Antibody response in the parotid fluid and serum of Irus monkeys (Macaca fascicularis) after local immunization with Streptococcus mutans.

The antibody response of Macaca fascicularis in parotid saliva and serum to local immunization by two routes with Streptococcus mutans was studied and compared over 1 year. Antibodies were titrated and classified by indirect immunofluorescent staining using specific antiglobulin conjugates. Antiglucosyltransferase activity was assayed by an enzyme inhibition test. Animals were immunized first by injecting formalin-killed bacterial cells and cell products subcutaneously into the vicinity of the four major salivary glands. The monkeys were next immunized by retrograde instillation of antigen into the parotid duct. Extensive subcutaneous local immunization gave a serum response only. After parotid duct immunization, high titers of immunoglobulin A (IgA) antibody, along with traces of immunoglobulin G (IgG) immunoglobulin M (IgM) antibody, appeared in the parotid saliva, and in the serum high titers of IgG antibody were present along with lower titers of IgA and IgM. IgA antibodies in parotid fluid were shown by double immunofluorescent staining to be associated with antigenic determinants which cross-reacted with an antiserum directed to human secretory component. Titers in parotid fluids and sera fell sharply when immunization was stopped. This response pattern was reproducible. High concentrations of antibody capable of inhibiting glucosyltransferase prepared from S. mutans were found in the sera, but relatively little was detected in the parotid fluids. Extensive immunization via the parotid duct resulted in transient functional impairment of the gland, as evidenced by diminished salivary flow rates. We conclude that parotid ductal immunization can be an effective method for stimulating a salivary secretory IgA antibacterial antibody response.     

Effects of local immunization with glucosyltransferase on colonization of hamsters by Streptococcus mutans.

Experiments were performed to study the effect of antibody to Streptococcus mutans glucosyltransferase (GTF) on the implantation of these organisms in hamsters. Salivary (immunoglobulin A) and serum (immunoglobulin G) antibodies to GTF and GTF-inhibiting activity were elicited by injection of GTF in Freund complete adjuvant in the salivary gland region. Sham-immunized and GTF-immunized groups were then orally challenged with approximately 10(7), 10(8), or 10(9) colony-forming units of cariogenic S. mutans 6715. The results were evaluated by systematically swabbing molars 4 days and approximately 4 weeks after challenge. In general, fewer GTF-immunized hamsters became infected with S. mutans after challenge with 10(7) or 10(8) organisms than did identically challenged sham-immunized hamsters. Of the animals that did become infected, fewer S. mutans colony-forming units were recovered from GTF-immunized hamsters. These results indicate that the presence of antibody to GTF can diminish the ability of S. mutans to implant in the oral cavity of immunized hamsters.     

Protective secretory immunoglobulin A antibodies in humans following oral immunization with Streptococcus mutans.

Ingestion of a vaccine containing killed Streptococcus mutans, originally isolated from each volunteer, daily for 10 consecutive days induced increased levels of specific secretory immunoglobulin A (sIgA) antibodies to S. mutans cells and two cell surface proteins, glucosyltransferase and surface antigen I/II, in parotid saliva and tears of four healthy males and in parotid saliva, tears, colostrum, and milk of a pregnant woman. In addition, these antibodies inhibited glucosyltransferase activity. Both IgA1 and IgA2 antibodies were induced. The levels of IgA antibodies in all secretions remained significantly above preimmunization levels for more than 50 days after oral administration of antigen. A second series of immunizations for 7 consecutive days resulted in even higher levels of sIgA antibodies, which peaked earlier and persisted longer than those observed after the primary immunizations. No increase in levels of antibodies in serum were detected in any subject. Antibodies reactive with human heart and kidney antigens could not be detected in saliva, tears, colostrum, milk, or serum samples collected at any time during the immunization regimen. The numbers of viable S. mutans organisms in dental plaque and whole saliva decreased after each series of immunizations, which correlated with increased levels of IgA antibodies in saliva, suggesting that IgA antibodies in saliva were responsible for the reduced adherence of this bacterium. These results indicate that ingested S. mutans antigen induces secretion of specific IgA1 and IgA2 antibodies in saliva, tears, colostrum, and milk, providing further evidence for the existence of a common mucosal immune system.     

