Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B, asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.     

Lipids of Bacteroides melaninogenicus

The lipids of Bacteroides melaninogenicus were readily extractable with chloroform-methanol. Three per cent of the fatty acids were not extractable. The neutral lipids contained 4% of the extractable fatty acids, the stench characteristic of these organisms, and 0.5 mumole of vitamin K(2) isoprenologues K(2)-35, K(2)-40, and K(2)-45 per g (dry weight). This is one-fifth to one-tenth of the vitamin K(2) level found in other bacteria. Ninety-six per cent of the extractable fatty acids were associated with the phospholipids (60 mumoles of lipid phosphate/g, dry weight), which consisted of the diacyl lipids phosphatidic acid, phosphatidyl serine, and phosphatidyl ethanolamine (with phosphatidyl glycerol and cardiolipin in one strain). The unusual phosphosphingolipids ceramide phosphorylethanolamine, ceramide phosphorylglycerol, and ceramide phosphorylglycerol phosphate accounted for 50 to 70% of the lipid phosphate. In protoheme-requiring strains, the protoheme concentration in the growth medium regulated the growth rate and the amount of enzymatically reducible cytochrome c. There were no gross changes in the lipid composition in cells containing different levels of enzymatically reducible cytochrome c.     

Ultraviolet Red Fluorescence of Bacteroides melaninogenicus

Under longwave ultraviolet light, Bacteroides melaninogenicus fluoresced vivid red on blood-agar plates as well as in chronic cutaneous ulcers and purulent drainage.     

Early diagnosis of crepitant gangrene caused by Bacteroides melaninogenicus.

A rapid non-invasive test for the presence of B. melaninogenicus in the wounds of crepitant non-clostridial gangrene is described. The wounds are viewed under an ultraviolet light, and the presence of bright red fluorescene indicates the probable presence of B. melaninogenicus.     

Casamino acids enhance growth of Bacteroides melaninogenicus.

Casamino Acids enhance the growth of Bacteroides melaninogenicus when added to various concentrations of Trypticase. Absence of a peptide, not amino acids, is responsible for the inability of Casamino Acids to support growth.     

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.     

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.     

The Classification of Bacteroides melaninogenicus and Related Species

One hundred and seventy-five strains of Bacteroides melaninogenicus , 17 strains of B. oralis and six strains of B. ochraceus were studied in a series of biochemical, chemical tolerance and antibiotic disc resistance tests and by the gas-liquid chromatographic analysis of the acid end products of metabolism. Strains of B. melaninogenicus ss. asaccharolyticus formed a distinct group with clear differences from other B. melaninogenicus strains. B. melaninogenicus ss. intermedius strains formed a homogeneous group that could be readily identified. B. ochraceus was distinguished from other Bacteroides spp. by its ability to grow in air enriched with CO₂. Bacteriodes melaninogenicus ss. melaninogenicus and B. oralis gave very similar patterns of results with the tests used and invariably were indistinguishable; the capacity to produce black-pigmented colonies on blood-containing media may not be a valid criterion for dividing these similar strains into two species.     

The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: Induction by vitamin K

An enzyme present in cell-free extracts of B. melaninogenicus grown with vitamin K is described which catalyzes the synthesis of 3-ketodihydrosphingosine from palmitoyl CoA and l-serine. Activity of the 3-ketodihydrosphingosine synthetase was measured as a function of time, palmitoyl CoA concentration, and serine concentration. The Bacteroides synthetase differs from corresponding enzymes from brain microsomes and from yeast in that it is present in the 100,000g supernatant of sonicated cells and is not associated with any particulate fraction.Extracts prepared from cells depleted of vitamin K showed only slight 3-ketodihydrosphingosine synthetase activity. Neither vitamin K₁, menadione, nor pyridoxal phosphate were effective in enhancing the activity in cell-free extracts of vitamin K-depleted B. melaninogenicus. However, induction of the enzyme activity in intact cells was demonstrated by the addition of vitamin K to a vitamin K-depleted culture. Synthetase activity was found to be increased 15 min following the addition of the vitamin, reached a maximum at 75 min, and thereafter remained constant. Both puromycin and rifampcin inhibit induction of the enzyme by vitamin K₁ suggesting that vitamin K induces de novo synthesis of the synthetase.     

