Basic Science Caries Res 1992;26:124-131
T. Ooshimaa T. Yoshidaa T. Hashidab A. Izumitania S. Sobuea S. Hamadac
Effects of Hyposalivation on the Oral Microflora of Rats Fed Sucrose or Wheat Flour Diets
Departments of Pcdodontics, Oral and Maxillofacial Radiology and Oral Microbiology, Osaka University Faculty of Dentistry, Yamadaoka. Suita-Osaka, Japan
Key Words
Abstract
Dental caries Experimental animal Hyposalivation Oral microflora Sucrose Wheat flour
The effects of hyposalivation on the induction of dental caries and on the com position of the oral microflora were examined in specific pathogen-free Sprague-Dawley rats fed either a sucrose or a wheat flour diet with or without inoculation of Streptococcus mutans. Significant dental caries was induced in hyposalivated rats fed diet 2000 containing 56% sucrose, irrespective of in fection by S. mutans. Diets containing 56% wheat flour did not induce dental caries in either hyposalivated or sham-operated rats, irrespective of infection by 5. mutans. Bacteriological examinations at the end of the experiment dem onstrated that the total numbers of lactobacilli and staphylococci increased in hyposalivated rats irrespective of the diet given, while the inoculated stain of S. mutans decreased significantly in hyposalivated rats. These findings suggest that some acidogenic microorganisms such as lactobacilli and staphylococci that can utilize sucrose or glucose but not wheat flour may also promote dental caries in hyposalivated rats.
Received: April 24.1991 Accepted after revision: September 12.1991
several animal species have been found to develop dental caries when infected with mutans streptococci isolated from human caries lesions and fed a high-sucrose diet, while other microorganisms could induce few or lower numbers of caries lesions in experimental animals [Ha mada et al., 1978; Ooshima et al„ 1981]. Collectively, these findings demonstrated that mutans streptococci are the most important microorganisms responsible for dental caries in normal experimental animals and man.
Takashi Ooshima Department of Pcdodontics, Osaka University Faculty o f Dentistry 1-8 Yamadaoka. Suita-Osaka 565 (Japan)
© 1992 S. Karger AG. Basel 0008-6568/92/0262-0124 S 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 1:49:23 AM
Dental caries is an infectious and transmissible disease although its etiology is multifactorial in nature. Mutans streptococci have been isolated from almost all active car ies lesions in humans [Hamada ct al„ 1976], and a signif icant correlation has been found between the number of mutans streptococci in saliva and caries prevalence [Zickert et al., 1983]. Furthermore, the development of dental caries is frequently preceded by colonization with high lev els of mutans streptococci [Ikeda et al., 1973], In addition.
Materials and Methods Bacteriological Examination and Cultivation S. mutans MT8148R (serotype c), which was made resistant to streptomycin, was used in some animal experiments [Ooshima ct al., 1983]. The organisms, grown in brain heart infusion broth (Difco Lab oratories, Detroit, Mich.) for 18 h at 37 °C, were collected by centrifu gation, washed three times with sterile saline, and resuspended to give a concentration of 2 x 10lu colony forming units (CFU) per milli liter of sterile saline for inoculation into the oral cavity of rats. During the experiment, oral swabs were taken from individual animals at weekly intervals which permitted semiquantitative micro biological comparisons (fig. 1). The swab was immediately immersed in 1 ml of sterile saline, thoroughly agitated, and ten-fold diluted with sterile saline. The dilutions were streaked on Mitis-salivarius (MS) agar (Difco) containing 500 ,ug of streptomycin per milliliter for the isolation of S. mutans MT8148R, MS agar plates for enumerating to tal streptococci, Trypticase-soy agar plates supplemented with 5% (v/v) sheep blood (Nippon Becton Dickinson Co., Tokyo. Japan) for total cultivable bacteria, Staphylococcus medium 111) (Difco) for total staphylococci, and Rogosa SL agar (Difco) for total lactobacilli. These plates were incubated for 2 days in an atmosphere of 95% N, and 5% CO, at 37 °C. followed by incubation for 1 day in air at 37 °C. The recoveries of the inoculated strains, total streptococci, total mi croorganisms, total staphylococci and total lactobacilli were eval uated by the number of colonies on the agar plates (CFU).
