Superoxide dismutase in Bacteroides fragilis and related Bacteroides species.

Superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides ovatus, and Bacteroides thetaiotaomicron. The strains were grown under anaerobic conditions in Trypticase soy broth, and the specific activity of SOD in the extracts was, in most strains, higher than in cell-free extracts of Escherichia coli B grown under anaerobic conditions. Isoelectric focusing of the extracts in polyacrylamide gel demonstrated distinct forms of SOD in the different species.     

Adherence of Bacteroides fragilis group species.

The ability of piliated and capsulated Bacteroides fragilis and Bacteroides ovatus to adhere to intestinal cells and mucus was investigated. The adherence of piliated and capsulated strains was at least five times greater than the adherence of their nonpiliated and noncapsulated or capsulated only counterparts. These data illustrate the importance of pili as promoters of adherence of B. fragilis group species to the gastrointestinal mucosa.     

Significance of encapsulated Bacteroides melaninogenicus and Bacteroides fragilis groups in mixed infections.

Organisms of the Bacteroides melaninogenicus and Bacteroides fragilis groups are often found mixed with facultatively anaerobic organisms in infections. The relative importance of these Bacteroides groups and facultative anaerobic pathogens in mixed infections was investigated in a subcutaneous abscess model in mice. This was determined by observing the effect of antimicrobial therapy directed against one or both organisms present in the abscess. Clindamycin or metronidazole was used for treatment of infections caused by Bacteroides species, and either gentamicin, penicillin, ampicillin, or oxacillin was used for treatment of infections caused by facultative flora. In almost all instances the aerobic counterparts in the infection were more important than the unencapsulated Bacteroides species. On the other hand, encapsulated B. melaninogenicus group organisms were found to be more important in abscess formation than were group A streptococci, Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, and Staphylococcus aureus. Encapsulated B. fragilis group organisms were found to be more important than or as important as Escherichia coli and group D streptococci and less important than S. aureus, group A streptococci, and K. pneumoniae in induction of subcutaneous abscesses. This study demonstrates that encapsulated Bacteroides species are a factor that should be considered in the treatment of mixed infections with antibiotics.     

Plasmid analyses in clinical isolates of Bacteroides fragilis and other Bacteroides species.

Plasmid analyses were performed on Bacteroides strains isolated from clinical specimens. Of 32 Bacteroides strains, 8 were found to contain plasmids. Seven of these eight strains were B. fragilis, and the other one was B. distasonis. Three of these eight strains harbored only a 3.0-megadalton plasmid. Two strains had only a 2.0-megadalton plasmid, and one had 2.0-, 3.0-megadalton plasmid. Of the remaining two strains, one had 2.0-, 3.0-, and 5.0-megadalton plasmids, and the other had 3.0- and 5.0-megadalton plasmids. Beta-Lactamase was produced by 93% of the clinical isolates. Seven of the eight plasmid-carrying strains were cadmium resistant, five were zinc resistant, four were mercury resistant, and two expressed a brick-red fluorescence under ultraviolet light. None of these traits could be associated with a plasmid after performing either curing experiments or genetic transfer experiments by cell-to-cell contact.     

Differentiation of Bacteroides fragilis species by gas chromatographic detection of phenylacetic acid.

Of 382 strains tested, 261 strains of the species Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, and B. distasonis produced phenylacetic acid; the remaining strains, belonging exclusively to the species B. vulgatus, failed to do so. This differentiation characteristic may be useful in routine clinical bacteriology.     

Serological study of trichloroacetic acid extracts of Bacteroides fragilis.

Immunodiffusion techniques were used on trichloroacetic acid extracts from 10 strains of Bacteroides fragilis in detecting precipitating antibodies against this species in immune rabbit sera. Species and even strain specificities were observed in these precipitin reactions. Multiple antigens were detected in the extracts from some strains, whereas only one precipitin band per extract developed during agar-gel diffusion tests of others. The antigen extracts were found to be both heat stable and resistant to hydrolysis by alpha-chymotrypsin. Four serological patterns were demonstrated in homologous and heterologous reactions with the B. fragilis. antigen-antibody systems used. The results showed that some strains were serologically distinct from others, indicating that the strains tested are of more than one serotype.     

Antigenic characteristics and serological identification of 10 black-pigmented Bacteroides species.

