The Campylobacter fetus S layer provides resistance to photoactivated zinc oxide nanoparticles.

The antimicrobial activity of metal-based compounds, including metal oxides, has resulted in numerous agricultural, industrial, and medical applications. Zinc oxide nanoparticles are toxic to Gram-positive and Gram-negative bacteria as well as to some fungi. In this study we assess the sensitivity of Campylobacter fetus, a Gram-negative bacterial pathogen of humans and animals, to ZnO nanoparticles and determine whether the S layer protects C. fetus from the antibacterial action of these nanoparticles. Broth and agar dilution assays revealed that ZnO nanoparticles at 100 µg/mL were bacteriocidal for C. fetus. Resazurin reduction assays confirmed the absence of metabolic activity, indicating that C. fetus cells had not entered into a viable but nonculturable state. Photoactivation of ZnO nanoparticles greatly enhanced their antibacterial activity, as evidenced by minimum bacteriocidal concentration (MBC) values decreasing to 16-62.5 µg/mL as a function of strain. MBC assays completed in the presence and absence of catalase revealed that H2O2, a product of ZnO nanoparticle photoactivation, contributed to C. fetus but not to C. jejuni cell death. S-layer-expressing C. fetus strains were more resistant to H2O2-mediated cell killing than were isogenic S-layer-deficient strains. These data indicate that C. fetus is sensitive to the antibacterial activity of ZnO nanoparticles and that the C. fetus S layer imparts protection against photoactivated nanoparticles.

Campylobacter fetus translocation across Caco-2 cell monolayers.

Campylobacter fetus is a recognized pathogen of cattle and sheep, though human infection has also been reported. Ingestion of contaminated food or water is a proposed route of transmission for both humans and animals. The subsequent detection of the organism from extra-intestinal and systemic locations implies an ability to translocate across epithelial barriers. To determine how C. fetus disseminates from the intestine, Caco-2 cells cultured on porous membrane supports, were used as model intestinal epithelial cell monolayers. C. fetus was found to translocate equally well in both apical-to-basolateral and basolateral-to-apical directions for up to 24 h without altering Caco-2 cell monolayer permeability as assessed by transepithelial resistance and absence of paracellular diffusion of FITC-inulin. Using modified antibiotic protection assays, C. fetus was also observed to invade and subsequently egress from Caco-2 cells. Caco-2 cell invasion and translocation occurred independently of C. fetus S layer expression. Scanning and transmission electron microscopy revealed the presence of C. fetus associated with both apical and basal surfaces as well as in intracellular locations. C. fetus was, however, never observed in paracellular locations nor associated with Caco-2 cells junctions. Neither C. fetus invasion nor translocation across Caco-2 cell monolayers was impacted by latrunculin A, though translocation was enhanced in the presence of cytochalasin D which disrupted tight junctions. Tubulin cytoskeleton disrupting agents, colchicine and vinblastine, did inhibit C. fetus translocation though entry into Caco-2 cells remained unaffected. Together, translocation without disrupting monolayer integrity, invasion and egression from Caco-2 cells, electron microscopy observations and the requirement of a functional tubulin cytoskeleton for translocation, support a transcellular mechanism of C. fetus translocation across Caco-2 cell monolayers. The ability to invade and subsequently egress would contribute to establishment of an infecting C. fetus population in the host, while the demonstrated ability to translocate across model intestinal epithelial barriers accounts for the observed in vivo recovery of C. fetus from extra-intestinal locations.

Fibronectin enhances Campylobacter fetus interaction with extracellular matrix components and INT 407 cells.

Campylobacter fetus is a recognized pathogen of cattle and sheep that can also infect humans. No adhesins specific for C. fetus have to date been identified; however, bacterial attachment is essential to establish an infecting population. Scanning electron microscopy revealed C. fetus attachment to the serosal surface of human colonic biopsy explants, a location consistent with the presence of the extracellular matrix (ECM). To determine whether the ECM mediated C. fetus adherence, 7 C. fetus strains were assessed in a solid-phase binding assay for their ability to bind to immobilized ECM components. Of the ECM components assayed, adherence to fibronectin was noted for all strains. Attachment to ECM components was neither correlated with S-layer expression nor with cell-surface hydrophobicity. Ligand immunoblots, however, identified the S-layer protein as a major site of fibronectin binding, and modified ECM binding assays revealed that soluble fibronectin significantly enhanced the attachment of S-layer-expressing C. fetus strains to other ECM components. Soluble fibronectin also increased C. fetus adherence to INT 407 cells. This adherence was inhibited when INT 407 cells were incubated with synthetic peptides containing an RGD sequence, indicating that integrin receptors were involved in fibronectin-mediated attachment. Together, this data suggests that C. fetus can bind to immobilized fibronectin and use soluble fibronectin to enhance attachment to other ECM components and intestinal epithelial cells. In vivo, fibronectin would promote bacterial adherence, thereby, contributing to the initial interaction of C. fetus with mucosal and submucosal surfaces.

