Association of the microbial flora of dental plaque and saliva with human root-surface caries.

Dental plaque was obtained from one or two sound root surfaces of subjects with different degrees of root-surface caries experience. From subjects with root-surface caries, plaque samples were also obtained from either one incipient or one more advanced lesion. Proportions of the total flora were determined for total streptococci and different streptococcal species, total and different Actinomyces species, and lactobacilli. A sample of saliva was obtained from about one-third of the subjects for determination of the concentrations of mutans streptococci and lactobacilli. The main observations were: (1) Subjects without root-surface caries or restorations (group I), as compared with subjects with root-surface caries with or without restorations (group II), were characterized by having a lower prevalence and proportion of mutans streptococci and a higher prevalence and proportion of A. naeslundii in plaque on sound root surfaces; (2) subjects in group I also tended to have a lower salivary concentration of mutans streptococci and lactobacilli than subjects in group II; (3) dental plaque on sound surfaces in group II subjects contained a lower proportion of mutans streptococci than plaque associated with incipient or advanced lesions; and (4) the prevalence and proportion of lactobacilli in plaque associated with sound as well as carious root surfaces were very low. The data reinforce findings from other studies and indicate that, as for coronal caries, the plaque and saliva populations of mutans streptococci specifically are correlated positively with the presence of root-surface caries.

A simplified diagnostic system for cultural detection and enumeration of Streptococcus mutans.

A simple dip-slide test (Cariescreen SM) based on MSB selective agar was devised for detection and quantitation of Streptococcus mutans in oral samples. For this test, a bacitracin tablet is dissolved in a vial containing buffered saline diluent. Paraffin-stimulated saliva is collected in this diluent vial. A dip slide which incorporates a modified MSB agar (minus bacitracin) is immersed briefly in the diluted saliva. After addition of a CO2-generating tablet, the screw-cap dip slide is closed tightly in the vial and incubated for two days at 37 degrees C and one day at room temperature. S. mutans populations in saliva are estimated by comparison with a colony density chart. Growth of reference strains of S. mutans was equivalent on Cariescreen SM dip slides and on MSB agar plates. Reference strains of Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis, and Streptococcus milleri did not grow on Cariescreen SM dip slides. Aliquots of saliva from 50 schoolchildren and 51 adults were tested by the dip-slide method and by conventional plating methods in MSB agar. Very good correlation (0.93) between the two methods was obtained. This simplified S. mutans detection system is suitable for use by clinical personnel in dental clinics or other non-laboratory settings for identification of subjects potentially at risk for caries.

Colonization of Syrian hamsters with streptomycin resistant Campylobacter jejuni.

A streptomycin resistant Campylobacter jejuni inoculated per os into two populations of Syrian hamsters (one endemically harboring C. jejuni, the other free of C. jejuni) established chronic colonization of the organism in both groups. Diet, steroid administration, age of hamsters or prior exposure to C. jejuni did not appreciably alter incidence of diarrhea or colonization of C. jejuni. The majority of hamsters sampled during the course of the experiment (1 to 22 weeks) shed streptomycin resistant C. jejuni in the feces. In four hamsters sampled at 14, 17, 19, and 22 weeks, post inoculation, streptomycin resistant C. jejuni were recovered in ileal, cecal, jejunal, duodenal and colonic contents (10(4) to 10(7) colony forming units/gram of intestinal content). The hamster appears to be a potentially useful model for the study of intestinal colonization of enteropathogenic C. jejuni. Hamsters shedding C. jejuni in their feces for extended periods of time should be considered a zoonotic threat to both pet owners and laboratory personnel.

Infectivity and natural transmission of the bacterium Streptococcus mutans in monkeys (Macaca fascicularis) at different ages.

