Periodontol (cid:149) September 2000
`
`Long-Term Treatment With Subantimicrobial
`Dose Doxycycline Exerts No Antibacterial
`Effect on the Subgingival Microflora
`Associated With Adult Periodontitis
`
`Clay Walker,* John Thomas, 1 Sonia Nango,* Jennifer Lennon,* Jeanne Wetzel, 1
`and Christopher Powala
`
`Background: The purpose of this study was to determine
`whether treatment with subantimicrobial dose doxycycline
`(SDD), 20 mg bid, exerted an antimicrobial effect on the
`microflora associated with adult periodontitis.
`Methods: Following the approval of the protocol and informed
`consent forms by the respective IRBs at the University of Florida
`and West Virginia University, 76 subjects with adult periodonti-
`tis were entered and randomly assigned to receive SDD or
`placebo. A split-mouth design was utilized, with each subject
`receiving subgingival scaling and root planing (SRP) in two
`quadrants immediately following baseline data collection, while
`the remaining two quadrants were left unscaled (non-SRP).
`Microbial samples were collected prior to treatment, after 3, 6,
`and 9 months of treatment, and after 3 months of no treatment.
`The samples were examined by microscopy and by enumera-
`tion on selective and non-selective media.
`Results: All treatments resulted in statistically significant
`decreases in the proportions of spirochetes and motile rods
`(P <0.05) and in an increase in the proportion of coccoid forms
`(P <0.0001) relative to baseline. No between-treatment differ-
`ences were detected between the SDD and placebo treatments
`in either the SRP or non-SRP design, with the exception of the
`small and large spirochetal groups. The spirochetal proportions
`present in the SDD group were significantly lower (P<0.05) than
`the paired placebo group during the 9-month treatment and was
`preceded by a significant decrease (P<0.01) in the proportion
`of microbiologic sample sites that bled on probing. No between-
`treatment differences were detected in any of the other micro-
`bial parameters.
`Conclusion: The microbial differences observed were attrib-
`uted to the anticollagenase and anti-inflammatory properties of
`SDD and not to an antimicrobial effect. J Periodontol 2000;71:
`1465-1471.
`
`Periodontitis/microbiology; doxycycline/therapeutic use;
`clinical trials, controlled.
`
`* Periodontal Disease Research clinics, University of Florida, Gainesville, FL.
`t college of Dentistry, West Virginia University, Morgantown, WV.
`ciic,< Pharmaceuticals, Inc., Newtown, PA.
`
`S ubantimicrobial dose doxycycline
`
`(SDD) consisting of 20 mg doxy-
`cycline hyclate§ bid has been
`approved as an adjunct to periodontal
`scaling and root planing (SRP) for the
`treatment of adult periodontitis. Doxy-
`cycline, like tetracycline and minocy-
`dine, in addition to being a broad-spec-
`trum antimicrobial agent, also has
`inhibitory activity on host-derived colla-
`genases and other matrix metallopro-
`teinases by mechanisms independent of
`its antimicrobial properties. Specifically,
`tetracyclines inhibit the activity of mam-
`malian neutrophil and osteoblast colla-
`genases that appear crucial in the
`destruction of Type I and II collagen
`found in the periodontal ligament." 2
`Apart from their anticollagenase activ-
`ity, tetracyclines are also reported to
`have anti-inflammatory properties and
`to be potent inhibitors of osteoclast func-
`tion. 3 Doxycycline is the most potent
`anticollagenase inhibitor of the commer-
`cially available tetracyclines with lC50
`values of 16 to 20 IIM for collagenases
`from PMNs, dental plaque, and gingival
`tissue. 4,5 Several short-term clinical stud-
`ies have reported that SDD resulted in a
`decrease in collagenase activity which
`was accomoanied by a beneficial and
`signil Amneal 1065
`Amneal v. Supernus
`1PR2013-00368
`
`§ Perios
`PA. (cid:9)
`
`

`

`JPeriodontol (cid:149) September 2000
`
`Long-Term Treatment With Subantimicrobial
`Dose Doxycycline Exerts No Antibacterial
`Effect on the Subgingival Microflora
`Associated With Adult Periodontitis
`Clay Walker,* John Thomas,", Sonia Nango,* Jennifer Lennon,* Jeanne Wetzel,
`and Christopher Powala’
`
`Background: The purpose of this study was to determine
`whether treatment with subantirnicrobial dose doxycycline
`(SDD), 20 mg bid, exerted an antimicrobial effect on the
`microflora associated with adult periodontitis.
