Pathogens in Relation to Periodontal Status

J Dent Res 74(11): 1789-1793, November, 1995 Salivary Levels of Suspected Periodontal Pathogens in Relation to Periodontal Status and Treatment B. von Troil-Lindenl*, H. Torkko2, S. Alaluusua3, H. Jousimies-Somer4, and S. Asikainent 'Department of Periodontology, 2lnstitute of Dentistry, 3Department of Pedodontics and Orthodontics, University of Helsinki, P.O. Box 41, Mannerheimintie 172, FIN0014 University of Helsinki, Finland; and 4Anaerobe Reference Laboratory, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland; *to whom correspondence should be addressed Abstract. The primary ecological niche for suspected periodontal pathogens seems to be the subgingival area, even though periodontal pathogens are also frequently recovered from saliva. The interrelationship of different periodontal conditions and the salivary levels of suspected periodontal pathogens is not known. In the present study, salivary levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, and Peptostreptococcus micros were determined by bacterial culture and related to clinical periodontal status in 40 subjects with either advanced, moderate, or initial/no periodontitis. Culture-positive subjects harbored the 5 bacterial species in mean numbers ranging from 2 x 105 to 6 x 107 colony-forming units (CFU)/mL saliva. A. actinomycetemcomitans was found in none and P. gingivalis in one of the subjects with initial periodontitis, whereas both species were found in 33% and 44%, respectively, of the subjects with moderate periodontitis and in 60% and 40%, respectively, of the subjects with advanced periodontitis. The mean numbers of CFU/mL of P. intermedia, C. rectus and P. micros were significantly higher in subjects with advanced periodontitis than in subjects with initial/no periodontitis. Ten patients with advanced periodontitis were treated mechanically and with adjunctive systemic metronidazole, and were re-examined 1 and 6 months after treatment. Periodontal treatment eradicated or significantly reduced the levels of salivary periodontal pathogens for half a year, whereas in untreated subjects, the levels and the detection frequencies generally remained fairly stable. In conclusion, the results showed that the salivary levels of periodontal pathogens reflect the periodontal status of the patient. Key words: periodontitis, periodontal bacteria, spouses, saliva, bacterial levels. Received July 7, 1994; Accepted August 22, 1995 Introduction The primary ecological niche for putative periodontal pathogens, including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, and Peptostreptococcus micros, seems to be the subgingival area (Tanner et al., 1979; Dzink et al., 1988; Lai et al., 1992; Rams et al., 1992). However, some of these bacteria are also common in saliva (van Winkelhoff et al., 1988b; Asikainen et al., 1991; Muller et al., 1993; Kononen et al., 1994b; von Troil-Linden et al., 1995a). Saliva is the probable vehicle for transmission of oral bacteria, and several studies using genetic intraspecies characterization of suspected periodontal pathogens have recently suggested the spread of micro-organisms between family members via saliva (DiRienzo and Slots, 1990; Alaluusua et al., 1993; Petit et al., 1993; Saarela et al., 1993; van Steenbergen et al., 1993; Kononen et al., 1994a; Preus et al., 1994). On the basis of our recent results on the periodontal status of and the occurrence of periodontal bacteria in married couples, we proposed that transmission of suspected periodontal pathogens may actually lead to colonization and even to the subsequent development of periodontitis in the recipient (von Troil- Linden et al., 1995a). Contrarily to subgingival samples, salivary samples allow for quantitative standardization of the sample size. Salivary samples are easy and rapid to obtain, and the sampling procedure does not require specially trained personnel. However, the interrelationship between different periodontal conditions and the salivary levels of suspected periodontal pathogens is not known. In the present study, we examined the relationship of these levels in groups of patients with advanced, moderate, or initial/no periodontitis. We also examined, longitudinally, whether the salivary levels are affected by treatment or remain stable when no periodontal treatment is given. This knowledge would be of value in the evaluation of the risk of transmitting periodontal pathogens between individuals 1789

