Oral Microbiology Immunology 2008: 23: 148–157
`Printed in Singapore. All rights reserved
`
`Ó 2008 The Authors.
`Journal compilation Ó 2008 Blackwell Munksgaard
`
`Microbiological changes
`associated with four different
`periodontal therapies for the
`treatment of chronic periodontitis
`
`Haffajee AD, Patel M, Socransky SS. Microbiological changes associated with four
`different periodontal therapies for the treatment of chronic periodontitis.
`Oral Microbiol Immunol 2008: 23: 148–157. Ó 2008 The Authors. Journal compilation
`Ó 2008 Blackwell Munksgaard.
`
`Background/aims: To examine subgingival microbiological changes in chronic peri-
`odontitis subjects receiving scaling and root planing (SRP) alone or with systemically
`administered azithromycin, metronidazole or a sub-antimicrobial dose of doxycycline.
`Methods: Ninety-two periodontitis subjects were randomly assigned to receive SRP
`alone or combined with azithromycin, metronidazole or sub-antimicrobial dose
`doxycycline. Subgingival plaque samples taken at baseline, 2 weeks, and 3, 6, and
`12 months were analyzed for 40 bacterial species using checkerboard DNA–DNA
`hybridization. Percentage of resistant species and percentage of sites harboring species
`resistant to the test antibiotics were determined at each time-point.
`Results: All treatments reduced counts of red complex species at 12 months, although no
`significant differences were detected among treatment groups for most species at all time-
`points. Both antibiotics significantly reduced counts of red complex species by 2 weeks.
`Percentage of resistant isolates increased in plaque samples in all adjunctive treatment
`groups, peaking at the end of administration, but returned to pretreatment levels by
`12 months.
`Conclusion: The significant reduction of red and orange complex species at 2 weeks in
`the subjects receiving SRP plus azithromycin or metronidazole may have contributed
`to a better clinical response in these treatment groups. Therapy did not appear to create
`lasting changes in the percentage of resistant isolates or sites harboring resistant species.
`
`A. D. Haffajee, M. Patel,
`S. S. Socransky
`Department of Periodontology, The Forsyth
`Institute, Boston, MA, USA
`
`Key words: antibiotics; antibiotic resistance;
`azithromycin; chronic periodontitis; metroni-
`dazole; periodontal pathogens; periodontal
`therapy; scaling and root planing; sub-anti-
`microbial dose doxycycline
`
`Anne D. Haffajee, Department of
`Periodontology, The Forsyth Institute, 140
`The Fenway, Boston, MA 02115, USA
`Tel: +(617) 892 8243;
`fax: +(617) 262 4021;
`e-mail: ahaffajee@forsyth.org
`Accepted for publication June 5, 2007
`
`In an earlier paper, the clinical effects of
`systemically administered azithromycin,
`metronidazole or sub-antibacterial dose
`doxycycline (SDD) as adjuncts to scaling
`and root planing (SRP) were examined
`(10). It was found that subjects receiving
`the systemically administered antibiotics,
`azithromycin or metronidazole, showed
`greater clinical improvement at 12 months
`post-therapy
`compared with
`subjects
`receiving SRP only and that the difference
`among groups was more marked at sites
`with initially deeper pockets. The dosage,
`
`duration, and mechanism of action of the
`three adjunctive agents are quite different
`and thus, the effects on the subgingival
`microbiota were examined. To date, there
`are few studies examining the changes in
`the subgingival microbiota in subjects
`receiving these adjunctive agents and no
`‘head to head’ comparison.
`Although it has been shown that bacterial
`species residing in biofilms are much more
`resistant to antibiotics than the same species
`in a planktonic state (1, 2, 11, 20), antibi-
`otics have been used frequently in the
`
`treatment of periodontal infections. Of the
`single antibiotics that have been evaluated,
`systemically administered metronidazole
`appeared to have the most consistent ben-
`eficial effect on clinical parameters of
`periodontal diseases and on subgingival
`plaque composition (29). Metronidazole is
`attractive for the treatment of patients with
`chronic periodontitis because the narrow
`spectrum of this agent is thought to work
`specifically on the anaerobic microbiota
`associated with periodontal diseases. In-
`deed, several investigations suggested that
`
`

`

`Microbial effects of antibiotic therapy
`
`149
`
`systemically administered metronidazole
`used as an adjunct to SRP in the treatment
`of chronic periodontitis offers a clinical
`benefit over SRP alone (8). Few studies
`have documented the microbiological
`changes associated with the systemic
`administration of metronidazole. Lindhe
`et al. (13) demonstrated that systemically
`administered metronidazole in combination
`with mechanical therapy was more effective
`in reducing spirochetes than SRP alone.