Specificity of monoclonal antibodies in local passive immunization against Streptococcus mutans

Local oral passive immunization in human subjects with a monoclonal antibody (MoAb) raised against the 185-kD antigen I/II from S. mutans significantly reduced or prevented oral colonization of an exogenous strain of the organism. In subjects sham-immunized with either saline or an unrelated MoAb, however, significantly greater proportions of S. mutans persisted for a longer duration than in those immunized with the specific anti-streptococcal MoAb. Recolonization of indigenous S. mutans after this organism was reduced to undetectable levels by an antimicrobial agent has also been completely prevented with specific MoAb. Indeed, S. mutans was not detected for a period of over 1 year, as compared with recolonization within 10-82 days in the control subjects. The specificity of MoAb in preventing colonization of the streptococci was studied with four MoAb. This revealed that: (1) the sub-class of antibody is not an essential factor, as both MoAb Guy's 1 and 13 prevented colonization, although Guy's 1 is an IgG2a and Guy's 13 is an IgG1 class of antibody; (2) serotype specificity is important, as MoAb Guy's 9, which only recognizes S. sobrinus (serotypes d and g), does not prevent colonisation by S. mutans (serotype c); (3) neither protein nor carbohydrate nature of the putative adhesin was a determining factor, because MoAb Guy's 1 recognizes a carbohydrate and Guy's 13 a protein determinant and both MoAb prevented adherence of S. mutans; and (4) epitope specificity appears to be the most important factor in preventing adherence of S. mutans, as MoAb Guy's 11 and 13 share the same serotype specificity and both recognize a protein determinant, yet only Guy's 13 prevents colonisation. The long duration of protection from re-colonization by indigenous S. mutans, lasting about 1 year after application of the specific MoAb was stopped, cannot be accounted for by functional MoAb remaining on the teeth. We suggest that initially the MoAb prevents colonization by S. mutans and that the ecological niche vacated by this streptococcus is filled by other organisms from the oral flora, thereby discouraging re-colonization by S. mutans.     

Effect of immunization on susceptibility to experimental Streptococcus mutans and Streptococcus sanguis endocarditis.

It has been asserted that humoral immunity is an important potentiating factor in pathogenesis of infective endocarditis, in that prior immunization to certain bacteria may predispose the host to endocarditis caused by those organisms. If so, possible future vaccination of humans with streptococcal antigens for the prevention of dental caries might increase the susceptibility of the population to streptococcal endocarditis. To examine this hypothesis further, we immunized rabbits with killed Streptococcus sanguis or Streptococcus mutans. After complement-fixing antibody had developed, the rabbits were tested for susceptibility to experimental infective endocarditis. Rabbits with high titers of complement-fixing antibody to the infecting organism developed streptococcal endocarditis less often (13%) than animals with lower titers (69%; P less than 0.0002). These findings do not support the hypothesis that pre-immunization predisposes to infective endocarditis and lend no credence to the concept that vaccination of human subjects against dental caries might increase their susceptibility to streptococcal endocarditis. On the contrary, the results of these experiments indicate that specific antibody can confer relative immunity to infective endocarditis.     

Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans.

Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenic Streptococcus mutans are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of S. mutans. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (n = 4) rinsed their mouths with immune milk and a control group (n = 4) rinsed with nonimmune milk. S. mutans levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of S. mutans in saliva and plaque. On the other hand, the numbers of S. mutans cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios of S. mutans to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling S. mutans in the human oral cavity.     

Specificity of natural antibodies reactive with Streptococcus mutans in monkeys.

