The pigmented oral BacterOides were characterized with regard to their pathogenic, collagenolytic, hemagglutinating and metabolic properties. Isolates were obtained from healthy and diseased gingival sulci of 396 children, adults and mongrel dogs. Fifty-three of the isolates proved to be members of the subspecies Ii. melaninogenicus ss. asaccharolyticus. The remainder were either 15. melaninogenicus ss. intermedius or melaninogenicus. All isolates designated asaccharolyticus were pathogenic in the guinea pig infectivity assay. The subspecies intermedius and melaninogenicus were not pathogenic. The subspecies asaccharolyticus possessed a cell bound oxygen sensitive collagenase, a cell bound and soluble oxygen sensitive hemagglutinin, and produced butyric and phenylacetic acids. These properties were unique to asaccharolyticus and were not detected with other organisms. All isolates required hemin. The pathogenic organisms possessed a heat sensitive toxin which induced fluid accumulation in ligated mouse ileal loops. Infectivity of 13. melaninogenicus asaccharolyticus was dependent on the presence of a second organism. An infective consortium consisting of 13. melaninogenicus asaccharolyticus and Klebsiella pneumoniae was defined. Neither organism was infective alone but the Klebsiella could be replaced by organisms of a number of different genera. The number and proportions of J3. melaninogenicus and K. pneumoniae required to establish a lesion was determined. The nature of the infection appeared to be determined by the length of the lag period preceding the initiation of growth of 15. melaninogenicus. A rapid onset of growth led to the severe spreading form of the disease whereas a slow initiation of growth resulted in the formation of a localized self limiting abscess. J3. melaninogenicus depends on the second or "helper" organism to produce a required growth factor which is not present at the inoculation site. The growth factor was shown to be succinate which was able to replace the hemin requirement. The dependency oh succinate produced by K. pneumoniae was demonstrated in agar medium, in liquid culture and in the infectivity assay. Any organism which produced succinate was able to stimulate growth of JJ. tnelahinogenicus on agar medium and could replace K.. pneumoniae as a member of the infectious consortium. The need for the second organism could be eliminated by inoculating 13. melaninogenicus together with agar-immobilized succinate or hemin. Growth of Ii. melaninogenicus is dependent on the presence of large quantities of succinic acid suggesting that the compound is used in energy metabolism and is not incorporated into cellular carbon. This assumption is supported by the observation that only 0.5% of the succinate carbon can be found in the cell, the remainder of the metabolized succinate is excreted as butyrate. Hemin blocks the metabolism of succinate. The fatty acid metabolites are qualitatively similar but quantitatively different in cells grown in hemin free succinate medium. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Bacteroides melaninogenicus strains 2D and K110 were characterized with regard to their pathogenic, collagenolytic, proteolytic, hemagglutinating and metabolic activities. Both strains were members of the subspecies 13. melaninogenicus ss. asaccharolyticus. They possessed a cell-bound oxygen-sensitive collagenase, a cell-bound and a soluble oxygen-sensitive hemagglutinin (HA), and a protease. Both strains produced butyric and phenylacetic acids and were infective in guinea pigs as characterized by their ability to produce necrotic lesions and to be transferred from one animal to another. Strain 2D required hemin for growth and its growth rate was influenced by the addition of free amino acids to the medium. The hemagglutinating and proteolytic activities of strain 2D were investigated further to determine their relationship to infection. The soluble HA was reversibly inhibited by Hg and activity was restored in the presence of reducing agents. Iodoacetic acid caused irreversible inhibition. The HA was sensitive to heat and pronase treatment. Treatment of the red blood cells (RBC) with neuraminidase enhanced HA activity while the presence of galactose in the reaction mixture inhibited it, suggesting the involvement of galactose residues on the RBCs in the reaction.. Adsorption of the HA to RBC followed by elution and gel filtration resulted in the recovery of 50% of the HA activity and a 52-fold purification. Protease production by _B. melaninogenicus strain 2D was dependent on the growth rate of the organism. The protease was reversibly inhibited by HgCl₂ and irreversibly inhibited by iodoacetamide and iodoacetic acid. The enzyme was insensitive to serine protease inhibitors and EDTA. The pH optimum for proteolytic activity was 7.0, which correlates with the pH of its natural environment, the gingival crevice. It is thus classified as a neutral sulfhydryl enzyme. A 774-fold purification of the cellular protease of 2D, with a 160% recovery of activity, was accomplished by precipitation with 60% ethanol, ultracentrifugation and gel filtration through Sephadex G-100 and Sepharose 2B in the presence of urea. Electrophoretic analysis of the protease on SDS-polyacrylamide gels revealed four distinct bands, each of which was shown to be associated with carbohydrate. In the absence of SDS only one band, which did not migrate into the gel, was obtained. Any attempts to further dissociate the protease resulted in the loss of activity. The protease was active against azocoll, azocasein, casein and N,N-dimethylcasein. No glycosidase, lipase, collagenase or HA activities were detected. Protein, carbohydrate and lipid were detected in the preparation. The soluble protease which amounted to 20% of the cellular protease of strain 2D was subjected to gel filtration on Sephadex G-100 and eluted in a single peak at the void volume. The properties of the soluble protease were identical to those of the cell associated enzyme, suggesting the presence of a single proteolytic enzyme which was released into the culture medium with cell lysis or due to shedding of outer membrane fragments. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate