Acid production by Actinomyces viscosus of root surface caries and non-caries origin during glycogen synthesis and degradation at different pH levels
K. Komiyama^ and R. L. Khandelwal' Departments of 'Oral Biology. 'Biochemistry. University of Saskatchewan. Saskatoon. Canada
Komiy;mia K. Kli;indclwal RL: Acid pioduclion by Actinotnyccs visco.'iti.s of root surface caries and non-caries origin during glycogen synthesis and degradation at different pll levels. .1 Oral Pathol Med 1992; 21: 343 7. Aclituwiycc.s viscosus strains, freshly isolated from root surface caries lesions and intact root surfaces, were studied for their glyeogen synthetic and degradative activities at pH 4.5. 5.0. and 7.0 in a pH-stat. At all three pH levels, root caries origin of .'(. V/.VCO.VK.V synthesized up to three times as much glycogen compared to non-root caries origin. Since root caries origin of .(. vl.sco.sit.s strains initially synthesized large amounts of glycogen. a longer period of time was required to deplete this polymer, resulting in an extended period of acid produetion. even at pH 4.5 and pH 5.0. This study suggests that the ability of A. iv.vro.w.v of root caries origin to synthesize large quantities of glycogen and subsequently degrade this stored polymer slowly with acid production, at acidic pH levels, may play an important role in the root caries process.
In the early studies of mierobiology of root surface caries. Actiiwimci's vi.icosus species was strongly implicated as an etiologic agent in human root surface caries (1 3). Since wide species variation in root surface plaque has been observed (4 13). our current knowledge of the relationship between initiation of root surface caries and a specific etiologic agent is limited. Thus, the importance of --(. i'/.vcr>.v(/.v. as a causative agent in the etiology ol root surface caries, has not been universally accepted. It has been known that glycogen metabolism by plaque bacteria contributes to their cariogenic potential (14. 15). In this respect, a great deal of attention has been focussed on the role of glycogen metabolistn by Streptococcus nmtatis in the pathogenicity of dental caries (16-21). Although, very little is known of glycogen metabolism by A. vi.sco.stts in relation lo root surface caries, we have recently observed that .4. viscosus strains, freshly i.solated from root caries lesions, synthesized much higher amounts of glycogen with production of acid, compared with the strains isolated from non-caries siles. al pH 7.0 (22). In
a subsequent study, we confirmed that A. viscosu.s of root caries origin consistently synthesized much higher amounts of glycogen with acid production, than those of non-caries origin, at different pH levels ranging tVom pH 5.0 to 7.0 (23). An important observation in this study was that the time required for glycogen degradation was much longer for the strain originating from root caries than for the strain originating from non-caries site, especially at pH 5.0. This resulted in an extended period of acid production for the strain of root caries origin. This study suggests that A. visco.tus originating from root caries is aciduric and that the glycogen metabolism by A. r/.vco.vi/.v may play an important role in the pathogenicity of root surface caries. In this study, however, we studied only one strain each of .4. vi.Kcostts of root caries and non-caries origin. By using a larger number ot strain, a more accurate representative ecologic survey dellning the role of glycogen metabolism by A. i7.vr(w;/.v in the root surface caries process may be established. Therefore, the present study examined five strains each of root
Key words: aciduricity: Actinomyces viscosus; caries, root: glycogen Kunio Komiyama. Dept. of Oral Biology. College of Dentistry. University of Saskatchewan. Saskatoon. Saskatchewan. Canada S7N OWO Accepted for publication February 4. 1992.
caries and non-caries origin of .4. vi.sco.Ku.< 0.025) by student t test.
Glycogen metabolism by A. viscosus at acidic pH (A) CARIES GROUP
for an extend period of time, and they still contained large amounts of glycogen after 90 min of incubation, as seen Fig. 1 A. In contrast, A. viscosus of noncaries origin virtually depleted their minimal glycogen stores during the first 5 to 10 min. after which the rates of glycogen degradation significantly slowed down at the acidic pH levels, as seen in Fig. 1B. The rates of glycogen degr. (.'ation by the root caries group were ^ ' to 2.5 folds faster than those of non-caries group (Table 3). This may be due to higher amounts of glycogen initially synthesized by A. vi.scosus of caries origin. The rates of total acid formation during glycogen degradation were 3 to 4 folds faster for the root caries group than for the non-caries group at all three pH levels (Table 3). At acidic pH levels, both the rates of glycogen degradation and acid productions were still enhanced by the root caries origin compared to the non-caries origin (Table 3).