Enzyme-linked immunosorbent assays were used to measure levels of natural antibody to defined antigens of Streptococcus mutans in sera from monkeys (Macaca fascicularis). The results suggest that most of the antibody in young monkeys that binds to whole bacteria is not specific to S. mutans.     

Use of monoclonal antibodies in local passive immunization to prevent colonization of human teeth by Streptococcus mutans.

Local passive immunization with monoclonal antibodies (MAbs) raised against streptococcal antigen (SA) I/II protects monkeys against colonization of teeth by Streptococcus mutans and the subsequent development of dental caries. In this study we extended the preclinical experiments to human subjects. In the first study of eight healthy subjects, four had anti-SA I/II MAb (immunoglobulin G2a [IgG2a]) and four had saline applied to their teeth on three occasions. A streptomycin-resistant S. mutans strain (Guy K2 strain, serotype c) was then implanted onto the teeth, and the organism was cultured sequentially from dental plaque and saliva up to 100 days after the first treatment with MAb. Decreased colonization by S. mutans was found in the dental plaque collected from smooth surfaces and fissures and in saliva of subjects whose teeth were treated with the MAb, as compared with the saline-treated control subjects. The experiment was then repeated on seven new subjects, and the effect of anti-SA I/II MAb was compared with that of an unrelated MAb to Campylobacter jejuni. The results again showed a consistently lower level of colonization of teeth in the anti-SA I/II MAb-treated subjects as compared with those sham immunized with the unrelated MAb. There was little difference in serum IgG, IgM, or IgA, gingival fluid IgG, or salivary IgA anti-SA I/II antibodies between the immunized and sham-immunized subjects, before and after the investigation. No side effects were observed, and the gingival and plaque indices remained unchanged. A sensitive radioimmunoassay failed to detect changes in anti-MAb (IgG2a) antibodies in any of the three fluids examined. We suggest that local passive immunization by means of MAb might be an alternative approach in the prevention of colonization of teeth by S. mutans and the development of dental caries.     

An investigation into the mechanism of protection by local passive immunization with monoclonal antibodies against Streptococcus mutans.

Local oral passive immunization with Streptococcus mutans-specific monoclonal antibody (MAb) (Guy's 13) prevented recolonization by indigenous S. mutans in human volunteers who had first been treated with a conventional antibacterial agent (chlorhexidine). The F(ab')2 fragment of the MAb was as protective as the intact immunoglobulin G, but the Fab fragment of the molecule failed to prevent recolonization of S. mutans. In subjects receiving the MAb Fab fragment, S. mutans levels in dental plaque and saliva reappeared at a similar rate to that found in sham-immunized subjects who received either saline or a nonprotective MAb. In vitro, MAb had no bacteriostatic or bacteriocidal effect on S. mutans. However, S. mutans grown in the presence of either intact immunoglobulin G MAb or the F(ab')2 fragment formed very long chains, which resulted in clumping of the cells. S. mutans grown with either saline or the MAb Fab fragment formed significantly shorter chains, more characteristic of streptococcal growth in liquid media. The results suggest that the two binding sites of the MAb molecule may be an essential feature for preventing streptococcal colonization but that the ability to bind to phagocytes and activate complement which resides in the Fc fragment is not essential. Protection against colonization by S. mutans lasting up to 2 years was observed in immunized subjects, although MAb was applied over a period of only 3 weeks. Furthermore, functional MAb was detected up to 3 days following application of MAb to the teeth. The long-term protection could not be accounted for by a persistence of MAb on the tooth surface, and we have suggested that it may be due to a shift in the balance of the oral flora which discouraged recolonization by S. mutans. However, examination of the proportions of Streptococcus sanguis and veillonella species in the recolonization experiments failed to reveal a significant change in the proportions of either organism, which returned to approximately the preexperimental levels in both the immunized and control groups. These findings confirm the in vivo functional specificity of the MAb to S. mutans but are not consistent with the suggestion that S. sanguis or veillonella take over the niche vacated by S. mutans, unless the shift in the proportion of these organisms cannot be detected by the method used.     