Surgical operation
Kill
0 2 55 |— |------------------------/ / -------1 21 2324 28 30 85 Rat age. days |— 1-|--------- 1— |-----------------------------/ / -------1 Administration of antibiotics
Experimental time
11*
Infection with S. mutans (experiment II alone)
UÜJ
Recovery of organisms and measurement of body weight
1_A
Diet
CE-2
♦
^Once a week ^ / // Test diet
♦
11
Fig. 1. Experimental design for examining the changes in the oral microflora in hyposalivatcd rats. Oral swabs were taken from individ ual animals at weekly intervals and the mandibles were removed ascptically at the end of the experiment for bacteriological examina tions. In experiment II, rats were inoculated with 5. mutans MT8148R to rats from day 24 to 28 of age, giving a total inoculum size of l.Ox 1010CFU per rat.
At the end of the experiments, the mandibles were removed aseptically. The jaws were immersed in 4.5 ml of sterile saline and ultrasonicated for 20 s at 20 W at maximum amplitude using a sonic oscil lator (model DR-20P, Torny Seiko Co., Tokyo) to disperse the dental plaque on the tooth surfaces. The suspensions were decimal diluted with sterile saline and streaked on agar plates as described above. Surgical Treatment for Hyposalivation Rats (30 days of age) were anesthetized by intraperitoneal in jection of pentobarbital solution (4 mg/100 g of body weight). The pa rotid ducts of rats in surgical groups were ligated and their subman dibular and sublingual glands were removed following dissection through a midline incision [Bowen ct al., 1988], The sham-operated controls had their major salivary glands exposed and (he wounds were closed by suture without impairing the salivary glands. Caries Induction in Rats Animal experiments for caries induction were carried out twice, using SPF Sprague-Dawley rats (male, 21 days of age; CLEA Japan, Osaka, Japan). The rats were derived from germ-free ancestors and mutans streptococci could not he detected in their indigenous oral microflora. All rats were first treated with tetracycline (4 mg/g pow dered diet CE-2; CLEA Japan) and penicillin G (4,000 U/ml of drink ing water) for 2 days [Ooshima ct al., 1990] (fig. 1). The rats in experi ment I received no bacterial inoculation, while the rats for experi ment II were infected with S. mutans MT8148R. This infection (2.0 x 10y CFU/rat) was repeated on 5 consecutive days (from day 24 to 28 of age). At 24 days of age and thereafter, rats were given ad lib itum the caries-inducing diet 2000 or the modified diet containing 56% (w/w) glucose or 56% (w/w) wheat flour in place of 56% (w/w) sucrose in diet 2000. The rats were randomly divided into six groups in experiment I, and four groups in experiment 11(15 rats per group). The diets given arc shown in tables 1-4. Groups A, C and E rats were sham-operated
115
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 1:49:23 AM
[t has been reported that hyposalivation as a result of salivary gland diseases, side effects of certain drugs, or ir radiation of salivary glands enhances the susceptibility to dental caries in humans and experimental animals [Dreizen et al., 1977; Edgar et al., 1981; Bowen ct al., 1986; Handclman et al., 1986; Ooshima et al., 1990,1991], Caries ac tivity is enhanced by the emergence of a cariogenic micro flora due to the diminution of salivary flow, paralleled by significant decreases in noncariogenic members of oral bacterial species [Llory et al., 1971,1972; Brown et al., 1975, 1976]. Our previous studies [Ooshima et al., 1990, 1991] have demonstrated that hyposalivation induced by either irradiation or surgical removal of salivary glands causes severe dental caries in specific pathogen-free (SPF) rats, irrespective of infection with oral streptococci including Streptococcus mutans. In addition, bacteriological exami nation demonstrated that the total number of lactobacilli increased significantly at the expense of the inoculated 5. mutans MT8148R. In our experimental system, wheat flour was used as a noncariogenic dietary component. Wheat flour had always induced only baseline levels of plaque formation and dental caries in many experiments in our laboratories [Ooshima et al., 1988, 1991], The pur pose of the present study is to examine the effect of hypo salivation on the induction of dental caries and on the composition of the oral microflora in rats fed either a su crose or a wheat flour diet.
Table 1. Caries induction in hyposalivated rats not infected with S. mulans and fed a diet containing 56% (w/w) carbohydrates Group
A B C D E F
Rats n
Carbohydrate in diet
Hyposalivation
Plaque index (mean ±SE )
Caries score (mean ±SE)
15 15 15 15 15 15
wheat flour wheat flour sucrose sucrose glucose glucose
—
0.28 ± 0.02 -i 0.55 ± 0.05 J 0.31 ±0.03 0.65 ± 0.04 J 0.40 ±0.03-i 0.75 ±0.08-*
4.6 ±0.1 0± 0 0±0 6.2 ±0.6 7.0 ± 0 .5 -i 0±0 41.9 ± 3.0 J 5.1 ± 0 .8 J 7 .5 ± 0 .6 > t , ± > * . 2.6 ± 1.1 J 37.9 ± 4.7 J
+ -
+ +
total
buccal + lingual
Salivary flow rate“ (mean ±SE ) pi/min
Weight gain (mean ±SE) g
218 ± 12 NDb 169 ± 7 ND 185 ± 11 ND
331.7 ±19.4-, 141.5 ±9.1 -* 323.2 ± 14.4-1 250.4 ±9.4 J 3 4 7 .6 i6 .6 -. > 248.8 ± 7.6 J
Statistical analyses were carried out between groups A and B, between groups C and D. and between groups E and F (***p < O.(X)l). a Flow rates were calculated by measuring the time between the excretion of l()() pi of whole saliva and that of 300 pi after the injection of pilocarpine. h Excretion of saliva was not detected 10 min after the injection of pilocarpine.
Statistical Analyses The caries scores and plaque indices from each group of rats were reduced by computing the means and standard errors. The bacteri ological counts were logl0-transformed prior to statistical analyses in order for the variances to be within the same order. For statistical analysis between each experimental group, the Mann-Whitney U test was used.
Results
Results of caries score and plaque index in experiment I in which rats were not infected with S. mulans are shown in table 1. Only low levels of dental caries were induced in
126
sham-operated rats fed sucrose, glucose or wheat flour diet, or in hyposalivated rats fed wheat flour diet. Howev er, hyposalivation due to surgical removal of salivary glands significantly enhanced the cariogcnic activity in rats fed a sucrose or glucose diet. Caries scores of the hy posalivated rats in groups D and F were significantly higher than those of sham-operated groups C and E, re spectively (p
T. Ooshimaa T. Yoshidaa T. Hashidab A. Izumitania S. Sobuea S. Hamadac
Effects of Hyposalivation on the Oral Microflora of Rats Fed Sucrose or Wheat Flour Diets
Departments of Pcdodontics, Oral and Maxillofacial Radiology and Oral Microbiology, Osaka University Faculty of Dentistry, Yamadaoka. Suita-Osaka, Japan
Key Words
Abstract
Dental caries Experimental animal Hyposalivation Oral microflora Sucrose Wheat flour
The effects of hyposalivation on the induction of dental caries and on the com position of the oral microflora were examined in specific pathogen-free Sprague-Dawley rats fed either a sucrose or a wheat flour diet with or without inoculation of Streptococcus mutans. Significant dental caries was induced in hyposalivated rats fed diet 2000 containing 56% sucrose, irrespective of in fection by S. mutans. Diets containing 56% wheat flour did not induce dental caries in either hyposalivated or sham-operated rats, irrespective of infection by 5. mutans. Bacteriological examinations at the end of the experiment dem onstrated that the total numbers of lactobacilli and staphylococci increased in hyposalivated rats irrespective of the diet given, while the inoculated stain of S. mutans decreased significantly in hyposalivated rats. These findings suggest that some acidogenic microorganisms such as lactobacilli and staphylococci that can utilize sucrose or glucose but not wheat flour may also promote dental caries in hyposalivated rats.
Received: April 24.1991 Accepted after revision: September 12.1991
several animal species have been found to develop dental caries when infected with mutans streptococci isolated from human caries lesions and fed a high-sucrose diet, while other microorganisms could induce few or lower numbers of caries lesions in experimental animals [Ha mada et al., 1978; Ooshima et al„ 1981]. Collectively, these findings demonstrated that mutans streptococci are the most important microorganisms responsible for dental caries in normal experimental animals and man.
