274
Microbiology of Healthy and Diseased Periodontal Sites in Poorly Controlled Insulin Dependent Diabetics Robert L. Mandell, * John Dirienzo, *
Haberf
Ralph Kent, * Kaumudi Joshipura,
*
and Jerome
A group of poorly-controlled insulin dependent diabetes mellitus (IDDM) patients examined in a cross-sectional design for total microbial levels, microbial incidence, and the percent levels of selected periodontal microorganisms. These organisms were selected on the basis of prior reports that associated them with either periodontal disease or health. One periodontally-healthy and one periodontally-diseased site were examined in each IDDM patient. Increased levels of the periodontal pathogens Prevotella intermedia, P. melaninogenica spp., Bacteroides gracilis, Eikenella corrodens, Fusobacterium nucleatum and Campylobacter rectus (formerly Wolinella recta) were found at the periodontal diseased sites. Increased prevalence of the organisms P. intermedia, P. melaninogenica spp., and C. rectus were found at the diseased sites. A significantly higher percentage of P. intermedia was found at the sites exhibiting deep pockets and attachment loss. / Periodontol 1992; 63:274-279. were
Key Words: Diabetes, insulin-dependent; periodontitis/microbiology.
The influence of diabetes on the risk of developing periodontal disease has been the subject of much discussion. According to a majority of authors, individuals suffering from insulin dependent diabetes mellitus (IDDM) are at increased risk for the development of periodontal disease.113 There is, however, a small group of other researchers who fail to find this increased risk.14"19 Recent research may explain these different findings based upon the size of the population studied as well as the role of other complicating factors such as smoking on the prevalence of periodontal disease in diabetic smokers versus diabetic nonsmokers.20 The microbiologie findings of diseased pockets in diabetics have been less extensively studied. Mashimo et al.21 examined 14 IDDM patients aged 13 to 27 years. The population consisted of 9 Periodontitis patients, 3 gingivitis patients, and 2 periodontally-healthy patients. The diseased patients, whose condition resembled juvenile Periodontitis, were found to harbor a flora with high levels of Actinobacillus actinomycetemcomitans, Capnocytophaga spp., and "Anerobic vibrios." Sastrowijoto et al.22 examined diabetic adults grouped by virtue of their glycosylated hemoglobin values. They examined 44 sites in 22 patients who were further divided into a near normal (Hb Ale 9.9%) group. They re* Forsyth Dental Center and Tufts School of Dental MA. Tufts School of Dental Medicine, Boston, MA. *
Medicine, Boston,
ported that the organisms A. actinomycetemcomitans, B. gingivalis, and B. intermedius were recovered at proportionally higher levels from the diseased versus control sites. The purpose of this investigation was to examine a group of suspected periodontal pathogens associated with healthy and diseased periodontal pockets in a group of IDDM patients with periodontal disease. MATERIALS AND METHODS
Subjects
Fifteen Caucasian patients (9 males, 6 females) with a mean age 34.6 years ( + /- 6.4; range 23 to 50 years) were selected for study. The mean duration of insulin dependent diabetes was 20.6 ( + /— 7.6) years. Daily insulin use was 41.0 ( + /- 18.6) units. Medical histories indicated that the mean levels of glycosalated hemoglobin was 10.4% (+/ 2.0) for 12 of the 15. Blood glycosalated hemoglobin levels were unavailable on the remaining 3 patients. Two of the 3 indicated that they were poorly controlled and the remaining patient was unaware of the level at the time of examination. These patients were a subset of a large ongoing epidemiological study and were eligible for microbiology sampling based upon having at least one pocket depth >5 mm and attachment loss of >2 mm (Table 1). A roster of patients who qualified for the microbiology study was generated from the epidemiology study. All patients on the list were contacted by mail and the first 15 who —
Volume 63 Number 4
MANDELE, DIRIENZO, KENT, JOSHIPURA, HABER
Table 1. Clinical Characteristics of Sampled Sites in 15 IDDM Patients with Periodontal Disease
(ii 15) =
=
Pocket depth* Attachment level*
Bleeding on probingt Rednesst
*Mean in
mm
5.8 (0.8) 2.8 (0.9) 100 93
15 13
(standard deviation).
