microbiological changes and clinical improvements were the association between the reductions of T. forsythia and the absence of pocket depths ≥ 5 mm at 12 months post-therapy. As the anticipated mechanism of action of metronidazole would be its antimicrobial potential, it may seem a bit paradoxical that participants treated with metronidazole fared statistically significant better on the clinical parameters CAL, PD and pocket persistence,6 whereas only modest effects were observed on the microbial parameters. However, as shown in Tables 2, 3 and Figure 2, trends in reduction of bacterial levels in the groups that received metronidazole were clear. It is not known what magnitude of numerical reduction in pathogenic species is necessary for beneficial clinical changes to occur, so even though the reductions in bacterial cell counts were “modest”, they might have been sufficient to lead to the PD reductions and CAL gains shown by Preus et al 2014.6 Another explanation, although more speculative, is that the periodontal microbiome may harbor microorganisms that was not in the CKB panel, but anyway could act as “key stone pathogens”,29 inasmuch as they could orchestrate inflammatory disease by remodeling a normally benign microbiome into a dysbiotic one.29,30 A more farfetched, although contributory explanation for the observed discrepancy between clinical and microbiological results could be found in the observation that metronidazole may exert an antiflogistic effect,31 in addition to its antimicrobial effect. Finally, the low baseline levels of the microbial species brought about by the pre-study hygiene phase might explain why changes would necessarily be smaller and at a greater risk of being masked by measurement errors than has been observed in traditional trials employing pre-intervention baseline observations as the basis for calculating change.

CONCLUSION: Observations from this clinical trial, following four treatment strategies among periodontitis patients maintaining a high standard of oral hygiene, suggested that the only microbial factor associated with the clinical outcomes of treatment after 12 months, in any of the four groups, was a statistically significant association between the reductions of T. forsythia, and being free from pockets ≥ 5 mm in the FDIS+MET group. ACKNOWLEDGEMENTS: There is no conflict of interest associated with this report, and the work was financed by the Norwegian Research Council, Oslo, Norway; grant # 185120 and contd’ # 229029

DISCLAIMER: There is no conflict of interest associated with this report.

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Corresponding Author; Hans R Preus, Dept of Periodontology, IKO, Faculty of Dentistry, PO 1109 Blindern, 0317 Oslo, Norway, +47 22 85 21 63 (tel.) (May be published), +47 22 85 23 96 (fax) (May be published), [email protected] Submitted November 4, 2014; accepted for publication February 16, 2015. Figure 1 – Consort Diagram. The flow of patients and microbiological diagnoses from Baseline through 3 and 12 months follow-up Figure 2 – The changes as a result of treatment in the log log10(count) values for the eight putative periodontal pathogens. Table 1. Baseline mean values (95% CI) of the log10(count) values for the eight periodontitis associated bacterial species. Given for each experimental group. Bacterial species 1 FDIS+MET 2 FDIS SRP+MET SRP n=44 n=45 n=44* n=45* P. gingivalis 4.4 (4.2;4.6) 4.4 (4.2;4.7) 4.5 (4.3;4.7) 4.3 (4.1;4.6) P. intermedia 4.8 (4.7;4.9) 4.8 (4.7;4.9) 4.9 (4.8;5.0) 4.7 (4.5;4.8) C. rectus 4.1 (4.0;4.2) 4.0 (3.9;4.1) 4.0 (3.9;4.1) 3.9 (3.7;4.0) T. forsythia 4.7 (4.5;4.9) 4.5 (4.3;4.7) 4.6 (4.4;4.8) 4.3 (4.1;4.5) F. nucleatum 4.6 (4.5;4.7) 4.5 (4.4; 4.6) 4.5 (4.4;4.6) 4.5 (4.4;4.6) T. denticola 4.9 (4.8;5.1) 4.9 (4.7;5.0) 4.9 (4.7;5.0) 4.7 (4.6;4.9) A. actinomycetemcomitans 4.1 (4.0;4.3) 4.1 (4.0;4.3) 4.1 (4.0;4.3) 3.9 (3.8;4.1) H. parainfluenzae 4.2 (4.0; 4.3) 4.2 (4.0;4.3) 4.2 (4.0;4.3) 4.0 (3.8;4.2) * 1 baseline sample missing

