Isolation and characteristics of a wheatbran-degrading Butyrivibrio from human faeces.

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H2, lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39-42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described.

The surface structure of Leptotrichia buccalis.

Leptotrichia buccalis shows a mosaic of surface structure on its outer membrane consisting of curved ridges 35 mm high and 22 nm apart, and erect on that surface. Fimbriae (common pili) are not present and nor is an S layer. The flap-like ridges consist of strings of macromolecules radiating from the cell surface. This ridge structure is not soluble in any of the usual chaotropes and can only be released when the outer membrane has been damaged or dispersed by extracting envelope preparations with 0.5% SDS at room temperature. The ridge is then found to be attached firmly to the peptidoglycan sacculus, which may be the point of origin of the structure. When so prepared the macromolecules forming the ridge can be removed from the sacculus by treatment with 6 M guanidine HCl, and SDS-PAGE analysis of the extract reveals a 210-kDa polypeptide as a major component and a 15-kDa minor component. The latter is probably a peptidoglycan-associated protein and much of it remains with the sacculus. Each string forming the ridge is of a volume consistent with being made of three elongated 210-kDa molecules, which are united in series by strong hydrophobic association and laterally with neighboring strings by slightly weaker forces. We confirm that L. buccalis causes haemagglutination and the bacteria are known to attach to various tissue cells. Human group A red blood corpuscles remove both of the proteins from solution, which supports the hypothesis that the ridges are adhesin structures. It is likely but not proven that the 210-kDa molecule is the adhesin.

Characterization of the cell wall of Butyrivibrio species.

Most Butyrivibrio strains have been isolated from the gastrointestinal tract of animals and have been classified as Butyrivibrio fibrisolvens. A few strains isolated from human feces are designated as Butyrivibrio crossatus, the other species in this genus. Butyrivibrio fibrisolvens strains are anaerobic, curved rods that produce butyrate, but numerous studies have shown that these strains display considerable variations in phenotypic properties and heterogeneity in DNA relatedness. Although over 60 strains have been characterized in these respects, the cell wall structure of only a few strains has been studied. In this study, cell wall related properties of 12 strains representative of five DNA relatedness groups were examined. All strains were very sensitive to penicillin and other antibiotics that interfere with cell wall synthesis. Although an occasional resistant strain was found, most strains were sensitive to a variety of protein synthesis antibiotics that included aminoglycosides and tetracycline. In contrast, all strains were highly resistant to nalidixic acid. Peptidoglycans were isolated from seven B. fibrisolvens strains and Lachnospira multiparus. Compositional analyses indicated molar ratios of 0.7:2:2:1:0.8 for muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid, respectively, in all peptidoglycans, which also showed a low degree of cross-linking. A trichloroacetic acid extractable galactosamine-containing polysaccharide copurified with the Butyrivibrio peptidoglycans. Electron microscopy of thin sections showed all strains to possess a Gram-positive type of cell wall that was atypically thin (12-18 nm). Most strains also displayed external (surface) polysaccharide layers. Cytoplasmic inclusions and granules were evident in many strains and were composed of polysaccharides, on the basis of cell composition analyses. The findings that Butyrivibrio strains have overall similarities in cell wall properties, but differences in DNA relatedness, suggest that these organisms should be classified as several more species in the same genus or family.

The hydrolysis of lucerne cell-wall monosaccharide components by monocultures or pair combinations of defined ruminal bacteria.

The defined ruminal bacterial strains Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, and Bacteroides ruminicola GA33 were grown, in monocultures or as combinations of pair strains, on isolated lucerne cell-walls (CW) as the sole carbohydrate substrate. Fibrobacter succinogenes S85 was the dominant strain determining extent of CW hydrolysis in all combinations with S85. The hydrolysis of cellulose, xylan, hemicellulose side-sugars, and total CW monosaccharides by pure S85 were: 58.8, 47.3, 66.9 and 57.0%, respectively. The strains combination S85 plus D1 comprised the highest complementary effect, increasing significantly the hydrolysis of cellulose and total CW monosaccharides by 16% and 13%, respectively, above the values obtained by pure S85. This complementation was expressed also in growth pattern of bacteria. The monocultures of FD1, D1 and GA33 had very little hydrolytic effect on lucerne cellulose, but higher effects on xylan and hemicellulose side-sugars. The combinations D1 plus GA33 and 7 plus GA33 were complementary in the hydrolysis of all CW polysaccharides. The combinations FD1 plus D1, FD1 plus GA33, and 7 plus D1 were complementary only with respect to hemicellulose hydrolysis. On the other hand, the cellulolytic combinations S85 plus FD1, S85 plus 7 and FD1 plus 7 demonstrated negative interactions in lucerne CW polysaccharides hydrolysis. Under scanning electron microscopy (SEM), S85 comprised the most dense layer of bacterial cell mass attached to and colonized on CW particles. The cell surface topology of the cellulolytic strains S85, FD1 and 7 attached to CW particles was specified by a coat of characteristic protuberant structures.

Surface characteristics of Wolinella recta ATCC 33238 and human clinical isolates: correlation of structure with function.