`Other investigators have shown that peri-
`odontal therapy that included systemically
`administered metronidazole led to clinical
`improvements and a reduction in the pro-
`portions or levels of several periodontal
`pathogens including Porphyromonas gin-
`givalis, Tannerella forsythia, Treponema
`denticola, Prevotella intermedia, members
`of the genera Campylobacter, Fusobacteri-
`um, Eubacterium nodatum, Peptostrepto-
`coccus
`micros
`Streptococcus
`and
`constellatus (5, 14–16, 31). Feres et al. (4)
`examined the changes in metronidazole
`resistance over time. The percentage of
`isolates resistant to metronidazole increased
`at 2 weeks, the end of antibiotic adminis-
`tration, but at 6 months post-therapy the
`levels were similar to pretreatment values.
`The effects of azithromycin on the oral
`microbiota have been examined in a limited
`number of studies. Smith et al. (23), in a
`randomized double-blind, placebo-con-
`trolled study of 46 chronic periodontitis
`subjects, demonstrated that subjects receiv-
`ing azithromycin as an adjunct
`to SRP
`showed a better clinical
`response than
`subjects not receiving this agent. In addi-
`tion, they found that compared with the
`placebo group, counts of spirochetes and
`‘pigmented anaerobes’ in subgingival pla-
`que
`samples were
`significantly more
`reduced in subjects receiving azithromycin
`at 22 weeks post-therapy (22).
`The effect of long-term SDD on the
`subgingival microbiota was examined by
`Thomas et al. (28). Subgingival plaque
`samples were taken before and after ther-
`
`apy that consisted of SRP or not, and
`20 mg twice daily (bid) doxycycline or
`placebo for 9 months and were evaluated
`using dark-field microscopy and culture.
`The E-test (AB Biodisk, Solna, Sweden)
`and minimum inhibitory concentrations
`(MICs) were used to determine doxycy-
`cline resistance. Morphotype distribution
`was somewhat similar among treatment
`groups at each time-point; there was no
`marked decrease in species recovery and
`no overgrowth of opportunistic species.
`Furthermore,
`the administration of SDD
`was not associated with the development
`of doxycycline-resistant species. Walker
`et al.
`(30) examined the effect of a
`9-month administration of SDD on the
`intestinal and vaginal microbiota in 69
`periodontally
`diseased
`subjects. No
`changes in the level of resistant species
`in either fecal or vaginal samples from
`these subjects were detected after long-
`term SDD administration.
`The purpose of the current study was to
`examine microbiological changes associ-
`ated with treatment groups receiving SRP
`alone or
`in combination with one of
`systemically administered azithromycin,
`metronidazole, or SDD. In addition, the
`effect of the different
`therapies on the
`proportion of antibiotic-resistant species
`post-therapy and the identity of
`these
`species was also examined.
`
`Material and methods
`Subject population
`
`Ninety-two subjects were recruited into the
`study. Subjects were >20 years of age, in
`good general health, had at least 20 natural
`teeth, including at least one molar tooth in
`each quadrant, and at least eight sites with
`pocket depth >4 mm. The subjects did not
`have a history of unsuccessful periodontal
`therapy that may have indicated ‘refrac-
`tory’ periodontitis and the history of the
`subjects precluded a diagnosis of aggres-
`sive periodontitis. Subjects were excluded
`
`if they had any known allergies to the test
`antimicrobial agents, had received peri-
`odontal therapy in the previous 3 months,
`were pregnant or nursing, or had systemic
`conditions that required antibiotic preme-
`dication or could influence the outcome of
`periodontal
`therapy. The subjects had a
`mean age of 46 years (range 22–77 years),
`36% were female, 63% were White, 26%
`were African American, 7% were Hispanic
`and 4% were Asian. The baseline clinical
`characteristics of the subjects are presented
`in Table 1. The study was approved by the
`Forsyth
`Institute
`Institutional Review
`Board. The nature of
`the study was
`described thoroughly to all subjects and
`each provided signed informed consent
`before entry into the study.