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Table 2. Mean reduction (95% CI) in the log10(count) values for the six bacterial species that reduced during the 12 months follow-up period. Given for each experimental group. Bacterial 1 FDIS+MET 2 FDIS SRP+MET SRP species BL-3 months BL-12 months BL-3 months BL-12 months BL-3 months BL-12 months BL-3 months BL-12 months P. gingivalis 0.34 (0.16;0.51) 0.28 (0.11;0.45) 0.22 (0.04;0.41) 0.25 (0.08;0.41) 0.25 (0.07;0.43) 0.19 (0.02;0.37) 0.24 (0.08;0.40) 0.16 (0.01;0.31) P. intermedia 0.26 (0.10;0.42) 0.22 (0.07;0.37) 0.03 (-0.11;0.16) 0.08 (-0.03;0.19) 0.14 (0.03;0.26) 0.12 (0.00;0.24) 0.14 (-0.01;0.28) 0.09 (-0.03;0.21) C. rectus 0.30* (0.19;0.42) 0.26* (0.14;0.39) 0.16 (0.06;0.27) 0.19 (0.08;0.31) 0.20 (0.06;0.34) 0.15 (0.01;0.28) 0.08 (-0.04;0.21) 0.04 (-0.08;0.17) T. forsythia 0.85 * (0.66;1.05) 0.73* (0.51;0.94) 0.41 (0.20;0.63) 0.38 (0.17;0.58) 0.53 (0.31;0.75) 0.41 (0.20;0.62) 0.47 (0.27;0.67) 0.20 (-0.02;0.43) F. nucleatum 0.23 (0.12;0.35) 0.20 (0.09;0.32) 0.09 (-0.04;0.21) 0.13 (0.02;0.25) 0.16 (0.04;0.26) 0.13 (0.02;0.23) 0.16 (0.05;0.26) 0.10 (-0.01;0.20) T. denticola 0.32 (0.18;0.46) 0.25 (0.09;0.41) 0.15 (0.02;0.28) 0.12 (0.01;0.24) 0.22 (0.08;0.35) 0.15 (0.02;0.29) 0.17 (0.05;0.30) 0.09 (-0.05;0.22) * Statistically significantly greater than estimates for SRP group. Table 3: The average reduction (95% CI) in the log10 count) for each of the eight periodontitis-associated species as a result of FDIS, respectively, Metronidazole treatment after 3 and 12 months, as determined by linear regression analysis. Bacterial species 3 months 12 months FDIS (ref= SRP) Metronidazole (ref=Placebo) FDIS (ref= SRP) Metronidazole (ref=Placebo) P. gingivalis 0.03 (-0.14;0.21) 0.06 (-0.11;0.23) 0.09 (-0.07;0.25) 0.04 (-0.12;0.20) P. intermedia 0.00 (-0.14;0.14) 0.12 (-0.01;0.26) 0.04 (-0.08; 0.17) 0.09 (-0.04;0.21) C. rectus* 0.09 (-0.03;0.21) 0.13† (0.01;0.25) 0.13†(0.01;0.25) 0.09 (-0.03;0.21) T. forsythia* 0.13 (-0.08;0.34) 0.25† (0.04;0.46) 0.26†(0.04;0.46) 0.28†(0.07;0.49) F. nucleatum 0.00 (-0.11;0.11) 0.07 (-0.04;0.18) 0.06 (-0.04;0.16) 0.05 (-0.05;0.16) T. denticola 0.04 (-0.09;0.17) 0.11 (-0.02;0.24) 0.07 (-0.07;0.20) 0.10 (-0.03;0.23) A. actinomycetemcomitans 0.03 (-0.08;0.14) 0.01 (-0.10;0.11) 0.01 (-0.11;0.13) -0.01 (-0.13;0.11) H. parainfluenzae 0.03 (-0.07;0.14) -0.01 (-0.12;0.08) 0.06 (-0.05;0.16) -0.02 (-0.13;0.08) * Statistically significant evidence for interaction between FDIS and Metronidazole †

Estimates in bold face were statistically significantly different from 0 at 5% significance level.