Strains of 10 black-pigmented Bacteroides species were serologically characterized using absorbed and unabsorbed rabbit antisera. An agglutination test using intact cells or heated cells (100 degrees C for 60 min) from each species and unabsorbed antisera revealed only homologous reactions with little or no reactivity in heterologous assays. Immunodiffusion tests using sonicated antigen demonstrated that Bacteroides gingivalis, B. endodontalis, B. asaccharolyticus, B. macacae, and B. levii are antigenically distinct. Strains of B. gingivalis, B. endodontalis, and B. asaccharolyticus were also clearly identified by the indirect immunofluorescent antibody method. B. intermedius, B. corporis, B. loescheii, B. melaninogenicus, and B. denticola possessed common antigens; however, species-specific antigens detectable with immunoabsorbed antisera were also demonstrated. B. intermedius strains isolated from the human oral cavity included at least two serogroups. In each black-pigmented Bacteroides species, lipopolysaccharide constituted one of the species-specific antigens.     

Experimental Bacteroides fragilis endocarditis in rabbits.

A serum-resistant strain of Bacterioides fragilis that did not produce heparinase was used to study the characteristics of B. fragilis endocarditis in the rabbit experimental model. The infective dose required to produce endocarditis in 50% of rabbits was significantly lower for rabbits with left-sided intracardiac catheters (log10 6.3 colony-forming units +/- 0.6/ml) as compared with right-sided intracardiac catheters (log10 7.7 colony-forming units +/- 0.8/ml). After 3 days of infection, bacterial titers of the tricuspid vegetations were significantly lower than titers of aortic vegetations (P less than 0.01), although at 5 days the titers were similar (P greater than 0.05). The weights of tricuspid vegetations, although similar at 3 days (P less than 0.05). There were no spontaneous deaths during 12 days of infection. In rabbits with the catheter removed before infection, bacterial titers were similar to those titers in rabbits with the catheter continuously in place. This model will permit study of various drug regimens for treatment of this disease.     

Detection of Bacteroides fragilis and Bacteroides melaninogenicus by direct immunofluorescence.

A new diagnostic kit, which contains a polyvalent antiserum for either Bacteroides fragilis or Bacteroides melaninogenicus, was tested for reliability and specificity on 146 clinical samples of different origin. A correlation between the culture and immunofluorescence was observed for B. fragilis in 87.39% of cases and for B. melaninogenicus in 81.48% of cases. When pure cultures were tested, aerobically as well as anaerobically, false-positive reactions were observed with staphylococci and Clostridium ramosum spores. The well-defined morphology of these bacteria and spores allows for the elimination of any diagnostic error. The method is rapid, and the margin for error is limited. The test gives a semiquantitative idea of the number of bacteroides organisms present in the clinical specimens even in the presence of a mixed flora.     

Lectinlike adhesins in the Bacteroides fragilis group.

Lectinlike adhesins were identified in the Bacteroides fragilis group by using sugars immobilized on agarose beads either with whole bacteria by direct microscopic examination or with soluble extracts by immunoaffinoelectrophoresis. These two methods allowed the identification of two sugars reacting with whole bacteria and with the corresponding extracts: alpha-D-glucosamine and D-galactosamine. Among eight strains tested representing seven species, the two strains of B. fragilis were equally adhesive and showed the greatest adhesions. The lectinlike adhesin was purified by affinity chromatography on glucosamine-agarose or galactosamine-agarose and showed one band at 70,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This lectinlike adhesin may help to elucidate the roles of the B. fragilis group in the colonization of intestinal surfaces and in the predominance of B. fragilis in infections alone and in synergy with other bacteria.     

Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides ovatus in clinical specimens by immunofluorescence with a monoclonal antibody to B. fragilis lipopolysaccharide.

A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluorescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroides fragilis lipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32 strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb. Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken from undefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificity of IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%, respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate with the occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used to establish an IF assay that can complement isolation in the detection of these three members of the B. fragilis group.     

Bacterial chromosomal restriction endonuclease analysis of the homology of Bacteroides species.

Chromosomal DNAs of selected Bacteroides organisms whose relatedness had been previously determined by "conventional" filter-annealing studies (J. L. Johnson, Int. J. Syst. Bacteriol. 28:245, 1978) were further analyzed by restriction endonuclease analysis coupled with the Southern hybridization procedure (E. M. Southern, J. Mol. Biol. 98:503, 1975). By comparing their EcoRI restriction fragment patterns in agarose gel electrophoresis, each Bacteroides strain could be clearly differentiated. As a simple and direct means for comparison purposes this method was particularly useful for differentiating genetically similar organisms such as Bacteroides strains of the same species which shared greater than 75% homology. In contrast, bacterial chromosomal restriction endonuclease analysis in conjunction with Southern hybridizations was most effectively used to determine the significance of low levels of homology (less than 24%) as this technique provided additional information on the nature and relative distribution of that homology when the areas of homology were displayed as reproducible bands in autoradiograms.     

Fluorescent antibody test kit for rapid detection and identification of members of the Bacteroides fragilis and Bacteroides melaninogenicus groups in clinical specimens.

The Fluoretec fluorescent antibody test kit (Pfizer Inc., New York, N.Y.), developed for the rapid detection of members of the Bacteroides fragilis and B. melaninogenicus groups, was evaluated by testing 58 stock cultures and 76 clinical specimens. The test reagents detected 100% of 40 B. fragilis and B. thetaiotaomicron stock culture strains, although only 22% of 18 B. vulgatus, B. distasonis, and B. ovatus strains showed positive fluorescence. The 76 clinical specimens were evaluated by examining fluorescent antibody-stained smears of 49 specimens of purulent material and smears of 27 blood cultures which were positive for gram-negative bacilli by Gram stain or subculture. The fluoretec reagent detected members of the B. fragilis group in 28 (97%) of the 29 specimens of purulent material and all (100%) of the 16 blood cultures in which these anaerobes were demonstrated by culture. Overall, the Fluoretec reagent detected members of B. fragilis group in 44 (98%) of the 45 clinical specimens which were shown by culture to contain these anaerobes. Two of the 76 clinical specimens gave positive fluorescence for members of the B. fragilis group but failed to yield these organisms by culture. Members of the B. melaninogenicus group were detected by culture in 15 specimens and in each case their presence was demonstrated by the Fluoretec reagent. No members of the B. melaninogenicus group were isolated from five clinical specimens that gave positive fluorescence with the B. melaninogenicus reagent.     

Rapid detection and identification of Bacteroides fragilis and Bacteroides melaninogenicus by immunofluorescence.

Bacteroides fragilis group and Bacteroides melaninogenicus group fluorescent-antibody kits were evaluated with 188 clinical specimens and 116 fresh aerobic and anaerobic bacterial isolates. Fluorescent-antibody and culture results corresponded in 88% of clinical specimens of the B. fragilis group and 94% of clinical specimens of the B. melaninogenicus group. There was greater than or equal to 90% correlation for both kits with colony smears. Antigen sharing by Bacteroides bivius, Bacteroides disiens, and B. melaninogenicus was demonstrated.     

Chemical and immunochemical analyses of Bacteroides fragilis lipopolysaccharides.

Lipopolysaccharides (LPSs) from 17 different Bacteroides fragilis strains were extracted in a two-step procedure. The first step was a hot phenol-water extraction of whole bacteria, resulting in a crude aqueous phase, which after lyophilization in a second step was extracted with a phenol-chloroform-light petroleum mixture. The resulting LPSs, which were essentially free from contaminating nucleic acid, proteins, and capsular polysaccharide, were investigated for their qualitative and quantitative sugar and fatty acid composition, immunochemical specificity by enzyme-linked immunosorbent inhibition assays, and particle weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the 17 strains, 13 had LPSs which all contained L-rhamnose, D-glucose, D-galactose, and D-glucosamine in approximately the same ratio. Three of these LPSs also contained D-galactosamine. The fatty acid composition was also similar in that the same fatty acids, although in slightly varying proportions, were found in all LPSs. The 13 strains also showed the same specificity in inhibition studies by enzyme immunoassay with rabbit anti-LPS antisera and LPS antigen. The LPS particle weights were also very similar, in the range of what is found for LPSs from rough mutant strains of enterobacteria. Our results suggest that most strains of B. fragilis have similar, if not identical, LPSs with relatively short polysaccharide chains.     

Reciprocal antimicrobial synergism between Escherichia coli and Bacteroides fragilis in the presence of metronidazole.