`
`Experimental design and treatment
`
`This was a randomized, single-blind study.
`Subjects were randomized to one of four
`treatment groups receiving either SRP
`alone or combined with systemically
`administered azithromycin at the dosage
`of 500 mg once daily for 3 days, system-
`ically administered metronidazole at
`the
`dosage of 250 mg thrice daily (tid) for
`14 days, or 20 mg doxycycline (SDD,
`PeriostatÒ; CollaGenex Pharmaceuticals,
`Newtown, PA) bid for 12 weeks. SRP was
`performed a quadrant at a time under local
`anesthesia at approximately weekly inter-
`vals. The adjunctive agents were started at
`the first SRP visit. Subjects were clinically
`monitored at baseline (before therapy) and
`at 3, 6, and 12 months post-therapy. In
`addition, all subjects received maintenance
`SRP at the three post-therapy monitoring
`visits.
`
`Microbiological assessments
`
`At each monitoring visit clinical measure-
`ments were taken as described in a com-
`panion paper
`(10). Subgingival plaque
`samples were taken from the mesial aspect
`
`Table 1. Mean (±SD) clinical and demographic features of subjects with data for the four monitoring visits in the four treatment groups at baseline
`
`Treatment group
`
`Azithromycin
`
`Metronidazole
`
`Periostat
`
`n
`Age
`No. missing teeth
`No. males
`No. current smokers
`% of sites with
`Plaque accumulation
`Gingival redness
`Bleeding on probing
`Suppuration
`Mean pocket depth (mm)
`Mean attachment level (mm)
`
`25
`
`47 ± 14
`2.40 ± 2.50
`15
`3
`
`62.49 ± 23.49
`64.32 ± 29.14
`34.29 ± 26.04
`0.94 ± 1.45
`3.11 ± 0.64
`3.42 ± 0.88
`
`24
`
`44 ± 11
`2.58 ± 2.12
`18
`3
`
`65.98 ± 20.90
`73.90 ± 21.38
`39.37 ± 28.02
`0.85 ± 2.05
`3.00 ± 0.45
`3.21 ± 0.78
`
`20
`
`47 ± 11
`2.35 ± 2.62
`10
`1
`
`66.52 ± 25.61
`66.09 ± 24.57
`41.11 ± 24.78
`2.52 ± 4.16
`3.33 ± 0.92
`3.47 ± 0.93
`
`SRP
`
`23
`
`43 ± 15
`1.83 ± 2.44
`16
`2
`
`64.26 ± 22.27
`63.95 ± 27.04
`32.62 ± 26.20
`0.21 ± 0.68
`2.92 ± 0.37
`3.03 ± 0.56
`
`Kruskal–Wallis
`P-value
`
`0.51086
`0.50788
`0.33237
`0.66845
`
`0.86874
`0.62136
`0.61425
`0.00246
`0.52465
`0.34108
`
`

`

`150
`
`Haffajee et al.
`
`Fig. 1. Mean counts (·105) of 40 test species at baseline, 2 weeks, and 3, 6, and 12 months in subjects in each of the four treatment groups. The species
`were ordered according to the complexes described by Socransky et al. (24). Counts for individual species from up to 28 sites in each subject were
`averaged within a subject and then across subjects in the four treatment groups for each time-point separately. Significance of differences over time was
`sought using the Friedman test *P < 0.05, **P < 0.01, ***P < 0.001, and adjusted for 40 comparisons (25).
`
`of each tooth (excluding the third molars)
`for a maximum of 28 teeth in each subject at
`baseline, 2 weeks, and 3, 6, and 12 months.
`After
`removal of supragingival plaque,
`subgingival biofilm samples were taken
`using individual sterile Gracey curettes
`from the mesial surface of each tooth and
`placed into separate Eppendorf tubes con-
`taining 0.15 ml Tris EDTA buffer (10 mm
`Tris–HCl, 1 mm ethylenediaminetetraace-
`tic acid, pH 7.6). Immediately after, 0.10 ml
`of 0.5 m NaOH was added to each sample.