The influence of oral immunization on local and systemic immune responses to heterologous antigens.

Oral immunization with 25 mg/day of ovalbumin resulted in a number of changes in non-specific immune responsiveness, including increases in antibody produced to T-dependent but not T-independent antigens and the abrogation of tolerance induced by both oral and intravenous routes. The effect was transient, sex- and strain-dependent. Evidence suggests that it is a consequence of cell-mediated immunity to the oral immunogen.     

Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene.

A recombinant Streptococcus lactis strain which carries the structural gene for a surface protein antigen (PAc) of 190,000 daltons from Streptococcus mutans serotype c was constructed for development of an oral vaccine against dental caries. The gene from S. mutans MT8148 joined to shuttle vector pSA3 was successfully transformed into S. lactis IL1403. A small amount of PAc was detected in the cell homogenate and cytoplasmic fraction of the recombinant S. lactis, but not in the culture supernatant of the recombinant, by Western immunoblotting and dot immunoblotting. The level of PAc-specific mRNA in the recombinant strain was lower than that in S. mutans MT8148. However, significant salivary immunoglobulin A and serum immunoglobulin G responses to PAc were induced in mice immunized orally with the recombinant S. lactis.     

Use of combined immunization routes in induction of a salivary immunoglobulin A response to Streptococcus mutans in Macaca fascicularis monkeys.

Various combinations of immunization routes were examined for ability to elicit or prolong (or both) a salivary secretory immunoglobulin A response to Streptococcus mutans strain Ingbritt (serotype c) in Macaca fascicularis monkeys. Intraductal (i.d.), per os (p.o.), and subcutaneous (s.c.) routes were utilized. Four groups of three to five monkeys each were immunized by the following schedules: group I--p.o., s.c., i.d.; group II--i.d., p.o., i.d.; group III--s.c., p.o., i.d.; and group IV--control. Immune responses in the serum and parotid fluid were quantitated by using passive hemagglutination assays with purified serotype-specific polysaccharide and by indirect immunofluorescent staining assays. Both s.c. and i.d., but not p.o., routes resulted in detectable serum antibody responses. Only i.d. immunization resulted in a measurable salivary response. Indirect immunofluorescent staining revealed specific secretory immunoglobulin A antibodies in the parotid fluid which correlated with passive hemagglutination titers. The p.o. procedures used in this study did not result either in a prolonged immune response or in measurable tolerance related to the humoral or secretory immune system.     

Oral adjuvants enhance IgA responses to Streptococcus mutans

The induction of immune responses to orally-administered trinitrophenyl (TNP)-haptenated Streptococcus mutans or its cell wall components and enhancement of immune responses with oral adjuvants has been studied in high IgA responsive C3H/HeJ mice and in gnotobiotic rats. Gastric intubation of TNP-S. mutans to LPS non-responsive C3H/HeJ or syngeneic, LPS responsive C3H/HeN mice induced IgA responses as determined by measuring splenic plaque-forming cell (PFC) responses and IgA anti-TNP antibodies in serum, saliva, and urine. Higher IgA responses always occurred in C3H/HeJ mice given oral S. mutans antigen than similarly treated C3H/HeN animals. Oral administration of the adjuvants concanavalin A or S. mutans cell wall peptidoglycan (PG) with antigen resulted in augmented IgA responses, especially in C3H/HeJ mice. On the other hand, oral administration of muramyl dipeptide (MDP) with antigen boosted anti-TNP responses in C3H/HeN, but not in C3H/HeJ, mice. Gnotobiotic rats given S. mutans whole cells (WC) or purified cell walls (CW) by the oral route exhibited a salivary IgA immune response which was potentiated greater than twofold when antigen was given with PG or MDP. In other studies, S. mutans WC or CW antigen in water-oil-water (W/O/W) emulsion or liposomes was administered by gastric intubation to rats. Significant salivary IgA responses were induced with these antigen-adjuvant preparations. Although rats given S. mutans WC or CW were protected from S. mutans challenge, the greatest degree of caries immunity was obtained in animals which received antigen and adjuvant and which exhibited significant salivary IgA antibody levels. In preliminary studies, it was observed that local injection of rats in the salivary gland region with a ribosomal preparation from S. mutans resulted in a significant salivary IgA response and caries immunity. The potential for soluble and lipid carrier adjuvants in oral vaccines for induction of protective antibodies to S. mutans is discussed.     