We have studied the antimicrobial action of metronidazole against Bacteroides fragilis and Escherichia coli both in pure cultures and in combination. Concentrations of metronidazole above the minimal inhibitory concentration (MIC) produced a marked bactericidal effect on B fragilis at six hours. E coli however showed only a slight and transitory decrease in population when exposed to concentrations of metronidazole close to its MIC. Kinetic studies of this drug's effect on mixed cultures of both organisms showed mutual bactericidal synergy between E coli and metronidazole against B fragilis and between B fragilis and metronidazole against E coli. These in vitro findings agree with the results obtained in vivo by other authors.     

Comparative serology of two clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron.

Antiserum prepared in rabbits against Bacteroides fragilis showed numerous bands when reacted with B. fragilis antigen in Ouchterlony plates. This antiserum also reacted with Bacteroides thetaiotaomicron and showed one band of apparent identity with B. fragilis. The band of identity common for both organisms was lost if the antigen was heated at 80 degrees C for 30 min. Antisera prepared against B. thetaiotaomicron did not react with B. fragilis.     

Effect of temperature on survival of Bacteroides fragilis subsp. fragilis and Escherichia coli in pus.

Approximately 40 to 60% of Bacteroides fragilis subsp. fragilis in pus from experimental intra-abdominal abscesses lost their viability within 24 h when stored at refrigeration temperature (4 degrees C) either aerobically or anaerobically. No viability loss of B. fragilis was noted when pus was stored at 25 degrees C. Only slight loss of viaability of B. fragilis was observed at 15 degrees C. Escherichia coli coexisting in pus with B. fragilis increased several 100fold in 24 h when stored at 25 degrees C, but no significant growth occurred when they were kept at 15 degrees C. Approximately 20 to 40% of E. coli lost their viability when such pus was stored at 4 degrees C. We suggest that 15 degrees C may be an alternative temperature for storage of anaerobic specimens in laboratories where some delay in routine processing is unavoidable.     

Surface components of Bacteroides fragilis involved in adhesion and haemagglutination.

The ability of 19 strains of Bacteroides fragilis to adhere to buccal epithelial cells (BEC) and to the human intestinal cell line HT-29 Clone 19A, and to agglutinate rabbit erythrocytes was compared. Adhesion to BEC was poor compared with that to the cell line. Adhesion to the latter was high for 21% of the strains, moderate for 37% and poor for 42%. Only 53% of the strains agglutinated rabbit red blood cells and only strain A459 did so strongly. Haemagglutination and adhesion of B. fragilis strain A459 were inhibited by sodium periodate, but not by proteases, heat or carbohydrates. These properties were not affected by protease which removed surface appendages. Periodate treatment did not remove the fimbriae or ruthenium red-staining layer, although the capsule was lost. This suggests that carbohydrate residues on the cell surface, possibly forming part of the capsule, are involved in adhesion and haemagglutination by this strain.     

Detection of Bacteroides fragilis in clinical specimens by PCR.

The direct detection of Bacteroides fragilis from clinical specimens was examined by using the PCR method for amplifying a specific fragment of the glutamine synthetase gene from B. fragilis. By this method, all five B. fragilis strains tested were detected, but DNAs from anaerobic bacteria of 24 other species tested, from aerobic bacteria of 12 species tested, and from human leukocytes were not amplified. Using the nested PCR method, we were able to detect as little as one bacterial cell or 100 fg of chromosomal DNA of B. fragilis. A total of 39 clinical specimens, which consisted of 19 bronchial aspirates, 10 percutaneous lung aspirates, 2 transtracheal aspirates, 6 pleural fluid specimens, and 2 pus specimens, were tested. All four culture-positive samples, of which two were bronchial aspirates, one was pleural fluid, and one was pus, were positive by PCR. Among 35 culture-negative samples, 2 bronchial aspirates were positive by PCR. One was from a patient whose two previous samples were positive by both culture and PCR. It had been submitted for culture several hours after collection, and clindamycin had been administered to the patient before collection of the specimen. The other bronchial aspirate positive by PCR was from a pneumonia patient who had also been administered clindamycin. We believe that B. fragilis was present in these two specimens but that either it was dead, it was below the level detectable by culture, or the process of anaerobic culture was unsuccessful. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobes in clinical specimens.