`Each sample was evaluated for its content of
`40 bacterial species using checkerboard
`DNA–DNA hybridization (26, 27). In brief,
`the samples were lysed and the DNA was
`placed in lanes on a nylon membrane using a
`Minislot device (Immunetics, Cambridge,
`MA). After fixation of the DNA to the
`membrane, the membrane was placed in a
`Miniblotter 45 (Immunetics), with the lanes
`of DNA at 90° to the lanes of the device.
`Digoxigenin-labeled whole genomic DNA
`probes to 40 subgingival species were
`hybridized in individual lanes of the Minib-
`lotter. After hybridization, the membranes
`were washed at high stringency and the
`DNA probes were detected using antibody
`
`to digoxigenin, conjugated with alkaline
`phosphatase and chemifluorescence detec-
`tion. Signals were detected using AttoPhos
`substrate (Amersham Life Sciences, Arling-
`ton Heights, IL) and were read using a
`Storm FluorImager (Molecular Dynamics,
`Sunnyvale, CA), a computer-linked instru-
`ment that reads the intensity of the fluores-
`cence signals resulting from the probe-
`target hybridization. Two lanes in each run
`contained standards at the concentration of
`105 and 106 cells of each species. The
`sensitivity of the assay was adjusted to
`permit the detection of 104 cells of a given
`species by adjusting the concentration of
`each DNA probe. Signals were evaluated
`using the Storm FluorImager and converted
`to absolute counts by comparison with the
`regression line determined from data from
`the standards on the same membrane.
`Failure to detect a signal was recorded as
`zero. A total of 10,913 subgingival samples
`were evaluated for the 92 subjects.
`
`Determination of antibiotic resistance
`
`from the
`Subgingival biofilm samples
`distal site of four selected teeth were taken
`
`at baseline and 12 months, and from two
`of the four selected teeth at 2 weeks (the
`third SRP visit) and 3 and 6 months in
`each subject. These samples were placed
`in separate tubes each containing 0.15 ml
`of Mycoplasma broth (BBL, Cockeysville,
`MD) supplemented with 1% glucose. One
`milliliter of whole saliva was collected into
`an empty tube at each time-point from
`each subject.
`The plaque and saliva samples were
`vortexed and serially diluted 10-fold.
`Then, 100 ll of each dilution was plated
`in duplicate on antibiotic-containing and
`non-selective agar plates. All plates were
`made with 2% trypticase soy agar and
`2.6% brain–heart infusion agar enriched
`with 5% sheep blood, 1% yeast extract,
`5 lg/ml hemin, 0.3 lg/ml menadione, and
`10 lg/ml N-acetylmuramic acid. Antibi-
`otic-containing plates were made up with
`the same basal medium supplemented with
`one of 4 lg/ml doxycycline, 2 lg/ml
`metronidazole or 2 lg/ml azithromycin.
`These values were chosen based on data
`for a wide range of species presented in
`NCCLS publications (18, 19). Samples
`taken from subjects in the SRP-only group
`
`

`

`Microbial effects of antibiotic therapy
`
`151
`
`Fig. 2. Mean counts (·105) of 40 test species at baseline and 2 weeks in subjects in each of the four treatment groups. The species were ordered
`according to the complexes described by Socransky et al. (24). Counts for individual species at each sampled site were averaged within a subject, and
`across subjects in the four treatment groups at baseline and 2 weeks separately. Significance of differences between baseline and 2 weeks was determined
`using the Wilcoxon signed ranks test; *P < 0.05, **P < 0.01, ***P < 0.001. The black asterisks represent significant differences between time-points
`without adjusting for 40 comparisons and the red asterisks represent significant differences between time-points after adjusting for 40 comparisons (25).
`
`the antibiotic-
`were plated on each of
`containing plates. Samples taken from
`subjects receiving an adjunctive agent
`were plated on plates containing that
`agent. The inoculated plates were then
`placed into an anaerobic chamber and
`incubated in an atmosphere of 80% N2,
`10% CO2 and 10% H2 at 35°C. After
`7 days of
`incubation,
`the plates were
`removed from the chamber, the colonies
`on the antibiotic and non-selective plates
`were counted, and the proportion of anti-
`biotic-resistant species was determined.
