Hirosuke Sagawa

Correlation between clinical symptoms and microorganisms isolated from root canals of teeth with periapical pathosis

Periapical pathosis cases were classified and the effect of bacteria on clinical symptoms was examined. A positive correlation between bacterial growth and clinical symptoms was found. Peptococcus magnus and Bacteroides species were commonly found in clinically acute cases, while oral streptococci and enteric bacteria were frequently isolated from clinically asymptomatic cases.

Localization and identification of root canal bacteria in clinically asymptomatic periapical pathosis

Twenty-one teeth with clinically asymptomatic periapical pathosis (class 3) were extracted and the isolation, identification, and localization of bacteria in the root apex were examined. Mixtures involving several bacteria were isolated from more than 60% of the cases. Scanning electron microscopy revealed bacterial masses to be associated with the apical part of the root canal, but not with the area of apical foramen or on the surface of root apex. Our results indicate that the bacteria in class 3 cases may be derived from organisms which colonized before or during endodontic treatment, but not from anachoresis. The bacteria-positive cases of asymptomatic periapical pathosis have the potential to progress to symptomatic periapical pathosis.

Lanthionine as an essential constituent of cell wall peptidoglycan ofFusobacterium nucleatum

Lanthionine, a sulfur-containing diamino acid which had not previously been reported as one of the main amino acids of any bacterial cell wall peptidoglycan, was demonstrated inFusobacterium nucleatum peptidoglycan isolated by sodium dodecyl sulfate extraction and protease digestion. Lysine, diaminopimelic acid, and ornithine were absent. Lanthionine seems to be an essential dibasic amino acid, involved in cross-linkages betwen stem peptide subunits inF. nucleatum.

Purification and chemical analysis of a bacteriocin from the oral bacterium Streptococcus mutans RM-10

Bacteriocin from supernatant of broth-grown cultures of Streptococcus mutans Rm-10 was purified by ammonium sulphate fractionation, precipitation at pH 3.1, and gel filtration on Sephadex G-200 and Sepharose 2B. The purified preparation was shown to be homogeneous by gel filtration and immunoelectrophoresis. A molecular weight of 973,000 was determined by equilibrium sedimentation with UV scanning. Electron microscopy of this purified preparation revealed a fibrous structure with a homogeneous morphology. The bacteriocin was essentially proteinaceous in nature, containing 90 μg phosphorus and 34 μg hexose per mg of protein, and was found to be rich in aspartic acid, glycine, alanine, valine, leucine and lysine.

Characterization and mode of action of a purified bacteriocin from the oral bacterium Streptococcus mutans Rm-10

The purified bacteriocin was sensitive to proteolytic enzymes such as trypsin, alpha-chymotrypsin and pronase, but resistant to papain and pepsin. Lowering the pH of the bacteriocin caused precipitation, and the Abs280 of the supernatant reached a minimum at pH 3.6, suggesting that this is the isoelectric point. Such a pH value coincides with minimum bacteriocin activity. The effect of the bacteriocin was bactericidal rather than bacteriolytic and concentration dependent. Treatment of Streptococcus faecalis ODU with Rm-10 bacteriocin led rapidly to the cessation of biosynthesis of macromolecules, DNA, RNA and protein. Electron microscopy showed bacteriocin fibres attached in aggregated bundles to target cells; after ultra-sonication or detergent treatment they were individually attached to the cell surface and, at the same time, the apparent bacteriocin activity had doubled. Bacteriocin activity was not altered by filter-sterilized saliva components. Oral rinsing with Rm-10 bacteriocin reduced the number of viable salivary bacteria after 20 min, suggesting a possible therapeutic use for Rm-10 bacteriocin.

Antimicrobial Susceptibilities of Eubacterium, Peptostreptococcus, and Bacteroides Isolated from Root Canals of Teeth with Periapical Pathosis

Eubacterium, Peptostreptococcus, and Bacteroides were isolated in high frequency from root canals with acute periapical inflammation. The antimicrobial susceptibilities of these strains were studied by determining minimum inhibitory concentrations of different agents. Although all three kinds of isolates were susceptible to penicillins, the isolates other than black-pigmented Bacteroides were less susceptible to cephems, tetracyclines, and macrorides with several resistant strains. All strains were uniformly resistant to aminoglycosides. Some differences in susceptibilities were observed among species of Eubacterium and Peptostreptococcus, while penicillins were effective for both species. Black-pigmented Bacteroides showed good susceptibilities to all agents except for aminoglycosides. The susceptibility of Bacteroides gingivalis was superior to that of Bacteroides intermedius. There were many resistant strains in non-black-pigmented but not in black-pigmented Bacteroides isolates. Penicillins were the most effective for Eubacterium, Peptostreptococcus, and Bacteroides, indicating that penicillins are suitable for treatment of root canals with acute apical periodontitis.