`Methods: Following the approval of the protocol and informed
`consent forms by the respective lRBs at the University of Florida
`and West Virginia University, 76 subjects with adult periodonti-
`tis were entered and randomly assigned to receive SDD or
`placebo. A split-mouth design was utilized, with each subject
`receiving subgingival scaling and root planing (SRP) in two
`quadrants immediately following baseline data collection, while
`the remaining two quadrants were left unscaled (non-SRP).
`Microbial samples were collected prior to treatment, after 3, 6,
`and 9 months of treatment, and after 3 months of no treatment.
`The samples were examined by microscopy and by enumera-
`tion on selective and non-selective media.
`Results: All treatments resulted in statistically significant
`decreases in the proportions of spirochetes and motile rods
`(P <0.05) and in an increase in the proportion of coccoid forms
`(P <0.0001) relative to baseline. No between-treatment differ-
`ences were detected between the SDD and placebo treatments
`in either the SRP or non-SRP design, with the exception of the
`small and large spirochetal groups. The spirochetal proportions
`present in the SDD group were significantly lower (P<0.05) than
`the paired placebo group during the 9-month treatment and was
`preceded by a significant decrease (P<0.01) in the proportion
`of microbiologic sample sites that bled on probing. No between-
`treatment differences were detected in any of the other micro-
`bial parameters.
`Conclusion: The microbial differences observed were attrib-
`uted to the anticollagenase and anti-inflammatory properties of
`SDD and not to an antimicrobial effect. J Periodontol 2000;71:
`1465-1471.
`
`Periodontitis/microbiology; doxycycline/therapeutic use;
`clinical trials, controlled.
`
`* Periodontal Disease Research Clinics, University of Florida, Gainesville, FL.
`t College of Dentistry, West Virginia University, Morgantown, WV.
`1’ CollaGenex Pharmaceuticals, Inc., Newtown, PA.
`
`S ubantimicrobial dose doxycycline
`
`(SDD) consisting of 20 mg doxy-
`cycline hyclate§ bid has been
`approved as an adjunct to periodontal
`scaling and root planing (SRP) for the
`treatment of adult periodontitis. Doxy-
`cycline, like tetracycline and minocy-
`dine, in addition to being a broad-spec-
`trum antimicrobial agent, also has
`inhibitory activity on host-derived colla-
`genases and other matrix metallopro-
`teinases by mechanisms independent of
`its antimicrobial properties. Specifically,
`tetracyclines inhibit the activity of mam-
`malian neutrophil and osteoblast colla-
`genases that appear crucial in the
`destruction of Type I and II collagen
`found in the periodontal ligament. 1 ’2
`Apart from their anticollagenase activ -
`ity, tetracyclines are also reported to
`have anti-inflammatory properties and
`to be potent inhibitors of osteoclast func-
`tion. 3 Doxycycline is the most potent
`anticollagenase inhibitor of the commer-
`cially available tetracyclines with lC50
`values of 16 to 20 l.LM for collagenases
`from PMNs, dental plaque, and gingival
`tissue. 4 ’5 Several short-term clinical stud-
`ies have reported that SDD resulted in a
`decrease in collagenase activity which
`was accompanied by a beneficial and
`significant improvement in attachment
`
`§ Periostat, CollaGenex Pharmaceuticals, Inc., Newtown,
`PA.
`
`1465
`
`

`

`Effect of Subantimicrobial Dose Doxycycline on Subgingival Microflora (cid:9)
`
`Volume 71 Number 9
`
`levels and probing depths. 6,7 More recently, a long-
`term, multi-centered clinical study compared the effi-
`cacy of a 9-month regimen of SDD following SRP to
`a placebo control and found that the use of SDD/SRP
`showed statistically significant improvements in attach-
`ment level and probing depth relative to SRP with a
`placebo. 8
`Substantial evidence indicates that the adjunctive
`use of SDD provides a significant benefit to SRP due
`to its anticollagenase and anti-inflammatory activities
`rather than to its antimicrobial activity. However, seri-
`ous concern has been expressed that even suban-
`timicrobial levels of doxycycline may exert a detri-
`mental effect on the subgingival flora. Such an effect
`could result in the disruption or suppression of the nor-
`mal flora and lead to its colonization or overgrowth by
`periodontal or opportunistic pathogens. The purpose
`of this study was to stringently evaluate the effects of
`a 9-month regimen of 20 mg doxycycline bid relative
`to a placebo control on the subgingival flora.