`For each sampled site that was plated on
`an antibiotic plate, the dilution with the
`number of colonies closest
`to 300 was
`washed off with 1 ml TE buffer into a
`tube. The optical density was adjusted to
`1.0 (approximately 109 cells); 0.01 ml of
`this suspension (about 107 cells) was put
`into a tube containing 140 ll TE buffer
`
`and 100 ll of 0.5 m NaOH was added.
`The samples were then analyzed for their
`content of 40 bacterial
`species using
`checkerboard DNA–DNA hybridization.
`
`Data analysis
`
`Data from a total of 92 subjects were
`available for analysis and included the
`counts of 40 bacterial species in up to 28
`subgingival biofilm samples at baseline,
`2 weeks, and 3, 6, and 12 months. The
`change in mean counts of bacterial species
`over time was the primary microbiological
`outcome evaluated. Data were averaged
`across sites within each subject and then
`averaged across subjects in each treatment
`group at each time-point separately. Since
`mean baseline values for individual spe-
`cies differed among treatment groups, the
`microbial changes among the four treat-
`
`ment groups were examined using AN-
`COVA adjusting for baseline microbial
`counts. The subject was the unit of anal-
`ysis. Significance of differences over time
`in the counts of the 40 individual species
`was determined using the Friedman test
`and adjusted for 40 comparisons (25).
`The proportion of resistant isolates was
`determined in saliva samples and in four
`subgingival plaque samples at baseline and
`12 months and in two of these samples at
`2 weeks and 3 and 6 months in subjects in
`the four treatment groups. Proportions of
`resistant isolates for the plaque samples
`were averaged across sites in each subject
`and then across
`subjects
`in the four
`treatment groups and at each time-point
`separately. The significance of differences
`over time in each group was sought using
`the Friedman test. Significance of differ-
`ences between test and control samples at
`
`

`

`152
`
`Haffajee et al.
`
`Fig. 3. Percentage of isolates resistant to 2 lg/ml azithromycin, 2 lg/ml metronidazole, or 4 lg/ml doxycycline in subgingival plaque samples (upper
`panels) and saliva samples (lower panels) taken at baseline, 2 weeks, and 3, 6, and 12 months from subjects in the four treatment groups. Antibiotic
`resistance in the SRP group was measured for all three systemic agents. Data were averaged within a subject at each time point and then across subjects in
`the four groups separately. The circles represent the mean and the whiskers the standard error of the mean. The shaded area represents the period of
`antibiotic administration in the test subjects. Significance of differences over time for each group separately was sought using the Friedman test,
`*P < 0.05, **P < 0.01, ***P < 0.001, and between test and control subjects using the Mann–Whitney test as indicated by the P-values at individual
`time-points.
`
`tested using the
`each time-point was
`Mann–Whitney test for each adjunctive
`agent separately. The mean percentage of
`sites colonized by resistant species in
`subgingival plaque samples was deter-
`mined by averaging the site data for each
`species across subjects in the four treat-
`ment groups at each time-point separately.
`Significance of differences over time was
`determined using the Friedman test and
`between test and control samples at each
`time-point using the Mann–Whitney test.
`
`Results
`
`Subject retention, patient compliance and
`adverse events were discussed in Haffajee
`et al. (10).
`
`Microbial changes in different treatment
`groups
`
`At baseline, virtually all subjects harbored
`each of the test species at some level in at
`
`least one sampled site. At levels >105, fewer
`subjects were positive for the test species,
`although the red complex species were, on
`average, still quite prevalent in subjects in
`each treatment group (Table 2). There were
`no significant differences among treatment
`groups in the prevalence of the individual
`test species at baseline. Figure 1 presents
`the mean counts of the 40 test species at each
`visit
`in the four
`treatment groups. On
`average, all treatment groups showed reduc-
`tions in counts of red complex species,
`which were maintained to 12 months.
`These changes were significant for T. for-
`sythia in all
`treatment groups and for
`P. gingivalis in the metronidazole and
`SRP-only groups. Other
`species were
`reduced markedly at the initial post-therapy
`visits, particularly members of the orange
`complex, although an increase in counts of
`many species had occurred by 12 months.
