Preemption of Streptococcus mutans 10449S Colonization by

INFECTION AND IMMUNITY, Jan. 1982, p. 138-142 0019-9567/82/010138-05$02.00/O Vol. 35, No. 1 Preemption of Streptococcus mutans 10449S Colonization by Its Mutant 805 J. M. TANZER,* J. FISHER, AND M. L. FREEDMAN Departments of Oral Diagnosis and Laboratory Medicine, University of Connecticut Health Center, Farmington, Connecticut 06032 Received 29 May 1981/Accepted 17 September 1981 Oral infection of rats with Streptococcus mutans mutant 805 was used to prevent the establishment of its highly virulent wild-type progenitor NCTC 10449S. The dose of wild-type cells required to colonize 100% of the specific pathogen-free Osborne-Mendel rats (21 to 43 days old) consuming caries test diet 2000 was greater than 4 x 105 but less than 4 x 106 cells. Therefore, the latter dose was used to challenge rats which had already been colonized by an oral dose of about 6 x 108 cells of mutant 805. This prior infection by 805 either completely protected rats from subsequent colonization by wild-type cells or greatly delayed and diminished their emergence. Rats in which wild-type cells became established showed much lower percentages of wild-type cells in their total recoverable floras than did rats that were not first infected by the mutant. Large doses of mutant 805, however, did not displace wild-type cells from rats once it became established. There was no evidence of reversion of the mutant, which is defective in intracellular polysaccharide synthesis and hence is less virulent than wild-type cells. The data indicate that the S. mutans cell which first colonizes rats gains the strongest ecological position and is difficult to displace. Also, they suggest the possible prophylactic utility of infection by this mutant of S. mutans. To explore whether some microorganisms, because of earlier colonization, may exclude or preempt Streptococcus mutans establishment in the oral cavity, we have studied plaque-forming, non-cariogenic, oral bacteria. This paper evaluates such a competitive infection with a mutant of S. mutans. Work with selected mutants of S. mutans has demonstrated that there are several determinants of its virulence (8, 16-19). Some of these mutants have lower virulence than their progenitors, because they either fail to infect or to persist in the oral cavities of test animals. Other mutants, however, successfully colonize, emerge, and persist in the oral ecology of the host, yet fail to demineralize the teeth appreciably. Among the latter mutants are those with defects in the formation of intracellular, iodophilic, glycogen-like polysaccharide, IPS (5, 19). One of these, isolate 805, is a stable, nitrosoguanidine-induced derivative of the highly virulent strain NCTC 10449S. The nature of its phenotypic defect has been characterized (1, 5), as has its reduced ability to cause caries in rats fed a cariogenic diet (19). We report that preemptive infection by mutant 805 can profoundly inhibit the establishment of the virulent wild-type cells (WT) in the oral cavities of rats, but cannot displace WT once it has become established. 138 MATERIALS AND METHODS Microorganisms. Spontaneously occurring streptomycin-resistant cells of strain NCTC 10449, NCTC 10449S, and its nitrosoguanidine-induced mutant 805 (5) were studied. After initial isolation and characterization, the strains were lyophilized until tested in experimental animals, whereupon they were cultured in fluid thioglycolate medium containing 20%o (vol/vol) meat extract (Difco Laboratories) and excess CaCO3. Animals, diets, and infection. Weanling specific pathogen-free Osborne-Mendel rats were used. This animal strain was reared and maintained free of S. mutans infection as previously detailed (15, 18). After the rats were weaned at 21 days of age, they were pooled, from several litters, in a single cage and given caries test diet 2000 containing 56% sucrose (10) and demineralized water ad libitum. The rats were then randomly grouped and infected at various times by pipetting fresh thioglycolate cultures of standardized numbers of late-logarithmic-phase cells of S. mutans into their mouths; the control rats remained uninoculated. All rats were then housed singly in openbottomed, suspended, stainless steel cages to minimize recycling or sharing of gut flora. To determine the infectious dose of WT for successful colonization of the oral cavities of 100lo of the test rats and to examine this value as a function of age, the rats were given a single infectious dose of microorganisms from 4 x 103 to 4 x 107 cells. Recovery of microorganims. To establish the presence and relative numbers of the streptomycin-resistant S. mutans among the total recoverable flora and

