Salam A. Syed

The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects

Sunnnary-Tooth surface plaque was removed from 11 institutional~ed individuats and cultured on agar plates in an anaerobic chamber. The recovery of organisms on a dilute trypticase yeast extract medium (MMIO), incubated anaerobically, averaged 33 & 26 per cent of the microscopic count. Growth on MMlO, under aerobicconditions averaged 8 i: 5 per cent. The anaerobe to aerobe recovery ratio on medium MM10 was about 4. Six hundred and seventy-one isoiates grew on subculture and were partially characterized. About half the isolates were not capable of lowering the pH in glucose broth below 5 * 5. Streptococci accounted for about 38 per cent of the isolates and were found in each subject. A sub-group of 15 strains grew in 40 per cent bile, formed NH3 from arginine, fermented salicin, but not inulin. These isolates possessed characteristics of Streptococcus sang& and Streptococcus mitis. Various Actinomyces species comprised about 14 per cent and Clo~tr~iam species accounted for 8 per cent of the cultivable flora. Bacteroides melaninogenicus, Fusobacterium species, and Veillonella species each were about 6 per cent of the isolates. The overall character of these plaque isolates suggested that a gingival crevice microflora containing several amino-acid fermenting species had colonized the tooth surfaces. These organisms would not be expected to produce a plaque capable of decalcifying enamel, which might explain why these subjects had a low caries experience.

Microbial colonization of dental implants in partially edentulous subjects

This study assessed the colonization of Brånemark dental implants by periodontopathic bacteria in four partially edentulous patients with a total of 10 implants. Marginal and subgingival plaque on the implants was sampled 14 days and 28 days after second-stage surgery and compared with plaque from three teeth closest to the implant sites sampled before second-stage surgery (baseline) and at 14 and 28 days. Slot immunoblot assay was used to determine the presence or absence of bacterial antigen to six different periodontopathic microorganisms. In general, colonization of marginal implant plaque occurred within 14 days, whereas subgingival colonization took longer and occurred within 28 days. It appears that Brånemark dental implants placed in partially edentulous patients may be colonized by disease-associated bacteria within 14 days of second-stage surgery.

Effects of repeated scaling and root planing and/or controlled oral hygiene on the periodontal attachment level and pocket depths in beagle dogs

A study was performed to evaluate the effect of initial and/or repeated scaling with or without controlled oral hygiene on the level of periodontal attachment and pocket depth in beagle dogs. The clinical results of this three-year longitudinal study have been reported previously (Morrison et al. 1979). The purpose of the present report is to present the microbiological results of subgingival plaque samples obtained from selected sites at the conclusion of the study. Eight beagle dogs with moderately developed periodontitis were divided into experimental and control animals. The experimental group received a thorough scaling and root planing at the start of the experiment following which the animals were subjected to daily toothbrushing and rubber cup and pumice prophylaxis every second week for 36 months. Four controi dogs were not subjected to any oral hygiene procedures for the entire period of the study. The teeth of two quadrants in each animal of the experimental and control group were scaled and root planed every six months. After three years subgingival plaques from the mesial aspect of the fourth premolar in each of the quadrants of each animal were collected by sterile curettes, processed anaerobically, and cultured in an anaerobic glove box. Significantly lower total viable colony forming units (CFU) as weil as significantly lower anaerohe/aerohe ratios were found in the subgingival plaques of the experimental animals. The total CFU of Bacteroides asaccharoiyticus v/as 25 times lower, and the proportion of CFU of this organism was nine times lower in the experimental sites when compared with the control sites. Repeated scaling every six months aiso lowered the total CFU and the proportion of Bacteroides asaccharolyticus in the suhgingival plaques of the experimental as well as the control animals. The data suggest that the level of Bacteroides asaccharolyticus as key organisms as well as the anaerobe/aerobe ratio are valuable microbiological parameters in evaluating the efficacy of periodontal therapy.

Benzoylarginine peptidase and iminopeptidase profiles of Treponema denticola strains isolated from the human periodontal pocket

Seven clinical isolates and the ATCC strain 35405 ofTreponema denticola, obtained from human periodontal pockets, were studied for peptidase activity with several chromogenic compounds as substrates. The cell sonicates of all strains hydrolyzed phenylazobenzyloxycarbonyl-l-prolyl-l-leucyl-glycyl-l-prolyl-d-arginine (a collagenase substrate), azocasein, and the 2-naphthylamines ofl-proline,l-hydroxyproline,l-pyrrolidine, and benzoyl-l-arginine, but the rates of hydrolysis varied considerably from strain to strain. Fast protein liquid chromatography on gel and anion exchange columns revealed further biochemical differences between the strains. The ATCC strain consistently produced several proline iminopeptidases, whereas four of the clinical isolates yielded high and three yielded low iminopeptidase activity. The ATCC strain and six clinical isolates displayed high benzoylarginine peptidase activity. The use ofN-l-prolyl-2-naphthylamine as substrate revealed more differences between the strains than other substrates. The substrate specificity of the enzymes discovered suggests that they may be important for the nutrition of the organism or in the protection of the organism against chemical defense factors present in the gingival pocket.

Dominance of iminopeptidase activity in the human oral bacterium Treponema denticola ATCC 35405

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 μM p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.

Evaluation of Kanamycin as an aid in the isolation of Bacteroides melaninogenicus from dental plaque

Abstract Dental plaque known to harbour Bacteroides melaninogenicus was removed from the gingival half of selected buccal tooth surfaces. The plaque was dispersed, diluted and cultured on medium containing 0 per cent, 0.05, 0.1 and 0.5 per cent Kanamycin. When the medium contained 0.05 per cent Kanamycin, only B. melaninogenicus were found and the number recovered was about 20 per cent of the B. melaninogenicus colonies isolated on the antibiotic free medium. Apparently certain strains of B. melaninogenicus are resistant to this level of Kanamycin and the addition of this antibiotic to a culture medium would facilitate the primary isolation of this organism from source material.

