Ornithine metabolism in the genus Rochalimaea.

Ornithine metabolism was studied in two strains of the trench fever rickettsia Rochalimaea quintana, Fuller and Guadalupe, and in the vole agent, a strain of Rochalimaea but not necessarily of Rochalimaea quintana. The metabolic activity of intact cells and cell-free extracts was measured by monitoring the evolution of 14CO2 from [1-14C]ornithine. Low levels of activity were obtained with all three strains, but requirements for the demonstration of this activity differed. With the cells of the Fuller and Guadalupe strains, the decarboxylation of ornithine was almost completely dependent on added pyruvate or succinate, presumably as sources of energy for transport. This enhancement was not prevented by the presence of chloramphenicol. The activity of the vole agent, on the other hand, required the complete medium. This activity was prevented by chloramphenicol added at the same time as the medium but not by chloramphenicol added after 1 h of incubation. In cell-free extracts, the demonstration of ornithine decarboxylase activity in the vole agent required prior induction with medium containing ornithine, whereas in the other two strains, the activity was constitutive. The activities of the extracts of the Fuller strain and the vole agent differed also in pH optimum, which was somewhat lower for the vole agent, and in the added pyridoxal phosphate requirement, which was greater for the Fuller strain. Comparable experiments with Rickettsia typhi and Rickettsia prowazekii failed to reveal evidence of ornithine metabolism.     

Ultrastructure of spirochetes isolated from Ixodes ricinus and Ixodes dammini

Two strains of Ixodes spirochetes, one isolated in the United States (B31) and the other in Sweden (G25), were examined by electron microscopy. Cells of strain G25 were 11-25 micron long with a wavelength of 2.1-2.4 micron and an amplitude of 0.4 micron. Eleven flagella were inserted subterminally at each end of the cell. Cells of strain B31 were similar but had eleven or seven flagella. Cytoplasmic tubules were not seen in cells of either strain. Although not identical, both strains showed ultrastructural details characteristic of the genus Borrelia.     

Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 X 1.2 micron in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 X 3.0 micron and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)-containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24 hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

Starvation and nutrient resuscitation of Klebsiella pneumoniae isolated from oil well waters

Klebsiella pneumoniae isolated from oil well waters reduced in size in response to nutrient starvation. The cells remained viable during starvation and later were able to grow rapidly when stimulated by nutrients. The heterotrophic potential, culture absorbance and extracellular polysaccharide production decreased during cell starvation whereas an initial increase in colony-forming units was observed on agar plates. Transmission electron microscopy (TEM) after 24 d revealed that the cells had changed to small rods or cocci between 0.5 by 0.25 micron and 0.87 by 0.55 micron. When transferred to half-strength brain heart infusion medium, TEM showed cell division and rod-shaped cells after 45 min and full resuscitation within 4 h. Cell response was much slower in sodium citrate medium and resuscitation took 8 h.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Demonstration by light and electron microscopy of capsules on gonococci recently grown in vivo.

A study by light microscopy, using Leishman's stain alone or Leishman's stain followed by nigrosin, showed the presence of capsules on gonococci of two strains subcultured from subcutaneous chambers in guinea pigs. With the Alcian blue method of preparation for electron microscopy, gonococci of both strains recently grown in vivo showed densely stained capsules on some cells, while others in the same preparation showed only irregular masses of dense material on their surfaces with strands connecting adjacent bacteria. Treatment with antiserum, complement and conglutinin revealed irregular masses and strands of extracellular material with fixatives that did not contain Alcian blue.     

Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero , causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood–brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild-type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild-type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inl B gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inl AB was complemented with a plasmid harbouring inl B only, whereas strains expressing inl A did not enter HUVECs. Thirdly, entry of wild-type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non-invasive mutants dramatically increased their ability to invade HUVECs. In laser-scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild-type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.     

Electron microscopy of adhesive interactions between Gardnerella vaginalis and vaginal epithelial cells, McCoy cells and human red blood cells

Exfoliated vaginal epithelial cells with attached bacteria, termed 'clue cells', which were procured from a patient with non-specific vaginitis, were stained with ruthenium red and examined by transmission electron microscopy. The attached bacteria appeared to adhere by means of an outer fibrillar coat. An epithelial tissue culture cell line (McCoy) and human red blood cells to which strains of Gardnerella vaginalis attached were similarly examined. The adherence of G. vaginalis to the epithelial cell line appeared to be mediated by an outer fibrillar coat while adherence to red cells appeared to be mediated by fimbriae. Transmission electron microscopy was performed on the Gardnerella strains used. Thin sections of tissue-culture-adherent strains revealed a dense outer fibrillar coat whereas the surface of the haemagglutinating strains showed fine fimbriae. Negative staining of haemagglutinating strains demonstrated fimbriae on a minority of organisms.     

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42–114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal–host interactions and adaptation to this ecological niche.     

