In vitro fungistatic effects of natural coniferous resin from Norway spruce (Picea abies).

Resins (rosin, pitch) are natural products of the coniferous trees and are antimicrobial against a wide range of microbes. The antifungal effectiveness of resin, purified from Norway spruce (Picea abies), was studied against human pathogenic fungi and yeasts with the agar plate diffusion tests and electron microscopy (EM). The fungistatic effect of these resin mixtures (resin salves) was tested against a set of Candida yeasts, dermatophytes, and opportunistic fungi. Transmission and scanning EM was done from samples of fungi (Trichophyton mentagrophytes). In agar diffusion tests, the resin was strongly antifungal against all dermatophytes tested, e.g., against all fungi of the genus Trichophyton, but it was not antifungal against the Candida yeasts or against the opportunistic fungi tested. According to EM, resin caused damages in the cell hyphae and cell wall structures. We conclude that, in the agar plate diffusion test, coniferous resins are strongly fungistatic against the dermatophytic fungi only.

Functions of S-layers.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Detection of Chlamydia pneumoniae in human nonrheumatic stenotic aortic valves.

OBJECTIVES: We sought to study the possible presence of Chlamydia pneumoniae in aortic valve stenosis (AVS). BACKGROUND: Inflammation and immune mechanisms are considered important for the pathogenesis of nonrheumatic AVS. All chlamydial species are able to cause heart infections, and seroepidemiologic studies have indicated an association between chronic C. pneumoniae infection and coronary artery disease. Furthermore, the organism has been demonstrated in atherosclerotic lesions. METHODS: Aortic valve specimens with varying degrees of macroscopic disease were obtained from 35 subjects--17 consecutive patients undergoing aortic valve replacement for treatment of nonrheumatic AVS and 18 age-matched subjects at autopsy. The possible presence of C. pneumoniae in aortic valves was studied by immunohistochemical analysis, polymerase chain reaction or transmission electron microscopy, or a combination of these. RESULTS: Positive immunohistochemical staining with C. pneumoniae specific antibody was found in 9 (53%) of 17 patients with advanced aortic valve disease requiring surgical treatment (group A), 8 (80%) of 10 cadavers with clearly macroscopic aortic valve pathology (group B) and 1 (12%) of 8 grossly normal cadaver control subjects (group C). Statistical significance with regard to the presence of C. pneumoniae was found when combined diseased subjects (groups A and B: total 17 of 27 subjects) were compared with group C (p = 0.018). However, when group A was compared with group C, there was only marginal statistical significance (p = 0.088). Finally, there was a strong statistical significance (p = 0.015) when groups B and C were compared. Chlamydia pneumoniae DNA was also found in three stenotic valves, and in two of the three tested valve specimens chlamydia-like particles were seen by electron microscopy. CONCLUSIONS: Chlamydia pneumoniae is frequently present in nonrheumatic AVS. Similarly, the high number of C. pneumoniae infections detected in the early lesions of "degenerative" AVS suggest that this pathogen may play an etiologic role in the development of this disease. The validity of this relation requires additional study.

Vinblastine-induced autophagic vacuoles in mouse liver and Ehrlich ascites tumor cells as assessed by freeze-fracture electron microscopy.

The structure of the membrane limiting apparently newly formed autophagic vacuoles was studied in vinblastine (VBL) induced autophagocytosis in mouse liver parenchymal cells and in Ehrlich ascites tumor cells. In the VBL-treated cells, the formation of autophagic vacuoles was far greater than in the controls as examined by thin section transmission electron microscopy. In freeze-fracture studies of both VBL-treated and control cells, only the P- and E-fracture faces of the outer limiting membrane of the autophagic vacuoles contained a few intramembrane particles (IMP). Their density appeared however, to be much lower than the IMP density on the P- and E-fracture faces of the endoplasmic reticulum or of the Golgi apparatus. The inner limiting membrane of the autophagic vacuoles was smooth. It is apparent that the membranes of endoplasmic reticulum or Golgi apparatus are not directly involved in autophagic vacuole formation.