Oral inoculation of adolescent monkeys with laboratory strains of Streptococcus mutans resulted in only sporadic implantation. Oral colonization of all strains, except possibly one, was not enhanced by dietary sucrose compared with glucose nor by precleaning of accessible tooth surfaces. Infant animals were more susceptible to implantation by laboratory-maintained strains of Strep. mutans than adolescent or adult animals. This difference was magnified when in-vivo maintained Strep. mutans strains were used. Strep. mutans was readily acquired by uninfected adolescent animals from infected cage mates. Strep. mutans-free adolescent monkeys exhibited significant-salivary IgA and serum-IgG response to Strep. mutans antigen, possibly representing a cross-reaction to indigenous Streptococcus sanguis. Uninfected infant animals showed no salivary or serum response to Strep. mutans. There was no change in salivary IgA antibody to Strep. mutans in adolescent animals after implantation or in recipient animals after acquisition of Strep. mutans.

Transmission of the oral bacterium Streptococcus mutans to young Macaca fascicularis monkeys from human nursery attendants.

Streptococcus mutans was detected in the mouths of 2 out of 7 infant monkeys that had been delivered by caesarian section and nursery-reared without contact with their mothers or other monkeys. Their human nursery attendants all harboured Strep. mutans biotype c (c serotype) as found in the monkeys. Bacteriocin studies also indicated similarity between the animal and human isolates. These findings suggest that the infant monkeys acquired Strep. mutans from the nursery personnel. Evidence for the reverse transmission of enterococci, from monkeys to man was also obtained.

Enhancement of experimental actinomycosis in mice by Eikenella corrodens.

The infectivity of Actinomyces israelii in a susceptible-weanling-mouse was increased by the presence of Eikenella corrodens in the inoculum. A minimal infecting dose of 1.7 X 10(7) CFU of A. israelii was required to establish chronic lesions after an intraperitoneal injection. When E. corrodens (3.8 X 10(7) CFU) was included in the inoculum, chronic lesions were established with a dose of 8.5 X 10(4) CFU of A. israelii. E. corrodens alone did not produce persistent lesions. Viable E. corrodens could be recovered from chronic mixed actinomycotic lesions in numbers that often equaled or exceeded the populations of A. israelii in the lesions. The duration of acute actinomycotic infections caused by A. viscosus was temporarily extended by the presence of E. corrodens. The cellular inflammatory response and overall morphology of mixed experimental lesions containing A. israelii and E. corrodens did not appear to be significantly different from those of pure-culture lesions containing A. israelii alone. E. corrodens cells could not be readily discerned in stained histological sections of mixed experimental lesions.

Persistence of associated gram-negative bacteria in experimental actinomycotic lesions in mice.

Mixed actinomycotic infections were established in a susceptible weanling mouse model by using combinations of Actinomyces israelii and Eikenella corrodens or A. israelii and Actinobacillus actinomycetemcomitans. Acute lesions caused by either of the gram-negative organisms alone were resolved within a few weeks; however, these organisms persisted up to 3 months in chronic lesions in combination with A. israelii.

Comparative histopathology of lesions produced by Actinomyces israelii, Actinomyces naeslundii, and Actinomyces viscosus in mice.

The histopathologic features of experimental actinomycotic lesions produced in mice by Actinomyces israelii, Actinomyces naeslundii, and Actinomyces viscosus were examined. In lesions caused by A israelii the outer edge of the bacterial granule exhibited an eosinophilic fringe with no evidence of penetration of polymorphonuclear leukocytes (PMNs) into the bacterial granule. Chronic lesions after 6 weeks contained lobulated advancing fronts as well as areas of resolution showing heavy penetration by phagocytic cells. The number of macrophages and plasma cells in these lesions increased with time. In contrast, lesions caused by A viscosus and A naeslundii showed cellular evidence of resolution during the early stages of the infection (3-6 weeks). The bacterial core was readily penetrated and fragmented by PMNs in early A viscosus lesions. In lesions caused by A naeslundii there was less penetration of the central core by PMNs, and the bacterial granule tended to retain its structural integrity. Elongated crystals of hyaloid material appeared in lesions caused by all species. These protein-rich bodies appeared to be associated with resolving areas of the lesions. The observed contrast in the histopathologic appearance of actinomycotic lesions caused by A israelii, A naeslundii, and A viscosus is suggestive of important differences in the immune response of the host to infections caused by these three species.