70 -
60-
pH7.0 pH5.0 pH4.5
50-
Q O)
a O
30 -
20-
O U
z UJ o
8
30
30-
60
90
I 120
(B) NON-CARIES GROUP
20-
10-
30
60
150
INCUBATION TIME (min.) rates of glycogen synthesis and acid production were still enhanced by A. viscosus of root caries origin compared to non-caries origin (Table 2). Gh'cogen degradation and total acid production - Both A. viscosus of root caries and non-caries groups rapidly uti-
Fig. I. A, effect of pH levels on glucose incorporated into glycogen (0-60 min) and glycogen degradation (60-150 min). Each point represents mean ( ± SD) of 5 strain of A. viscosus originating from root caries lesion. B, effect of pH levels on glucose incorporated into glycogen (0-60 min) and glycogen degradation (60 150 min). Each point represents means ( ± SD) of 5 strains of A. vi.'ico.ius originating from non-root caries sites.
lized glycogen, albeit much faster in the first 5 to 10 min at all 3 pH levels (Figs. lA and B). However, because of higher amounts of glycogen initially synthesized by .4. viscosus of root caries origin, a longer period of time was required to degrade this stored polymer
Table 2. Rates of glycogen synthesis and total acid formation during glycogen synthesis by A. viscosus of root caries and non-caries group Glycogen (|jmol glucose incorporated into glycogen/mg D N A / h ± S D ) pH levels pH 7.0 pH 5.0 pH 4.5
Non-caries group Root caries group 17.6 ±3.9 10.1 ±2.4 8.6±2.1
345
61.2 ±15.7* 37.2±8.1* 27.5±4.8*
Total acid total acid/mg D N A / h ± S D ) Non-caries group Root caries group 311.3±43.7 160.6 ±33.2 68.3 ±20.7
823.2 ±70.8* 431.3 ±55.6* 197.6±41.4*
SD: Standard Deviation. All values for A. viscosus of root caries origin were significantly higher than for those of nonearies origin (* / ' < 0.001) by student t test.
Discussion Although A. viscosus species has been suggested as one of the etiologic agents in root surface caries, the role of glycogen metabolism by this organism has not been critically evaluated. SAXTON (28), using electron microscope autoradioraphy, reported that the proportion of glycogen synthesizing organisms was relatively low (16%) at the saliva interface but increased to 52% in the plaque adjacent to the tooth, and that the only organisms which synthesized glycogen within the deeper regions of the plaque at very low glucose concentration were filamentous forms. He suggested that an acidic environment would be prolonged at the tooth surface through the catabolism of the intracellular polysaccharides. and that the filamentous forms, although numerically few, probably contributed significantly to this environment during the absence of exogenous sugars. Recently. HAMILTON & ELLWCX)D (29) reported that a strain of A. viscosus growing in continuous culture synthesized glycogen. and this glycogen synthesis followed the pathway which has been observed in other bacteria. In the present study. A. viscosus strains isolated plaques over root surface caries lesions, not only synthesized large amounts of glycogen at acidic pH levels of 4.5 and 5.0. but also degraded this stored polymer slowly for an extended period of time with the produc-
346
KOMIYAMA AND KHANDELWAL
Table 3. Rates of glycogen degradation and total acid formation during glycogen degradation by A. vi.scosus of root caries and non-caries group Glycogen glycogen/mg DNA/h + SD) pH levels pH 7.0 pH 5.0 pH 4.5
Non-caries group Root caries group
7.9 ±2.7 5.4±1.8 4.0+1.4
16.8 + 3.2** I2.3±2.9*** 9.8 + 2.5***
Total acid total acid/mg DNA/h + SD) Non-caries group Root caries group 103.5±35.3 43.6 ±27.6 26.3 ±8.8
310.1 ±49.8* I60.7±38.8** 104.6 + 31.4**
SD: Standard Deviation. All values for A. vi.scosus of root caries origin were significantly higher than for those of noncaries origin (* /'
K. Komiyama^ and R. L. Khandelwal' Departments of 'Oral Biology. 'Biochemistry. University of Saskatchewan. Saskatoon. Canada