1790 von Troil-Linden et al. J Dent Res 74(11) 1995 Table 1. Demographic characteristics of the 40 study subjects, from 20 married couples, who were divided into probands (Groups 1 and 2) and spouses (Groups 3 and 4) and further grouped based on clinical periodontal status Number of Subjects Age Sex Group Clinical Periodontal Status Total Treated Mean (years) (SD) Male/Female Probands 1 Advanced periodontitis 10 10 55.5 (8.1) 9/1 2 Initial/no periodontitis 10 4 47.5 (9.4) 2/8 Spouses 3 Moderate periodontitis 9 5 54.3 (9.6) 2/7 4 Initial/no periodontitis 11 0 49.4 (9.6) 7/4 and in the design of maintenance programs for treated periodontitis patients. Materials and methods Subjects The present study was comprised of 40 subjects (Table 1), a group of 20 married couples, included in our studies on the transmission of periodontal bacteria between spouses (Saarela et al., 1993; von Troil-Linden et al., 1995a). The experimental study protocol was approved by the Ethical Committee of the Institute of Dentistry, Helsinki. All subjects were informed about the study design and procedures, and a written consent was given by each participant. To avoid comparisons between related subjects, the study population was divided into probands and spouses. The probands and the spouses were further grouped according to their periodontal status: Groups 1 and 2 were comprised of the probands with either advanced or initial/no periodontitis, respectively, and Groups 3 and 4 were comprised of their spouses exhibiting either moderate or initial/no periodontitis. The three disease categories, advanced, moderate, or initial/no periodontitis, were classified according to the severity and the extent of radiographic alveolar bone loss in the entire dentition. In subjects with advanced periodontitis, alveolar bone loss exceeded or was equal to 1/3 of the root length, and in those with moderate or initial/no periodontitis, less than 1/3 of the root length. The groups of moderate and initial/no periodontitis were further differentiated according to the extent of alveolar bone loss: In subjects with moderate periodontitis, the alveolar bone loss affected > 30% of the examined tooth surfaces, and in subjects with initial/no periodontitis, < 30%/0%, respectively. Probing pocket depths were assessed by means of a WHO probe (and a force of approximately 20 g). Study protocol All subjects were clinically examined on three occasions (B.v.T.- L.). Salivary samples were obtained from each subject at all examinations. Ten subjects with advanced periodontitis (Group 1) were treated (B.v.T.-L.). They were examined at baseline and at 1 month and 6 months after treatment. Twenty-one of the 30 subjects in the moderate or initial/no periodontitis groups were not treated but were only examined at baseline and at 1 month and 6 months after baseline. Periodontal treatment Ten subjects with advanced periodontitis received information and instruction in oral hygiene measures at the first or second mechanical treatment session, and the hygiene level was reinforced at later appointments. Mechanical treatment included scaling and root planing, soft tissue curettage, and gingivoplasty where indicated, and was given at 4 to 7 appointments within 1 to 3 weeks in all subjects, except within 6 weeks in one subject receiving amoxycillin (AmorionR, Orion, Espoo, Finland) prophylaxis (3 g) 1 hr before treatment because of a hip prosthesis inserted 3 months earlier. The subjects rinsed their mouths twice a day with chlorhexidine solution (0.2% Hibitane DentalR, ICI, Macclesfield, England) for 1 min, placed chlorhexidine gel (1% Hibitane gelr, ICI) interdentally with an interdental brush during the mechanical treatment period, and continued to do so for 1 wk after the mechanical treatment period was completed. In addition, 500 mg metronidazole (ElyzolR, Dumex, Copenhagen, Denmark) was given per os twice a day for 7 days, starting on the day of the first mechanical treatment session. The change in the proportion of sites with periodontal pockets 2 4 mm deep was applied for evaluation of the clinical response to treatment. Because of ethical reasons, nine of the 30 subjects in the moderate or initial/no periodontitis groups were treated mechanically or received periodontal prophylaxis at some time during the six-month study. Thus, these nine subjects were excluded from the 30 subjects in whom the stability of bacterial salivary levels was originally planned to be evaluated. Bacterial samples As previously reported (von Troil-Linden et al., 1995a), paraffinstimulated saliva was collected for 1 min, and a 0.3-mL aliquot was transferred to vials containing 2 ml VMGA III transport medium (Moller, 1966) with glass beads. A. actinomycetemcomitans, P. gingivalis, P. intermedia, C. rectus, and P. micros were cultured within 24 hours in the Microbiology Laboratory of the Institute of Dentistry, University of Helsinki. From a total of 118 samples taken for the present study, most samples (94%) were cultured on the day of sampling, and only 6% were cultured on the following day (within 24 hours). In a separate pilot study as an internal quality control, we assessed the ability of VMGA III transport medium to maintain the viability of the examined bacterial species. Nine salivary samples were cultured immediately and 24 hours after sampling. No significant differences in bacterial counts were observed between the two regimens. The bacterial species were cultured and identified as