Proteolytic and oxidoreductase activity of Treponema denticola ATCC 35405 grown in an aerobic and anaerobic gaseous environment

The cells of a human oral spirochete, Treponema denticola ATCC 35405, and of seven clinical isolates of this organism obtained from the subgingival dental plaque of periodontitis patients were studied for their ability to grow in an aerobic and an anaerobic environment, and for their profile of peptidohydrolase and oxidoreductase enzymes. The growth yield of aerobically grown cultures was either comparable to or higher than that of anaerobically grown ones regardless of whether prereduced broth, freshly prepared broth or oxidized broth was used. However, elimination of certain supplements from the growth media resulted in poor growth regardless of the nature of the gaseous environment. The microscopic morphology and motility of the cells were not affected by differences in the gaseous atmosphere. Quantitative studies on several peptidohydrolase activities suggest that anaerobically grown cells displayed higher specific activity especially toward N alpha-L-prolyl-2-naphthylamine, indicating that increased synthesis of proline iminopeptidase enzymes (or enzyme) of the cells was associated with anaerobic growth conditions. The formation of enzymes hydrolysing N alpha-benzoyl-DL-arginyl-2-naphthylamine (and the corresponding p-nitroaniline) was not affected to the same extent. Growth experiments suggest that T. denticola ATCC 35405 is a facultatively anaerobic spirochete instead of an obligate anaerobe as reported in previous literature. The quantitative enzyme studies suggest that the gaseous growth atmosphere of the cells can exert a selective effect on the activity levels of certain peptidolytic enzymes of this organism. Such effects were not observed when the whole cells were studied by means of qualitative or semi-quantitative enzyme tests. The activities of catalase, peroxidase and superoxide dismutase of the cells were low and variable. Because of this, it was not possible to relate these oxidoreductase activities to the composition of the gaseous atmosphere.

Hydrolysis of the Leu-Gly bond of phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-D-Arg (a substrate of microbial collagenases) by treponemes isolated from the subgingival plaque of periodontitis patients

Cell extracts prepared from several oral treponemes isolated from the subgingival plaque of periodontitis patients showed high enzyme activity toward phenylazobenzyl-oxycarbonyl-l-prolyl-l-leucylglycyl-l-prolyl-d-arginine (a compound used as a substrate for microbial collagenases). One major enzyme hydrolyzing this substrate at the Leu-Gly bond only was partially purified from an unspeciated treponeme (strain US),Treponema denticola ATCC 35405, and 29 different clinical isolates ofT. denticola. TheTreponema US enzyme also hydrolyzed furylacryloyl-l-leucylglycyl-l-prolyl-l-alanine (another substrate of bacterial collagenases) at the Leu-Gly bond. This enzyme also hydrolyzed various collagens and collagen-derived peptides. These treponemal proteases were sensitive to metal chelators andp-chloromercury compounds. The results indicate that human oral treponemes contain enzymes that readily hydrolyze in chromogenic protease substrates the Leu-Gly bond only that is the cleavage site of these substrates also by “true” microbial collagenases.

In-vitro evaluation in man of immunostimulation by subfractions of Actinomyces viscosus

Abstract Actinomyces viscosus was fractionated into cell wall (PEL) and intracellular supernatant (SUP) fractions following ultrasonication. In vitro lymphocyte transformation induced by each fraction was assessed using the lymphocytes obtained from subjects with minimal periodontal disease. In kinetic experiments, a detectable blastogenic response to both fractions was measured by the fourth day, with a peak at day 7. Nuclease treatment enhanced the immunostimulatory activity of the PEL fraction 5.5-fold over untreated PEL; it had no effect on the activity of SUP. Although pronase treatment had no effect on the PEL fractions, it abrogated the ability of SUP to activate lymphocytes. Fractions of nuclease-treated and untreated SUP were batch-eluted from DEAE-sepharose columns with increasing concentrations of NaCl, producing 3 major stimulatory subfractions which accounted for 75 per cent of the activity of unfractionated SUP. Comparisons of lymphocyte responses to the DEAE-fractionated SUP indicated that not all individuals responded equally to each subfraction. As there was a differential responsiveness to the various antigenic components, detailed evaluation of the antigens of A. viscosus is warranted to define differences in antigen responsiveness in individuals with differing severities of periodontal disease.

Hydrolysis of γ-glutamyl linkages by Fusobacterium nucleatum

The cell extracts of two human oral strains (FN2 and FN3) ofFusobacterium nucleatum displayed exceptionally highγ-glutamylpeptidase activity as determined withN-γ-l-glutamyl-2-naphthylamine as substrate. This activity was so dominant that the hydrolysis of otherN-aminoacyl-2-naphthylamines progressed at a rate <10% of the former. Two major enzymes (I and II) were partially purified from FN2. I had a molecular weight of 115,000 and did not hydrolyzeγ-glutamylcysteinylglycine (glutathione). II had a molecular weight of 70,000 and rapidly liberated only glutamic acid from glutathione. Strain FN3 contained several enzymes hydrolyzingγ-glu-2NA. Direct anion exchange chromatography of FN3 cell extracts separated one enzyme that liberated both glutamic acid and glycine from glutathione, one that was inactive against glutathione (but hydrolyzedγ-glu-2NA), and one that liberated only glutamic acid. Althoughγ-glu-2NA was a good synthetic substrate, glutathione was hydrolyzed at least 500 times faster by an enzyme present in both strains. These results indicate that the presence ofγ-glutamylpeptidase activity is very characteristic of theseF. nucleatum strains.