Electron Transport System of the Protoheme-requiring Anaerobe Bacteroides melaninogenicus

Protoheme is essential for the growth of some strains of Bacteroides melaninogenicus. At low concentrations in the growth medium, protoheme determines the doubling time, total cell yield, and amount of cytochrome per bacterium. At high protoheme concentrations, the doubling time, total cell yield, and amount of enzymatically reducible cytochrome appear to remain nearly constant, and protoheme is accumulated by the cell. The accumulated protoheme can support the growth of the bacterium for at least eight generations in a protoheme-free medium. When growth and cytochrome content are proportional during growth at low protoheme concentrations, the bacteria incorporate 10 to 20% of the total available protoheme into a membrane-bound respiratory system. This respiratory system includes cytochrome c, a carbon monoxide-binding pigment, and possibly flavoproteins. The pigments can be reversibly reduced by reduced nicotinamide adenine dinucleotide or endogenous metabolism and can be oxidized anaerobically by fumarate or by shaking in air. Electron transport is inhibited by 2-n-nonyl-4-hydroxy-quinoline-N-oxide.     

Succinate as a Growth Factor for Bacteroides melaninogenicus

Rumen strains of the obligate anaerobe Bacteroides melaninogenicus normally require medium supplemented with both heme and vitamin K. Sodium succinate was found to be an additional growth factor in that this compound can replace the requirement for heme in the presence of vitamin K, allowing good growth of the organism, and succinate can also partially replace the requirement for vitamin K in the presence of heme. The addition of succinate to a medium supplemented with both vitamin K and heme increases the growth rate of the culture. This ability to stimulate growth was specific for succinate, and cells grown without heme but with vitamin K and succinate were insensitive to cyanide. These experiments demonstrate a central role for succinate in the metabolism of B. melaninogenicus.     

The M band. Studies with fluorescent antibody staining

The M band can be extracted from fibrils suspended in 5 mM Tris buffer, pH 8.0, for 15 min. The M band is completely removed only from fibrils of sarcomere lengths greater than 2.1 µ. Extraction does not alter the fluorescent antimyosin staining pattern of the A band, thus providing strong evidence that no alteration of the structural integrity of the thick filament has occurred. Fluorescent antibody staining of the M band of unextracted fibrils can be prevented specifically by absorbing the fluorescent antibody with extracted M band material prior to staining. This verifies the specificity of the extraction procedure.     

Serological Properties of Lipopolysaccharide from Oral Strains of Bacteroides melaninogenicus

Lipopolysaccharide (LPS) extracted with phenol-water from four oral strains of Bacteroides melaninogenicus was found to be serologically active in precipitation and complement fixation tests and sensitized sheep erythrocytes to agglutination. Except for the capacity to inhibit indirect hemagglutination, the serological activity was destroyed by oxidation with periodate. The isolated LPS was antigenic in rabbits, giving rise to low- and high-molecular-weight antibodies. Cross-reactivity experiments revealed the presence in LPS of both type-specific and group-reactive antigenic determinants.     

Characterization of Bacteroides asaccharolyticus and B. melaninogenicus oral isolates

Experiments were designed to characterize a number of oral "pigmented" Bacteroides isolates with regard to their pathogenicity in an experimental model system and a number of other properties. T these include fatty acid determination, hemagglutination studies, collagenase and protease activities, and vitamin K dependency. Oral B, asaccharolyticus and B, melaninogenicus isolates differed from one another in phenylacetic acid production, hemagglutination, collagenase activity, and pathogenicity. All B. asaccharolyticus were found to be pathogenic in the vivo mixed infection model and this property could be correlated with biochemical enzymatic activities.     

Influence of amino acids on the growth of Bacteroides melaninogenicus.

Addition of individual amino acids to a Trypticase-yeast extract-hemin medium affected growth rates and final yields of an asaccharolytic strain and a saccharolytic strain of Bacteroides melaninogenicus. L-Aspartate or L-asparagine produced maximal growth enhancement for both strains. L-[14C]aspartate was fermented by resting cells of the asaccharolytic strain. L-Cysteine or L-serine also enhanced growth for the saccharolytic strain. However, growth of the saccharolytic strain was inhibited by L-lysine, L-glutamate, L-glutamine, L-isoleucine, L-leucine, and L-proline; growth of the asaccharolytic strain was inhibited by DL-valine and L-serine. Both strains were inhibited by L-histidine, DL-methionine, L-tryptophan, L-arginine, and glycine.     

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.