The e

eet o f suera

ate o n t h e g r o w t h o f c a r i o g e n i c

D a v i d T . B r o w n , D D S , M S , a C h r i s H. M i l l e r , P h D , b a n d D i a n e E. M a u p i n , B S c

Indiana University, School of Dentistry, Indianapolis, Ind. Sucralfate, an a l u m i n u m salt o f s u c r o s e o c t a s u l f a t e , has b e e n s h o w n to be effective in r e d u c i n g the d i s c o m f o r t o f radiation t h e r a p y - i n d u c e d oral mucositis. This s t u d y w a s done to d e t e r m i n e w h e t h e r s u e r a l f a t e could be u s e d as a nutritional source for d e n t a l c a r i e s - p r o d u c i n g o r g a n i s m s . T h r e e Streptococcus s t r a i n s w e r e cultured in a defined m e d i u m . S u e r a l f a t e p o w d e r w a s e v a l u a t e d for its ability to be u s e d as a c a r b o h y d r a t e food source b y t h e s e o r g a n i s m s . T h e addition o f s u c r a l f a t e alone did not s t i m u l a t e the o r g a n i s m ' s growth. The addition o f s u c r a l f a t e and g l u c o s e r e s u l t e d in l e s s g r o w t h than the addition o f $1ucose alone. I n c r e a s i n g the s u c r a l f a t e concentration f r o m 1% to 10% in the $1ueose-containing c u l t u r e s r e s u l t e d in s t a t i s t i c a l l y significant g r o w t h inhibition (p < 0.02). S u c r a l f a t e a p p e a r s to h a v e no c a r i o g e n i c p o t e n t i a l and m a y h a v e s o m e e a r i o s t a t i c potential. (J PROSTHET DENT 1991:66:256-60.)

M u c o s i t i s is a common sequela in oncology patients who have received some types of chemotherapy. Oral mucosat inflammation or desquamation may occur with alkylating agents, antimetabolities, and antibiotics. The serious mucosal ulcerations produced by these antineoplastic drugs can result in severe pain and discomfort and can interfere with normal nutritional maintenance. 1 Head and neck cancer patients who are receiving radiation also frequently experience oral mucositis. The severity of this mucositis is dependent on the radiation field dose and volume, Denuding of the superficial mucosal layers begins after 12 to 14 days of radiation treatments. The mucoSitis steadily progresses until approximately the fourth week when it becomes intense with large surfaces of denuded oral and oropharyngeal mucosa (Fig. 1). Eating, maintaining hydration, and maintaining adequate nutrition become difficult problems for the patient. Mouth rinses, gargles, and topical anesthetics can provide some short term alleviation of symptoms and allow easier consumption of food and beverages. Topical anesthetics may be required prior to oral hygiene procedures. 2 Sucratfate (Carafate, Marion Labs, Kansas City, Mo.), an aluminum salt of sucrose octasulfate, has been found to be an effective agent for peptic ulcer disease and esophageal ulcerationsfl 4 Sucralfate apparently forms a complex

with proteins and inhibits their hydrolysis by forming an adherent, protective coating that binds to active ulcerated surfaces. The use of sucralfate suspensions in patients with chemotherapy-induced mucositis has been reportedfi 6 Within 2 days after beginning sucralfate suspension rinses, resolution of symptoms were reported for most patients. 5 Sucralfate suspensions were also used in a preliminary clinical trial with radiation-induced and chemotherapyinduced stomatitis patients. 7 Within 24 houri of beginning rinses four times daily and as needed, the patients experienced pain alleviation, rapid return of normal oral function, and complete resolution of stomatitis without complications. Because radiation therapy to the head and neck is associated with rapidly progressing dental caries, and because sucralfate has a disaccharide base, its potential for promoting or inhibiting the growth of oral cariogenic microorganisms should be evaluated before oral use. This study was done to determine whether sucralfate could (1) be used as a carbohydrate food source for growth of oral streptococci or (2) inhibit the growth of oral streptococci. MATERIAL Bacterial

