Identification of Prevotella in pedal osteomyelitis of a diabetic patient.

Osteomyelitis in a diabetic patient with a nonhealing foot ulcer, multiple medical conditions, and recurrent hospitalization for antibiotic therapy was found to be associated with gram-negative bacteria Prevotella melanginoganica and Prevotella melaninoganica hemagglutinating variant. Those organisms were identified due to the morphologically distinct features in electron microscopy and sequencing of the genes after Polymerase chain reaction amplification from the pathological material. The bacteria invaded the bone and resided in osteocyte, osteoblast, and endothelial cells. The bacteria are usually associated with periodontal plaques, causing inflammation and destruction of gingival tissue and resorption of the alveolar bone. This is the first ultrastructural and molecular study of a diabetic bone lesion with anaerobic bacterial infection.

Morphological changes in the bacterium Bacteroides melaninogenicus subspecies melaninogenicus isolated from the human mouth and grown in culture without added blood components.

Striking polymorphism in the cellular morphology could be induced by removal of blood components from the liquid growth medium, but the cells of Bacteroides gingivalis and B. melaninogenicus subspecies intermedius did not exhibit polymorphism when grown under these conditions. The major changes observed with light microscopy were an increase in cell size and extreme polymorphism. Electron microscopy of the polymorphic forms of B. melaninogenicus subspecies melaninogenicus strains showed that such cells lacked both the outer cell membrane and peptidoglycan layer. Serum promoted the growth of these strains, suggesting that some blood component is either conducive to the synthesis of the cell wall or masks an unknown inhibitor for cell-wall synthesis contained in the medium.

Ultrastructure of Bacteroides species: Bacteroides asaccharolyticus, Bacteroides fragilis, Bacteroides melaninogenicus subspecies melaninogenicus, and B. melaninogenicus subspecies intermedius.

Representative strains of two subspecies of Bacteroides melaninogenicus (subspecies melaninogenicus and subspecies intermedius) and Bacteroides asaccharolyticus as well as B. asaccharolyticus strain 536B isolated from a human perirectal abscess and Bacteroides fragilis ATCC 25285 were examined by glutaraldehyde-osmium fixation, ruthenium red fixation and staining, and thorium hydroxyde staining as well as by the physical preparative techniques of critical point drying--transmission electron microscopy (CPD--TEM) and scanning electron microscopy (SEM). All strains, with the exception of B. fragilis 25285, possessed an electron-dense material external to their outer membranes. Ruthenium red staining further revealed a layer, external to the surface of the outer membrane, that was distinct for each species examined. Thorium hydroxide, as well as CPD--TEM and SEM, showed the cells to be interconnected by thin fibers that not only connected adjacent cells but also traversed several microns to connect cell aggregates.

Immunochemical characterization of surface antigens of Bacteroides melaninogenicus.

Major surface antigens of Bactmbrane complex by gentle methods, purified, and characterized immunochemically. A lipopolysaccharide (LPS) was found to be chemically distinct from the LPS of facultative gram-negative bacteria in that it lacked two core sugars, 2-keto-3-deoxyoctonate and heptose, as well as beta-hydroxymyristic acid, the predominant fatty acid in the lipid A moiety. The LPS was further atypical in that it had a very low level of biologic activity. A capsular polysaccharide was demonstrated morphologically by electron microscopy with ruthenium red staining and a ferritin-labeled antibody technique. This antigen was shown to be subspecies-specific by indirect immunofluorescence. Antibody to the capsular polysaccharide was measured by an enzyme-linked immunospecific assay. The presence of a relatively impotent LPS and a surface capsular antigen may partly explain the rarity of bacteremia and septic shock due to B. melaninogenicus subspecies asaccharolyticus and the common association of this organism with abscess formation.

Identification of a subspecies-specific capsular antigen from Bacteroides melaninogenicus subspecies asaccharolyticus by immunofluorescence and electron microscopy.

An indirect fluorescent antibody test was developed with the use of hyperimmune rabbit antiserum to a purified capsular polysaccharide of Bacteroides melaninogenicus subspecies asaccharolyticus. All of 23 strains of B. melaninogenicus subspecies asaccharolyticus were fluorescence-positive in this test. All 11 strains of Bacteroides melaninogenicus subspecies intermedius tested and three strains of Bacteroides melaninogenicus subspecies melaninogenicus were fluorescence-negative. Thirty-one strains of other bacterial species were also fluorescence-negative. The indirect fluorescent antibody test demonstrated the presence of a subspecies-specific capsular antigen from B. melaninogenicus subspecies asaccharolyticus. The capsular antigen was further demonstrated by electron microscopy with ruthenium red, a polysaccharide-staining material.

Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets.

This investigation examined the ability of cells of Bacteroides melaninogenicus subsp. asaccharolyticus 381 to adhere to surfaces that might be important for its initial colonization of the mouth and its subsequent colonization in periodontal pockets. Of 48 asaccharolytic strains of B. melaninogenicus, 47 agglutinated human erythrocytes, whereas none of 20 fermentative strains, which included reference cultures of the subspecies intermedius and melaninogenicus, were active. Electron microscopy indicated that both asaccharolytic and fermentative strains possessed pili; hence, the presence of pili did not correlate with the hemagglutinating activities of B. melaninogenicus strains. Both asaccharolytic and fermentative B. melaninogenicus strains suspended in phosphate-buffered saline adhered in high numbers to buccal epithelial cells and to the surfaces of several gram-positive bacteria tested, including Actinomyces viscosus, A. naeslundii, A. israelii, Streptococcus sanguis, and S. mitis. B. melaninogenicus subsp. asaccharolyticus 381 also attached, but in comparatively low numbers, to untreated and to saliva-treated hydroxyapatite. Addition of clarified whole saliva to suspensions of strain 381 almost completely eliminated adherence to buccal epithelial cells and to hydroxyapatite surfaces, but saliva had no detectable effect on attachment to gram-positive plaque bacteria. Both fermentative and nonfermentative strains of B. melaninogenicus also attached in high numbers to crevicular epithelial cells derived from human periodontal pockets, but normal human serum strongly inhibited attachment. Serum also inhibited attachment of strain 381 to saliva- and serum-treated hydroxyapatite, but it had little effect upon attachment to gram-positive bacteria. These observations suggested that salivary and serum components would strongly inhibit the attachment of B. melaninogenicus cells to several oral surfaces, but not to the surfaces of certain gram-positive bacteria commonly present in human dental plaque. This was confirmed by an in vivo experiment in which streptomycin-labeled cells of B. melaninogenicus 381-R were introduced into the mouths of two volunteers. After 10 min, several hundred-fold higher numbers of the organism were recovered from preformed bacterial plaque present on teeth than from clean tooth surfaces or from the buccal mucosa and tongue dorsum. High numbers of B. melaninogenicus cells were also recovered from preformed plaque after 150 min, but virtually no cells of the organism were recovered from the other surfaces studied. These data suggest that the presence of dental plaque containing Actinomyces and other gram-positive bacteria may be essential for the attachment and colonization of B. melaninogenicus cells after their initial introduction into the mouth. Similarly, the presence of subgingival plaque containing gram-positive bacteria may be necessary for its secondary colonization in periodontal pockets.