John B. Macdonald

The microbiota of the gingival crevice area of man. I. Total microscopic and viable counts and counts of specific organisms.

Abstract Total microscopic, total aerobic and anaerobic viable counts, as well as counts of aerobic and facultative streptococci, fusobacteria, spirochetes and Bacteroides melaninogenicus per g wet weight of gingival debris were determined in normal and periodontally-involved individuals. It was found that the total microscopic counts approximated 1.7 × 10 11 organisms per g wet weight, indicating that gingival debris is composed almost entirely of bacteria. Total viable counts performed aerobically averaged 1.6 × 10 10 , while total anaerobic viable counts averaged 4 × 10 10 . This suggests that the majority of the bacterial population is obligately anaerobic. All organisms which grew aerobically were facultative. Spirochetes were the only group of organisms enumerated which increased significantly in periodontal disease.

The microbiota of the gingival crevice area of man—II: The predominant cultivable organisms

Abstract All of the cultivable organisms present in high dilutions of debris from the gingival crevice area of man have been isolated, partially characterized, and their relative proportions ascertained. No significant quantitative or qualitative differences were observed in the microbiota of normal and periodontally-involved individuals. The gingival crevice microbiota was found to be comprised as follows: 28.8 per cent gram positive facultative cocci; 20.2 per cent gram positive anaerobic rods; 16.1 per cent gram negative anaerobic rods; 15.3 per cent gram positive facultative rods; 10.7 per cent gram negative anaerobic cocci; 7.4 per cent gram positive anaerobic cocci; 1.2 per cent gram negative facultative rods; and 0.4 per cent gram negative facultative cocci. Anaerobes comprised 54.3 per cent, gram positive organisms 71.5 per cent, and bacillary forms 52.8 per cent of the cultivable organisms.

Biochemical characteristics of Bacteroides melaninogenicus: A study of thirty-one strains

Abstract Thirty-one strains of Bacteroides melaninogenicus were studied. All strains were actively proteolytic, attacking reconstituted neutral salt-extracted collagen and gelatin, and producing H 2 S. Most strains produced indol. The strains all required or were stimulated by haemin. None reduced nitrates and none formed catalase. Colony form was usually smooth, but rough variants were found: all strains produced black colonies, given an excess of haemin. Some strains were nonfermentative; some fermented only glucose, lactose and galactose; some fermented several carbohydrates other than lactose and galactose and hydrolysed starch. Some strains have an absolute requirement for menadione or related naphthoquinones.

A survey of the bacterial flora of the periodontium in the rice rat

Abstract A survey of the bacteria recoverable from the periodontium of normal rice rats and rice rats with periodontal destruction was undertaken. Forty-five strains of bacteria isolated from fourteen rice rats were examined by appropriate tests to permit generic classification. Recurring strains included enterococci, coli-aerogenes species, Actinomyces, Lactobacilli, aerobic diphtheroids, Staphylococci, and possibly Actinobacillus species. Bacteroides and Fusobacterium were recovered infrequently and spirochaetes were never seen.

Quantitative studies of the bacterial flora of the periodontium in rice rats

Abstract Eighteen 21-week old rice rats were scored for periodontal disease according to the method of Gupta and Shaw (1956). The molar teeth and periodontium of one side of the heads were weighed, and ground in broth. Dilutions determined in preliminary trials were plated in 0·1 ml amounts on each of five plates of the following media: horse blood agar, Na azide agar, nutrient agar, EMB agar, Rogosa SL agar, serum tellurite agar. The blood agar was incubated anaerobically for 5 days, other media aerobically for 3 days. Colony counts were made at 10 × magnification with a stereoscopic microscope. Total counts from growth on blood agar incubated anaerobically ranged from 3·6 × 10 7 to 3·6 × 10 10 per total sample. Counts averaged 3·4 × 10 8 for twelve animals with periodontal scores of 57 or less, and more than 1·6 × 10 10 for six animals with scores exceeding 100. Enterococci were the most numerous organisms, counts ranging from 2·0 × 10 6 to 3·5 × 10 10 . High counts, averaging 1·4 × 10 10 , were associated with high periodontal disease scores. Actinobacillus counts ranged from 1·0 to 5·6 × 10 8 for the four rice rats with highest periodontal scores. Except for one animal, remaining Actinobacillus counts averaged 6·9 × 10 6 . The only other regularly occurring organisms were diphtheroids ranging in counts from 3·0 × 10 5 to 7·3 × 10 7 and unrelated to periodontal disease. Staphylococci, coliforms and lactobacilli each appeared on plates from over half the animals, usually in the ranges 10 6 –10 7 , 10 4 –10 5 , 10 3 –10 6 , respectively. Fusobacterium , although observed only twice, numbered 1·2 × 10 8 in one animal.

Pathogenicity Experiments with the Flora of the Periodontium in Rice Rats

Periodontal disease in the rice rat has been shown to be histologically comparable to periodontal disease in man (Gupta and Shaw, Oral Surg., 9:592, 1956). However, it has been demonstrated that the resident flora of the periodontium in the rice rat differs strikingly from that of man (Macdonald, Socransky, and Sawyer, Arch. Oral Biol., 1:1, 1959). Since the gingival flora of periodontal disease in man is capable of producing characteristic mixed anaerobic infections in the subcutaneous tissues of the guinea pig (Rosebury and Foley, J.A.D.A., 26:1791, 1939), it appeared worthwhile to determine whether or not the periodontal flora in the rice rat would produce similar infections. The molar teeth and associated periodontium from 11 rice rats, all with periodontal disease, were removed, and in each instance diluted 100 fold in heart infusion broth and ground in a glass tissue grinder. The 11 suspensions were injected subcutaneously into the groin of 350-gm. guinea pigs. Each suspension was injected into two animals in doses of 1.0 and 1.5 ml. In addition, 6 of the suspensions were cultured anaerobically as unstreaked drops on blood agar plates for 5 days. The growth from 2 drops was suspended in 3-ml. heart infusion broth and injected in 1-ml. amounts into 2 guinea pigs. None of the total of 34 guinea pigs developed typical mixed infection of the type seen when gingival scrapings from man are used as inoculum (Rosebury and Foley, J.A.D.A., 26:1791, 1939). Seventeen guinea pigs developed no lesions in 7 days; 5 showed small abscesses (less than 1 cm. in diameter) filled with caseous exudate. All 12 guinea pigs injected with uncultured or cultured suspensions from 3 of the rice rats developed spreading acute cellulitis with little exudate. Escherichia coli was recovered in almost pure culture from these lesions. Pure culture of the organism in each instance was found to be capable of eliciting similar inflammatory lesions when injected into guinea pigs. These results provide evidence of additional fundamental differences between the flora of periodontal disease in man and in the rice rat. Quantitative studies of the rice-rat periodontal flora have demonstrated that bacterial counts increase with increases in the severity of periodontal disease; the higher counts are largely attributable to increased numbers of enterococci (Socransky, Macdonald, Sawyer, and Auskaps, Arch. Oral Biol., in press). The findings suggested the possibility of special significance of enterococci in the pathogenesis of periodontal disease in the rice rat. It therefore appeared desirable to determine the pathogenicity of these organisms in the subcutaneous tissues of this animal. Five strains of enterococci isolated from the oral cavity of rice rats were grown in trypticase soy broth. The cells were suspended in saline to give concentrations of 1, 5, and 10 X 109 cells/ml. Each strain was injected subcutaneously into the groin of young adult rice rats. One rice rat was inoculated with 1 ml. of bacterial suspension at each dosage level. Three of the strains, in addition, were heat-killed (70° C. for 60 min.) and inoculated into rice rats in the same dosage as above. All the animals were examined daily and sacrificed at 4 days. No lesions developed in the control group injected with heat-killed enterococci. All but one of the 15 rice rats injected with viable cultures developed local abscesses containing a small amount of very thick exudate from which enterococci were cultured. One animal died during the first 24 hours and had inflammation and adhesions at the site of infection. These findings indicate that the enterococci are capable of infecting the subcutaneous tissues of the rice rat and lend further support to the view that these organisms may be significant in the pathogenesis of periodontal disease in the rice rat.