`
`MATERIALS AND METHODS
`Study Design
`Clinical and microbial data were collected at the Uni-
`versity of Florida and West Virginia University from
`subjects with adult periodontitis during a 9-month treat-
`ment period followed by a 3-month no-treatment
`period. Microbiological samples of subgingival plaque
`were collected prior to the initiation of treatment (base-
`line), after 3, 6, and 9 months of treatment, and at 3
`months post-treatment. A total of 76 subjects (38 at
`each study site) with adult periodontitis who met the
`inclusion and exclusion criteria set forth in the exper-
`imental protocol were entered into the placebo-con-
`trolled, double-blind treatment phase.
`The design of the study was as follows: A split-
`mouth design was used where two quadrants in each
`subject received scaling and root planing (SRP) while
`the opposite two quadrants did not (non-SRP). The
`quadrants selected to receive SRP were required to
`have a minimum of two sites with probing depth (PD)
`and loss of attachment level (AL) of ~5 but :~9 mm and
`that bled on probing. The non-SRP quadrants may or
`may not have met this criteria. Each subject was then
`randomly assigned to receive either SDD or placebo
`treatment. Thus, in effect, there were four treatment
`groups: SRP-SDD, non-SRP-SDD, SRP-placebo, and
`non-SRP-placebo. SRP-placebo was considered as a
`positive control, while non-SRP placebo was a true
`negative control. Thus, the study was considered to
`consist of two parallel experiments. SRP-SDD and non-
`SRP-SDD were paired as were non-SRP-SDD and non-
`SRP-placebo so that the SDD was the variable tested.
`All subjects who completed the 9-month treatment
`phase were invited to continue in a 3-month no-treat-
`ment phase. Of the 67 subjects who completed the
`
`9-month treatment phase, 27 of 36 and 26 of 29 sub-
`jects at the University of Florida and West Virginia Uni-
`versity, respectively, returned for sampling at the end
`of the 3-month no-treatment period.
`A total of 4 sites, distributed in a minimum of 3
`quadrants (4 quadrants were selected where possible),
`with PD ~5 mm but :!~8 mm were selected in each sub-
`ject for microbial sampling; two sites were from the
`SRP quadrants and two from the non-SRP quadrants.
`These sample sites were retained throughout the study.
`Plaque samples were collected using sterile endodon-
`tic paper points as previously described. 9 The two
`microbial samples collected from the SRP sites were
`pooled by subject and then processed, as were the
`two samples from the non-SRP sites.
`Microbial Enumeration
`Immediately following collection, samples were trans-
`ported to the microbiology laboratories. The samples
`were gently sonicated to dispense adherent plaque and
`then processed. Each sample was examined by direct
`microscopy and by culture on selective and non-selec-
`tive media.
`Microscopy. A 10 ill aliquot of the sample was
`removed under anaerobic conditions and placed on a
`clean slide for examination at 1,000x by dark-field
`microscopy. Eight distinct cellular morphotypes were
`distinguished and enumerated as previously de-
`scribed. 10
`Selective and non-selective media. Following a
`series of 10-fold dilutions in pre-reduced, anaerobic-
`sterilized Ringers solution, performed under strict
`anaerobic conditions, 0.1 ml aliquots were dispensed
`onto agar plates and spread with sterile glass rods.
`The following taxa were enumerated on selective and
`non-selective media: total anaerobic counts, total fac-
`ultative counts, total Streptococcus, total Actinomyces,
`Actinobacillus actinomy cetemcomi tans, Eikenella cor -
`rodens, Porphyromonas gin givalis, Prevotella interme-
`dia, Bacteroides forsythus, enteric bacteria, Staphylo-
`coccus aureus, and Candida. Estimates of obligate
`anaerobic bacteria were determined by subtracting the
`total facultative count from the total anaerobic count.
`If the facultative count was greater than the anaerobic
`count, a zero value was entered for the obligate anaer-
`obes. Bacteria tentatively identified as P intermedia
`are, in reality, P intermedia sensu lacto since P inter-
`media was not differentiated from P nigrescens.
`
`Statistical Analyses
`The study was considered to consist of two parallel
`experiments, each of which was designed to test for dif-
`ferences between doxycycline treatment and a placebo
`control. One design sought for differences following
`conventional periodontal treatment consisting of
`mechanical scaling and root planing (SRP), and the
`second sought for differences without the initial pen-
`
`1466
`
`

`

`Periodontol
`
`(cid:149) September 2000 (cid:9)
`
`Walker, Thomas, Nang(cid:243), Lennon, Wetzel, Powala
`
`odontal therapy of scaling and root planing (non-SRP).