:!ssistant Professor, Department of Prosthodontics. '~ ~ofessor and Chairman, Department of Oral Microbiology. 'i ,aboratory Supervisor, Department of Oral Microbiology. ~1/24228

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AND

METHODS

strains

Three Streptococcus strains were obtained for this study. Streptococcus mutans 25175 (NCTC 10449) was purchased from the American Type Culture Collection in Rockville, Md. Streptococcus mutans 6715 was obtained from P.H. Keyes, formerly at the National Institute for

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Fig. 1. Typical oral mucositis that occurs during radiation therapy for head and neck cancer. Dental Research, Bethesda, Md., and Streptococcus sanguis I IUOM l l M was a human isolate from the Indiana University Oral Microbiology Department stock culture collection. Upon their receipt or isolation, organisms were cultured for 18 hours at 37 ° C in trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) containing 0.25% glucose and excess CaC03. Multiple 18-hour subcultures were prepared in the same media to which sterile glycerol was added to a final concentration of 10% after growth and before freezing at - 2 0 ° C. These tubes served as stock cultures from which all bacterial inocula originated. Purity of the cultures was periodically monitored by streaking samples on blood agar and observing gramstained smears of the colonies.

Preparation of growth media All test bacteria were cultured in a chemically defined medium (FMC) previously tested for its ability to support growth of oral streptococci, s The medium was prepared by mixing stock solutions of 20 amino acids, eight vitamins, three purine and pyrimidine bases, and 12 mineral salts, adjusting the pH to 7, and filter-sterilizing as previously described. 8 This medium served as the unsupplemented basal FMC medium. Glucose-supplemented FMC medium was prepared by adding appropriate concentrations of glucose to the basal FMC medium before pH adjustment and filter sterilization. Sucralfate, [alpha-D-glucopyranoside, beta-D-fructofuransyl-, octakis-(hydrogen sulfate), aluminum complex] was added to the basal FMC medium or to the glucose-supplemented FMC medium as pure sucralfate powder or as Carafate tablets that had been pulverized. Since sucralfate is only minimally soluble in water, it was aseptically added after the basal medium had been filtersterilized and placed in sterile culture tubes. The final pH of the sucralfate-supplemented FMC medium was asepti-

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Fig. 2. Growth of S. mutans NCTC 10449 in presence of sucralfate powder and in presence of 5 % glucose powder. All cultures were incubated for 18 hours at 37 ° C. cally adjusted to 7 with sodium hydroxide before use. Contamination of the sucralfate-supplemented FMC medium was not observed. Bacterial growth did not occur in this uninoculated medium, which was incubated for 18 hours at 37 ° C.

Analysis of bacterial growth Stock cultures of the test bacteria were inoculated (0.2 ml per 10 ml of medium) into 10 ml of FMC medium supplemented with 0.25% glucose. After incubation for 18 hours at 37 ° C, 0.2 ml was aseptically transferred to 10 ml of unsupplemented FMC medium or to FMC medium supplemented with 5 % glucose, various concentrations of sucralfate or combinations of glucose and sucralfate. The cultures were incubated at 37 ° C for 18 hours and the final pH values and viable cell counts were determined. Viable cell counts were expressed as colony-forming units (CFU) per millileter of culture. CFU per millileter were measured by serially diluting each well-mixed culture in tenfold increments to 10 -7 in sterile reduced transport fluid. 9 One-tenth millileter aliquots of each dilution were spread on the surfaces of Mitis-salivarius agar (Difco Laboratories, Detroit, Mich.), which is selective for the growth of streptococci. After incubation for 3 days at 37 ° C in an atmosphere of 85% N2, 10% H2 5% C02 (anaerobe chamber Model 1024, Forma Scientific, Marietta, Ohio), the number of CFU present on each plate was determined with the aid of a stereomicroscope. These data were multiplied by the appropriate dilution factor to determine viable cell concentrations expressed as CFU per miUileter. The general linear model was used to analyze each organism's CFU total per concentration of sucralfate. The Tukey-Student range test was used to evaluate CFU totals

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BROWN, MILLER, AND MAUPIN

Fig, 3, Growth of S. mutans 6715 in presence of sucralfate powder and in presence of 5 % glucose and sucralfate powder. All cultures were incubated for 18 hours at 37 ° C.