Selected characteristics of the surface of Wolinella recta ATCC 33238 and three W. recta clinical isolates (CI) were studied as well as the adherence of these strains to human gingival fibroblasts (HGF). W. recta ATCC 33238 and the CI were examined by electron microscopy, electrophoresis, isoelectric focusing, and adherence to HGF. Electron microscopic examination of CI revealed the presence of a periodic paracrystalline layer external to and associated with the outer membrane. This surface layer (S layer) was not observed on ATCC 33238. Whole cells and outer envelope protein profiles of the CI revealed major bands of 159- to 138-kilodalton proteins which were barely detectable in ATCC 33238. Repeated in vitro subculturing of the CI on solid or liquid medium resulted in both the physical loss of this layer and the loss of the high-molecular-weight proteins. Low-passage-number CI demonstrated 40 to 60% less adherence to HGF than ATCC 33238. These observations suggest that short term in vitro-subcultured W. recta strains possess surface characteristics which are significantly different from those of their long-term in vitro-subcultured counterparts. These differences may have significant effects on host cell interactions.

Bacteremias caused by Selenomonas artemidis and Selenomonas infelix.

We report two different cases of bacteremia caused by two recently described Selenomonas species, Selenomonas artemidis and Selenomonas infelix. Both species are normally found in human buccal flora. S. artemidis bacteremia appeared in a patient (number 1) who presented with an air-fluid pulmonary cavity and clinical conditions consistent with an anaerobic lung abscess. While the patient improved with antibiotic therapy, cultures of respiratory secretions yielded Mycobacterium tuberculosis. This case demonstrated a strong possibility of a coexisting lung abscess due to S. artemidis. S. infelix bacteremia appeared in a cancer patient (number 2) with heart disease during preterminal acute respiratory distress. It was more difficult in this case to assess the clinical impact of the Selenomonas organisms on the patient.

Ultrastructural relationship of cell envelope layers in Wolinella recta.

The cell envelope of Wolinella recta was studied by electron microscopy. In thin sections the surface layer (S-layer) seemed to be in close contact to the outer membrane (OM). A hexagonally arranged pattern of the S-layer was revealed by freeze-etching. Unlike in other oral bacteria with an S-layer, the cleavage occurred along the S-layer by freeze-fracture, suggesting an exceptionally tight relationship between the two outermost layers. The convex surface of the S-layer was usually revealed by this technique. When cleavage occurred through the outer membrane, no periodicity was seen in the arrangement of the intramembranous particles.

The characterization and ultrastructure of two new strains of Butyrivibrio.

Strains B-385-1 and 2-33 are numerically important components rumen bacterial populations , but they have remained (taxonomically) undefined. In spite of some resemblance to Selenomonas ruminantium in their cell size and in their formation of tufts of flagella, they more closely resemble Butyrivibrio fibrisolvens in the subpolar location of their flagella, in their guanine + cytosine content, and in most biochemical characteristics, including butyrate formation. Cells of these strains stain Gram negative, as do both Selenomonas and Butyrivibrio, but their cell walls closely resemble those of Butyrivibrio in their Gram-positive type of molecular architecture and in their cleavage pattern in freeze-etching. Cells of these strains and of B. fibrisolvens have a very thin (ca. 12 nm) peptidoglycan cell wall; thus, they fail to retain the crystal violet complex of the Gram stain and stain Gram negative. This important structural characteristic of their cell walls places strains B-385-1 and 2-33 within the genus Butyrivibrio and certain morphological and biochemical characteristics distinguish them from B. fibrisolvens.

Specialized cell surface structures in cellulolytic bacteria.

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.

Comparative ultrastructure of certain Actinomyces species, Arachnia, Bacterionema and Rothia.

A comparative ultrastructural study was carried out on several species of Acinomyces and related Gram-positive rods including Arachnia, Bacterionema, Rothia and Leptotrichia. A total of 52 well characterized strains were examined by transmission electron microscopy. Particular attention was paid to the ultrastructure of the cell periphery, that is the plasma membrane, periplasmic space, the cell wall per se and extramural structures, including surface "fuzz". In addition, the ultrastructure of various features of the cytoplasm were also examined. A good correlation appeared to exist between certain ultrastructural characteristics of the microorganisms and their taxonomic position as determined by other criteria. It should be noted, however, that the ultrastructure of certain strains differed markedly from that of the remaining strains of the species. This observation raises some doubt on the appropriateness of the current classification for these particular strains. The ultrastructural features of the cell periphery were found to be particularly stable for all strains grown under standard conditions. For this reason, ultrastructural features of the cell wall and associated structures are probably a more reliable source of morphological criteria for identification purposes than the ultrastructural characteristics of the cytoplasmic components which tend to be more variable. The results suggest that certain ultrastructural features are useful criteria for the identification and classification of these Gram-positive rods.

Leptotrichia buccalis hemagglutination in cell binding and salivary inhibition studies.

The characteristic hemagglutination (HA) of Leptotrichia buccalis was used for measuring its attachment to various human cells and for determining if saliva contained hemagglutination inhibition (HI) factors. The microbial strain utilized displayed the characteristic EM morphology of L. buccalis. Sonicated preparations of the organism were tested for HA activity before and after adsorption with human cells. Buccal epithelial cells, red blood cells (RBC), HeLa and embryonic kidney cells all bound the HA fragments of the organisms. The bacterial fragments on the cells could be observed by fluorescent antibody testing. The fragments were released from the cells used for adsorption with chelators and upon addition of CaCl2 the HA activity returned. Whole saliva displayed hemagglutination inhibition activity in a manner suggesting a binding site interaction. The similarity of the HA activity of F. nucleatum is discussed as are the relationships of cell binding to colonization of the organisms and immunopathology to host cells.