`With this in mind, the resulting data sets were ana-
`lyzed with the subject as the statistical unit to detect
`if differences existed at any sample period between
`doxycycline -treated and placebo-treated subjects.
`The factoral ANOVA and Fisher’s PLSD test were uti-
`lized to determine if statistically significant differences
`were present between the paired treatment groups at
`each sample period. The repeated measures ANOVA
`was used for longitudinal analyses to test for differ-
`ences within a treatment. If differences were detected
`longitudinally, the paired t test was used to detect the
`location of the differences. In cases where outliers were
`suspected, e.g., microbial culture counts that could
`influence parametric analyses, the Wilcoxon signed
`rank, a non-parametric version of the paired t test,
`was used to verify statistical significance. Since the
`paired t test and Wilcoxon signed rank require
`matched samples from the same subject and the 3-
`month post-treatment data were derived from fewer
`subjects than the 9-month data set, it was necessary
`to construct a new data set limited to those subjects
`who consented to participate in the 3-month no-treat-
`ment phase for analyses seeking differences in the
`latter.
`A total of 78 subjects were entered at the two study
`sites with the expectation that a minimum of 65 sub-
`jects would complete the 9-month treatment phase of
`the study. This sample size, if equally split, had a 90%
`power of detecting a difference of 1 log 10 in microbial
`counts between SDD and the paired treatment. All sta-
`tistical comparisons were based on P !!~0.05.
`
`RTISU (.=iS
`Microscopic Enumeration
`Differences between and within treatment groups were
`analyzed for each of the following morphological
`groups: small, intermediate, and large spirochetes;
`motile rods; coccoid forms; non-motile rods; fusiforms;
`and filamentous rods.
`Between-treatment differences. No between-treat-
`ment differences were detected for any morphological
`group other than the spirochetes. In the SRP design,
`the proportion of small spirochetes (Table 1) present
`at the 3- and 6-month sample periods and the pro-
`portion of large spirochetes (Table 2) present at the 6-
`month sample were significantly lower in the SDD
`group than in the placebo group (P<0.05). In the non-
`SRP design, the proportions of both the small and large
`spirochetal groups present at the 9-month sample were
`significantly lower in the SDD group than in the placebo
`group (P<0.05).
`Within-treatment differences. Differences within a
`treatment were analyzed using the paired t test to
`detect if the treatment had any significant effect on a
`particular morphologic group. Both the SDD and
`
`Table I.
`Mean Percentage of Small Spirochetes
`Relative to Total Microscopic Flora for SDD
`and Placebo Treatment Groups in SRP and
`Non-SRP Design
`
`Treatment Phase
`
`Design (cid:9)
`
`Treatment
`Group (cid:9)
`
`Baseline Months Months Months (cid:9)
`
`SDD (cid:9)
`
`10.35 (cid:9)
`
`4.32*t 6. 28t 495t (cid:9)
`
`SRP (cid:9)
`
`Placebo (cid:9)
`
`10.36 (cid:9)
`
`9,59* 10.40* (cid:9)
`
`5.89t (cid:9)
`
`SDD (cid:9)
`
`9.98 (cid:9)
`
`6.26 (cid:9)
`
`7.36 (cid:9)
`
`Non-SRP (cid:9)
`
`Placebo (cid:9)
`
`11.42 (cid:9)
`
`8.91 (cid:9)
`
`9.54 (cid:9)
`
`6.58* (cid:9)
`
`9,79* (cid:9)
`
`3 Months
`Post
`
`6.98
`
`8.57
`
`8.53
`
`10.25
`
`* Statistically significant differences (P<0.05) between SDD and placebo
`treatment groups.
`Statistically significant within-treatment differences (P !~0.05) relative to
`baseline.
`
`Table 2.