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DOI: 10.1902/jop.2015.140620

Table 4. Results of regression analyses of the influence of the 12-month species reductions on the 12-month reductions in mean pocket depth (linear regression), respectively, the persistence of pockets ≥ 5 mm at 12 months (logistic regression). Predictor Outcome variable Reduction in log10(counts) of species Pocket depth reduction (mm) Persistence of pockets ≥ 5 mm at 12 months BL-12 months

P. gingivalis P. intermedia C. rectus T. forsythia F. nucleatum T. denticola A. actinomycetemcomitans H. parainfluenzae

β§ (95% CI) -0.08 (-0.22, 0.05) 0.12 (-0.06, 0.30) 0.03 (-0.15, 0.21) 0.01 (-0.09, 0.11) 0.05 (-0.15, 0.26) -0.01 (-0.18, 0.16) -0.10 (-0.29, 0.09) 0.16 (-0.04, 0.37)

β$ (95% CI) -0.21 (-0.77, 0.34) 0.05 (-0.67, 0.77) 0.02 (-0.69, 0.73) -0.43† (-0.85, -0.01) -0.15 (-0.99, 0.68) 0.01 (-0.65, 0.67) 0.25 (-0.52, 1.01) 0.46 (-0.37, 1.29)

§ Determined by linear regression; $ Determined by logistic regression; †

Estimates in bold face were statistically significantly different from 0 at 5% significance level.

*

Corsodyl 2mg/ml mouth rinse, SmithKline Beecham, Brentford, UK.

†

Corsodyl Gel 1%, Smithcline Beecham, Brentford, UK.

‡

Flagyl 400 mg, Sanofi Aventis, Lysaker, Norway.

§

LM syntette, LM-Instruments, Parainen, Finland.

§

Roeko paper points, Medium, Coltene/Whaledent, Langenau, Germany

||

Nunc Store-it Cryo-Tubes, 1.8ml (working volume), Thermo Fischer Scientific, Roskilde, Denmark.

¶

10 mM Tris HCl, 1 mM EDTA, pH 7.6.

#

FDC381, originally obtained from Dr Socransky, Forsyth Dental Center (FDC), Boston, MA, US

**

ATCC25611

††

ATCC33238

‡‡

ATCC43037

§§

FDCY4

||||

ATCC10953

¶¶

OMGS3271

##

CCUG 12836/OMGS3725

***

clinical isolate OMGS3863

†††


‡‡‡


§§§

CCUG46390/OMGS3751

‖‖‖

the High-Prime labelling kit, Roche Diagnostics, Mannheim, Germany

¶¶¶

CDP-Star, Roche Diagnostics, Mannheim, Germany

###

LumiImager Workstation, Boehringer-Mannheim, Mannheim, Germany

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DOI: 10.1902/jop.2015.140620

Assessed for eligibility (n=292)

Enrollment

Excluded (n=108) Not meeting inclusion criteria (n=98) Refused to participate (n=6) Other reasons (n=4)

Randomized (n=184)

Allocated to intervention (n=45) Received intervention (n=45) Microbiol.Diagnoses (n=45) Did not receive intervention(n=0)

SRP + MET

SRP + PLACEBO

Allocated to intervention (n=46) Received intervention (n=46) Microbiol.Diagnoses (n=44) Did not receive intervention(n=0)

Allocated to intervention (n=47) Received intervention (n=47) Miicrobiol.Diagnoses (n=45) Did not receive intervention (n=0)

Follow-up, 3 months f

Allocated to intervention (n=46) Received intervention (n=46) Microbiol.Diagnoses (n=44) Did not receive intervention (n=0)

FDIS + PLACEBO

Lost to follow-up (n = 2) Discontinued intervention (n=1) Microbiol. Diagnoses (n= 42)

Lost to follow-up (n = 0) Discontinued intervention (n=1) Microbiol. Diagnoses (n = 44)



Follow-up 12 months

Allocation, baseline

FDIS + MET

Analyzed (n=44) Excluded from analysis (n=0) Microbiol. Diagnoses (n = 44)




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DOI: 10.1902/jop.2015.140620

80 70 60 50 40 30 20 Baseline 3 months 12 months

0 2

3

4

5

6

80 70 60 50 40 30 20 10 0

7

C. rectus, log(count) 100

Baseline 3 months 12 months

90 80 70 60 50 40 30 20 10 0

3

4

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6

3

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5

6

F. nucleatum, log(count)

70 60 50 40 30 20 10

7


90 80 70 60 50 40 30 20 10

7

3

4

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7

6

13

70 60 50 40 30 20 Baseline 3 months 12 months

10

7

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90

90


10

2

3

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3

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6

7

A. actinomycetemcomitans, log(count) 100

T. forsythia, log(count)

T. denticola, log(count)

80

100

0 2

90

0 2

P. intermedia, log(count)

0 2

80

P. gingivalis, log(count)

Cumulative percent subjects


100

100


90

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10

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90


100

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100



Figure 2. The changes as a result of treatment in the log log10(count) values for the eight putative periodontal pathogens.


10 0

7

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H. parainfluenzae, log(count)

7

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