Ultrastructure of rumen bacterial attachment to forage cell walls.

The degradation of forage cell walls by rumen bacteria was investigated with critical-point drying/scanning electron microscopy and ruthenium red staining/transmission electron microscopy. Differences were observed in the manner of attachment of different morphological types of rumen bacteria to plant cell walls during degradation. Cocci, constituting about 22% of the attached bacteria, appeared to be attached to degraded plant walls via capsule-like substances averaging 58 nm in width (range, 21 to 84 nm). Many bacilli appeared to adhere to forage substrates without distinct capsule-like material, although unattached bacteria with capsules were observed occasionally. Certain bacili appeared to be attached to degraded tissue via small amounts of extracellular material, but others apparently had no extracellular material. Bacilli with a distinct morphology due to an irregularly folded, electron-dense outer layer or layers (about 15 nm thick) and without fibrous extracellular material consituted about 37% of the attached bacteria and were observed to adhere so closely to degraded plant walls that the bacterial shape conformed to the shape of the degraded zone. In the rumen ecosystem, bacteria appeared to adhere to plant substrates during degradation by capsule-like material and by small amounts of extracellular material, as well as by the other means not observable by electron microscopy.     

Adherence to HEp-2 cells and replication in macrophages of Salmonella derby of human origin.

Adherence to a HEp-2 cell monolayer was tested for in four strains of Salmonella derby which were isolated from patients with diarrhea. One strain, SB1, was highly adherent and another strain, SB4, was nonadherent. The other two strains exhibited moderate adherence. Further in vitro study of invasion of HEp-2 cells by S. derby and its replication in murine peritoneal macrophages was carried out using SB1 and SB4. Thin section electron micrographs revealed that SB1 invaded HEp-2 cells but SB4 did not. The number of viable bacteria within macrophages was determined at intervals after inoculation of bacteria. The result indicates that SB1 can replicate in the macrophages but SB4 cannot. Flagella and fimbriae were compared by electron microscopy between SB1 and SB4, and their lipopolysaccharides and outer membrane proteins were also compared with each other by SDS-polyacrylamide gel electrophoresis. The presence of a 41 kDa protein in the outer membranes of SB1 was only the difference detected, suggesting that this protein could be a factor required for adherence of this serovar to epithelial cells.     

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.     

Ultrastructure of methanotrophic yeasts.

The cellular structure of two yeast strains capable of growth on methane was investigated by electron microscopy. Microbodies were observed in cells of Sporobolomyces roseus strain Y and Rhodotorula glutinis strain CY when grown on methane but rarely when grown on glucose. The size of the microbodies and the number observed per cell in a thin section did not increase with culture age. No crystalline organization was observed within these organelles. Similar microbodies were also observed in cells of R. glutinis CY grown on hexadecane. The plasma membranes of both methane and hexadecane-grown cells exhibited increased invagination compared to that of glucose-grown cells. Catalase activity was detected in the microbodies of alkane-grown cells by using 3,3'-diaminobenzidine as a cytochemical stain. The data presented suggest that microbodies, and the catalase contained within them, play a role in eucaryotic methane metabolism.     

Freeze-etching observations of trophozoites of pathogenic Entamoeba histolytica.

Pathogenic Entamoeba histolytica trophozoites were studied by the freeze-etching (FE) technique of electron microscopy. Surface replicas of intact cell membranes were highly convoluted with numerous invaginations, evaginations, and undulations. Sperical depressions and elevations varying from 0.5 mu to 1.0 mu in diameter were commonly present on the external cell membrane and appeared to represent an extracellular secretory mechanism of trophozoites. Cleaved surfaces of amebae exhibited a granular and lumpy cytoplasm in which there were many vesicles and vacuoles that ranged in diameter from 0.2 mu to 9.0 mu. Some vacuoles contained tightly enveloped bacteria, while others contained bacteria and host cytocomponents. Occasional vesicles and vacuoles appeared to be fused to each other. Replicas of FE nucleus were enclosed by double nuclear membranes which were fenestrated by numerous sperical pores measuring approximately 640 A in diameter and spaced at intervals of 650 A. Counts of nuclear pores were possible and indicated 35 pores per square micron on the nuclear envelope. Golgi apparatus, mitochondria and well formed endoplasmic reticulum were absent in FE replicas. This was in agreement with electron microscope observations on thin sections previously reported by other investigators.     

Internalization of iron-transferrin complex by murine L1210 leukemia cells and rat reticulocytes demonstrated by a minibead probe

To determine if the cellular uptake of iron is associated with internalization of iron-transferrin (TF) complex by the cell, we synthesized a visual probe in which TF is covalently bound to amide-modified latex minibead, submicrometer in size (0.345 micron). Incubation of the probe with L1210 leukemia cells and rat reticulocytes led to the binding of the probe to the cell surface visualized and semiquantified by scanning and transmission electron microscopy. The binding was inhibited by preincubation with nonderivatized iron-TF complex. Internalization of the probe occurred through clathrin-coated pits and vesicles. Minibeads derivatized by nontransport proteins or glycine as well as nonderivatized minibeads did not appreciably bind to the cells and were not internalized. Ethylamine, an inhibitor of receptor-mediated endocytosis abolished the internalization but not the binding of the probe which, then, accumulated on the cell surface. These findings provide direct evidence for internalization of TF during the iron uptake.     