`Indeed, several orange complex species
`approached or exceeded baseline values at
`the 12-month monitoring visit, a finding
`
`particularly noticeable in the SDD group.
`The data in this figure indicated that the
`major decrease in bacterial counts occurred
`between baseline and 2 weeks. Figure 2
`highlights the data at these two visits for
`subjects who were subset
`into the four
`treatment groups. The data indicated that
`subjects receiving systemically adminis-
`tered azithromycin or metronidazole dem-
`onstrated significant reductions in red and
`many orange complex species at 2 weeks.
`There were no statistically significant dif-
`ferences among treatment groups for any of
`the test species examined at any time-point
`with the exception of Actinomyces israelii,
`Streptococcus gordonii, Streptococcus in-
`termedius, and P. gingivalis at the 6 months
`post-therapy visit.
`
`Percentage of the microbiota resistant to
`the test antibiotics
`
`Figure 3 presents the percentage of the
`cultivable microbiota that was resistant to
`
`

`

`Microbial effects of antibiotic therapy
`
`153
`
`Table 2. Proportion (%) of subjects positive for the 40 test species at mean levels> 105 at baseline
`
`Treatment
`
`AZ
`
`Species
`A. gerencseriae
`A. israelii
`A. naeslundii 1
`A. naeslundii 2
`A. odontolyticus
`V. parvula
`S. gordonii
`S. intermedius
`S. mitis
`S. oralis
`S. sanguinis
`A. actinomycetemcomitans
`C. gingivalis
`C. ochracea
`C. sputigena
`E. corrodens
`C. gracilis
`C. rectus
`C. showae
`E. nodatum
`F. nucleatum ss nucleatum
`F. nucleatum ss polymorphum
`F. nucleatum ss vincentii
`F. periodonticum.
`P. micros
`P. intermedia
`P. nigrescens
`S. constellatus
`T. forsythia
`P. gingivalis
`T. denticola
`E. saburreum
`G. morbillorum
`L. buccalis
`N. mucosa
`P. acnes
`P. melaninogenica
`S. anginosus
`S. noxia
`T. socranskii
`
`20
`24
`24
`68
`4
`48
`0
`0
`0
`0
`4
`4
`12
`24
`12
`4
`16
`28
`16
`16
`48
`4
`68
`20
`12
`44
`48
`0
`64
`40
`24
`4
`8
`12
`72
`0
`16
`0
`32
`4
`
`MET
`
`25
`25
`17
`63
`0
`46
`0
`0
`0
`0
`0
`4
`8
`17
`8
`4
`8
`42
`13
`4.17
`46
`17
`58
`29
`13
`33
`42
`0
`63
`58
`25
`8
`8
`8
`71
`4
`17
`0
`17
`13
`
`SDD
`
`SRP
`
`55
`45
`45
`70
`30
`60
`15
`0
`15
`15
`15
`25
`20
`55
`30
`10
`45
`50
`35
`25
`50
`22
`65
`45
`25
`45
`60
`5
`70
`60
`35
`15
`10
`30
`55
`10
`35
`10
`40
`20
`
`26
`26
`30
`61
`4
`39
`9
`0
`0
`4
`13
`9
`4
`26
`0
`0
`22
`30
`13
`26
`48
`9
`65
`30
`30
`55
`52
`4
`61
`57
`30
`9
`0
`22
`57
`0
`26
`4
`30
`13
`
`azithromycin, metronidazole, or doxycy-
`cline before antibiotic therapy and at
`2 weeks, and 3, 6, and 12 months after
`initial therapy in subgingival plaque and
`saliva samples from subjects in the four
`treatment groups. Before therapy, the per-
`centage of species resistant to azithromy-
`cin was about 22% in both the test and
`control samples. Forty-six and 37% were
`resistant to metronidazole and 8 and 9%
`were resistant to doxycycline in the test
`and control samples, respectively. In all
`samples from subjects receiving systemic
`agents, antibiotic resistance increased at
`2 weeks with significant differences be-
`tween the test and control samples for
`azithromycin and doxycycline. For both of
`these antibiotics, the significant difference
`between test and control samples remained
`to 6 months, but by 12 months the per-
`centage of resistant species were reduced
`in the test samples and levels approached
`those detected before therapy. At no time-
`point was there a significant difference
`
`between test and control subjects in the
`percentage of species resistant to metroni-
`dazole. The data for the saliva samples
`were similar to those observed for the
`plaque samples except that the pretherapy
`proportions of resistant species were high-
`er in the saliva samples (Fig. 3). Further-
`more,
`there was an increase in the
`percentage of resistant species in the 12-
`month samples in subjects in the SDD
`group so that
`levels were significantly
`higher
`than those observed in control
`saliva samples.