VOL. 35, 1982 to detect possible extraneous non-streptomycin-resistant S. mutans, the teeth of all animals were swabbed before and after infection and at various intervals. The cotton swabs were immediately placed into buffered yeast extract, vortex mixed, and plated on appropriate media (see below) within 1 h. The problems of sampling plaque from the teeth of small animals and of reading plate counts of streptococci are well known (15; J. M. Tanzer, ed., Animal Models in Cariology, in press). Previous data (19) suggested that 805 and its progenitor had similar ecological distributions and similar recoveries from the mouths of rats. Nonetheless, to verify that swabbing did not bias the detection of mutant or WT, plaque was also scraped from the opening to the central fissure of the mandibular first molars at the time of sacrifice of the animals. Plaque scrapings were diluted and spread on plates, as were the swab samples, and the results of the two methods were compared. Cultures were plated on Mitis Salivarius (MS) agar containing Chapman tellurite (Difco Laboratories), MS supplemented with 200 p.g of streptomycin per ml (MSS), trypticase soy agar supplemented with 5% sheep blood (BA; BBL Microbiology Systems), and a complex medium (9) supplemented with 5% glucose, 5% sucrose, and 100,ug of streptomycin per ml solidified with 1.5% agar (GSS). After incubation, recoveries were expressed as the percentage of colony-forming units on BA which grew on MSS agar or of iodophilic or non-iodophilic colonies which grew on GSS agar. The WT and mutant microorganisms could be readily differentiated from each other and from other microorganisms: (i) both grew differentially with typical frosted-glass morphology on MS; (ii) both grew selectively and with typical frosted-glass morphology on MSS and GSS; and (iii) only the WT stained reddish-brown upon flooding GSS plates with iodine reagent. This differentiation of the WT from 805 could be carried out with absolute reliability by incubating GSS plates for 48 h in candle jars and flooding the plates with a 37 C solution of 0.2% 12 in 2% KI. By this method, a single iodophilic colony-forming unit could be detected among far more than 1,000 non-iodophilic colony-forming units. Hence, streptomycin-labeled, iodophilic, and noniodophilic infectants with the morphology of S. mutans could be expressed as percentages of the total S. mutans or total facultative flora. Also, the presence of non-streptomycin-resistant S. mutans infectants could be detected on MS agar. Periodically, the identities of selected reisolates which were streptomycin-resistant and S. mutans-like in morphology were confirmed by well-established biochemical-physiological and morphological techniques (16). Statistical analyses of percentage recoveries were carried out by using arcsin transformation to improve the normalcy of distribution (12) and, thus, to enable parametric analysis by t test. Data were also analyzed by the nonparametric rank sum test of Wilcoxon (14). Recoveries of more than 0 but <1% were treated as though they were 1.0o. RESULTS Determination of 1D1om. For specific pathogenfree Osborne-Mendel rats between 21 and 43 days of age, the infectious dose of WT required PREEMPTION OF S. mutans COLONIZATION 139 UN INFECTED UNINFECTED 805 INFECTED + WT 805 + WT INFECTED 805/WT 0O 15/O 27/jo 34/1,7 41/2 V V V V FIG. 1. Experimental design for study of preemptive infection. The calendar at the bottom of the figure begins with the weaning date. Oral cultures were taken with reference to the days elapsed since 805/WT infection: 0/0 signifies culture before both 805 and WT infection, 15/0 signifies culture 15 days after 805 but before WT infection, 27/10 signifies culture 27 days after 805 and 10 after WT, etcetera, until the termination of the experiment at 48/31 calendar days. to colonize 