j Dent Res 7401) 1995 Salivary Levels of Periodontal Pathogens 1791 Table 2. Means (standard deviations) of colony-forming units/ml saliva of suspected periodontal pathogens at baseline in subjects with advanced (Group la) or initial/no periodontitis (Group 2) Group 1 Group 2 Subjects with Advanced Periodontitis (N = 10) Subjects with Initial/No Periodontitis (N = 10) Mean SD Detection Mean SD Detection Bacterial Species x 106 x 106 Frequency (%) x 106 x 106 Frequency (%) A. actinomycetemcomitans 0.1 0.2 6 (60) 0 0 0 (0) P. gingivalis 4.8 10.0 4 (40) 0 0 0 (0) P. intermedia 16.0b 30.0 10 (loo)d 1.7b 2.7 4 (40)d C. rectus 29 Ob 72.0 10 (l1o)d 0.5b 1.0 4 (40)d P. micros 42.0c 88.0 7 (70) 1.2c 2.5 3 (30) a Group definitions as in Table 1. b Student's t test: p < 0.01. c Student's t test: p < 0.05. d Fisher's exact test: p < 0.01. previously described (von Troil-Lind6n et al., 1995a). Briefly, A. actinomycetemcomitans was cultured on TSBV agar (Slots, 1982), C. rectus on Hammond's selective medium (Hammond and Mallonee, 1988), and P. gingivalis, P. intermedia, and P. micros on supplemented brucella blood agar plates. TSBV-agar plates were incubated at 37 C in 5% CO2 for 3 to 5 days. Hammond's medium and brucella agar plates were incubated at 37 C in anaerobic jars filled with mixed gas (85% N2, 10% H2, 5% C02) for 7 to 10 days. Identification of the bacterial species was based on Gram staining, colony morphology, pigmentation, motility, and enzymatic reactions (catalase, oxidase, ol-fucosidase, P-Nacetyl-glucosaminidase, lipase, urease, alkaline phosphatase, nitrate reductase, and MUG and CAAM tests). The numbers of colony-forming units (CFU)/mL saliva were assessed for each species. The detection thresholds for A. actinomycetemcomitans, C. rectus, and for the bacterial species cultured on brucella blood agar plates were 7.7 x 102, 7.7 x 104, and 7.7 x 105 CFU/mL saliva, respectively. Statistical analysis Among groups, statistical significances were determined by Student's t test for differences in means, by Fisher's exact test for differences in frequency distribution, and by paired t test for changes from baseline to 1 and 6 months after treatment. For microbiological variables, a transformation [, unit of x is 106)] was used to compensate for biases caused by abnormally distributed microbiological data. Probability of equal to or less than 0.05 was considered statistically significant. Results The demographic characteristics of the subjects in the four groups are shown in Table 1. The age of the subjects ranged between 38 and 70 years, and no statistically significant difference was observed in the mean ages of the four study groups. When the salivary levels of the five bacterial species were related to the periodontal conditions, it was seen that A. actinomycetemcomitans was found in none and P. gingivalis in only one of the subjects with initial periodontitis (Groups 2 and 4), whereas the detection frequencies of these species ranged between 33% and 60% in the subjects with advanced (Group 1) or moderate (Group 3) periodontitis (Tables 2 and 3). The detection frequencies and the mean salivary numbers of P. intermedia and C. rectus, but only the mean salivary numbers of P. micros, were significantly higher in Group 1 than in Group 2 (Table 2). No statistically significant differences were found in the detection frequencies or the mean salivary numbers of P. intermedia, C. rectus, and P. micros between Groups 3 and 4 (Table 3). At baseline, A. actinomycetemcomitans and P. gingivalis were detected in six and four subjects out of ten in Group 1, respectively, whereas 1 month after periodontal treatment, these species were eliminated or suppressed below detection level in all subjects (Fig. 1). Six months after treatment, A. actinomycetemcomitans could not be detected in four of the six subjects harboring the species at baseline, but was found in higher numbers than at baseline in the two remaining subjects (R.J. and Y.L., Fig. 2). Six months after treatment, P. gingivalis could not be detected in the four subjects positive at baseline, but was found in one subject (R.J.) negative for this species at baseline (Fig. 2). At baseline, P. intermedia, C. rectus, and P. micros were detected in ten, ten, and seven subjects out of ten in Group 1, respectively, and three, two, and one subjects, respectively, still harbored the species 1 month after treatment. The mean salivary numbers of these three species decreased statistically significantly (p values ranged between 0.002 and 0.017 in the paired t test) from baseline numbers 1 month after treatment (Figs. 1 and 2). Six months after treatment, P. intermedia, C. rectus, and P. micros were detected in six, three, and two subjects, respectively. All subjects harboring the three species 6 months after treatment also harbored the species at baseline. No statistically significant differences could be found in the mean salivary numbers of P. intermedia and P. micros between the baseline examination and 6 months after treatment, except for C. rectus (p value = 0.02). In the group of subjects with no periodontal treatment, no statistically significant differences were seen in the mean salivary numbers or the detection frequencies of the studied species during the six-month follow-up period, except for a statistically significant decrease in the mean numbers of P. micros between baseline and 1 month after baseline (Fig. 1). The mean proportions of periodontal pockets 2 4 mm deep decreased statistically significantly from baseline at 1