Fig. 4. Growth of S. sanguis IUOM l l M in presence of sucralfate powder and in presence of 5% glucose and sucralfate powder. All cultures were incubated for 18 hours at 37 ° C.

for S. mutans 6715, for sucralfate powder, and for pulverized Carafate tablets.

on growth of the oral streptococci. Figs. 2 through 4 show that in general the final cell concentrations decreased as the concentration of sucralfate increased in cultures of all three bacteria. In addition, final culture pH values were less acidic with increasing concentrations of sucralfate, an indication of increasing inhibition of cell growth as the sucralfate concentration increased. To test the effects of the form of sucralfate on growth of S. mutans 6715, the experiment described in Fig. 3 using sucralfate powder was repeated with the use of pulverized Carafate tablets (Fig. 5). Similar results were obtained except that overall growth in the unsupplemented and in glucose-free FMC media was higher. For all three organisms, there was a statistically significant decrease in the number of CFU as the concentration of sucralfate increased (S. mutans 10449, p = 0.02; S. mutans 6715, p = 0.007; and S. sanguis, p = 0.011). The use of sucralfate as pulverized Carafate tablets with S. m u t a n s 6715 resulted in a significant decrease in CFU as the concentration of sucralfate increased (p = 0.007). There was no significant difference between the powdered sucralfate and pulverized Carafate tablets CFU totals for S. mutans 6715.

RESULTS The effects of sucralfate on the growth of S. mutans NCTC 10449. S. mutans 6715. and S. sanguis IUOM l l M are shown in Figs. 2 through 4. respectively, and Table I. Growth of the oral streptococci in the unsupplemented, chemically defined basal medium (FMC) was poor as expected. After 18 hours of incubation there was little or no change in pH (Table I), and final cell concentrations (CFU per ml) were 4.55 × 107 for S mutans NCTC 10449, 1A1 × 105 for S. rnutans 6715 and 3.42 × 107 for S. sanguis tUOM llM. The addition of 571 glucose to the FMC medium resulted in excellent growth of all three bacteria with final pH values below 5 (Table It and final cell concentrations between two and three orders of magnitude higher than in the unsupplemented basal medium. Sucralfate in the form of either an anhydrous powder or pulverized tablet was added to the FMC basal medium to determine whether this substance could be used for growth by the streptococci. The presence of 1% to 10 % sucralfate in the otherwise unsupplemented basal medium with S. m~.tans NCTC 10449 (Fig. 2) and S. sanguis IUOM l l M ~Fig. 4} resulted in final cell concentrations lower than ~hose observed for cultures in the basal medium only. With ,~ rautans 6715 (Fig. 3) the final cell concentrations in the .~tmralfate media did not differ more than one order of ~sagnitude from those observed in the basal medium only. "3one of the bacteria produced final culture pH values of ~ess than 6.33 at any sucralfate concentration (Table I). Sucralfate was also added to FMC medium supplemented with excess glucose to detect any inhibitory effects

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DISCUSSION Radiomucositis changes in the oral cavity may become evident after a dose of 2000 to 2500 centiGray (cGy) has been reached. Pain and dysphagia are usually continuous by the time a dose of 3000 to 3500 cGy has been applied. As the dose reaches 5000 to 6000 cGy, the patient experiences extreme difficulty in eating and maintaining his nutrition. Analgesics and anesthetics are usually required for the patient to eat with even minimal comfort. At this point

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EFFECT O F S U C R A L F A T E