Humoral and cell-mediated responses to a ribosomal preparation from Streptococcus mutans.

Streptococcus mutans 6715 ribosomes disrupted in a Braun homogenizer were isolated in sodium dodecyl sulfate by differential centrifugation. This preparation contained 80% RNA and 20% protein, and carbohydrate was not detected by phenol-sulfuric acid and methyl pentose assays. The sedimentation coefficient of the ribosomes was 70S. After dialysis in 0.01 M phosphate buffer containing 10(-4) M MgCl2, the ribosomes dissociated into 54S and 32S particles. Leukocytes from rabbits immunized intramuscularly with the ribosomal preparation showed transformation and migration indices of 13.0 and 0.71, which were significantly different (P less than 0.05) from the respective indices of 0.9 and 0.98 in nonimmunized animals. Hyperimmune serum from these rabbits agglutinated representative Formalin-killed strains of all seven serotypes of S. mutans, inhibited adherence of live S. mutans 6715 to glass, and agglutinated S. mutans 6715 ribosomes adsorbed upon erythrocytes. These findings suggested that animals immunized with S. mutans ribosomes may be protected from caries caused by any of the seven serotypes of this organism.     

Protective salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal immunization with S. mutans antigen I/II coupled to the B subunit of cholera toxin.

The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/II), was chemically coupled to CTB (Ag I/II-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 micrograms) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag I/II and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/II response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag I/II and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag I/II responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/II-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.     

Human T-cell responses to the glucosyltransferases of Streptococcus mutans

We previously reported differential humoral responses to glucosyltransferases (GTFs), with significantly higher saliva and serum antibody levels to GtfD than to GtfB or GtfC. To test the hypothesis that cellular immune responses to these molecules also may differ, peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses in young adults and children with distinct genetic backgrounds were determined using purified recombinant GtfC and GtfD. PBMCs from all of the volunteers responded to GtfC and -D, but responses were directed predominantly towards GtfD and were major histocompatibility class II antigen dependent. A predominant T-cell response to GtfD, over GtfC, was detectable at various antigen concentrations ranging from 1 to 20 microg/ml and correlated with the differential serum immunoglobulin G (IgG) and salivary IgA antibody responses to the GTFs. Therefore, in naturally sensitized humans, Streptococcus mutans GTFs stimulate differential humoral and cellular immune responses, with the secreted form of GtfD eliciting a stronger response than the cell wall-associated form of GtfC.     

Fundus lesions after carotid injection of Streptococcus mutans in monkeys.

Beige mice, which are a homolog of the Chediak-Higashi syndrome, and mice treated with 89Sr to destroy the bone marrow provide animal models of defects in mononuclear phagocyte and natural killer cell functions. The innate resistance of these mice to viruses such as herpes simplex and encephalomyocarditis viruses, however, is normal. Moreover, treatment of the mice with immunomodulators such as Propionibacterium acnes (formerly designated Corynebacterium parvum) and pyran produced a significant increase in resistance to encephalomyocarditis virus. The antiviral effect of P. acnes in 89Sr-treated mice was exhibited during marked monocytopenia and without evidence for an inflammatory influx of macrophages into the peritoneal cavity. Treatment with P. acnes was also effective in increasing the resistance of beige mice to infection with Listeria monocytogenes. Thus, immunomodulators can be effective in mice that exhibit impaired macrophage and natural killer cell functions.     