`Mean Percentage of Large Spirochetes
`Relative to Total Microscopic Flora for SDD
`and Placebo Treatment Groups in SRP and
`Non-SRP Design
`
`Treatment Phase
`
`Design (cid:9)
`
`Treatment
`Group (cid:9)
`
`Baseline Months Months Months (cid:9)
`
`3 Months
`Post
`
`SDD (cid:9)
`
`3.34 (cid:9)
`
`0,72t (cid:9)
`
`0.1 3*’ 058t (cid:9)
`
`0.81 t
`
`SRP (cid:9)
`
`Placebo (cid:9)
`
`4.29 (cid:9)
`
`SDD (cid:9)
`
`3.22 (cid:9)
`
`I ,99t (cid:9)
`
`0.81 I t (cid:9)
`
`I. 13‘t 1.74t (cid:9)
`
`1.06t
`
`0.56k (cid:9)
`
`0.56*t (cid:9)
`
`0.47t
`
`Non-SRP (cid:9)
`
`Placebo (cid:9)
`
`3.13 (cid:9)
`
`2.28 (cid:9)
`
`1.25t (cid:9)
`
`I 79*1’ (cid:9)
`
`1.76
`
`* Statistically significant differences (P<0.05) between SDD and placebo
`treatment groups.
`1’ Statistically significant within-treatment differences (P!~0.05) relative to
`baseline.
`
`placebo treatments, regardless of SRP or non-SRI’
`design, produced statistically significant reductions in
`both the intermediate and large spirochetal groups
`(Tables 2 and 3). In the SRP design, the SDD treatment
`yielded significant reductions in small spirochetes, rel-
`ative to baseline, for all 9 months of treatment, while
`the placebo treatment demonstrated only significant
`reductions at the 9-month sample period (Table 1).
`Significant reductions in the proportion of motile rods
`were detected for all treatments at all sample periods
`relative to baseline (Table 4). Significant increases
`(P <0.0001) were found in the proportion of coccoid
`forms, relative to baseline, for all sample periods (Table
`5). No significant changes were noted during any treat-
`
`1467
`
`(cid:9)
`(cid:9)
`

`

`Effect of Subantimicrobial Dose Doxycycline on Subgingival Microflora (cid:9)
`
`Volume 71 Number 9
`
`Table 3.
`Mean Percentage of Intermediate Spirochetes
`Relative to Total Microscopic Flora for SDD
`and Placebo Treatment Groups in SRP and
`Non-SRP Design
`
`Treatment Phase
`
`Design (cid:9)
`
`Treatment
`Group (cid:9)
`
`Baseline Months Months Months (cid:9)
`
`3 Months
`Post
`
`SDD (cid:9)
`
`15.57 (cid:9)
`
`3.86* (cid:9)
`
`1.88* (cid:9)
`
`2.40*
`
`SRP (cid:9)
`
`Placebo (cid:9)
`
`13.43 (cid:9)
`
`5.05* (cid:9)
`
`377’ (cid:9)
`
`5,63* (cid:9)
`
`SDD (cid:9)
`
`13.56 (cid:9)
`
`3.80* (cid:9)
`
`2.85* (cid:9)
`
`5.86’ (cid:9)
`
`4.70.*
`
`2.87*
`
`Non-SRP (cid:9)
`Placebo (cid:9)
`13.94 (cid:9)
`5.38 (cid:9)
`* Statistically significant within-treatment differences (P!~0.001) relative to
`baseline.
`
`4.00.* (cid:9)
`
`6.33* (cid:9)
`
`4. 32*
`
`Table 4.
`Mean Percentage of Motile Rods Relative
`to Total Microscopic Flora for SDD and
`Placebo Treatment Groups in SRP and
`Non-SRP Design
`
`Treatment Phase
`
`Design (cid:9)
`
`Treatment
`Group (cid:9)
`
`SDD (cid:9)
`
`6.93 (cid:9)
`
`Baseline Months Months Months (cid:9)
`1.55* (cid:9)
`
`1.86* (cid:9)
`
`2.29* (cid:9)
`
`3 Months
`Post
`
`3.21 *
`
`175*
`
`SRP (cid:9)
`
`Placebo (cid:9)
`
`SDD (cid:9)
`
`6.63 (cid:9)
`
`8.64 (cid:9)
`
`1.29* (cid:9)
`
`1.23* (cid:9)
`
`193* (cid:9)
`
`1.91* (cid:9)
`
`1.79* (cid:9)
`
`2.64* (cid:9)
`
`2 . 81*
`
`Non-SRP (cid:9)
`Placebo (cid:9)
`6.53 (cid:9)
`3.46 (cid:9)
`* Statistically significant within-treatment differences (P!~0.05) relative to
`baseline.
`
`3.89* (cid:9)
`
`1.66* (cid:9)
`
`2.88*
`
`ment in the proportion of non-motile rods, fusiforms,
`or filamentous rods present at any sample period.
`
`Culture Enumeration
`As with the microscopic parameters, data analyses
`were conducted to detect statistically significant dif-
`ferences both between and within the treatment groups.