Komiy;mia K. Kli;indclwal RL: Acid pioduclion by Actinotnyccs visco.'iti.s of root surface caries and non-caries origin during glycogen synthesis and degradation at different pll levels. .1 Oral Pathol Med 1992; 21: 343 7. Aclituwiycc.s viscosus strains, freshly isolated from root surface caries lesions and intact root surfaces, were studied for their glyeogen synthetic and degradative activities at pH 4.5. 5.0. and 7.0 in a pH-stat. At all three pH levels, root caries origin of .'(. V/.VCO.VK.V synthesized up to three times as much glycogen compared to non-root caries origin. Since root caries origin of .(. vl.sco.sit.s strains initially synthesized large amounts of glycogen. a longer period of time was required to deplete this polymer, resulting in an extended period of acid produetion. even at pH 4.5 and pH 5.0. This study suggests that the ability of A. iv.vro.w.v of root caries origin to synthesize large quantities of glycogen and subsequently degrade this stored polymer slowly with acid production, at acidic pH levels, may play an important role in the root caries process.
In the early studies of mierobiology of root surface caries. Actiiwimci's vi.icosus species was strongly implicated as an etiologic agent in human root surface caries (1 3). Since wide species variation in root surface plaque has been observed (4 13). our current knowledge of the relationship between initiation of root surface caries and a specific etiologic agent is limited. Thus, the importance of --(. i'/.vcr>.v(/.v. as a causative agent in the etiology ol root surface caries, has not been universally accepted. It has been known that glycogen metabolism by plaque bacteria contributes to their cariogenic potential (14. 15). In this respect, a great deal of attention has been focussed on the role of glycogen metabolistn by Streptococcus nmtatis in the pathogenicity of dental caries (16-21). Although, very little is known of glycogen metabolism by A. vi.sco.stts in relation lo root surface caries, we have recently observed that .4. viscosus strains, freshly i.solated from root caries lesions, synthesized much higher amounts of glycogen with production of acid, compared with the strains isolated from non-caries siles. al pH 7.0 (22). In
a subsequent study, we confirmed that A. viscosu.s of root caries origin consistently synthesized much higher amounts of glycogen with acid production, than those of non-caries origin, at different pH levels ranging tVom pH 5.0 to 7.0 (23). An important observation in this study was that the time required for glycogen degradation was much longer for the strain originating from root caries than for the strain originating from non-caries site, especially at pH 5.0. This resulted in an extended period of acid production for the strain of root caries origin. This study suggests that A. visco.tus originating from root caries is aciduric and that the glycogen metabolism by A. r/.vco.vi/.v may play an important role in the pathogenicity of root surface caries. In this study, however, we studied only one strain each of .4. vi.Kcostts of root caries and non-caries origin. By using a larger number ot strain, a more accurate representative ecologic survey dellning the role of glycogen metabolism by A. i7.vr(w;/.v in the root surface caries process may be established. Therefore, the present study examined five strains each of root
Key words: aciduricity: Actinomyces viscosus; caries, root: glycogen Kunio Komiyama. Dept. of Oral Biology. College of Dentistry. University of Saskatchewan. Saskatoon. Saskatchewan. Canada S7N OWO Accepted for publication February 4. 1992.
caries and non-caries origin of .4. vi.sco.Ku.< 0.025) by student t test.
Glycogen metabolism by A. viscosus at acidic pH (A) CARIES GROUP
for an extend period of time, and they still contained large amounts of glycogen after 90 min of incubation, as seen Fig. 1 A. In contrast, A. viscosus of noncaries origin virtually depleted their minimal glycogen stores during the first 5 to 10 min. after which the rates of glycogen degradation significantly slowed down at the acidic pH levels, as seen in Fig. 1B. The rates of glycogen degr. (.'ation by the root caries group were ^ ' to 2.5 folds faster than those of non-caries group (Table 3). This may be due to higher amounts of glycogen initially synthesized by A. vi.scosus of caries origin. The rates of total acid formation during glycogen degradation were 3 to 4 folds faster for the root caries group than for the non-caries group at all three pH levels (Table 3). At acidic pH levels, both the rates of glycogen degradation and acid productions were still enhanced by the root caries origin compared to the non-caries origin (Table 3).