tSites displaying the characteristic.
responded to the mailing and could make pointment were included in the study.
a
scheduled ap-
Clinical Indices The dichotomous indices of bleeding on probing (BOP) and gingival redness (GR) were recorded for all sampled sites.23 Pocket depths were measured from the gingival margin to the probe tip. The measurements were made to the nearest 0.5 mm by a single examiner (R.L.M.) using a Michigan 0 probe. Attachment level was derived from the pocket depth by a second measure made from the cemento-enamel junction (CEJ) to the gingival margin. These measurements were then subtracted to arrive at the attachment level or loss measure.23
Sampling and Media Subgingival plaque samples were taken from the base of the periodontal pockets with a sterile Morse (00) sealer inserted into the depth of the pocket. The tip was aseptically transferred to prereduced one-quarter strength Ringer's solution, sonicated for 5 seconds* at settings previously determined to give the highest recoverable bacterial counts24 and then 10-fold dilutions were made under an 02-free atmosphere.25 0.1 ml samples were plated on duplicate plates of selective and elective media to enumerate the following organisms: 1) Actinobacillus actinomycetemcomitans,26 a modified media consisting of 4 ug/ml malachite green (plain base form) and 128 ug/ml bacitracin in 5% sheep's blood Trypticase soy agar;§ 2) Eikenella corrodens;27 3) Streptococcus sanguis I, 5. oralis (formerly S. sanguis II), and S. intermedius, Mitis-Salivaris agar11 representative colonies were isolated and identified;28 4) Fusobacterium nucleatum;29 5) Capnocytophaga spp.;30 6) Bacteroides spp.31 representative colonies were transferred and further differentiated based upon long-wave UV light,32 MUG test for lactose utilization,33 and CAMM test,34 to allow rapid presumptive identification of P. gingivalis, P. intermedia, and P. melaninogenica spp. (includes P. loeschi and P. denticola (formerly B. loeschi, B. denticola); 7) B. gracilus, Campylobacter concisus, C. rectus, and C. curvus (formerly W. curva), were isolated on a newly developed se* 5 1
Heat System Ultrasonic, Plainview, NY. TSAB, BBL Cockeysville, MD. Difco Laboratories, Detroit, MI.
Table 2. Mean Counts* of Bacteria Isolated from 15 Diabetics With Disease
Diseased
Healthy (n 15) 2.8 (0.4) 0.6 (0.5)
275
Bacteria A. actinomycetemcomitans P. gingivalis P. intermedia P. melaninogenica spp. B. gracilis Capnocytophaga spp. C. concisus C. curvus C. rectus E. corrodens F. nucleatum S. intermedius S. sanguis I S. oralis Total counts
Diseased
4.7E2 2.5E3 8.7E5 1.3E4 2.0E6 5.0E5 0 9.7E3 4.3E6 1.4E6 4.4E5 1.3E5 2.6E5 1.9E5 7.3E7
Healthy (2.0E3)
(4.6E2) (1.8E3) (3.4E5)t (7.7E3)í (1.3 6) (3.2E5)
2.7E3 0 8.0E4
5.0E3 5.8E3 1.2E4 2.8E3 4.8E3 1.8E4 2.3E4 2.9E4 5.1E4 4.0E4 6.8E4 9.6E6
(8.6E3) (3.2E6)t (1.2E6)t (2.3 5) (6.1E4) (1.6E5) (1.2E5) (3.4E7)t
(4.2E4) (5.0E3) (2.8E3) (9.2E3) (2.0E3) (4.0E3) (8.2E3) (2.0E4) (1.1E4) (4.0E4) (1.7E4) (4.4E4) (6.0E6)
'Counts (standard error) expressed in scientific notation. tSignificantly elevated by Wilcoxon signed rank test P
Microbiology of Healthy and Diseased Periodontal Sites in Poorly Controlled Insulin Dependent Diabetics Robert L. Mandell, * John Dirienzo, *
Haberf
Ralph Kent, * Kaumudi Joshipura,
*
and Jerome