`Between-treatment differences. With one single
`exception, no statistically significant differences
`(P
`>0.300) were detected between SDD and placebo treat-
`ments in either the SRP or non-SRP design at any sam-
`ple period for the total cultivable bacterial mass (total
`anaerobic counts, total facultative counts, or obligate
`anaerobes), normal flora (total streptococci, total actin-
`omyces), putative periodontal pathogens (P gingivalis,
`P intermedia, B. forsythus, A. actinomycetemcomitans,
`or E. corrodens), or opportunistic pathogens (Candida,
`
`1468
`
`enteric bacteria, or S. aureus). The only exception was
`that the total facultative counts were significantly higher
`(P = 0.0146) in the placebo treatment compared to
`the SDD treatment group in the SRP design at the 6-
`month sample period. No differences were detected
`between treatments at any other sample period (P
`>0.3000).
`Within-treatment differences. The means of the
`colony forming units (CFUs) for total anaerobic counts,
`facultative counts, and obligate anaerobes obtained at
`each sample period for each treatment are given in Fig-
`ures 1 through 3. Statistically significant increases were
`detected with the paired t test in both the total anaer -
`obic counts and the obligate anaerobes present at 3
`months relative to baseline for the SDD and placebo
`treatments in both designs. Significant increases were
`also detected at 6 months, relative to baseline, for both
`the SDD and placebo treatments in the non-SRP design.
`However, when these data were reanalyzed using the
`Wilcoxon signed rank test to minimize the effects of
`extreme outliers, statistically significant increases were
`detected only in the placebo treatment in the non-SRP
`design for the total anaerobic counts and the obligate
`anaerobes at 3 and 6 months relative to baseline
`(P
`<0.02). Significant increases were noted in the number
`of facultative counts present at 6 months relative to
`baseline for the placebo treatment in both the SRP and
`non-SRP designs, but these increases were not statis-
`tically significant when reanalyzed using the Wilcoxon
`signed rank test. No statistically significant differences
`were detected within the SDD or placebo treatment
`groups in the SRP and non-SRP design by either the
`paired t test or Wilcoxon signed rank test in any of the
`following microbial groups: streptococci, Actinomyces,
`P gingivalis, P intermedia, B. forsythus, A. actino-
`mycetemcomitans, E. corrodens, Candida, enterics, or
`S. aureus.
`
`Clinical Parameters Associated With Microbial
`Sample Sites
`Since statistically significant microbial decreases, either
`between or within treatments, during the 9-month treat-
`ment period were associated with motile groups (spiro-
`chetes and motile rods) that have been used as indi-
`cators of disease activity, the clinical indices obtained
`for the microbiology sample sites at each sample
`period were analyzed.
`Between-treatment differences. No statistically sig-
`nificant differences were detected between the SDD
`and placebo treatments in the SRP design for either AL
`or PD at any sample period. However, in the SRP
`design, the percentage of BOP sites (Fig. 4) in the
`SDD group was significantly lower (P<0.01) than the
`placebo group at all sample periods following baseline.
`In the non-SRP design, significant gains (P<0.01) in
`AL were present in the SDD group at 3, 6, and 9
`
`(cid:9)
`(cid:9)
`

`

`Periodontol (cid:149) September 2000 (cid:9)
`
`Walker, Thomas, Nang(cid:243), Lennon, Wetzel, Powala
`
`Table S.
`Mean Percentage of Coccoid Forms Relative
`to Total Microscopic Flora for SDD and
`Placebo Treatment Groups in SRP and
`Non-SRP Design
`
`Treatment Phase
`
`Design (cid:9)
`
`Treatment (cid:9)
`Group (cid:9)
`
`9 (cid:9)
`6 (cid:9)
`3 (cid:9)
`Baseline Months Months Months (cid:9)
`
`3 Months
`Post
`
`SDD
`
`14.87
`
`3353* 3704* 41.36*
`
`3445*
`
`lxi
`
`1x10 7
`
`lXlO6
`
`D
`LI-
`0
`C a
`
`C
`
`0
`0
`a
`
`Ca
`0
`I-
`
`SRP (cid:9)
`
`Placebo
`
`13.11
`
`26.18*. 31.31 *
`
`37 . 20*
`
`3359*
`
`1x105
`
`SDD
`
`14,23
`
`34.76
`
`3905*
`
`Non-SRP (cid:9)
`
`Placebo
`
`13.20
`
`24.04* 30.54
`
`39,32
`
`30.92
`
`3933*
`
`3153*
`
`* Statistically significant within-treatment differences (P’zO.00OI) relative to
`baseline.
