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Summary1. Differences in the vitamin requirements of susceptible strains of S. mitis, their artificially-induced resistant variants and a naturally-resistant S. fecalis, have been determined. 2. The greatest difference was in the PGA requirements, the mitis strains grew in its absence; the resistant mutants required PGA. 3. The induced resistance of the trained S. mitis strains appears to be the same as that of the natural resistance of S. fecalis, and may be due to the loss of ability to synthesize PGA, the synthesis of which is inhibited by sulfonamides.

Tube dilution studies concerned with Thiostrepton were performed on a series of strains of each of the following species of microorganisms: various nonhemolytic streptococci (twelve strains), hemolytic streptococci (ten strains), Staphylococcus aureus (ten strains), Staphylococcus albus (ten strains), and seventeen other selected microorganisms. Tube dilution studies with Thiostrepton against the above-mentioned organisms indicated a marked inhibitory capacity of this antibiotic in extremely low concentration against sensitive microorganisms. The minimum inhibitory concentration of Thiostrepton against the various microorganisms studied after twenty-four hours was as follows: For Staphylococcus aureus, a range of 0.0300 mcg. to 0.0035 mcg. per milliliter, with a median of 0.0150 mcg. per milliliter; for Staphylococcus albus, a range of 0.0625 mcg. to 0.0156 mcg. per milliliter, with a median of 0.0312 mcg. per milliliter; for Streptococcus pyogenes Group A, a range of 0.1250 mcg. to 0.0019 mcg. per milliliter, with a median of 0.0039 to 0.0156 mcg. per milliliter; for nonhemolytic streptococci—Streptococcus faecalis (five strains), a range of 0.5000 mcg. to 0.0078 mcg. per milliliter, with a median of 0.0156 mcg. per milliliter; for Streptococcus mitis (five strains), a range of 0.5000 mcg. to 0.0156 mcg. per milliliter, with a median of 0.0250 mcg. per milliliter; for Streptococcus salivarius (two strains), a range of 1.0000 mcg. per milliliter for one organism and 0.0125 mcg. per milliliter for the other. Eighteen other microorganisms tested, including three E. coli, five A. aerogenes, four salmonella species, four proteus species, one Pseudomonas aeruginosa, and one Mycobacterium phlei were resistant to Thiostrepton in a concentration of one mcg. per milliliter. Five organisms, including one each of pneumococcus Type 1, pneumococcus Type 2, pneumococcus Type 3, Neisseria catarrhalis, and Corynebacterium xerose, were found highly sensitive to Thiostrepton. Further laboratory and clinical studies with Thiostrepton are indicated.

The salivary flora of 204 children and adults who received systemic antibiotics by mouth over a long period of time was compared to 140 siblings and spouses who had not. Adults and children received 200,000 units of penicillin daily for the prophylaxis of recurrent rheumatic fever. Another group of children received 1–3 million units of penicillin daily plus 1.0 g of tetracycline occasionally for the treatment of chronic respiratory disease. The numbers of viable bacteria from representative salivary microbial categories were compared using selective and enriched media. There was little or no difference in the total cultivable flora on aerobic and anaerobic incubation, the Veillonella, Lactobacillus , salt-tolerant staphylococci and Candida organisms. The facultative streptococcal population was 24 per cent less for patients receiving antibiotics than those who did not, and was the same for subjects receiving antibiotics on the two dosage levels and for adults and children receiving antibiotics. The reduction in the streptococcal population was attributed to Streptococcus salivarius .

Summary. The survival of bacteria in specimens of human faeces transported and stored at different temperatures was examined quantitatively. No significant losses of bacteroides, bifidobacteria, viridans streptococci, Streptococcus salivarius, enterococci, enterobacteria, and clostridia were observed when 10% (w/v) suspensions of faeces in broth containing 10% (v/v) of glycerol were frozen for one month at −25, −78 or −196°; lactobacilli showed significant losses at −196°; veillonellae showed significant losses at −25°. At −12° veillonellae and enterobacteria showed significant losses. Most bacteria survived well for 24 h in undiluted faeces sent by post.