T a b l e I. Final pH values of 18-hour cultures of test

bacteria in supplemented and unsupplemented FMC medium* Medium

S. m u t a n s

S. m u t a n s

supplement

NCTC 1 0 4 4 9

6715

S. s a n g u i s I U O M 11 M

None 1% Sucralfate 2.5% Sucralfate 5% Sucralfate 7.5% Sucralfate 10% Sucralfate 5 % Glucose 5% Glucose and 1% sucralfate 5% Glucose and 2.5% sucralfate 5% Glucose and 5% sucralfate 5% Glucose and 7.5% sucralfate 5% Glucose and 10% sucralfate

7.16 7.33 7.36 6.90 6.54 6.39 4.56 4.91

7.07 7.32 7.37 6.75 6.54 6.33 4.58 5.70

7.18 7.32 7.28 6.84 6.84 6.43 4.80 5.37

5.30

5.76

5.60

5.79

5.50

5.63

6.64

5.89

5.64

6.28

6.25

6.10

Fig. 5. Growth of S. m u t a n s 6715 in presence of pulverized Carafate tablets and in presence of 5 % glucose and pulverized Carafate tablets. All cultures were incubated for 18 hours at 37 ° C.

*Data derived from some cultures described in Figs. 2 t h r o u g h 4.

the mucositis covers the entire mucosal surfaces that are within the field of radiation. The mucosal surface is generally covered with a white pseudomembrane, which if d.;.sturbed, will bleed easily. Patients also experience a profound xerostomia at this stage of treatment. 2 Sucralfate has been demonstrated to relieve the discomfort from radiomucositis rapidly and effectively.5, 7 The sucralfate compound is described as having an affinity for ulcerated mucosa. This coating is apparently more effective at protecting the denuded surfaces than the pseudomembrane Scanlon 2 describes. The bacteria used in this study to test the effects of sucralfate on microbial growth were selected because S. sanguis has been shown to occur regularly in human plaque. 1° The growth medium used was a chemically defined medium that permits maximum growth of the test streptococci only when a fermentable substrate (glucose or sucrose) is added to the medium. Thus, various substrates can be added to this medium to determine whether they can serve as a fermentable substrate to support growth of the test bacteria. Glucose instead of sucrose was used as the standard fermentable substrate because sucrose stimulates clumping of S. m u t a n s during growth and makes it impossible to measure growth accurately by viable cell counts. The range of sucralfate concentration tested (0% to 10%) was selected because mucositis therapy has used 10% suspensions of the agent. Our results show that the addition of sucralfate powder or ground Carafate tablets alone to the defined medium did not appreciably stimulate the growth of the S. m u t a n s strains cultured as shown by the number of CFU and the

THE JOURNAL OF PROSTHETIC DENTISTRY

pH measurements of the cultures. This implies that neither the sucralfate powder nor the Carafate tablet was a usable carbohydrate nutritional source for the microorganisms. When growth-stimulating glucose was present in the culture medium, the sucralfate powder or Carafate tablets had an inhibitory effect on the growth of both S. m u t a n s strains and the S. sanguis strain (Figs. 2 through 5). The number of CFU was lower when sucralfate and glucose were added than when glucose alone was added to the medium. There was a direct, statistically significant relationship between the amount of growth inhibition and the concentration of sucralfate or Carafate tablets. It appears that sucralfate interferes with the oral microorganism's ability to use glucose as a food source. The pH values of the test cultures also showed a direct relationship between inhibition and sucralfate concentration. Table I shows that as the concentration of sucralfate increased in the presence of a constant amount of glucose, the pH of the culture medium was less acidic (because of fewer acid-producing organisms). Since no significant difference in growth was noted between the cultures with sucralfate powder and the cultures with pulverized Carafate tablets, the materials used to formulate the tablet (D and C Red No. 30 Lake, FD and C Blue No. 1 Lake, magnesium stearate, microcrystalline cellulose, and starch)11 were apparently not responsible or involved in the growth inhibition. Because sucralfate has negligible acid-neutralizing capacity, 11 the pH increase with increasing sucralfate concentrations is not attributable to a direct effect of sucralfate on the acids. The possibility exists that the aluminum complex may be the origin of the growth inhibition. Jenkins 12 cites a study in which a population with a low caries incidence was as-

259

BROWN, MILLER, AND MAUP1N

molybde'n to have primarily through the ability of the aluminum to reduce enamel dissolution, there m a y be a direct action of aluminum on the microo~ that contributes to the cariostatic potential o f thi~ element. echanism microor gauismsin t~e presence of glucose.