A group of poorly-controlled insulin dependent diabetes mellitus (IDDM) patients examined in a cross-sectional design for total microbial levels, microbial incidence, and the percent levels of selected periodontal microorganisms. These organisms were selected on the basis of prior reports that associated them with either periodontal disease or health. One periodontally-healthy and one periodontally-diseased site were examined in each IDDM patient. Increased levels of the periodontal pathogens Prevotella intermedia, P. melaninogenica spp., Bacteroides gracilis, Eikenella corrodens, Fusobacterium nucleatum and Campylobacter rectus (formerly Wolinella recta) were found at the periodontal diseased sites. Increased prevalence of the organisms P. intermedia, P. melaninogenica spp., and C. rectus were found at the diseased sites. A significantly higher percentage of P. intermedia was found at the sites exhibiting deep pockets and attachment loss. / Periodontol 1992; 63:274-279. were
Key Words: Diabetes, insulin-dependent; periodontitis/microbiology.
The influence of diabetes on the risk of developing periodontal disease has been the subject of much discussion. According to a majority of authors, individuals suffering from insulin dependent diabetes mellitus (IDDM) are at increased risk for the development of periodontal disease.113 There is, however, a small group of other researchers who fail to find this increased risk.14"19 Recent research may explain these different findings based upon the size of the population studied as well as the role of other complicating factors such as smoking on the prevalence of periodontal disease in diabetic smokers versus diabetic nonsmokers.20 The microbiologie findings of diseased pockets in diabetics have been less extensively studied. Mashimo et al.21 examined 14 IDDM patients aged 13 to 27 years. The population consisted of 9 Periodontitis patients, 3 gingivitis patients, and 2 periodontally-healthy patients. The diseased patients, whose condition resembled juvenile Periodontitis, were found to harbor a flora with high levels of Actinobacillus actinomycetemcomitans, Capnocytophaga spp., and "Anerobic vibrios." Sastrowijoto et al.22 examined diabetic adults grouped by virtue of their glycosylated hemoglobin values. They examined 44 sites in 22 patients who were further divided into a near normal (Hb Ale 9.9%) group. They re* Forsyth Dental Center and Tufts School of Dental MA. Tufts School of Dental Medicine, Boston, MA. *
Medicine, Boston,
ported that the organisms A. actinomycetemcomitans, B. gingivalis, and B. intermedius were recovered at proportionally higher levels from the diseased versus control sites. The purpose of this investigation was to examine a group of suspected periodontal pathogens associated with healthy and diseased periodontal pockets in a group of IDDM patients with periodontal disease. MATERIALS AND METHODS
Subjects
Fifteen Caucasian patients (9 males, 6 females) with a mean age 34.6 years ( + /- 6.4; range 23 to 50 years) were selected for study. The mean duration of insulin dependent diabetes was 20.6 ( + /— 7.6) years. Daily insulin use was 41.0 ( + /- 18.6) units. Medical histories indicated that the mean levels of glycosalated hemoglobin was 10.4% (+/ 2.0) for 12 of the 15. Blood glycosalated hemoglobin levels were unavailable on the remaining 3 patients. Two of the 3 indicated that they were poorly controlled and the remaining patient was unaware of the level at the time of examination. These patients were a subset of a large ongoing epidemiological study and were eligible for microbiology sampling based upon having at least one pocket depth >5 mm and attachment loss of >2 mm (Table 1). A roster of patients who qualified for the microbiology study was generated from the epidemiology study. All patients on the list were contacted by mail and the first 15 who —
Volume 63 Number 4
MANDELE, DIRIENZO, KENT, JOSHIPURA, HABER
Table 1. Clinical Characteristics of Sampled Sites in 15 IDDM Patients with Periodontal Disease
(ii 15) =
=
Pocket depth* Attachment level*
Bleeding on probingt Rednesst
*Mean in
mm
5.8 (0.8) 2.8 (0.9) 100 93
15 13
(standard deviation).