Takashi Ooshima Department of Pcdodontics, Osaka University Faculty o f Dentistry 1-8 Yamadaoka. Suita-Osaka 565 (Japan)
© 1992 S. Karger AG. Basel 0008-6568/92/0262-0124 S 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 1:49:23 AM
Dental caries is an infectious and transmissible disease although its etiology is multifactorial in nature. Mutans streptococci have been isolated from almost all active car ies lesions in humans [Hamada ct al„ 1976], and a signif icant correlation has been found between the number of mutans streptococci in saliva and caries prevalence [Zickert et al., 1983]. Furthermore, the development of dental caries is frequently preceded by colonization with high lev els of mutans streptococci [Ikeda et al., 1973], In addition.
Materials and Methods Bacteriological Examination and Cultivation S. mutans MT8148R (serotype c), which was made resistant to streptomycin, was used in some animal experiments [Ooshima ct al., 1983]. The organisms, grown in brain heart infusion broth (Difco Lab oratories, Detroit, Mich.) for 18 h at 37 °C, were collected by centrifu gation, washed three times with sterile saline, and resuspended to give a concentration of 2 x 10lu colony forming units (CFU) per milli liter of sterile saline for inoculation into the oral cavity of rats. During the experiment, oral swabs were taken from individual animals at weekly intervals which permitted semiquantitative micro biological comparisons (fig. 1). The swab was immediately immersed in 1 ml of sterile saline, thoroughly agitated, and ten-fold diluted with sterile saline. The dilutions were streaked on Mitis-salivarius (MS) agar (Difco) containing 500 ,ug of streptomycin per milliliter for the isolation of S. mutans MT8148R, MS agar plates for enumerating to tal streptococci, Trypticase-soy agar plates supplemented with 5% (v/v) sheep blood (Nippon Becton Dickinson Co., Tokyo. Japan) for total cultivable bacteria, Staphylococcus medium 111) (Difco) for total staphylococci, and Rogosa SL agar (Difco) for total lactobacilli. These plates were incubated for 2 days in an atmosphere of 95% N, and 5% CO, at 37 °C. followed by incubation for 1 day in air at 37 °C. The recoveries of the inoculated strains, total streptococci, total mi croorganisms, total staphylococci and total lactobacilli were eval uated by the number of colonies on the agar plates (CFU).
Surgical operation
Kill
0 2 55 |— |------------------------/ / -------1 21 2324 28 30 85 Rat age. days |— 1-|--------- 1— |-----------------------------/ / -------1 Administration of antibiotics
Experimental time
11*
Infection with S. mutans (experiment II alone)
UÜJ
Recovery of organisms and measurement of body weight
1_A
Diet
CE-2
♦
^Once a week ^ / // Test diet
♦
11
Fig. 1. Experimental design for examining the changes in the oral microflora in hyposalivatcd rats. Oral swabs were taken from individ ual animals at weekly intervals and the mandibles were removed ascptically at the end of the experiment for bacteriological examina tions. In experiment II, rats were inoculated with 5. mutans MT8148R to rats from day 24 to 28 of age, giving a total inoculum size of l.Ox 1010CFU per rat.
At the end of the experiments, the mandibles were removed aseptically. The jaws were immersed in 4.5 ml of sterile saline and ultrasonicated for 20 s at 20 W at maximum amplitude using a sonic oscil lator (model DR-20P, Torny Seiko Co., Tokyo) to disperse the dental plaque on the tooth surfaces. The suspensions were decimal diluted with sterile saline and streaked on agar plates as described above. Surgical Treatment for Hyposalivation Rats (30 days of age) were anesthetized by intraperitoneal in jection of pentobarbital solution (4 mg/100 g of body weight). The pa rotid ducts of rats in surgical groups were ligated and their subman dibular and sublingual glands were removed following dissection through a midline incision [Bowen ct al., 1988], The sham-operated controls had their major salivary glands exposed and (he wounds were closed by suture without impairing the salivary glands. Caries Induction in Rats Animal experiments for caries induction were carried out twice, using SPF Sprague-Dawley rats (male, 21 days of age; CLEA Japan, Osaka, Japan). The rats were derived from germ-free ancestors and mutans streptococci could not he detected in their indigenous oral microflora. All rats were first treated with tetracycline (4 mg/g