Epidemiology and pathogenesis of Bacillus cereus infections.

Bacillus cereus is a causative agent in both gastrointestinal and in nongastrointestinal infections. Enterotoxins, emetic toxin (cereulide), hemolysins, and phoshpolipase C as well as many enzymes such as beta-lactamases, proteases and collagenases are known as potential virulence factors of B. cereus. A special surface structure of B. cereus cells, the S-layer, has a significant role in the adhesion to host cells, in phagocytosis and in increased radiation resistance. Interest in B. cereus has been growing lately because it seems that B. cereus-related diseases, in particular food poisonings, are growing in number.

Radiation sensitivity of Bacillus cereus with and without a crystalline surface protein layer.

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.

Electron microscopic visualization of extracellular polysaccharides on the cell wall of some streptococci.

The thickness of the acid polysaccharide capsule varied remarkably among the studied streptococci. In some strains, projections of the capsule and sloughing of the polysaccharide were observed in the medium.

Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes.

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.

Characterization of Prevotella intermedia and Prevotella nigrescens isolates from periodontic and endodontic infections.

The occurrence and surface properties of prevotella intermedia and P. nigrescens in healthy sites and in periodontic and endodontic infections were studied among 73 strains, tentatively identified as P. intermedia. Fifteen strains were from necrotic root canal infections, 41 were from periodontal samples, and 17 isolates were obtained from healthy gingival sites. Identification of isolates as either P. intermedia or P. nigrescens was based on differences in malate and glutamate dehydrogenase electrophoretic mobilities which allowed unambiguous separation of P. intermedia and P. nigrescens. The majority of strains from periodontal samples were P. intermedia (29 of 41 strains). In endodontic samples only 4 out of 15 isolates were P. intermedia, while all except 1 of 17 strains from healthy gingival sites were identified as P. nigrescens. SDS-PAGE of whole cell proteins revealed 31 and 38 kDa proteins in P. nigrescens which were not detected in P. intermedia. Surface biotinylation of cells, followed by Western blotting and detection by alkaline phosphatase conjugated extravidin, showed strong staining of the 31 kDa protein in P. nigrescens indicating that this protein is located on the surface of the cell. Corresponding staining was not seen in P. intermedia. Fimbria-like projections were observed using electron microscopy of negatively-stained cells of P. nigrescens. The results show that P. intermedia and P. nigrescens may have different site specificities and surface properties and thus emphasize the need for accurate identification of these two species for the evaluation of their role in the pathogenesis of oral infections.

On the differences between the BaSO4 particles and additives in media for the double contrast examination of the stomach.

Sizes and shapes of the particles in double contrast media for the stomach have been studied with scanning electron microscope. EZ-HD deviated most from the other media because of its large particles (even 70 micrometers in diameter) which were also variable in shape. The additives soluble in water or methanol raise the viscosity of the media considerably (with the exception of EZ-HD) thus lowering their greatest possible densities. The observations explain the differences in the results obtained with patients in clinical examinations with various contrast media.

Ultrastructure of the outer membrane of Salmonella typhimurium bacteriocin-resistant mutants deficient in the 33K protein.

Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles.

Cotton bacterial endotoxin assessed by electron microscopy.

A piece of bale cotton was incubated in nutrient broth. Electron microscopic inspection of the cotton and the broth showed Gram-negative bacteria with long flagella, loosely attached to the cotton fibres. Large amounts of endotoxin liberating from these bacteria were visible in the growth medium.

Crystalline cell surface layer of Mycobacterium bovis BCG.

A paracrystalline surface layer (S layer) was found as the outermost layer of the cell wall of five Mycobacterium bovis BCG strains. An oblique arrangement of the subunits in the S layer was only clearly seen in thin-sectioned and shadowed preparations, and the unit constant was about 5.5 nm.