Highly diluted feces, obtained from healthy adult individuals, was plated on blood-agar plates which were incubated both aerobically and anaerobically. From the anaerobic plates containing 30 to 60 colonies, every colony was subcultured. Nearly all isolates were obtained in pure culture and partially characterized. It was found thatBacteroides species were the most predominant organisms, being present in numbers approximating 1010 per gram wet weight. Selected bacteria present in lower numbers were determined by plating appropriate dilutions of feces on selective media. It was found that coliforms, streptococci and lactobacilli were regularly present in concentrations of 106 − 108 organisms per gram wet weight material, whileVeillonella, Streptococcus salivarius, Bacteroides melaninogenicus and staphylococci were present in lower numbers. Fusobacteria were only found in one sample, whileNeisseria were not detected in any of the samples. Wet mounts of fecal material, inspected by darkfield microscopy, did not reveal the presence of spirochetes. Anaerobes outnumbered facultative bacteria by a factor of 40, indicating that the human adult fecal flora is predominantly anaerobic. Total microscopic counts indicate that bacteria comprise approximately 30% of the mass of human feces.

In 20 edentulous persons wearing full dentures the prevalence of Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius was studied in samples of the bacterial plaque on the dentures and in samples of the saliva. Streptococcus sanguis could be recovered from 17 and Streptococcus mutans from 14 of the subjects. These organisms constituted a higher percentage of the streptococcal flora in the denture plaque than in saliva. Seven subjects refrained from using their dentures for 2 days after which the number of these streptococci was so few in saliva that they could not be recovered with the methods used. Streptococcus salivarius was present in all 20 subjects and constituted a higher percentage of the streptococcal flora in the saliva than in the denture plaque. The composition of the streptococcal flora on the dentures was similar to that previously found on teeth. The accumulated evidence on the ecology of Streptococcus sanguis and Streptococcus mutans indicate that these streptococci can only be established in the mouth if solid surfaces such as on teeth and dentures are present.

The type specific immunological properties of certain non-hemolytic streptococci, including Str. salivarius type I and type II, present in the respiratory tract of human beings appear to be dependent upon the presence of capsular polysaccharides. The levans formed from sucrose by Str. salivarius (encapsulated S cells or non-encapsulated R variants), or by cell-free enzymes derived from these microorganisms, are indistinguishable immunologically and show no evidence of type specificity. Such levans appear to be immunologically distinct from and unrelated to the capsular polysaccharides of the microorganisms which produce them.

ConclusionsA medium has been developed on which a specific oral streptococcus can be selectively isolated from mixed bacterial populations. The value of this selective medium as a detector of oral contamination of restaurant eating utensils is indicated by the distribution studies, which show that this bacterium is found normally in the human mouth and on articles which have come in direct contact with human lips.

SummaryAn improved technic for demonstrating the presence of oral streptococci on washed glasses is presented. The procedure involves primary enrichment of swab cultures in azide broth followed by inoculation onto sucrose-crystal violet-postassium tellurite agar. The simplicity of the technic, the ease of preparation of the media involved and the readily distinguishable colonies formed by S. salivarius on the selective agar substrate makes this procedure applicable to routine examination of restaurant ware. The data obtained by such routine examinations has public health significance which cannot be too strongly emphasized.

The most numerous cultivable bacteria present on the dorsum of the tongues of six individuals were isolated and partially characterized. On the basis of 409 isolates examined, the tongue was found to contain a relatively constant microflora which was composed of the following microbial groups: facultative streptococci, 38.3 per cent; Veillonella, 14.5 per cent; facultative and anaerobic diphtheroids, 13.0 per cent and 7.4 per cent respectively; micrococci-staphylococci, 6.5 per cent; Bacteroides, 5.3 per cent; Peptostreptococcus-Peptococcus, 4.2 per cent; Neisseria, 2.3 per cent; vibrios, 2.1 per cent; Fusobacterium, 0.8 per cent; unidentified gram negative rods and cocci, 3.2 per cent and 2.6 per cent respectively. The recovery of total facultative streptococci, other than Streptococcus salivarius, was favoured by anaerobic incubation. The recovery of S. salivarius was either indifferent or slightly favoured by aerobic incubation.

SUMMARY: Extraction of Streptococcus salivarius NCTC 8606 with formamide yielded a mixture of carbohydrate antigens which was fractionated by alcohol precipitation into a type antigen (containing rhamnose, glucose, galactose) and a group-like (z) antigen. The type antigen is related to type antigen III in group F streptococci. After partial hydrolysis five disaccharides and two trisaccharides were isolated and identified. Five of these were inhibitory in the quantitative precipitation of the type antigen. Two other strains of Streptococcus salivarius contained different group-like antigens and lacked the type antigen isolated from strain NCTC 8606.