`
`lxlO9 ?
`
`C/)
`
`LL
`(3
`C 1x10 8
`a
`a
`E
`
`C
`
`B lxlO
`
`0
`
`0
`Q) a
`C
`
`Ca
`0
`I-
`
`1x10 6
`
`lxi
`
`BL (cid:9)
`
`3 months (cid:9)
`
`6 months (cid:9)
`Sample period
`
`9 months 3 months post
`
`’Ll SOD/SAP (cid:9)
`
`Piacebo/SRP (cid:9) (cid:149) SOD (cid:9)
`
`53 Placebo
`
`Figure
`Total cultivable anaerobic counts (means) obtained for each treatment
`at each sample period.
`
`months and fewer sites bled on probing (P<0.005) at
`6 and 9 months. No differences were detected in PD
`at any sample period.
`Within-treatment differences. Statistically signifi-
`cant (P<0.0001) increases in AL and decreases in PD
`were detected at 3, 6, and 9 months, relative to base-
`line, regardless of treatment or design. No significant
`differences were detected between either the 3-, 6-, or
`9-month measurements relative to each other. Signif-
`icant decreases in proportion of BOP sites (Fig. 1)
`were noted at 3, 6, and 9 months, relative to baseline,
`for the SDD group in the SRP design (P <0.0005) and
`in the non-SRP design (P<0.01). Significant decreases
`
`BL (cid:9)
`
`3 months (cid:9)
`
`6 months (cid:9)
`Sample period
`
`9 months 3 months post
`
`E. SOD/SRP (cid:9)
`
`Placebo/SRP (cid:9) (cid:149) SDD (cid:9)
`
`5J Placebo
`
`Fig i:rO 2..
`Total cultivable facultative counts (means) obtained for each treatment
`at each sample period (*statistical significance between treatments,
`P <0.02).
`
`lxi
`
`a
`
`LL
`C.) 11(10
`C
`Ce a
`E
`0,
`
`lxlO
`
`Ca
`
`iii
`
`1x106
`
`1X10
`
`5
`
`BL (cid:9)
`
`3 months (cid:9)
`
`6 months (cid:9)
`Sample period
`
`9 months 3 months post
`
`LI SOD/SAP (cid:9)
`
`Piacebo/SRP (cid:9) (cid:149) SDD (cid:9)
`
`C.J Placebo
`
`Figora .3.
`Obligate anaerobic counts (means) obtained for each treatment at
`each sample period (*statistical significance relative to baseline,
`P <0.02).
`
`in BOP sites were noted in the placebo group in the
`SRP design at 3 and 6 months (P <0.001) relative to
`baseline but not at 9 months, and in the placebo group
`in the non-SRP design at 3 months (P<0.005) but not
`at 6 or 9 months.
`
`U 11 115
`The principal objective of this investigation was to
`determine whether SDD exerted any detectable effect
`on the subgingival flora that could be attributed to
`
`1469
`
`

`

`Effect of Subantimicrobial Dose Doxycycline on Subgingival Microflora (cid:9)
`
`Volume 71 Number 9
`
`§
`
`r
`
`biI)i (cid:9)
`
`a) a>
`’a
`0
`0
`m
`0
`C
`0
`
`0
`0
`0
`0
`
`bL (cid:9)
`
`3 months (cid:9)
`
`6 months (cid:9)
`
`9 months 3 months post
`
`H SDD/SRP (cid:9)
`
`Placebo/SRP
`
`Placebo
`
`Sample period
`
`(cid:149) SOD (cid:9)
`
`Percentage of microbial sample sites bleeding an probing for each
`treatment at each sample period (*statistical significance between
`treatments, P <0.01; trelotive to baseline, P <0.0005; trelative to
`baseline, P <0.01;brelative to baseline, P <0.005),
`
`antimicrobial activity. Doxycycline is normally given
`at a daily dose of 100 mg, following a loading dose of
`200 mg, which yields biologically active levels of 8
`to 16 .tg/ml in the gingival crevicular fluid and
`around 4 ig/ml in the blood. 11 Studies in human vol-
`unteers have demonstrated that 20 mg doxycycline
`bid yields steady-state serum concentrations of 0.6 to
`0.8 ig/ml (unpublished data). This level is consider-
`ably below the minimal inhibitory concentration (MIC)
`determined in vitro for the vast majority of the bacte-
`ria isolated from the subgingival flora. 12’13 Since sub-
`gingival plaque exists as a biofilm rather than in a
`planktonic state, 14 even higher drug concentrations
`are probably necessary for in vivo inhibition. Even so,
`the possibility exists that levels obtained with SDD
`might be inhibitory for certain bacteria that are exquis-
`itely sensitive to the tetracyclines. Therefore, in this
`study, a comprehensive microbial examination of the
`subgingival flora was conducted by microscopy and
`culture enumeration in an attempt to detect differences
`between and within treatments that could be con-
`tributed to an antimicrobial effect.
