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.

Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

Hard metal alveolitis accompanied by rheumatoid arthritis.

Hard metal lung diseases (HML) are rare, and complex to diagnose. We describe the case of a patient with allergic alveolitis accompanied by rheumatoid arthritis. A sharpener of hard metal by trade, our patient was a 45-year-old, nonsmoking Caucasian female who experienced symptoms of cough and phlegm, and dyspnea on exertion. Preliminary lung findings were inspiratory rales in both basal areas, decreased diffusion capacity and a radiological picture resembling sarcoidosis. A high-resolution computed tomography scan indicated patchy alveolitis as well. An open lung biopsy revealed non-necrotizing granulomas consisting of epitheloid cells and surrounded by lymphocytes, plasma cells and a few eosinophils. These cells also occupied the thickened alveolar interstitium. Macrophages in the alveolar spaces, some of them multinuclear, contained dust particles. Hard metal alveolitis is clinically well known and, in this patient, has been described histologically. After the patient had quit working with hard metal and following corticosteroid therapy, pulmonary symptoms and signs were relieved. During this recovery period, however, she contracted rheumatoid arthritis.

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.

Surface structure, hydrophobicity, phagocytosis, and adherence to matrix proteins of Bacillus cereus cells with and without the crystalline surface protein layer.

Nonopsonic phagocytosis of Bacillus cereus by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied. Two reference strains (ATCC 14579(T) and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant. Both young and old cultures of the reference strains of B. cereus were resistant to PMN ingestion. Preincubation of PMNs with the phagocytosis-resistant strains of B. cereus did not affect the phagocytosis of the sensitive strain. Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface. In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells. No S-layer was detected on the reference strains. Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342. One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin. In contrast, reference strains (hydrophobicity, -1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of B. cereus OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin. Heating the cells at 84 degreesC totally abolished binding to both proteins. Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of B. cereus cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.

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.

Release and activation of human neutrophil matrix metallo- and serine proteinases during phagocytosis of Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola.

The phagocytic ingestion of reference strains and clinical isolates of Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola by polymorphonuclear leukocytes (PMNs) and the concomitant release of PMN granule proteinases were studied by specific functional and immunological assays. PMNs were incubated with the microorganisms anaerobically at 37 degrees C for indicated time periods. The suspensions and pellets were used for phagocytic ingestion assay and electron microscopic study, respectively. The supernatants were used for the measurements of the amounts and activities of the released PMN enzymes including PMN gelatinase (MMP-9), collagenase (MMP-8), serine proteases (elastase and cathepsin G), and lactate dehydrogenase (LDH). Both fluorescence microscopy and transmission electron microscopy showed that F. nucleatum, P. gingivalis and T. denticola were ingested by the PMNs in comparable numbers. However, measurements of the enzymes released from the triggered PMNs revealed major differences among the three species. High amount of elastase was released from the PMNs triggered by F. nucleatum, but not by P. gingivalis or T. denticola. The treatment of PMNs with P. gingivalis whole cells resulted in the release of gelatinase partly in the 82 kD active form, suggesting proteolytic activation of the degranulated 92 kD proMMP-9. The 82 kD active form of gelatinase was not detected upon triggering the PMNs with F. nucleatum and T. denticola. The PMN-bacteria interaction did not result in release of LDH from triggered PMNs indicating the proteinase release was not due to the PMN cell death. The results show that the susceptibilities of the 3 potentially periodontopathogenic microorganisms, F. nucleatum, P. gingivalis and T. denticola to phagocytic ingestion are not directly related to the amounts and activities of PMN enzymes released during the bacteria-PMN interactions. As PMN degranulation is considered as one of the major pathogenic mechanisms in periodontitis, the observed differences among the microorganisms may be important virulence characteristics of these species.

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.

Demonstration of Chlamydia pneumoniae in the walls of abdominal aortic aneurysms.