Gnotobiotic rats infected orally with various human cariogenic Streptococcus mutans strains or a Streptococcus salivarius strain exhibited greater alveolar loss of bone than control rats. The same strains also showed considerable differences in their cariogenic activity. There was a positive correlation between the extent of bone loss and carious surfaces.

Sucrose labeled in the fructosyl ( 3 H) and glucosyl ( 14 C) moieties was used to quantitate extracellular polysaccharide production and degradation by cariogenic and noncariogenic oral streptococci. All of the strains produced glucan and fructan. Streptococcus salivarius produced primarily fructan, whereas S. mutans and S. sanguis produced more glucan than fructan. The cariogenic streptococci could degrade the fructan produced by noncariogenic strains. Although the soluble glucans from all of the strains were sensitive to dextranase, the insoluble glucan from S. mutans could be distinguished from the S. sanguis insoluble glucan by its greater resistance to this enzyme.

The identity of the polysaccharide antigens of Streptococcus mg and streptococci with group antigen F and type antigen III is shown by precipitation reactions with formamide extracts, cross-absorption tests, sugar composition and by the inhibition of the quantitative precipitation reaction. On this basis Streptococcus mg can be classified as an f III strain. The relationship between Streptococcus mg and Streptococcus salivarius type I is explained by their common reactions with anti-III serum. Inhibition of the quantitative precipitations with simple sugars suggest that the determinant groups of the type III and the ‘salivarius’ antigen are not quite identical, although both antigens contain a β-glucosidic and a galactosidic group. A second unrelated antigen has been found in some ‘salivarius’ strains alone (represented by strain 51) or together with type III antigen. The quantitative precipitation of the strain 51/anti-strain 51 system was only slightly inhibited by rhamnose. The qualitative composition of the sugar components of the formamide extracts of Streptococcus mg, f III, both ‘salivarius’ types of 0 III are compared.

The cultures confirmed the findings of direct smear examinations although the relative number of the various organisms was affected by culturing. Doderlein bacillus grows very scantily, and the gram-positive nonhemolytic streptococcus cultures luxuriantly. No group of strict anaerobes was found, although the gram-positive streptococcus and the gram-negative bacillus grew readily under anaerobic as well as aerobic conditions. The only organisms found with any degree of consistency in all patients with Trichomonas vaginalis vaginitis were the Doderlein bacillus and the gram-positive nonhemolytic streptococcus. These organisms were likewise found in about the same proportions in all cases in which no Trichomonas vaginalis were found. The streptococcus, in agreement with the experience of Curtis, appeared in a diplococcic form in direct smear examination and was either oval or lancet-shaped. It formed chains when grown on artificial culture media. A number of streptococci isolated from our cultures were subcultured in sugar (lactose, mannite, salicin, and saccharose) and according to Holman's 9 classification, belonged to the following groups: Streptococcus ignavus, Streptococcus salivarius, and Streptococcus nonhemolyticus III. Other bacteria found less consistently in both groups were B. coli, a staphylococcus, B. mucosus, diphtheroids, a small gram-negative diplococcus (not gonococcus), a small gram-negative bacillus not of the typhoid-coli group, a thin gram-negative bacillus (Vibrio), a large gram-positive spore-bearer, and one large gram-positive hemolytic streptococcus. No cases with gonorrheal infection were found among the group with Trichomonas vaginalis vaginitis and none were used for control cases. Direct smear examination, verified by cultures, revealed the presence of yeasts in only 6 of our 59 patients. One of these had Trichomonas vaginalis vaginitis; another had a previous history of trichomonas, but she was apparently cured; 2 had Trichomonas vaginalis vaginitis complicating pregnancy; and 1, who showed no evidence of trichomonas infection, had a profuse leucorrhea. It is apparent from this study that yeasts were not responsible for the vaginitis in the group of women under our observation. It is also obvious that inasmuch as the same group of bacteria was found in the vaginal secretion in the presence of Trichomonas vaginalis as in that without the parasite, the flagellate is responsible for the altered clinical picture. From our clinical and laboratory studies of more than one hundred cases of Trichomonas vaginalis vaginitis over a period of approximately eighteen months, we believe the following conclusions may be drawn

SUMMARY: A survey of 91 strains of Streptococcus salivarius showed that 72 could be classified as type I or II. Only type I strains reacted with group K antiserum. The component responsible for type specificity is a cell-wall polysaccharide composed in each case of galactose, glucose, rhamnose and a trace of glucosamine. However, differences in polysaccharide structure are indicated by differences in the rate of release of soluble carbohydrate by dilute acid. Soluble cell-wall products were obtained by digesting wall with a Streptomyces enzyme preparation and used for serological studies. Galactose was the most effective monosaccharide inhibitor of both type I and type II precipitation. Further investigation suggested that type I specificity depends on the grouping O-β-D-galactopyranosyl-(I → 6)-D-galactose.