100% of the rats consuming test diet 2000 was measured at 4 x 105 to 4 x 106 cells. Doses of 4 x 103 colonized no rats, whereas 4 x 10 and 4 x 105 cells colonized, on an average, 33% of the rats. However, because 4 x 106 cells of WT reliably colonized all rats within this age range, this dose was selected as the challenge for subsequent interference studies and is referred to as ID10o. Preemptive infection by 805. Four groups of singly caged rats were compared (Fig. 1): those uninfected by either 805 or WT, those infected by 805 only, those infected by WT only, and those infected by WT after infection by 805. Half of the rats were infected with 805 by means of a single oral dose of approximately 6 x 108 cells 3 days after weaning and provision of diet 2000. 75 -." SO - E (A 25 UNINFECTED a,) 0 I- Eli- aii o Oral swabbing Tooth scraping FIG. 2. Mean recoveries (±standard error of the mean) of 805 and WT as percentages of total S. mutans in doubly infected rats. Comparison of oral swabbing and tooth scraping recovery methods. 48/31

140 TANZER, FISHER, AND FREEDMAN ILl Z c 100 50s *z o I.- 100- a E 75- Em I- 0 w o 2S MA 21 o _30 C 20- C B A I 0-0-0-0 O I> A A 27A0 34j7 4V24 4W431 L - 0oo-0 0 & 80s 27/lo 34/17, 41;%4 INFECT. IMMUN. m 0 L. - g ck 80S 15/W 27/10 48/31 805 0WT FIG. 3. The recovery of NCTC 10449S WT and mutant 805 from rats. (A) The percentages of rats with positive recoveries of WT after infection by WT only (0) or by 805 followed by WT (0). (B) Mean recoveries (+standard error of the mean) of WT as percent-. After 17 days, half of the uninfected rats and half of the 805-infected rats were orally challenged with the ID10o of the WT. There were eight animals in each of the four groups in the typical experiment. In no case was (i) an uninfected rat observed to harbor either streptomycin-susceptible or streptomycin-resistant S. mutans, (ii) a rat infected by 805 only seen to harbor an iodophilic S. mutans, and (iii) a rat infected by WT only seen to harbor a non-iodophilic S. mutans. Thus, there was no evidence of extraneous or cross-contamination of the rats, of reversion of 805 to WT phenotype, or of spontaneous mutation from WT to 805 phenotype. The oral-swabbing and plaque-scraping methods gave the same mean values for recoveries of 805 and WT as percentages of total recovered S. mutans (Fig. 2). Moreover, when the data derived from the two methods were compared for individual rats, the correlation coefficient was statistically significant at P < 0.05. Prior infection by 805 delayed, and, in some cases, completely inhibited successful colonization by WT, at least for the duration of the experiment (Fig. 3A). Thus, by 27/10 calendar days all rats which had not been first infected by 805 were successfully infected by the WT after challenge by its ID100 (see legend to Fig. 1 for an explanation of calendar notation). By contrast, only 12% of the 805-infected rats showed WT infection. Later, at 34/17 and 41/24, 50% of the 805-infected rats evidenced infection by WT, and 62% evidenced infection at 48/31. In rats previously infected by 805 and subsequently infected by WT, the WT represented a far lower percentage of total S. mutans than if the rats had not been first colonized by 805 (Fig. 3B). Thus, rats infected by WT only gave recoveries of WT as 100lo of the total S. mutans, whereas the mean WT percentage among those doubly infected which gave a positive recovery ascended to only 23% by the end of the experiages of total S. mutans from rats infected by WT only (0) or from those infected by 805 followed by WT. The recoveries from the latter group are presented both as the group mean (O) and as the mean for those rats which gave positive recoveries (-). (C) Mean recoveries (±standard error of the mean) of WT and of 805 as percentages of total recoverable flora. Those rats not inoculated by either 805 or WT showed negative recoveries for these streptomycin-resistant infectants and negative recoveries of non-streptomycin-resistant extraneous S. mutans. The data for these animals are not plotted. Symbols: O, WT recoveries from rats infected by WT only; *, WT recoveries from rats infected first by 805, then by WT; A, 805 recoveries from rats infected by 805 only; A, 805 recoveries from rats first infected by 805, then by WT.