1792 von Troil-Linden et al. J Dent Res 74(11) 1995 Table 3. moderate (Group 3a) and initial/no periodontitis (Group 4) Means (standard deviations) of colony-forming units/ml saliva of suspected periodontal pathogens at baseline in subjects with Group 3 Group 4 Subjects with Moderate Periodontitis (N = 9) Subjects with Initial/No Periodontitis (N = 11) Mean (SD) Detection Mean SD Detection Bacterial Species x 106 x 106 Frequency (%) x 106 x 106 Frequency (%) A. actinomycetemcomitans 0.4 1.3 3 (33) 0 0 0 (0) P. gingivalis 8.3 14.0 4 (44) 5.6 19.0 1 (9) P. intermedia 2.6 3.0 8 (89) 7.5 19.0 6 (55) C. rectus 1.3 2.4 8 (89) 0.9 2.1 5 (46) P. micros 7.0 21.0 2 (22) 1.6 4.5 4 (36) a Group definitions as in Table 1. month and 6 months after periodontal treatment (Table 4). The subjects who did not receive periodontal treatment showed no statistically significant differences in the mean proportions of periodontal pockets 2 4 mm deep during the six-month follow-up period (Table 4). Discussion In the present study, the salivary numbers of suspected periodontal pathogens were determined by bacterial culture and related to clinical periodontal status in non-family subjects. The ability of periodontal treatment to reduce bacterial levels in saliva was also examined, with the bacterial levels in untreated subjects used as controls. Twenty married couples comprised the subject group in the present study. To avoid biased results, we compared bacteriological findings in only unrelated subjects, since our earlier findings suggested similarity in the occurrence of salivary putative periodontal pathogens in spouses (von Troil-Linden et al., 1995a). The subjects were divided into three disease categories-advanced, moderate, and initial/no periodontitis-based on severity and extent of radiographic alveolar bone loss. The results showed significant differences in detection frequencies and mean salivary numbers of the bacterial species between subjects included in the groups of advanced or initial/no periodontitis, whereas no significant differences were seen between the groups of moderate or initial/no periodontitis. Periodontal treatment eradicated or suppressed the studied bacterial species under detection level from saliva for 6 months, except P. intermedia, which was detected 6 months after treatment in 60% of the subjects harboring this species at baseline. However, if the species were still recovered 6 months after treatment, their salivary levels were generally lower than at baseline. In only a few subjects were comparable levels found at baseline and 6 months after treatment. Some individual variation in the numbers of the five bacterial species, most probably due to clustering near the detection treshold, occurred in the subjects with no periodontal treatment during the study period, but, taken as a group, the mean salivary numbers and the detection frequencies of the species remained the same during the study period. For example, in the case of C. rectus, in six out of nine (67%) subjects, the numbers were between 8 x 104 (the detection treshold) and 6 x 105 (data not shown). The salivary numbers of other oral bacterial species, such as mutans streptococci, have been reported to be fairly stable (Zickert et al., 1982; Alaluusua et al., 1990). However, studies comparing salivary levels of suspected periodontal pathogens before and after periodontal treatment could not be found in the literature. Previous reports on the time interval for subgingival recolonization with periodontopathic bacteria after periodontal treatment vary considerably and seem to depend more on whether the patients received oral hygiene instructions and how often they were recalled for maintenance care (Haffajee et al., 1988a; Renvert et al., 1990; Socransky and Haffajee, 1993) than on whether antibiotics were administered (van Oosten et al., 1987; Loesche et al., 1992) or not (Magnusson et al., 1984; van Winkelhoff et al., 1988a; Rosenberg et al., 1989; Sbordone et al., 1990; Pedrazzoli et al., 1991). These studies report that subgingival periodontal pathogens were Table 4. Clinical response to periodontal treatment in 10 subjects with advanced periodontitis and follow-up of 21 subjectsa with no periodontal treatment Proportions of Periodontal Pockets. 4 mm deep (%) Group Clinical Periodontal Status Number of Subjects Mean SD Mean SD Mean SD 1 Advanced periodontitis 10 50.1b 15.1 14.6b 5.5 17.7b 9.3 2-4 Moderate, initial/no periodontitis 21 7.3 12.6 6.1 10.0 5.5 9.2 a Four subjects with moderate and 17 with initial/no periodontitis. b Paired t test: p < 0.001.