Antibody and clinical responses in volunteers to immunization with malaria peptide-diptheria toxoid conjugates.

Twenty residue peptides from the 185-200-kD and 45-kD merozoite surface antigens of the malaria parasite Plasmodium falciparum were covalently linked to diphtheria toxoid as a carrier and used to immunize human volunteers with aluminium hydroxide as an adjuvant. Significant antibody levels were elicited by two boosting injections. The antibodies reacted with acetone-methanol fixed merozoite membranes in an immunofluorescence assay, but no inhibition of merozoite reinvasion could be detected in in vitro cultures containing the antibodies. Antibody levels against the immunizing peptides declined markedly within 77 days after the third injection. No hypersensitivity was observed against the peptides. However, the volunteers developed hypersensitivity against diphteria toxoid, and in particular a pronounced type III (Arthus) hypersensitivity after three injections with the toxoid. This effect might appear to limit the use of peptide-diphtheria toxoid conjugates for human immunization. Several biochemical, haematological and immunological tests done on the volunteers showed no other adverse effects from the immunizations.     

Antibody responses of splenectomized patients with non-Hodgkin's lymphoma to immunization with polyvalent pneumococcal vaccines.

The serum antibody responses of splenectomized patients with non-Hodgkin's lymphoma (NHL) who had been immunized with a polyvalent pneumococcal vaccine (Pneumovax 23) were evaluated by an enzyme-linked immunosorbent assay with the 23-valent pneumococcal vaccine as the antigen. A response to immunization, defined as a twofold-or-higher rise of the prevaccination titer of antibodies against Streptococcus pneumoniae polysaccharide, was elicited in 5 of 11 patients with NHL. No significant difference in the level of antibodies against S. pneumoniae polysaccharide between lymphoma patients and patients who had undergone splenectomy for other reasons was detected (P = 0.83 and 0.87 before and after vaccination, respectively). NHL patients who did not respond to the first immunization received a booster dose of the polysaccharide vaccine. This injection did not increase the pneumococcal-antibody titer significantly (P = 0.7). We conclude that vaccination with pneumococcal polysaccharides in splenectomized patients with NHL elicits an adequate antibody response in 45.4% of the cases and should therefore be administered. Revaccination of the nonresponders does not further increase the pneumococcal-antibody levels.     

Amino acid requirements of Streptococcus mutans and other oral streptococci.

The amino acid requirements of Streptococcus mutans strains AHT, OMZ-61, FA-1, BHT, GS-5, JC-2, Ingbritt, At6T, OMZ-176, 6715, Streptococcus salivarius HHT, Streptococcus sanguis OMZ-9, and strain 72x46 were determined in a chemically defined medium. When grown anaerobically in the presence of sodium carbonate (or bicarbonate for a few strains), few amino acids were required. All strains tested required cystine (or cystine) as a nutrient. Three strains (S. mutans OMZ-176, FA-1, and BHT) required glutamate (and/or glutamine). A third amino acid (lysine for S. mutans FA-1 and histidine for S. mutans OMZ-176) was required by two of the three strains which required glutamate. The amino acids mentioned above were required for all conditions of incubation (and inoculum) tested. The requirements for several other amino acids were conditional, that is, dependent on the incubation conditions and inoculum used. For example, when carbonate was not added, glutamate was required by S. mutans GS-5. Aerobic incubations, with carbonate or bicarbonate added, resluted in requirements for glutamate and leucine by several strains. With these incubation conditions, one strain required isoleucine (S. mutans FA-1), another valine (S. mutans AHT), and a third tyrosine (72x46). Aerobic incubations in the absence of carbonate or bicarbonate further increased the number of amino acids required by several strains. Furthermore, when stationary-phase cultures replaced exponentially growing cultures as an inoculum, several strains required additional amino acids, presumably for the initiation of growth.