Ultrastructure of Gram-negative cotton bacteria with different pulmonary toxicities.

The shedding of outer membrane material was observed by electron microscopy in typical gram-negative cotton bacteria, except Agrobacterium sp. This finding is in accordance with the relative pulmonary toxicities of these bacteria. The presence of capsules did not seem to be correlated with the acute pulmonary toxicity of the bacteria.

Freeze-fracturing of oral epithelial cells in pemphigus.

Biopsies from the oral mucosa of three patients (two with pemphigus vulgaris and one with pemphigus erythematosus) were taken for both freeze-fracturing and thin-sectioning studies. Oral mucosa from a clinically healthy volunteer was used as control. The samples collected from both the involved and uninvolved oral mucosa of pemphigus patients showed widened intercellular spaces and fewer than normal desmosomes. Thin sections showed a random distribution of tonofilaments. However, in freeze-fracture replicas tonofilaments were arranged in thick bundles with a slightly coiled orientation. In freeze-fracture replicas of samples from a pemphigus patient in remission no difference was found in the number of particles on both fracture faces.

Effect of polymyxin on the outer membrane of Salmonella typhimurium: freeze-fracture studies.

Polymyxin-caused projections on the cell surface of Salmonella typhimurium were seen as depressions in the outer concave fracture face and as protrusions in the outer convex fracture face, indicating participation of both leaflets of the outer membrane in these projections.

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.

Biotypes of Arcanobacterium haemolyticum.

Colony morphology, beta hemolysis on horse blood agar, beta-glucuronidase activity, and ability to ferment sucrose and/or trehalose defined two biotypes of Arcanobacterium haemolyticum. One, the smooth type, grew as smooth, beta-hemolytic colonies and was beta-glucuronidase negative but often fermented sucrose and/or trehalose, while the other, the rough type, grew as rough colonies and was nonhemolytic, beta-glucuronidase positive, and negative for sucrose and trehalose fermentation. About 75% of the A. haemolyticum strains studied (n = 138) were of the smooth type. The smooth type predominated in wound infections, while the rough type was isolated almost exclusively from respiratory tract specimens; thus, 84% of the smooth-type strains were derived from wounds and 91% of the rough-type strains were isolated from respiratory tracts.

Eubacterium yurii subspecies margaretiae is resistant to nonopsonic phagocytic ingestion.

We have previously shown that strains of Eubacterium yurii are hydrophobic, as compared with human polymorphonuclear leukocytes (PMNs), possibly because of a crystalline surface layer (S-layer) covering the cell envelope of this potential endo-perio pathogen. The aim of the present study was to investigate the phagocytic ingestion by PMNs of the three E. yurii subspecies, with special attention to bacterial surface structures and hydrophobicity. Type strains of subspp. margaretiae, yurii, and schtitka, together with three clinical isolates from necrotic root canals, were studied. All strains were hydrophobic when tested by a two-phase partition method. E. yurii subspp. margaretiae strains ATCC43715T, ES4C, and ES14B-8E were resistant to PMN ingestion in the absence of opsonins, whereas strains of the two other subspecies were readily ingested. The presence of a resistant strain (subsp. margaretiae ATCC43715T) did not inhibit the ingestion of a sensitive strain (subsp. schtitka ATCC43716T). Ingestion of E. yurii subsp. margaretiae strains required opsonization by normal human serum or specific antibodies. Electron microscopy revealed an S-layer in all strains and fimbria-like structures in the subspp. margaretiae and yurii strains. The antiserum prepared against the S-protein of E. yurii subsp. margaretiae ATCC43715T showed only slight cross-reactivity with other E. yurii strains and indicated the presence of strain-specific rather than species- or subspecies-specific antigens in the S-protein of E. yurii subsp. margaretiae ATCC43715T. The results suggest that the mere presence of the S-layer or fimbria-like structures cannot explain the susceptibility to ingestion by the PMNs.(ABSTRACT TRUNCATED AT 250 WORDS)