Oral colonization and pathogenicity of Actinomyces israelii in gnotobiotic rats.

Reference strains of A. israelii were orally implanted in germ-free rats after multiple inoculations. Early colonization was associated with areas of interdental hair and bedding impactions. Largest populations of organisms were isolated from teeth, as compared to the tongue, cheek, or palate. Examination of molar teeth with the scanning electron microscope revealed bacterial accumulations in the occlusal fissures, but not on smooth or approximal surfaces. Extensive plaque formation after 100-150 d was accompanied by root-surface caries and bacterial invasion of the pulp. There was no evidence of bacterial penetration of the dentin underlying surface enamel.

Comparative pathogenicity of Actinomyces species in mice.

The comparative pathogenicity of different species of Actinomyces was studied in a susceptible weanling-mouse model. After the intraperitoneal injection of strains of Actinomyces israeli, A. naeslundi, A. viscosus and Arachnia propionica, numerous abscesses developed in the intestine, mesentery, liver, and at the site of injection. Lesions were not produced by A. odontolyticus. A. naeslundi and A. viscosus produced acute lesions that resolved after a few weeks. Abscesses produced by rough strains of A. israeli and Arach. propionica persisted and led to a slowly progressive chronic infection. Viable organisms were always recovered from the lesions. Spread of the lesions by extension into other areas, including the thoracic cavity, led to the death of the animal after approximately 1 year. This study demonstrated a clear difference in the pattern of infection produced by the different species of Actinomyces as well as Arach. propionica.

Development of a selective medium for detection and enumeration of Actinomyces viscosus and Actinomyces naeslundii in dental plaque.

A selective medium (CFAT) was developed for the detection and enumeration of Actinomyces viscosus and Actinomyces naeslundii in dental plaque. Neutral acriflavin and potassium tellurite were used in combination with the known selective agents cadmium and fluoride to eliminate most of the competing plaque flora. Composition of CFAT per liter was as follows: Trypticase soy broth (BBL Microbiology Systems), 30 g; glucose, 5 g; agar, 15 g' cadmium sulfate, 13 mg; sodium fluoride, 85 mg; neutral acriflavin, 1.20 mg; potassium tellurite, 2.50 mg; basic fuchsin, 1.25 mg; defibrinated sheep blood, 50 ml. A. viscosus reference strains of human origin grew on CFAT without reduction in numbers under an atmosphere of 90% air-10% CO2. Animal strains of A. viscosus were inhibited at the level of cadmium in CFAT. Two of six A. naeslundii strains did not grow on CFAT. Improved recovery of A. viscosus and A. naeslundii from dental plaque occurred on CFAT, as compared with two other selective media which contained either cadmium sulfate or sodium fluoride, respectively, as selective agents. CFAT was more selective with regard to much of the extraneous gram-positive flora. Bacterionemia, Neisseria, yeasts, and streptococci were virtually eliminated.

Simple and convenient method for culturing anaerobic bacteria.

A simple and convenient method for culturing anaerobic bacteria is described. Cultures can be grown in commercially available flasks normally used for preparation of sterile external solutions. A special disposable rubber flask closure maintains anaerobic conditions in the flask after autoclaving. Growth of a variety of anaerobic oral bacteria was comparable to that obtained after anaerobic incubation of broth cultures in Brewer Anaerobic Jars.

Bacteriocin-like activity within the genus Actinomyces.

Actinomyces strains were tested for evidence of bacteriocin-like antagonism. Prevalence of inter-strain antagonism was low, and, when present, zones of inhibition were generally small and poorly defined. Strains of A. viscosus were very susceptible to bacteriocin-like inhibition by S. mutans. Prevalence of inhibition was high, and zones of inhibition tended to be large and well defined. Inhibitors produced by S. mutans were generally heat stable, nondialyzable, and sensitive to proteolytic enzymes. Individual strains of A. viscosus could be differentiated on the basis of sensitivity patterns to a battery of S. mutans strains.