BACKGROUND: Seroepidemiologic studies have indicated an association between chronic Chlamydia pneumoniae infection and coronary heart disease. The organism, which is a common respiratory pathogen, has been demonstrated in atherosclerotic lesions of the aorta and coronary arteries. Abdominal aortic aneurysms are frequently associated with atherosclerosis, and inflammation may actually be an important factor in aneurysmal dilatation. Hence it could be assumed that C. pneumoniae may play a role in maintaining an inflammation and triggering the development of aortic aneurysms. METHODS AND RESULTS: Specimens from abdominal aortic aneurysm were examined for the presence of C. pneumoniae by immunohistochemical analysis, the polymerase chain reaction amplifying omp 1 gene, transmission electron microscopy, and culture methods with histologically atherosclerosis-negative human aortic tissues used as a control group. Chlamydial lipopolysaccharide and C. pneumoniae specific antigens were found by immunohistochemistry in 12 and 8 of 12 aneurysm specimens, respectively, and C. pneumoniae DNA could be demonstrated in 6 of 6 aneurysm specimens studied. Furthermore electron microscopy revealed the presence of Chlamydia-like elementary bodies in three of four aneurysm specimens tested. None of the control samples gave positive reaction in the polymerase chain reaction, and C. pneumoniae antigens were not detected in any of them. CONCLUSIONS: C. pneumoniae is frequently found in the vessel wall of abdominal aortic aneurysm. The potential etiopathogenetic role of C. pneumoniae in the development of these aneurysms remains to be studied.

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.

Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope.

Interferon-inducible Mx proteins belong to the family of large GTPases and are highly homologous with dynamins within their GTP-binding domain. Cytoplasmically localized human MxA protein mediates resistance to influenza and several other viruses, whereas human MxB protein has not been found to have any antiviral activity. Here we show that MxB protein is found both in the cytoplasm and in the nucleus, where it is localized in a granular pattern in the heterochromatin region beneath the nuclear envelope. Transfection experiments in COS cells of N-terminally deleted MxB constructs revealed a functional nuclear localization signal within the first 24 N-terminal amino acids. Nuclear 78-kDa and cytoplasmic 76-kDa forms of MxB protein were found in all of the cell lines studied and in human peripheral blood mononuclear cells. MxB protein proved to be a functional GTPase with activity comparable to that of MxA protein. N-terminally truncated (delta1-82) MxB protein lacking both the nuclear localization signal and a proline-rich domain had almost completely lost its GTPase activity. Analysis of peripheral blood mononuclear cells suggested that MxB protein expression is strictly regulated by interferon-alpha. This is the first documentation that human Mx protein resides in the nucleus. It also emphasizes that there are considerable differences in the localization and structure of functional domains within Mx proteins.

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.

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.

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)

S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence.

The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found.

Hydrophobicities of human polymorphonuclear leukocytes and oral Bacteroides and Porphyromonas spp., Wolinella recta, and Eubacterium yurii with special reference to bacterial surface structures.

The hydrophobicities of human polymorphonuclear leukocytes (PMNLs) and Bacteroides buccae, B. oris, B. oralis, B. veroralis, B. buccalis, B. heparinolyticus, B. intermedius, B. denticola, B. loescheii, B. melaninogenicus, Porphyromonas gingivalis, P. endodontalis, Wolinella recta, and Eubacterium yurii were studied by the hexadecane method. The majority of the strains were equally or less hydrophobic than the PMNLs. Only in the case of E. yurii and the only strain of B. buccalis were all strains more hydrophobic than the PMNLs. However, some strains of B. intermedius, B. oris, B. denticola, and P. gingivalis were also more hydrophobic than the PMNLs. With the exception of B. intermedius and species with a crystalline surface protein layer (S-layer), the strains of all other species with a thick capsule were more hydrophilic than the strains with little or no extracellular polymeric material. All strains of the S-layer species were either quite hydrophilic or hydrophobic depending on the species, totally irrespective of the presence of the capsule. The results suggest that the S-layers of oral anaerobic bacteria may be important determinants of cell surface hydrophobicity.

Ingestion of Bacteroides buccae, Bacteroides oris, Porphyromonas gingivalis, and Fusobacterium nucleatum by human polymorphonuclear leukocytes in vitro.

The phagocytic ingestion of clinical isolates and reference strains of Bacteroides buccae, Bacteroides oris, Porphyromonas gingivalis and Fusobacterium nucleatum by polymorphonuclear leukocytes (PMNs) was studied. Special attention was focused on the hydrophobicity of the strains. B. buccae strains, less or equally hydrophobic than PMNs, were poorly ingested without opsonization. Hydrophobic, but not hydrophilic, strains of B. oris and both hydrophilic and hydrophobic P. gingivalis and F. nucleatum strains were readily ingested without opsonization. Hydrophobicity thus contributes to the adherence of bacteria to PMNs in some, but not all, species tested. Normal human serum enhanced the ingestion of B. buccae, but failed to do so after heat-inactivation. Heat-inactivation of the immune serum to B. buccae strain ES57 did not reduce opsonic activity suggesting that specific antibodies enhanced the ingestion of B. buccae.