pow dered diet CE-2; CLEA Japan) and penicillin G (4,000 U/ml of drink ing water) for 2 days [Ooshima ct al., 1990] (fig. 1). The rats in experi ment I received no bacterial inoculation, while the rats for experi ment II were infected with S. mutans MT8148R. This infection (2.0 x 10y CFU/rat) was repeated on 5 consecutive days (from day 24 to 28 of age). At 24 days of age and thereafter, rats were given ad lib itum the caries-inducing diet 2000 or the modified diet containing 56% (w/w) glucose or 56% (w/w) wheat flour in place of 56% (w/w) sucrose in diet 2000. The rats were randomly divided into six groups in experiment I, and four groups in experiment 11(15 rats per group). The diets given arc shown in tables 1-4. Groups A, C and E rats were sham-operated
115
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 1:49:23 AM
[t has been reported that hyposalivation as a result of salivary gland diseases, side effects of certain drugs, or ir radiation of salivary glands enhances the susceptibility to dental caries in humans and experimental animals [Dreizen et al., 1977; Edgar et al., 1981; Bowen ct al., 1986; Handclman et al., 1986; Ooshima et al., 1990,1991], Caries ac tivity is enhanced by the emergence of a cariogenic micro flora due to the diminution of salivary flow, paralleled by significant decreases in noncariogenic members of oral bacterial species [Llory et al., 1971,1972; Brown et al., 1975, 1976]. Our previous studies [Ooshima et al., 1990, 1991] have demonstrated that hyposalivation induced by either irradiation or surgical removal of salivary glands causes severe dental caries in specific pathogen-free (SPF) rats, irrespective of infection with oral streptococci including Streptococcus mutans. In addition, bacteriological exami nation demonstrated that the total number of lactobacilli increased significantly at the expense of the inoculated 5. mutans MT8148R. In our experimental system, wheat flour was used as a noncariogenic dietary component. Wheat flour had always induced only baseline levels of plaque formation and dental caries in many experiments in our laboratories [Ooshima et al., 1988, 1991], The pur pose of the present study is to examine the effect of hypo salivation on the induction of dental caries and on the composition of the oral microflora in rats fed either a su crose or a wheat flour diet.
Table 1. Caries induction in hyposalivated rats not infected with S. mulans and fed a diet containing 56% (w/w) carbohydrates Group
A B C D E F
Rats n
Carbohydrate in diet
Hyposalivation
Plaque index (mean ±SE )
Caries score (mean ±SE)
15 15 15 15 15 15
wheat flour wheat flour sucrose sucrose glucose glucose
—
0.28 ± 0.02 -i 0.55 ± 0.05 J 0.31 ±0.03 0.65 ± 0.04 J 0.40 ±0.03-i 0.75 ±0.08-*
4.6 ±0.1 0± 0 0±0 6.2 ±0.6 7.0 ± 0 .5 -i 0±0 41.9 ± 3.0 J 5.1 ± 0 .8 J 7 .5 ± 0 .6 > t , ± > * . 2.6 ± 1.1 J 37.9 ± 4.7 J
+ -
+ +
total
buccal + lingual
Salivary flow rate“ (mean ±SE ) pi/min
Weight gain (mean ±SE) g
218 ± 12 NDb 169 ± 7 ND 185 ± 11 ND
331.7 ±19.4-, 141.5 ±9.1 -* 323.2 ± 14.4-1 250.4 ±9.4 J 3 4 7 .6 i6 .6 -. > 248.8 ± 7.6 J
Statistical analyses were carried out between groups A and B, between groups C and D. and between groups E and F (***p < O.(X)l). a Flow rates were calculated by measuring the time between the excretion of l()() pi of whole saliva and that of 300 pi after the injection of pilocarpine. h Excretion of saliva was not detected 10 min after the injection of pilocarpine.
Statistical Analyses The caries scores and plaque indices from each group of rats were reduced by computing the means and standard errors. The bacteri ological counts were logl0-transformed prior to statistical analyses in order for the variances to be within the same order. For statistical analysis between each experimental group, the Mann-Whitney U test was used.
Results
Results of caries score and plaque index in experiment I in which rats were not infected with S. mulans are shown in table 1. Only low levels of dental caries were induced in
126
sham-operated rats fed sucrose, glucose or wheat flour diet, or in hyposalivated rats fed wheat flour diet. Howev er, hyposalivation due to surgical removal of salivary glands significantly enhanced the cariogcnic activity in rats fed a sucrose or glucose diet. Caries scores of the hy posalivated rats in groups D and F were significantly higher than those of sham-operated groups C and E, re spectively (p