tSites displaying the characteristic.
responded to the mailing and could make pointment were included in the study.
a
scheduled ap-
Clinical Indices The dichotomous indices of bleeding on probing (BOP) and gingival redness (GR) were recorded for all sampled sites.23 Pocket depths were measured from the gingival margin to the probe tip. The measurements were made to the nearest 0.5 mm by a single examiner (R.L.M.) using a Michigan 0 probe. Attachment level was derived from the pocket depth by a second measure made from the cemento-enamel junction (CEJ) to the gingival margin. These measurements were then subtracted to arrive at the attachment level or loss measure.23
Sampling and Media Subgingival plaque samples were taken from the base of the periodontal pockets with a sterile Morse (00) sealer inserted into the depth of the pocket. The tip was aseptically transferred to prereduced one-quarter strength Ringer's solution, sonicated for 5 seconds* at settings previously determined to give the highest recoverable bacterial counts24 and then 10-fold dilutions were made under an 02-free atmosphere.25 0.1 ml samples were plated on duplicate plates of selective and elective media to enumerate the following organisms: 1) Actinobacillus actinomycetemcomitans,26 a modified media consisting of 4 ug/ml malachite green (plain base form) and 128 ug/ml bacitracin in 5% sheep's blood Trypticase soy agar;§ 2) Eikenella corrodens;27 3) Streptococcus sanguis I, 5. oralis (formerly S. sanguis II), and S. intermedius, Mitis-Salivaris agar11 representative colonies were isolated and identified;28 4) Fusobacterium nucleatum;29 5) Capnocytophaga spp.;30 6) Bacteroides spp.31 representative colonies were transferred and further differentiated based upon long-wave UV light,32 MUG test for lactose utilization,33 and CAMM test,34 to allow rapid presumptive identification of P. gingivalis, P. intermedia, and P. melaninogenica spp. (includes P. loeschi and P. denticola (formerly B. loeschi, B. denticola); 7) B. gracilus, Campylobacter concisus, C. rectus, and C. curvus (formerly W. curva), were isolated on a newly developed se* 5 1
Heat System Ultrasonic, Plainview, NY. TSAB, BBL Cockeysville, MD. Difco Laboratories, Detroit, MI.
Table 2. Mean Counts* of Bacteria Isolated from 15 Diabetics With Disease
Diseased
Healthy (n 15) 2.8 (0.4) 0.6 (0.5)
275
Bacteria A. actinomycetemcomitans P. gingivalis P. intermedia P. melaninogenica spp. B. gracilis Capnocytophaga spp. C. concisus C. curvus C. rectus E. corrodens F. nucleatum S. intermedius S. sanguis I S. oralis Total counts
Diseased
4.7E2 2.5E3 8.7E5 1.3E4 2.0E6 5.0E5 0 9.7E3 4.3E6 1.4E6 4.4E5 1.3E5 2.6E5 1.9E5 7.3E7
Healthy (2.0E3)
(4.6E2) (1.8E3) (3.4E5)t (7.7E3)í (1.3 6) (3.2E5)
2.7E3 0 8.0E4
5.0E3 5.8E3 1.2E4 2.8E3 4.8E3 1.8E4 2.3E4 2.9E4 5.1E4 4.0E4 6.8E4 9.6E6
(8.6E3) (3.2E6)t (1.2E6)t (2.3 5) (6.1E4) (1.6E5) (1.2E5) (3.4E7)t
(4.2E4) (5.0E3) (2.8E3) (9.2E3) (2.0E3) (4.0E3) (8.2E3) (2.0E4) (1.1E4) (4.0E4) (1.7E4) (4.4E4) (6.0E6)
'Counts (standard error) expressed in scientific notation. tSignificantly elevated by Wilcoxon signed rank test P