`
`Percentage of sites harboring resistant
`species
`
`Figures 4–6 present the percentage of sites
`colonized by the 40 test species that were
`resistant
`to azithromycin, metronidazole,
`and doxycycline. Irrespective of treatment
`group, there were no significant differences
`over time in the percentage of sites harbor-
`ing resistant species. The most commonly
`
`detected resistant species in each treatment
`group were the streptococci. Resistant
`strains of Veillonella parvula were detected
`in over 50% and 80% of sites in subjects in
`the SDD and azithromycin groups respec-
`tively. In general, a wider range of species
`was found to be resistant in the azithromy-
`cin and SDD groups.
`Resistant isolates of many of the test
`species were found in a very small percent-
`age of sampled sites including periodontal
`pathogens such as T. forsythia, P. gingiva-
`lis, E. nodatum, and Campylobacter spe-
`cies. The percentage of sites harboring
`resistant strains of Fusobacterium was also
`very low in the metronidazole group. There
`were a small number of significant differ-
`ences between test and control samples at
`various time-points for each of the test
`antibiotics. However, when adjustments
`were made for multiple comparisons, these
`differences were no longer significant.
`
`Discussion
`
`The goal of the present investigation was
`to describe the subgingival microbial
`changes that took place as a result of four
`different treatment modalities, SRP alone
`or
`in conjunction with the antibiotics,
`azithromycin or metronidazole or SDD.
`The choice of adjunctive agents was based
`on their different modes of action and the
`different dosage regimens required, which
`ranged from 3 days for azithromycin to
`3 months for SDD. As described in Haf-
`fajee et al. (10),
`there were differences
`among treatment groups in the clinical
`changes at 1 year post-therapy.
`Irrespective of the treatment modality
`employed, there was a significant decrease
`in mean counts of many of the test species,
`particularly members of the red and orange
`complexes, species associated with the
`etiology of periodontal infections. It has
`been shown that a decrease in the counts of
`this segment of the subgingival microbiota
`is associated with clinical improvements
`(9). This was confirmed in the current
`investigation because a decrease in mean
`counts of many of the test species was
`significantly associated with an improve-
`ment in attachment level and a reduction in
`probing pocket depth (10). The most
`marked decrease in counts occurred be-
`tween the pretreatment visit and the
`2-week sampling. At this time-point, two
`quadrants of SRP had been completed, as
`well as administration of both azithromy-
`cin and metronidazole. This is in accord
`with data in the literature suggesting that
`treatment of part of
`the mouth has a
`beneficial effect on ‘untreated’ segments
`
`

`

`154
`
`Haffajee et al.
`
`Fig. 4. Mean (±SEM) percentage of sites colonized by species resistant to 2 lg/ml azithromycin at baseline, 2 weeks, and 3, 6, and 12 months in
`subjects who received systemically administered azithromycin as part of their therapy. Samples were taken from two to four sites at each time-point and
`resistant species were averaged across subjects for each time-point separately. The pink lines represent the resistance profiles for samples tested for
`azithromycin resistance from subjects receiving SRP only (control group). Significance of differences over time was sought using the Friedman test. There
`were no statistically significant differences over time in either the test or control samples. Significance of differences between test and control samples at
`each time-point was tested using the Mann–Whitney test, *P < 0.05, **P < 0.01, ***P < 0.001, without adjusting for 40 comparisons. After adjustment
`there were no significant differences between test and control samples for any of the species at any time-point.
`
`(12, 21). It may also suggest that system-
`ically administered antibiotics have a
`marked effect on the subgingival microbi-
`ota either in the presence or absence of
`SRP, as suggested by Lopez et al. (17).