Cell wall-associated protein A as a tool for immunolocalization of Staphylococcus aureus in infected human airway epithelium.

Staphylococcus aureus is a common human pathogen involved in non-bronchial diseases and in genetic and acquired bronchial diseases. In this study, we applied an immunolabeling approach for in vivo and in vitro detection of S. aureus, based on the affinity of staphylococcal protein A (SpA) for the Fc region of immunoglobulins, especially IgG. Most strains of S. aureus, including clinical strains, can be detected with this labeling technique. The approach can be used for detection and localization with transmission electron microscopy or light-fluorescence microscopy of S. aureus in infected tissues such as human bronchial tissue from cystic fibrosis (CF) patients. The methodology can also be applied to cell culture models with the aim of characterizing bacterial adherence to epithelial cells in backscattered electron imaging with scanning electron microscopy. Application to the study of S. aureus adherence to airway epithelium showed that the bacteria did not adhere in vivo to intact airway epithelium. In contrast, bacteria adhered to the basolateral plasma membrane of columnar cells, to basal cells, to the basement membrane and were identified beneath the lamina propria when the epithelium was injured and remodeled, or in vitro when the epithelial cells were dedifferentiated.     

Evidence for the Colonization of Lactic Acid Bacteria in the Gastrointestinal Tract of Suckling Mink

Lactic acid bacteria are considered indigenous members of the gastrointestinal microflora in a number of animal species (Savage 1977a). Some intestinal strains of lactobacilli and streptococci are aWe to adhere to stratified squamous epithelium of some animals (Tannock et al. 1987), in the non-secreting part of the stomach of piglets (Barrow et al. 1980, Fuller et al. 1978) and rodents (Tannock et al. 1982), and in the crop of poultry (Fuller 1978). The presence of lactic acid bacteria in the digestive tract is believed to be of beneficial value to the host animal (Fuller 1989). The production of organic acids in the stomach or the crop helps maintaining a low pH which may be important for inhibiting the colonization of potentially pathogenic bacteria, particularly in the newborn animal (Barrow et al 1980, Fuller 1977, Fuller 1978). The adhesion of lactobacilli to squamous epithelium is host specific: strains capable of adhering to the epithelium of piglets are usually not able to adhere in rodents or poultry and vice versa (Fuller 1978, Lin & Savage 1984, Tannock et al 1982). Adhesion of lactic acid bacterial strains to other epithelia than stratified squamous epithelium has been reported. Thus, the attachment of lactobacilli to cells from the secreting epithelium of the murine stomach (Kotarski & Savage 1979), to intestinal cells of humans (Goldin & Gorbach 1987), and to columnar epithelial cells of piglets and calves (Mäyrä-Mäkinen et al 1983) has been demonstrated using in vitro methods. In another study the in vivo attachment of Enterococcus faecium to duodenal epithelium of gnotobi-otic chickens was demonstrated (Fuller et al 1981). Recent research indicated that in adult mink lactic acid bacteria are not indigenous members of the intestinal flora, and they do not attach to epithelium in any part of the gastrointestinal tract (Federsen & Jørgensen 1992). The present paper presents evidence that Gram positive cocci may colonize the gut of suckling mink kits and attach to the gut mucosa.     

Vaccinia virus E2L null mutants exhibit a major reduction in extracellular virion formation and virus spread

The vaccinia virus E2L (VACWR058) gene is conserved in all sequenced chordopoxviruses and is predicted to encode an 86-kDa protein with no recognizable functional motifs or nonpoxvirus homologs. Although the region immediately upstream of the open reading frame lacked optimal consensus promoter motifs, expression of the E2 protein occurred after viral DNA replication. Transfection studies, however, indicated that the promoter was weak compared to well-characterized intermediate and late promoters. The E2 protein was present in mature virions purified from infected cells but was more abundant in extracellular enveloped forms. Despite the conservation of the E2L gene in chordopoxviruses, deletion mutants could be isolated from both the WR and IHD-J strains of vaccinia virus. These null mutants produced very small plaques in all cell lines tested, reduced amounts of mature infectious virions, and very low numbers of extracellular virions. Nevertheless, viral protein synthesis appeared qualitatively and quantitatively normal. The defect in extracellular virus formation was corroborated by electron microscopy, which also showed some aberration in the wrapping of virions by cisternal membranes. Extracellular virions that did form, however, were able to induce actin tail formation.