Isolation, partial purification and preliminary characterization of a bacteriocin from Streptococcus mutans Rm-10

An extracellular bactericidal substance was isolated from the supernatant of Streptococcus mutans Rm-10 culture fluid and partially purified with 60% ammonium sulfate precipitation, differential centrifugation, and gel filtration on Sephadex G-200. There was a good correlation of the sensitivy profiles of indicator strains whether assayed on solid medium or with purified material from cell-free culture fluid, indicating that the same inhibitory substance is produced on solid medium and in broth. Vapor from organic solvents such as chloroform, acetone, ethanol, and ether as well as heat treatment at 100°C for 30 min had little effect on the bactericidal factor. It was sensitive to trypsin and pronase and resistant to deoxyribonuclease, ribonuclease, lysozyme, and phospholipase C. The inhibitor was not infective, and electron microscopic studies failed to reveal phage or phage-like particles in concentrated solutions of the bactericidal material. The results indicate that the extracellular bactericidal substance is indeed a bacteriocin. Activity in broth cultures reached a maximum only after exponential growth had ceased. It was active against other streptococcal strains as well as strains of Actinomyces naeslundii, A. viscosus, Bacillus subtilis and Staphylococcus aureus, but not against strains of Fusobacterium nucleatum and Escherichia coli.

The elimination of bacteriocin production in the oral bacterium Streptococcus mutans.

Abstract Bacteriocin production by some strains of Streptococcus mutans was eliminated when producing strains were exposed to various curing agents such as sodium dodecyl sulphate, acriflavine, acridine orange, ethidium bromide, rifampicin and growth at 42 °C, suggesting that bacteriocin synthesis by some strains of Strep, mutans is regulated by plasmids. Revertants from the non-bacteriocin-producing mutants were not found. Cured cells were susceptible to the bacteriocin produced by the parent strains, whereas bacteriocin-producing parent strains were resistant to their own bactericidal action. The biological properties of mutants not capable of producing bacteriocin were compared with those of the parent strains, and coordinate loss was observed in some, but not all cases.

Humoral immune responses in experimental gingivitis in rats

Serum IgG, IgM, and IgA antibody levels to extracts of rat dental plaque and five oral bacteria (Haemophilus actinomycetemcomitans Y-4, Bacteroides gingivalis 381, Bact. intermedius ATCC 25261, Capnocytophaga sp. M-12, Eikenella corrodens ODU) were determined by ELISA. In addition, the presence of rat dental plaque and oral bacterial components in the inflamed gingival tissue was studied using immunofluorescence techniques. Serum and gingival tissue samples were obtained from ODU plaque-susceptible and plaque-resistant rats. In several susceptible rats, IgG, IgM, and IgA antibodies against dental plaque and oral bacteria were detected. There was a correlation between the levels of IgG antibody to dental plaque and pocket probing depth, but not between pocket probing depth and the levels of IgM and IgA. Furthermore, components of rat dental plaque and oral bacteria were detected in the inflamed gingival tissue.

Characterization of leukocyte chemotactic activity of bacteriocin from Streptococcus mutans Rm-10.

Bacteriocins have several biological activities in addition to their antibacterial effect. We investigated the chemotactic properties and mode of action of purified streptococcal bacteriocin. Bacteriocin purified from a culture supernatant ofStreptococcus mutans (S. mutans) Rm-10 induced chemotaxis of human polymorphonuclear leukocytes and monocytes. Following purification, leukocyte migration appeared in one bacteriocin fraction, and this migration was dependent on the concentration gradient in dilution ranges from 1/960 to 1/15. Chemotactic activity of the bacteriocin was heat labile and trypsin sensitive. Moreover, preincubation of bacteriocin with varying dilutions of its antiserum prepared in rabbits resulted in a significant loss of the chemotactic activity.