A single serological type of non-hemolytic streptococcus, designated streptococcus MG, has been isolated from the lungs of fatal cases of primary atypical pneumonia, from the sputa of patients with this disease, and occasionally from the respiratory tracts of normal human beings. Certain biological characteristics of this microorganism have been studied. All of the 59 strains isolated have been shown to belong to a homogeneous bacteriological group with characteristics which serve to distinguish it from any other well defined species of streptococcus. It has been suggested that this microorganism probably represents a distinct and hitherto undifferentiated species of streptococcus.

Streptococcus strain 1A, which closely resembled Streptococcus salivarius was isolated from human dental plaque. It was found to form gelatinous plaque deposits and to induce dental

In a previous paper, one of us1 described several pleomorphic strains of Streptococcus salivarius in tonsillitis, and in the course of routine work on meningococcus carriers, we made note of the relative frequency of pleomorphic streptococci and determined their classification. The organisms formed chains uniformly, under anaerobic conditions they remained small, and pleomorphism was constant under aerobic conditions. They had the same morphologic and cultural characteristics as many of the streptococci recently described by Rosenow and his associates2 in poliomyelitis. In view of their interesting results we thought it worth while to compare the results of animal inoculations and immunological reactions. This seemed desirable as our results suggest an extensive distribution of the organisms. The cultural characteristics of the strains we studied are given in Table 1.

An improved system is presented for measurement of interactions between a number of individual bacterial species. In the procedure, steady-state populations are fed into a common-mixed-culture vessel. Generation times of each species are determined under these conditions and contrasted with generation time in an identical situation in pure culture. Populations for generation time are determined with the aid of differential media. The mechanical system includes three types of peristaltic pumps for media feed and a unit for measurement, recording, and/or control of pH. A new type of anaerobic continuous-culture vessel which can be inoculated, sampled, and fed continuously is also described. A functional test of a three-part system including Streptococcus salivarius, Veilloncella alcalescens, and Staphylococcus aureus is presented. An unusual feature was the finding that, under certain conditions, the generation time of S. Salivarius was less than 10 min.

Reciprocal transformation reactions to streptomycin resistance have been demonstrated between pneumococcus and 2 strains of viridans streptococci, which by the usual bacteriological criteria are distantly related to pneumococcus. Desoxyribonucleic acid (DNA) extracts of a strain of streptomycin-resistant Streptococcus salivarius have been found capable of transforming pneumococcus and two strains of viridans streptococci but the strain itself was not transformed by autologous or heterologous DNA extracts. The two strains of viridans streptococci were more efficient than pneumococcus as receptors of both autologous and heterologous transforming principles. It is inferred, therefore, that efficiency of transformation does not necessarily indicate closeness of relationship among bacteria. The efficiency of transforming principles (TP's) from three streptococcal strains to induce streptomycin resistance in pneumococcus increased following their reduplication in pneumococcus. At the same time the efficiency of two of these TP's to transform streptococcus was reduced. Resistance to optochin (ethylhydrocupreine), a characteristic of most streptococci, can be transferred to pneumococcus, a naturally susceptible species, by transformation reactions.

Of the 18 oral streptococci studied, 6 were cariogenic strains of Streptococcus mutans , while the other 12 were of unknown cariogenicity. All strains were grown aerobically and the vitamin requirements were determined by omitting in turn each of 8 B-group vitamins from a complete synthetic medium. Single transfer was used to show the non-essential nature of some of the vitamins. Pantothenate and nicotinic acid were essential or stimulatory for the growth of all strains but none required folic or p -amino-benzoic acids. The use of 2 other synthetic media suggested that the vitamin requirements of most strains were not influenced by the composition of the medium. With the possible exception of Streptococcus salivarius , the vitamin requirements do not indicate species or group divisions, nor do they appear to be related to cariogenicity. Moreover, these observations cannot account for the preferential colonization of various sites in the oral cavity by the streptococcal species.

