In vitro studies of growth and competition between S. salivarius TOVE-R and mutans streptococci.

Streptococcus salivarius TOVE-R has previously been reported as a successful competitor with Streptococcus mutans 10449S and Streptococcus sobrinus 6715-13 WT on the teeth of rats. We studied, in vitro, some possible bases for this competition, including hydrogen peroxide or catalase production, bacteriocin or enocin production, and growth rates. Growth rates were measured spectrophotometrically in both complex and defined media. We studied conditions of aerobic and anaerobic incubation; glucose and sucrose medium supplementation at three concentrations each; various initial pH's; singly- or doubly-inoculated cultures; and half-strength, normal, or double-strength broth used both fresh and as culture filtrates. TOVE-R grew as well as did mutans streptococci at acid pH, decidedly better at alkaline pH, and nearly twice as fast near neutral pH. The average doubling time for TOVE-R was about 0.5 hr, while that for the mutans streptococci was about 1.0 hr. When TOVE-R was grown together with a mutans streptococcus, the growth rate observed for the doubly-inoculated culture was equal to or less than that of TOVE-R alone, never greater. The presence and proportions of both organisms in mixed cultures were confirmed by plate counts, direct specific immunofluorescence, and Nomarski interference microscopy. There was no evidence, by any of the methods employed, to indicate the production of an inhibitory substance against mutans streptococci by TOVE-R, or vice versa. Also, there was no evidence that the faster growth rate of TOVE-R could be attributed to nutrient limitation of the mutans streptococci.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of ecological emergence of mutans streptococci naturally transmitted between rats and consequent caries inhibition by Streptococcus salivarius TOVE-R infection.

The ability of Streptococcus salivarius strain TOVE-R to inhibit the ecological emergence of virulent representatives of the most prevalent human mutans streptococci on the teeth of specific pathogen-free Osborne-Mendel rats was studied. Rats which were infected by TOVE-R, or either S. mutans 10449S or S. sobrinus 6715-13WT, or uninfected were transiently co-caged so as to allow natural fecal transfer of organisms due to coprophagy. The infectants were differentially recovered from swabs of the teeth over the time course of the experiments and from sonified teeth at termination. Data were expressed on both relative (percentage) and absolute (CFU) bases. Initial oral colonization of rats by TOVE-R inhibited the ecological emergence of fecally transmitted S. mutans 10449S and S. sobrinus 6715-13WT. There was a generally inverse relationship between the percentages and absolute numbers of TOVE-R and the mutans streptococci on the teeth, which strongly suggested their competition for tooth sites. Absolute numbers of total recoverable flora from the teeth upon sonification were correlated with caries scores, thus suggesting that total recoverable flora counts substantially reflect cavitation status. TOVE-R itself induced no apparent caries activity and its transmission to rats already infected by 10449S or its colonization of rats before 10449S infection inhibition caries induction by this S. mutans strain; similar anticaries effects were not statistically significant for TOVE-R against 6715-13WT in these experiments. These data on the inhibition of the ecological emergence of the mutans streptococci supplement the already reported ability of TOVE-R to preempt initial colonization of teeth and partially displace the colonization of teeth by the mutans streptococci.

Competitive displacement of mutans streptococci and inhibition of tooth decay by Streptococcus salivarius TOVE-R.

The ability of Streptococcus salivarius TOVE-R to displace virulent representatives of the most prevalent human mutans streptococci from the teeth of rats, and thereby to inhibit caries, was studied. Streptococcus mutans 10449S- or Streptococcus sobrinus 6715-13WT-infected specific-pathogen-free rats consuming a high-sucrose diet were inoculated by TOVE-R. The infectants were differentially recovered from swabs of the teeth over the time course of infection and from sonically treated material of extracted teeth and excised tongues. Despite initial colonization of the teeth by the mutans streptococci, TOVE-R colonized the teeth, unlike other essentially nonvirulent plaque formers already described. It did not colonize the tongues of the rats. TOVE-R emerged and persisted as a prominent member of the plaque ecology. There was an associated decline in the mutans streptococci on the teeth, and this decline was associated with significant inhibition of the caries component attributable to 10449S infection (56%) and to 6715-13WT infection (52%). TOVE-R did not reliably inhibit the component of fissure caries attributable to the nonmutans indigenous flora of the rats. TOVE-R itself induced no detectable decay. The data suggest the potential therapeutic utility of TOVE-R to inhibit caries by displacement of mutans streptococci from the teeth. These results supplement the already reported ability of TOVE-R to preempt initial colonization of teeth by the mutans streptococci.

Glucose-sucrose-potassium tellurite-bacitracin agar, an alternative to mitis salivarius-bacitracin agar for enumeration of Streptococcus mutans.

An agar medium for selective recovery and enumeration of Streptococcus mutans was developed as an alternative to mitis salivarius-bacitracin (MSB) agar. Combinations of dyes, antibiotics, and tellurite were added to a nonselective medium which, because of its sucrose content, allowed easy recognition of S. mutans colonies. Candle jar incubation for 2 days, by comparison with anaerobic incubation, reduced background flora but did not diminish S. mutans recoveries from clinical samples. Quantitative comparisons were made of the simultaneous recoveries of a number of authentic S. mutans serotype representatives and fresh clinical isolates, using various glucose-sucrose-potassium tellurite-bacitracin (GSTB) formulations and mitis salivarius, MSB, and blood agars. Mitis salivarius counts were not detectably different from blood counts, but counts on MSB were distinctly lower. A formulation of the new medium containing 5% glucose 5% sucrose, 0.001% potassium tellurite, 0.3 U of bacitracin per ml (hence GSTB), and 2% agar gave recoveries nearly equal to those on mitis salivarius agar and much greater than those on MSB. The medium yielded readily recognized S. mutans colonies and facilitated detection of intracellular polysaccharide formers upon flooding with I2 reagent. Freshly isolated serotype c, E, and f colonies could often be distinguished from serotype d and g colonies, a distinction made reliable by testing for intracellular polysaccharide. A study of 300 salivary samples revealed GSTB to give significantly higher recoveries than MSB. About 72% of all samples were substantially underestimated for S. mutans with MSB, and 6.7% of samples were falsely negative for S. mutans with MSB. Recovery of background flora on GSTB was as low or lower than on MSB, and both types of agar could be stored for at least 9 weeks without notable change of selectivity. Thus, GSTB agar appears to be simple and reliable to use and requires no anaerobic incubation. Caution is voiced about interpretation of data previously reported which evaluated S. mutans on MSB agar.