The~resutts ofthis study indi'cate that sucratfate suspensions are not used as a carbohydrate food source for oral caries-producing organisms. Chemotherapy-induced or radiation-induced mucositis patients who are using sucralfate rinses would receive the benefit of inhibition of streptococcal organisms as well as increased oral comfort.

SUMMAItY Three orat Streptococcus strains were cultured in a chemically defined minimal medium. Sucralfate powder was evaluated for its ability to be used as a carbohydrate source by these microorganisms. The addition of sucralfate alone did not stimulate growth of the organisms. The addition of sucratfate and glucose resulted in less growth than the addition of glucose alone. Increasing the concentration of sucralfate in the glucose-containing cultures resulted in significant additional inhibition of the organisms. Sucralfate, despite its structural relationship to sucrose, appears to have no cariogenic potential and may have some carlostatic potential: It appears that Carafate suspensions used for the care of radiation-induced and chemotherapyinduced mucositis patients do not stimulate growth of carbohyd~ate fermenting oral streptococci. These solutions, in addition toaUeviating the discomfort of the mucositis, may

potentially inhibit the growth of organisms responsible for the ca~es associated with radiation therapy.

REFERENCES 1. Matvias FM. Chemotherapy and its dental manifestations. In: Beumer J, Curtis TA, Firtell DN, eds. Maxillofacial rehabilitation: prosthodontic and surgical considerations. St. Louis: CV Mosby, 1979;11-22. 2. Scanlon PW. Manifestations and complications of radiotherapy. In: Laney WR, Gibilisco JA, eds. Diagnosis and treatment planning in prosthodontics. Philadelphia: Lea & Febiger, 1983;327-45. 3. Fisher RS. Sucralfate: a review of drug tolerance and safety. J Clin Gastroenterol 1981;3(suppl):181-4. 4. Roark G. Treatment of postsclerotherapy esophageal uloers with sucralfate. Gastrointest Endosc 1984;30:9-10. 5. Ferraro JM, Mattern JQA. Sucralfate suspension for stomatitis. Drug Intell Clin Pharm 1984;18:153. 6. Soloman MA. Oral sucralfate suspension for mucositis. N Engl J Med 1986;315:459-60. 7. Adams S, Toth B, Dudley BS. Evaluation of sucralfate as a compounded oral suspension for the treatment of stomatitis. Clin Pharmacol Ther 1985;37:178. 8. Terleckyi B, Willett N, Shockman G. Growth of several cariogenic strains of oral streptococci in a chemically defined medium. Infect Immun 1975;11:649-55. 9. Loesche WJ, Hackett RN, Syed SA. The predominant cultivable flora of tooth surface palque removed from institutionalized subjects. Arch Oral Biol 1972;17:1311-25. 10. Newburn E. Cariology. 2nd ed. Baltimore: Williams & Wilkins, 1983. 11. Carafate product insert. Kansas City, Mo: Marion Laboratories Inc, 1988. 12. Jenkins GN. The physiology and biochemistry of the mouth. 4th ed. Oxford: Blackwell Scientific Publ, 1978;453. 13. Kleber CJ, Putt MS. Aluminum and dental caries: a review of the literature. Clin Prev Dent 1984;6.'14-25. 14. Putt MS, Kleber CJ: Effect of pH concentration and treatment time of aluminum solutions on acid dissolution of enamel. J Dent Res 1986; 65:1356-8. Reprint requests to: DR. DAVIDT. BROWN SCHOOLOF DENTISTRY INDIANAUNIVERSITY 1121 WEST MICHIGANST. INDIANAPOLIS, IN 46202

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AUGUST 1991 VOLUME66 NUMBER 2