VOL. 35, 1982 ment. The differences between the recoveries of WT from the doubly infected and the singly infected group were thus highly significant. Figure 3C shows the recoveries of 805 and WT as a percentage of the total recoverable flora. The mean recoveries of 805 from rats infected by 805 only or by 805 followed by WT were the same throughout the experiment. Thus, there was no evidence of 805 displacement from the oral ecology by the WT. The recovery of the WT from the WT-only group was 4% of the total recoverable flora at 27/10 and ascended to 24% of the total recoverable flora at 48/31. By contrast, the recovery of the WT from the group first infected by 805 was 'C1% at 27/10 and only 2% at 48/31. Hence not only did WT yield a higher percentage recovery by 48/31 than did 805 (P < 0.05), but prior infection by 805 greatly reduced the ability of WT to emerge in the oral flora when compared with its level in animals infected by WT only (P < 0.001). Displacement infection by 805. Experiments were conducted to test whether 805 could displace WT after the latter had successfully colonized rats. Mutant 805, even after three oral inoculations of about 6 x 108 cells each, on alternate days, failed to displace established WT and failed to become reliably established itself. DISCUSSION It appears that the S. mutans cell which first colonizes rats gains the strongest ecological position and is difficult to displace. It is noteworthy that (i) 805 successfully competes for establishment and persistence within the mouths of the rats with their indigenous non-s. mutans flora and (ii) weakly virulent 805 successfully prevents or reduces the establishment of its highly virulent progenitor. These observations suggest the potential prophylactic utility of 805. Because the inception of most carious lesions begins in rodents, like humans, in the early period after the teeth erupt and the cariogenic diet is presented (2, 4), protection during the early posteruptive period might inhibit most of the decay experience during the lifetime of the host. The present model, however, which uses a poorly virulent microorganism to preemptively infect rats, is not useful for assessing the inhibition of caries. Before WT challenge, time has elapsed during which both the non-s. mutans indigenous flora of the animals and the exposure to the weakly virulent 805 could have produced low levels of decay. Therefore, to test protection against caries by nonvirulent S. mutans, other models must be used to quantitate the caries protection by 805. Such studies are in progress. The natural occurrence and the prophylactic utility of competitive infections in a variety of PREEMPTION OF S. mutans COLONIZATION 141 disease states have been recognized and used (3, 11, 13). Others have also recognized the possibility of using specific mutants of S. mutans to inhibit virulent S. mutans infection (6). ACKNOWLEDGMENTS This work was supported by Public Health Service grant DE 03758 from the National Institute of Dental Research. The authors thank F. N. Woodiel and V. Young for technical assistance. LITERATURE CITED 1. Bhrkhed, D., and J. M. Tanzer. 1979. Glycogen synthesis pathway in Streptococcus mutans NCTC-10449 and its glycogen synthesis defective mutant 805. Arch. Oral Biol. 24:67-74. 2. Carlos, J. r., and A. M. Gittelsohn. 1965. Longitudinal studies of the natural history of caries. Arch. Oral Biol. 10:739-751. 3. Davkdon, J. N., and D. C. Hirsh. 1976. Bacterial competition as a means of preventing neonatal diarrhea in pigs. Infect. Immun. 13:1773-1774. 4. Fltzgerald, R. J., and R. H. Larson. 1967. Age and caries susceptibility in gnotobiotic rats. Helv. Odontol. Acta 11:49-52. 5. Freedman, M. L., J. M. Tanzer, and R. L. Elfert. 1976. 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142 TANZER, FISHER, AND FREEDMAN tion in adhesion and virulence of Streptococcus mutans, p. 211-212. In M. T. Parker (ed.), Pathogenic streptococci. Reedbooks Ltd., Chertsey, England. 18. Tanzer, J. M., M. L. Freedman, R. J. Fitzgerald, and R. H. Larsn. 1974. Diminished virulence of glucan synthesis-defective mutants of Streptococcus mutans. Infect. Immun. 10:197-203. INFECT. IMMUN. 19. Tanzer, J. M., M. L. Freedman, F. N. Woodiel, R. L. EUfert, and L. A. Rinehlmer. 1976. Association of Streptococcus mutans virulence with synthesis of intraceilular polysaccharide, p. 597-616. In H. M. Stiles, W. J. Loesche, and T. C. O'Brien (ed.), Microbial aspects of dental caries. Information Retrieval, Ltd., New York, N.Y.