Robert J. Fitzgerald

Basic Biological Sciences Cariogenicity of Human Plaque Lactobacilli in Gnotobiotic Rats

Thirty-two strains of Lactobacilli, comprising eight species and obtained from human dental plaque or other sources, were tested for cariogenic potential in gnotobiotic Sprague-Dawley rats receiving modified cariogenic Diet 2000. Seventeen of these strains were moderately to highly cariogenic, and only one, a strain of Lactobacillus lactis, was scored as non-cariogenic. The molar fissures were the predominant site of caries attack. Primary lesions of the smooth surfaces were infrequently seen, although these surfaces were often secondarily involved by the extension of fissure lesions. The preferential location of caries in the sheltered fissure sites was attributed to their inability to form adherent plaque deposits on the smooth surfaces of the teeth. Caries production by a strain of L. salivarius was equally severe on sucrose- or glucose-containing versions of Diet 2000, but glucose was less supportive of caries development than sucrose for a strain of L. fermentum. It is concluded that a wide variety of lactobacilli may be cariogenic under favorable environmental conditions.Thirty-two strains of Lactobacilli, comprising eight species and obtained from human dental plaque or other sources, were tested for cariogenic potential in gnotobiotic Sprague-Dawley rats receiving modified cariogenic Diet 2000. Seventeen of these strains were moderately to highly cariogenic, and only one, a strain of Lactobacillus lactis, was scored as non-cariogenic. The molar fissures were the predominant site of caries attack. Primary lesions of the smooth surfaces were infrequently seen, although these surfaces were often secondarily involved by the extension of fissure lesions. . The preferential location of caries in the sheltered fissure sites was attributed to their inability to form adherent plaque deposits on the smooth surfaces of the teeth. Caries production by a strain of L. salivarius was equally severe on sucrose- or glucose-containing versions of Diet 2000, but glucose was less supportive of caries development than sucrose for a strain of L. fermentum. It is concluded that a wide variety of lactobacilli may be cariogenic under favorable environmental conditions.

Comparison of Extracellular Protein Profiles of Seven Serotypes of Mutans Streptococci Grown under Controlled Conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.

Characterization of two strains of cariogenic lactobacilli.

Two strains of lactobacilli that initiate dental caries in conventional animals were exained for their physiological and serological characteristics. The strain designated V CL-25 was identified as Lactobacillus fermentum and belonged to serological group F. The strain designated IV CL-37 was a Lactobacillus salivarius, but it could not be further identified as either of the known subspecies, nor did it belong to serological group G.

Biochemical properties of Streptococcus sobrinus reisolates from the gastrointestinal tract of a gnotobiotic rat

Streptococcus sobrinus strain 6715-13-201 was inoculated into the oral cavity of a gnotobiotic rat and then reisolated from different portions of the gastrointestinal tract. Fourteen isolates, selected on the basis of their colonial morphology, were then screened for their ability to adhere to saliva-coated hydroxyapatite (SHA) in vitro, and their ability to produce extracellular polysaccharide from sucrose, and low pH in glucose broth. Certain isolates were also tested for their cariogenic potential as monoinfectants in gnotobiotic rats. All isolates differed in their abilities to adhere to SHA, with most showing an increased level of adhesion in the presence of sucrose, but this did not correlate with their ability to be aggregated by dextran. Most isolates were capable of producing glucosyltransferases (with only one exception) and dextranases (also one exception). There was more variability in the production of dextranase inhibitor. No isolate was capable of producing dextranase inhibitor in the absence of dextranase production. There were no correlations between the ability of isolates to adhere in vitro or produce/utilize polysaccharides and their ability to produce caries in vivo. Due to the differences between strains in their abilities to adhere, produce polysaccharides, utilize polysaccharides or produce a low pH and the lack of correlation between any of these parameters and cariogenicity, the results suggest that the ability of strains to colonize and produce caries depends on a number of different characteristics, no one of which is essential.

Potassium requirement of oral streptococci.

Potassium (K+) is considered to be an essential mineral for all living cells, including microorganisms. Unstimulated, mixed human saliva contains approximately 800 Mug K+/ml and about 230 Mug Na+/ml; whereas stimulated, mixed saliva has about 570 ug K+/ml and 570 ,Mg Na+/ml (ALTERMAN and DITrMER (eds), Metabolism, 1968). Most lactic acid bacteria have requirements of up to 1,000 Mug K+/ml of medium for maximal growth (McLEOD and SNELL, J Biol Chem 170: 351-365, 1948). In the present report, the specific requirements for two species of oral streptococci that colonize the dentition were determined. Streptococcus mutans and S sanguis cultures were grown in a chemically defined medium (COWMAN, PERRELLA, and FITZGERALD, Appl Microbiol 27: 86-92, 1974) made K+-deficient by neutralizing and buffering with sodium salts (final concentration of Na+ was 805 ,ug/ml) and to which increasing amounts of potassium chloride (KCl) were added. The final concentration of K+ in the media was verified by flame photometry. Inoculated tubes of medium were incubated under 90% N2-10% CO2 for 16 hours at 35 C. After the third consecutive transfer, the growth density attained by each culture was measured at 600 nm. Stock cultures were maintained in Trypticasea-GlucoFe Broth (COWMAN, PERRELLA, and FITZGERALD, Appl Microbiol 27: 86-92, 1974) that contained approximately 200 Mug K4/ml of medium. In the absence of a sufficient external K+ concentration, S mutans and S sanguis were unable to maintain an adequate level of growth in the defined medium, as shown in the illustration, for a representative number of strains. When as