`No statistical or microbiological differences in any
`of the microbial parameters enumerated were detected
`between SDD and placebo treatments in either the SRP
`or non-SRP design, with the exception of the spiro-
`chetes. In both designs, the small and large spirochetal
`groups were found to be significantly lower at certain
`periods in the SDD treatment than in the correspond-
`ing placebo group. There are several possible expla-
`nations for the suppression of the spirochetes in the
`SDD groups. One is that the levels of doxycycline
`obtained in the periodontal pocket are inhibitory for
`
`1470
`
`these organisms. Although the large spirochetes have
`not been cultivated and their sensitivity to the tetra-
`cyclines is unknown, it is generally thought that the
`small spirochetes are relatively sensitive to the tetra-
`cyclines, although resistance has been reported. 15
`Therefore, it might be argued that the suppression of
`the spirochetes was due to the antimicrobial activity
`of doxycycline. However, other bacterial groups are
`equally sensitive, if not more so. Almost all isolates of
`P gingivalis are inhibited in vitro by !~0.25 g/ml of the
`tetracyclines. 2’13 ’16 Neither we nor a number of other
`investigators have been successful in isolating wild-
`type strains of this organism with naturally occurring
`resistance to the tetracyclines. In the study reported
`here, there were no differences between treatments at
`either West Virginia University or the University of
`Florida in the numbers of P gingivalis recovered at
`any sample period. This tends to argue against the
`possibility that the decrease in the relative proportion
`of the spirochetes was due to antimicrobial activity,
`since corresponding decreases in the numbers or pro-
`portions of P gingivalis were not found.
`Another possibility that has been advanced is that
`the decrease in spirochetes was due to the periodon-
`tal pocket becoming more aerobic. Since the spiro-
`chetes are thought to have a relatively low redox (Eh)
`requirement for growth, 17 an increase in the Eh of the
`pocket might favor the growth of more oxygen-sensi-
`tive species at the expense of the spirochetes. How-
`ever, this would most likely occur following mechani-
`cal disruption of the structure of the plaque biofilm. If
`this were the case, one would not expect to find treat-
`ment differences between SDD and placebo treatments
`in the SRP design, since both groups received peri-
`odontal scaling prior to the adjunctive treatment reg-
`imen.
`The most likely explanation for the observed spiro-
`chetal differences between treatments is probably
`related to an improvement in the health of the peri-
`odontal pocket. There was significantly less inflam-
`mation as determined by the proportion of sites bleed-
`ing on probing in both SDD groups relative to placebo.
`The proportion of bleeding sites was significantly lower
`in the SDD/SRP group than the placebo group at 3, 6,
`and 9 months (P <0.005) and in the SDD/non-SRP
`group at 6 and 9 months (P<0.005). Within-treatment
`analyses revealed statistically significant improvements
`for all treatments in AL, PD, and BOP. Concurrently
`with these improvements in clinical indices, within-
`treatment analyses detected statistically significant
`decreases in spirochetes and motile rods with corre-
`sponding increases in coccoid forms. Since micro-
`scopic motility and bleeding on probing are often use-
`ful as indicators of disease activity, it seems reasonable
`to expect some relationship between the two. There-
`fore, we think the most logical explanation for the
`
`

`

`Periodontol (cid:149) September 2000 (cid:9)
`
`Walker, Thomas, Nang(cid:243), Lennon, Wetzel, Powala
`
`8.
`
`human adult periodontitis gingiva. J C/in Periodontol
`1995;22:100-109.
`6. Golub LM, Lee NM, Greenwald RA, et al. A matrix met-
`alloproteinase inhibitor reduces bone-type collagen
`degradation fragments and specific collagenases in gin-
`gival crevicular fluid during adult periodontitis. Inflamm
`Res 1997;46:310-319.
`7. Crout RJ, Lee NM, Schroeder K, et al. T