The Campylobacter fetus S layer is not essential for initial interaction with HEp-2 cells.

In vitro adherence assays were used to determine whether the S layer mediated interactions between Campylobacter fetus subsp. venerealis strains and HEp-2 cells. At multiplicity of infection ratios ranging from 0.1:1 through 100:1, quantitation of bacterial adherence by light microscopy revealed that S layer deficient isogenic C. fetus 809K and C. fetus 810K were not less efficient in their attachment to HEp-2 cells; either S layer deficient C. fetus strains interacted with HEp-2 cells in greater numbers than the corresponding wild-type parent strains 809 and 810 or there was no significant difference in adherence levels between wild-type and mutant strains. Adherence of C. fetus strains to HEp-2 cells increased most during the first 2 h of a 22-h incubation period with only a slight increase in C. fetus cell numbers occurring subsequent to 2 h. At each assay point throughout this 22-h time period, equivalent numbers of wild-type and S layer deficient C. fetus strains were observed associated with HEp-2 cells. Prior to 2 h, adherence levels of all C. fetus strains exceeded those of Escherichia coli AB264 and Salmonella typhimurium SL1344. And, unlike S. typhimurium, C. fetus did not undergo significant replication following initial adherence to HEp-2 cells. Campylobacter fetus did not adhere to HEp-2 cells in a localized or aggregative pattern but were randomly distributed over individual HEp-2 cells and at no time during the assay with C. fetus were changes in HEp-2 cell morphology apparent. These data suggest that the S layer is not essential for mediating initial interactions between C. fetus and HEp-2 cells.

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.

HIV and serious mental illness: reducing the risk.

Persons with SMI residing in community mental health center group homes received an educational intervention program on HIV/AIDS. As with virtually all such approaches provided for this population, the intervention was generalized from programs used with other populations, for example, users of intravenous drugs, gay men, and adolescents. Assessment of pre- and postintervention knowledge indicated no increase in accurate information. Further, consumers were initially uncertain regarding their risk for HIV infection; this attitude, too, remained unchanged. The research design employed did not compare persons with SMI with a normative sample receiving the same information, and assessed with the same instruments, limiting hypotheses about the generalizability of existing interventions. However, the data seems to suggest its potential inefficacy. Several findings are germane to effective educational intervention techniques.

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.

How to reduce the risk of HIV infection for the seriously mentally ill.

1. The professional literature increasingly indicates the need to educate persons with serious mental illness regarding HIV/AIDS. 2. Community-based organizations currently responding to the AIDS epidemic are poorly equipped to respond to the special needs of persons with serious mental illness. 3. Persons with serious mental illness are concerned by HIV/AIDS. Some, however, will incorporate education into their pathology via delusional systems or misunderstandings. 4. Effective programs for this population can be developed following community health nursing principles.

Freeze-substitution studies of bacteria.

Typically, models of bacterial structure combine biochemical data obtained from bulk analyses of cell populations with electron microscopic observation of individual cells. Recent development of a battery of cryotechniques specific for biological electron microscopy have begun to supercede routine procedures such as conventional thin sectioning. One of these cryotechniques, freeze-substitution, combines the advantages of ultrarapid freezing with standard microtomy methods. This technique is particularly well suited to the examination of bacterial structure and has yielded additional ultrastructural information consistent with biochemical data but often challenging models of cell structure obtained from conventional microscopical methods. In addition to retaining more accurately the spatial distribution of cell components, freeze-substitution has been successfully combined with immunochemical labelling techniques and has enabled identification and localization of specific molecules both within the cell and on the cell surface. In this review, I describe current ideas on bacterial ultrastructure, modified in accordance with new data obtained from recent freeze-substitution studies.

Surface layers of bacteria.

Since bacteria are so small, microscopy has traditionally been used to study them as individual cells. To this end, electron microscopy has been a most powerful tool for studying bacterial surfaces; the viewing of macromolecular arrangements of some surfaces is now possible. This review compares older conventional electron-microscopic methods with new cryotechniques currently available and the results each has produced. Emphasis is not placed on the methodology but, rather, on the importance of the results in terms of our perception of the makeup and function of bacterial surfaces and their interaction with the surrounding environment.

Freeze-substitution of gram-negative eubacteria: general cell morphology and envelope profiles.