A total of 108 strains of viridans streptococci from subacute bacterial endocarditis, and 99 strains from human throats and extracted teeth have been studied, and approximately two-thirds of all the strains so far have been differentiated into a number of serological groups. The similarity between strains from these sources is evident from the fact that approximately 50 per cent of the reacting strains from each source fall into two groups I and II, and that three groups I, II, and IV, contain streptococci from both sources in approximately the same distribution. Fifteen vaginal strains of viridians streptococci failed, with the exception of one strain, to react with groups I–IV of the endocarditis serums. More than three-fourths of the strains from both sources were Streptococcus salivarius as determined by Sherman's criteria. However, no correlation between the biochemical and serological classification could be made. It may be concluded that the viridans streptococci are amenable to classification by serological methods. The results so far obtained do not indicate a serological difference between strains of viridans streptococci isolated from subacute bacterial endocarditis, and those isolated from human throats and extracted teeth. The greater definitive value of serological methods in the identification of a particular strain is indicated.

Complete replacement of sucrose by glucose resulted in a higher incidence of dental caries in gnotobiotic rats inoculated with Lactobacillus casei, Streptococcus salivarius, Streptococcus faecalis, or Streptococcus FA-1. Caries was observed only in the occlusal sulci and there were no observable accumulations of dental plaque. A series of thirteen experiments was performed. There was considerable variation from one experiment to another. Mean scores of 18.0 were obtained in one experiment only to be followed by a mean score of zero although the same strain of rats, the same diet, and the same micro-organism were used in a repeat experiment. Biologic variation is a factor which must be considered even in gnotobiotic experiments.

Sodium fluoride (0.48 mM) completely inhibited intracellular polyglucose synthesis by resting cells of Streptococcus salivarius incubated anaerobically with glucose. Inhibition was complete if fluoride was preincubated with the cells before the addition of the substrate or if added during active synthesis. However, the degradation of the intracellular material in the absence of exogenous glucose continued at an appreciable rate at 15 times this NaF concentration. The degree of fluoride inhibition of both polyglucose degradation and synthesis was increased as the pH of the reaction mixture decreased. The rate and quantity of the intracellular polysaccharide synthesized during subsequent incubations with glucose in the absence of NaF was not reduced significantly by preincubating the cells with NaF up to 2.4 mM. However, both parameters were reduced by prior exposure to 4.8 mM NaF.Fluoride-resistant cells, obtained either by stepwise selection or by ultraviolet-induced mutation, synthesized polyglucose in the presence of 2.4 mM NaF at rates and amounts only slightly less than those of control cells incubated in buffer free of this inhibitor. Furthermore, the ability of resistant cells to synthesize polyglucose in the presence of high NaF levels was retained despite growth for 300 generations in medium free of sodium fluoride. The ultraviolet-induced strains synthesized polyglucose at greater rates and amounts than the stepwise selected cells, suggesting that they differ in the nature of their resistance.

The proportions of Streptococcus salivarius, and other zooglea-producing streptococci of the total facultative streptococci on Mitis-salivarius agar were determined in samples obtained from the lingual surfaces of both central upper incisors of nine subjects within 30–120 min after thorough prophylaxis. The percentages of Strep. salivarius on the tooth surfaces were much lower than those in samples of saliva or of the tongue tip obtained simultaneously. On the other hand, the percentages of other zooglea-forming streptococci, most of which were considered to be Streptococcus sanguis, were very high on the tooth surfaces but much lower in the saliva and tongue tip samples. The observed differences in proportions of these organisms in the initial phase of plaque formation are considered to be due to differences in ability to adhere to the tooth surface rather than in ability to grow on the tooth surface. These observations seem to provide an explanation for the relative proportions of Strep. salivarius and Strep. sanguis in mature dental plaque.

Tongue and plaque samples from seven of twenty-two caries-susceptible persons produced a thick, gelatinous, plaque on wires. Neither tongue nor plaque samples from the remaining fifteen subjects produced a similar type of plaque in vitro. The formation of this characteristic artificial plaque was attributable to a Streptococcus salivarius variant. The tongue appeared to be a major source of these variants. Following 2 weeks of tongue brushing, these organisms could no longer be isolated from either tongue or “newly” formed plaque. These results suggest that numbers and types of plaque organisms are altered by a regimen of daily tongue brushing.

Three different strains of the common oral inhabitant, Streptococcus salivarius, were atomized into the Naval Biomedical Research Laboratory’s Programmed Environment, Aerosol Facility controlled at 82 F (28 C) and at eight relative humidity values ranging from 0 to 92%. The ability of these organisms to remain viable under such conditions, with a negligible drop in viable count during an observation period of 12 hours, is indicative of the hazard one may encounter in a dental office.