70 -
60-
pH7.0 pH5.0 pH4.5
50-
Q O)
a O
30 -
20-
O U
z UJ o
8
30
30-
60
90
I 120
(B) NON-CARIES GROUP
20-
10-
30
60
150
INCUBATION TIME (min.) rates of glycogen synthesis and acid production were still enhanced by A. viscosus of root caries origin compared to non-caries origin (Table 2). Gh'cogen degradation and total acid production - Both A. viscosus of root caries and non-caries groups rapidly uti-
Fig. I. A, effect of pH levels on glucose incorporated into glycogen (0-60 min) and glycogen degradation (60-150 min). Each point represents mean ( ± SD) of 5 strain of A. viscosus originating from root caries lesion. B, effect of pH levels on glucose incorporated into glycogen (0-60 min) and glycogen degradation (60 150 min). Each point represents means ( ± SD) of 5 strains of A. vi.'ico.ius originating from non-root caries sites.
lized glycogen, albeit much faster in the first 5 to 10 min at all 3 pH levels (Figs. lA and B). However, because of higher amounts of glycogen initially synthesized by .4. viscosus of root caries origin, a longer period of time was required to degrade this stored polymer
Table 2. Rates of glycogen synthesis and total acid formation during glycogen synthesis by A. viscosus of root caries and non-caries group Glycogen (|jmol glucose incorporated into glycogen/mg D N A / h ± S D ) pH levels pH 7.0 pH 5.0 pH 4.5
Non-caries group Root caries group 17.6 ±3.9 10.1 ±2.4 8.6±2.1
345
61.2 ±15.7* 37.2±8.1* 27.5±4.8*
Total acid total acid/mg D N A / h ± S D ) Non-caries group Root caries group 311.3±43.7 160.6 ±33.2 68.3 ±20.7
823.2 ±70.8* 431.3 ±55.6* 197.6±41.4*
SD: Standard Deviation. All values for A. viscosus of root caries origin were significantly higher than for those of nonearies origin (* / ' < 0.001) by student t test.
Discussion Although A. viscosus species has been suggested as one of the etiologic agents in root surface caries, the role of glycogen metabolism by this organism has not been critically evaluated. SAXTON (28), using electron microscope autoradioraphy, reported that the proportion of glycogen synthesizing organisms was relatively low (16%) at the saliva interface but increased to 52% in the plaque adjacent to the tooth, and that the only organisms which synthesized glycogen within the deeper regions of the plaque at very low glucose concentration were filamentous forms. He suggested that an acidic environment would be prolonged at the tooth surface through the catabolism of the intracellular polysaccharides. and that the filamentous forms, although numerically few, probably contributed significantly to this environment during the absence of exogenous sugars. Recently. HAMILTON & ELLWCX)D (29) reported that a strain of A. viscosus growing in continuous culture synthesized glycogen. and this glycogen synthesis followed the pathway which has been observed in other bacteria. In the present study. A. viscosus strains isolated plaques over root surface caries lesions, not only synthesized large amounts of glycogen at acidic pH levels of 4.5 and 5.0. but also degraded this stored polymer slowly for an extended period of time with the produc-
346
KOMIYAMA AND KHANDELWAL
Table 3. Rates of glycogen degradation and total acid formation during glycogen degradation by A. vi.scosus of root caries and non-caries group Glycogen glycogen/mg DNA/h + SD) pH levels pH 7.0 pH 5.0 pH 4.5
Non-caries group Root caries group
7.9 ±2.7 5.4±1.8 4.0+1.4
16.8 + 3.2** I2.3±2.9*** 9.8 + 2.5***
Total acid total acid/mg DNA/h + SD) Non-caries group Root caries group 103.5±35.3 43.6 ±27.6 26.3 ±8.8
310.1 ±49.8* I60.7±38.8** 104.6 + 31.4**
SD: Standard Deviation. All values for A. vi.scosus of root caries origin were significantly higher than for those of noncaries origin (* /'