Freeze-substitution was performed on strains of Escherichia coli, Pasteurella multocida, Campylobacter fetus, Vibrio cholerae, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas salmonicida, Proteus mirabilis, Haemophilus pleuropneumoniae, Caulobacter crescentus, and Leptothrix discophora with a substitution medium composed of 2% osmium tetroxide and 2% uranyl acetate in anhydrous acetone. A thick periplasmic gel ranging from 10.6 to 14.3 nm in width was displayed in E. coli K-12, K30, and His 1 (a K-12 derivative containing the K30 capsule genes), P. multocida, C. fetus, P. putida, A. salmonicida, H. pleuropneumoniae, and P. mirabilis. The other bacteria possessed translucent periplasms in which a thinner peptidoglycan layer was seen. Capsular polysaccharide, evident as electron-dense fibers radiating outward perpendicular to the cell surface, was observed on E. coli K30 and His 1 and P. mirabilis cells. A more random arrangement of fibers forming a netlike structure was apparent surrounding cells of H. pleuropneumoniae. For the first time a capsule, distinct from the sheath, was observed on L. discophora. In all instances, capsular polysaccharide was visualized in the absence of stabilizing agents such as homologous antisera or ruthenium red. Other distinct envelope structures were observed external to the outer membrane including the sheath of L. discophora and the S layers of A. salmonicida A450 and C. crescentus CB15A. We believe that the freeze-substitution technique presents a more accurate image of the structural organization of these cells and that it has revealed complex ultrastructural relationships between cell envelope constituents previously difficult to visualize by more conventional means of preparation.

Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria.

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acid and wall components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetone-treated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (ie., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.

Effect of chemical fixatives on accurate preservation of Escherichia coli and Bacillus subtilis structure in cells prepared by freeze-substitution.

Five chemical fixatives were evaluated for their ability to accurately preserve bacterial ultrastructure during freeze-substitution of select Escherichia coli and Bacillus subtilis strains. Radioisotopes were specifically incorporated into the peptidoglycan, lipopolysaccharide, and nucleic acids of E. coli SFK11 and W7 and into the peptidoglycan and RNA of B. subtilis 168 and W23. The ease of extraction of radiolabels, as assessed by liquid scintillation counting during all stages of processing for freeze-substitution, was used as an indicator of cell structural integrity and retention of cellular chemical composition. Subsequent visual examination by electron microscopy was used to confirm ultrastructural conformation. The fixatives used were: 2% (wt/vol) osmium tetroxide and 2% (wt/vol) uranyl acetate; 2% (vol/vol) glutaraldehyde and 2% (wt/vol) uranyl acetate; 2% (vol/vol) acrolein and 2% (wt/vol) uranyl acetate; 2% (wt/vol) gallic acid; and 2% (wt/vol) uranyl acetate. All fixatives were prepared in a substitution solvent of anhydrous acetone. Extraction of cellular constituents depended on the chemical fixative used. A combination of 2% osmium tetroxide-2% uranyl acetate or 2% gallic acid alone resulted in optimum fixation as ascertained by least extraction of radiolabels. In both gram-positive and gram-negative organisms, high levels of radiolabel were detected in the processing fluids in which 2% acrolein-2% uranyl acetate, 2% glutaraldehyde-2% uranyl acetate, or 2% uranyl acetate alone were used as fixatives. Ultrastructural variations were observed in cells freeze-substituted in the presence of different chemical fixatives. We recommend the use of osmium tetroxide and uranyl acetate in acetone for routine freeze-substitution of eubacteria, while gallic acid is recommended for use when microanalytical processing necessitates the omission of osmium.

Responding to the needs of the terminally ill through laughter and play.

Care for the terminally ill has greatly expanded. Humor and play, however, remain largely unexplored, surrendered to the cultural expectation of dignity and respect for the dying. This paper suggests several uses of humor and play with the dying, and the benefits of these interventions with patients, families, and care givers.

Toxic shock syndrome: odontogenic origin.

Although the majority of reported cases of toxic shock syndrome (TSS) in the United States continue to be associated with tampon use, TSS also occurs in postpartum women and in patients with pharyngitis, infected surgical wounds, cutaneous and subcutaneous infections, and infections of other body sites. The article presents the case of a 23-year-old black man in whom TSS developed secondary to an odontogenic infection.

Elimination of mandibular labial undercut with autogenous bone graft from a maxillary tuberosity.

A brief review of various grafts and implants that may be used to eliminate labial undercuts was described. A technique for elimination of the mandibular labial undercut with autogenous bone from the maxillary tuberosity was presented.