`For many species there was a continued
`decrease in levels to 3 or even 6 months,
`with a slight
`rebound detected at
`the
`12-month visit. It
`is interesting that at
`12 months, mean pocket depth and attach-
`ment level measurements were still show-
`ing improvement overall
`in subjects in
`each of the four treatment groups, with the
`exception of the SDD group (10). One
`may speculate that the rebound in micro-
`bial counts might continue and be associ-
`ated with a subsequent worsening of
`the clinical condition. Unfortunately, the
`present study was not of sufficient duration
`to answer this question.
`This study was in accord with the
`findings described by Haffajee et al. (7)
`and Cugini et al. (3), who found that red
`complex species were significantly re-
`duced by SRP. The current study extended
`
`these findings and indicated that subjects
`in the three treatment groups receiving
`adjunctive agents also showed a reduction
`in red complex species and that
`these
`reductions, over time, were significant for
`T. forsythia in the azithromycin group and
`for T. forsythia and P. gingivalis in the
`metronidazole group. The findings for the
`adjunctive groups were consistent with
`those in the literature. Sefton et al. (22)
`described reductions in spirochetes and
`anaerobic ‘black-pigmented’ species at
`22 weeks
`after
`azithromycin
`therapy,
`while Feres et al. (4) described significant
`reductions in counts and proportions of red
`complex species at 12 months after initial
`therapy consisting of SRP and 2 weeks of
`systemically administered metronidazole.
`The antibiotic-treated subjects in the cur-
`rent investigation exhibited a better clinical
`response, suggesting that a rapid decrease
`in subgingival counts of periodontal patho-
`gens may be crucial for successful peri-
`odontal therapy and long-term periodontal
`stability. However, there were few signif-
`
`icant differences in the subgingival micro-
`biota
`at 12 months
`among treatment
`groups.
`It has been suggested that SDD does not
`act as an antibiotic and thus microbial
`changes would be limited, as suggested by
`the findings of Thomas et al. (28). In the
`current
`investigation there were reduc-
`tions, some significant, in the mean counts
`of several species at 2 weeks post-therapy
`in the SDD group, a pattern of change
`similar
`to that observed in the other
`treatment groups. However, there was an
`increase in counts of many species in the
`SDD group at 6 months, the first monitor-
`ing visit after cessation of the medication.
`Indeed,
`the only statistically significant
`differences among groups, after adjusting
`for multiple comparisons, was observed at
`the 6-month visit
`for mean counts of
`A. israelii, S. gordonii, S. intermedia, and
`P. gingivalis primarily the result of the
`much higher levels of these species in the
`SDD group at
`this time-point. Further,
`counts of several orange complex species
`
`

`

`Microbial effects of antibiotic therapy
`
`155
`
`Fig. 5. Mean (±SEM) percentage of sites colonized by species resistant to 2 lg/ml metronidazole at baseline, 2 weeks, and 3, 6, and 12 months in
`subjects who received systemically administered metronidazole as part of their therapy. Samples were taken from two to four sites at each time-point and
`resistant species were averaged across subjects for each time-point separately. The pink lines represent the resistance profiles for samples tested for
`metronidazole resistance from subjects receiving SRP only. Significance testing was as described in Fig 4.
`
`exceeded baseline values at 12 months in
`the SDD group.
`the percentage of
`Not
`surprisingly,
`resistant
`isolates
`increased in subjects
`receiving adjunctive agents immediately
`after taking these agents, primarily because
`of a decrease in susceptible species. Levels
`had returned to baseline or close to
`baseline values at 12 months, with the
`exception of the high levels of species
`resistant to doxycycline observed in the
`saliva samples of subjects in the SDD
`group. The pretherapy levels, the rise in
`resistant species during antibiotic admin-
`istration and the subsequent post-therapy
`decline in resistance levels were similar to
`the findings of Feres et al. (4, 6). The data
`from the present investigation indicated a
`similar percentage of
`resistant
`isolates
`pretherapy and at 12 months post-therapy
`but the nature of the resistant species could
`not be determined. The question as to
`whether the same strains of a given species
`were resistant pre- and post-therapy to the
`administered agents or whether new, resis-
`tant strains or strains resistant to multiple
`antibiotics had emerged could not be
`answered.
`
`A large percentage of sites harbored
`strains of V. parv