J Dent Res 74(11) 1995 Iit -n I5I log (I +X) log (I+x) () ().8 No periodontal treatment 2 0.6- { A. actinomycetemcomitans X P. gingivalis E * P. intermedia 0.4 - - P. micros D0.4 Salivary Levels ofperiodontal Pathogens I3 T 2- -a -O A. actinomycetemcomitans * P. gingivalis * P. intennedia 1- A C. rectus P. micros 0-3 Ty 2-1 - 0-,.C rectus A. actinomycetemcomitans P. gingivalis.. E. S. M. S. R. J. M. V. Y. L. -- M.S. * R.J. 0- M.V. -- H.E. +- O.R. 1793 ( s Baseline month 6 months Figure 1. Mean numbers of colony-forming units/ml saliva for 5 bacterial species in ten periodontally treated subjects and in 21 subjects with no periodontal treatment. eliminated or reduced in proportions or counts immediately after therapy, but that a re-population of subgingival sites occurred in 3 weeks to 6 months. In patients afflicted with localized juvenile periodontitis, the eradication or suppression of subgingival A. actinomycetemcomitans seems possible with meticulous mechanical debridement in combination with systemic administration of antimicrobial agents (Slots and Rosling, 1983; van Winkelhoff et al., 1989; Saxen and Asikainen, 1993). The present eradication of salivary bacterial counts of most species and the reduction in pocket depths after treatment prevailed for the six-month follow-up period in most of the subjects, but not in all. Trying to explain the individual variation, we examined the changes in bacterial numbers for each treated subject separately (Fig. 2) and related the bacterial findings to the clinical status (data not shown). Six months after treatment, A. actinomycetemcomitans could be detected in two subjects (Y.L. and R.J.) in higher numbers than at baseline and P. gingivalis in only one subject (R.J.), who did not harbor P. gingivalis at detectable levels in his salivary samples at baseline. At the end of the six-month follow-up period, the latter subject harbored all studied bacterial species, except C. rectus, in higher numbers than at baseline. Although the proportions of periodontal pockets. 4 mm deep and numbers of putative periodontal pathogens decreased 1 month after treatment in these two subjects (Y.L. and R.J.), 6 months after treatment they had regained increasing proportions of periodontal pockets. 4 mm deep 1 log (l+x) 3 r 2- Baseline I month 6 months ---i-- - --O- -U - - --O -9--- -a---- 0 E. S. M. S. R. J. M. V. H. E. I. K. T.L. Y. L. 0. R. E. S. M. S. R. J. M. V. H. E. t. K. T. L. Y. L. 0. R. IM..S. Ill. J. NI. V H. F. l K. I. R Figure 2. Individual changes in colony-forming units/ml saliva for 5 bacterial species in culture-positive, periodontally treated subjects.

1794 von Troil-Linden et al. and high numbers of putative periodontal pathogens in saliva (Fig. 2). About 10 to 20% of adult periodontitis patients do not respond favorably to therapy (Haffajee et al., 1988b; Slots and Rams, 1992), which is in accordance with the present findings. Whether treatment failure is due to external or endogenous re-infection with periodontal pathogens or to relapse because of exceptionally low host resistance is not yet known. Many viruses and bacteria, including respiratory viruses and Corynebacterium diphtheriae and Mycobacterium tuberculosis (Schuster, 1990), are shed from the respiratory tract and oral surfaces into saliva and are able to maintain their viability for prolonged periods in this secretion while protected from drying (Merchant, 1991). The infective ability of saliva is likely to increase with growing numbers of micro-organisms as well as with frequency of exposure. Our present study emphasizes the high salivary levels of periodontal pathogens in patients with advanced periodontitis. Furthermore, our earlier and contemporary studies suggest that salivary and subgingival periodontal pathogens are genotypically similar within an individual and are transmissible and infective in spouses (Saarela et al., 1993; von Troil-Linden et al. 1995a,b). In the present study, we showed that the salivary levels of periodontal pathogens reflected the periodontal status of the patient, and that treatment of periodontitis generally eliminates or decreases the salivary levels of A. actinomycetemcomitans, P. gingivalis, C. rectus, and P. micros to undetectable for a period of half a year. However, reemergence of periodontal pathogens in saliva seems to occur after therapy in some patients. 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