Effect of Growth Medium on Amino Acid Pools of Oral Streptococci

Krezeminiski (J Dent Res 54: 183, 1975) reported that the intracellular amino acid pool of Streptococcus mutans grown aerobically in a Tryptosea medium consisted predominately of glutamic and aspartic acids as well as alanine and glycine. The present study compares the composition of the amino acid pools of S mutans and S sanguis after growth in media differing in the available source of organic nitrogen. S mutans KI-R (type d) and S sanguis 903-1600 were grown under 90% N10% C02 (1.5% 02) for 15 hours at 37 C in Todd-Hewitt Brotha (TH) containing 0.5% lactalbumin hydrolysate,a Trypticaseb-Glucose Broth (TG), and a defined amino acid (AA) medium (COWMAN ET AL, Appl Microbiol 27: 86-92, 1974) . Cells were collected by centrifugation at 15,000 x g and washed twvice with 0.85 M NaCl at 20 C. Cells were extiactecl twice with 20 ml of 95% ethanol at 70 C to remove amino acids; the extracts were pooled anid concentrated to 2 ml and deproteinated as described by Harris et al (Can J Biochem 39: 439, 1961) The amino acid fraction after drying wvas dissolved in 5.0 ml of 0.2 N sodium citrate buffer (pH, 2.2) and 200-gl portions were analyzed using an amino acid analyzer.c Growth yields were determined by drying portions of the cell suspensions to constant weight. All tests were performed three times. (;rowth yields for the tvo organisms were compai able for the same medium. TG gave the hiighest yields (88 to 92 mg/ 100 ml) followed by TH and AA in an approximate ratio of 1:0.5:0.2. The total free amino acid (FAA) content varied with both the medium and organism, and was highest in cells from the AA medium. The relative distribution of the principal intracellular FAA under the test conditions used is given in the table. Those amino acids present at less than 5.0 mole % in all samples were omitted. The

Effects of chlorhexidine gluconate in drinking water on dental caries and oral microorganisms in the Syrian hamster.

Hamsters infected with Streptococcus sobrinus (formerly Streptococcus mutans) strain 6715-1119 were given high-sucrose diet 2000. After 5 days, they were presented with 0.05 or 0.1 per cent w/v chlorhexidine gluconate (CHX) continuously in their drinking water, which was well-tolerated. Compared to infected but untreated controls, their caries scores were less by 84 and 97 per cent respectively after 42 days. Coronal dental plaque deposits were reduced in a dose-related fashion. Culture plates of oral swabs showed reductions in Strep. sobrinus and total streptococci after 10 and 35 days of CHX. Hamster oral streptococci were more sensitive to CHX than were lactobacilli but neither was completely eliminated. The incidence of stained tooth deposits after 0.1 per cent CHX was slightly but not significantly greater than in controls.

Microbes and Their Products in Plaque

To most investigators studying the biologic aspects of caries, the overriding factor appears to be the periodic attack on the tooth surface of large amounts of acid produced by the microorganisms of the plaque. In our deliberations during this workshop, we have learned of some of the physiochemical factors that may tend to have modifying influences on the development of the caries lesion by potentiating the demineralization process, by acting to slow the process, or by actually reversing it. The ultimate significance of many of these factors remains to be established in actual disease situations, either in animals or in humans. In my own view, it is difficult to conceive how processes such as remineralization can normally have more than a transitory role in the face of the abundance of acid that is produced in the active cariogenic plaque in children or animals. Except in the special cases of teeth exposed to fluoride or treated with relatively large amounts of phosphates, the microbial composition of the plaque and its biochemical activities on dietary carbohydrates seems to far outweigh any other factor in the overall process of caries development. It is in this area (plaque ecology and metabolism) that I personally would seek caries control procedures of a lasting nature. In caries in adults, which ordinarily is a much slower process than that in children, the relative numbers of Streptococcus mutans and frequency of isolation in plaque deposits are lower than those observed in children. This would indicate that qualitative changes have occurred in the plaque microflora; this results in a plaque that poses a less severe threat to the tooth from the standpoint of acidogenic potential. In this situation, it is easier to conceive that the various process might be influenced by more subtle factors as, for example, the crystalline state of the enamel mineral or the presence of selective diffusion barriers that regulate the passage of anions and cations in the tooth-plaque-saliva system. Another mitigating factor could be reduction of the effective H+ activity in plaque by organisms that produce weaker acids than lactic acid or that can metabolize the lactic acid produced by other organisms to less acidic products. As Dr. Briner has demonstrated, the potential usefulness of remineralizing solutions in vivo depends on prior eradication or inhibition of the plaque acidogenic microflora. The reason for this is that demineralization normally proceeds at a more rapid rate than remineralization when plaque and fermentable substrates are available. These observations indicate the necessity of including microbial plaque control procedures in any systematic program to control caries.