George Tortora

Autologous fibrin gel: bactericidal properties in contaminated hepatic injury.

Fibrin glue is an effective hemostatic agent in a variety of clinical situations; its utility is limited by potential transmission of viral infection. We studied the bactericidal properties of fibrin gel (FG) in a murine contaminated hepatic injury model and in vitro by agar plate culture method. Intra-abdominal abscess formation and adhesion rate were assessed following controlled liver injury in association with abdominal contamination with 10(7) Bacteroides fragilis and hepatorrhaphy (H, n = 15) or FG (n = 12). Animals treated by hepatorrhaphy had a significantly greater intra-abdominal abscess rate (15/15 vs. 4/12, p less than 0.05) and adhesion rate (14/15 vs. 6/12, p less than 0.05) than animals treated with FG. Fibrin gel is bactericidal to Bacteroides fragilis, Enterobacter faecium, Escherichia coli, and Staphylococcus aureus but has no effect against Klebsiella pneumoniae or Pseudomonas aeruginosa; the plasma component appears active. Fibrin gel demonstrates significant improvement in adhesion formation and intra-abdominal abscess rate when compared with suture hepatorrhaphy. Fibrin gel appears protective in contaminated hepatic injury.

Exosporial membrane plasticity of Clostridium sporogenes and Clostridium difficile

This investigation examines the morphological alterations of the exosporial membranes of Clostridium sporogenes ATCC 3584 and Clostridium difficile ATCC 43594 and 9689 endospores in relation to their possible function during germination in the attachment/colonization process of these pathogenic bacteria. There is no reported function for the exosporial membrane, nor exosporial appendages, of clostridial endospores. Advances in high resolution, scanning electron microscopy (SEM) permit the examination of these delicate, morphological projections on intact spores in the process of attachment. The morphological plasticity of the exosporial membrane projections during activation and germination was examined to determine whether the appearance of these exosporial projections coincided with attachment of the spores to the nutritive substrate, and whether this attachment could be altered by physical agitation, cation competition with Ba2+, chelation with EDTA, or treatment with colchicine. Following incubation, activated spores could not be removed from the agar surface by agitation in water (pH 7.2 or 9.1), nor by agitation in buffer or colchicine, indicating that some form of adherence or attachment to the agar had taken place. When agitated in the presence of Ba2+ or EDTA in phosphate buffered saline or EDTA in water, all activated spores detached from the agar and exhibited decreased exosporial projections and minimal, if any, attachment structures to the agar surface. Activated clostridial spores were found to attach to agar by delicate extensions of the exosporium that could be disrupted by EDTA or Ba2+ exposure, but were unchanged when shaken in buffer or water.

Pseudomonas putrefaciens bacteremia associated with shellfish contact

Observation chez un homme ayant pris des bains dans des baies ou se fait la recolte de coquillages et ayant ingere ceux-ci

Potato Dextrose Agar Antifungal Susceptibility Testing for Yeasts and Molds: Evaluation of Phosphate Effect on Antifungal Activity of CMT-3

ABSTRACT The broth macrodilution method (BMM) for antifungal susceptibility testing, approved by the National Committee for Clinical Laboratory Standards (NCCLS), was found to have deficiencies in testing of the antifungal activity of a new type of antifungal agent, a nonantibacterial chemically modified tetracycline (CMT-3). The high content of phosphate in the medium was found to greatly increase the MICs of CMT-3. To avoid the interference of phosphate in the test, a new method using potato dextrose agar (PDA) as a culture medium was developed. Eight strains of fungi, including five American Type Culture Collection strains and three clinical isolates, were used to determine the MICs of amphotericin B and itraconazole with both the BMM and the PDA methods. The MICs of the two antifungal agents determined with the PDA method showed 99% agreement with those determined with the BMM method within 1 log2 dilution. Similarly, the overall reproducibility of the MICs with the PDA method was above 97%. Three other antifungal agents, fluconazole, ketoconazole, and CMT-3, were also tested in parallel against yeasts and molds with both the BMM and the PDA methods. The MICs of fluconazole and ketoconazole determined with the PDA method showed 100% agreement within 1 log2 dilution of those obtained with the BMM method. However, the MICs of CMT-3 determined with the BMM method were as high as 128 times those determined with the PDA method. The effect of phosphate on the antifungal activity of CMT-3 was evaluated by adding Na2HPO4 to PDA in the new method. It was found that the MIC of CMT-3 against a Penicillium sp. increased from 0.5 μg/ml (control) to 2.0 μg/ml when the added phosphate was used at a concentration of 0.8 mg/ml, indicating a strong interference of Na2HPO4 with the antifungal activity of CMT-3. Except for fluconazole, all the other antifungal agents demonstrated clear end points among the yeasts and molds tested. Nevertheless, with its high reproducibility, good agreement with NCCLS proposed MIC ranges, and lack of interference of phosphate, the PDA method shows promise as a useful assay for antifungal susceptibility testing and screening for new antifungal agents, especially for drugs that may be affected by high (supraphysiologic) phosphate concentrations.

A Chemically Modified Tetracycline (CMT-3) Is a New Antifungal Agent

ABSTRACT Several chemically modified tetracycline analogs (CMTs), which were chemically modified to eliminate their antibacterial efficacy, were unexpectedly found to have antifungal properties. Of 10 CMTs screened in vitro, all exhibited antifungal activities, although their efficacies varied. Among these compounds, CMT-315, -3, and -308 were found to be the most potent as antifungal agents. The MICs of CMT-3 against 47 strains of fungi in vitro were determined by using amphotericin B (AMB) and doxycycline as positive and negative controls, respectively. The MICs of CMT-3 were generally found to be between 0.25 and 8.00 μg/ml, a range that approximates the blood levels of this drug when administrated orally to humans. Of all the yeast species tested to date, Candida albicans showed the greatest sensitivity to CMT-3. The filamentous species most susceptible to CMT-3 were found to be Epidermophyton floccosum, Microsporum gypseum, Pseudallescheria boydii, a Penicillium sp., Scedosporium apiospermum, a Tricothecium sp., and Trichophyton rubrum. Growth inhibition of C. albicans by CMT-3, determined by a turbidity assay, indicated a 50% inhibitory concentration of 1 μg/ml. Thirty-nine strains, including 20 yeasts and 19 molds, were used to measure viability (the ability to grow after treatment with a drug) inhibition by CMT-3 and AMB. CMT-3 exhibited fungicidal activity against most of these fungi, especially the filamentous fungi. Eighty-four percent (16 of 19) of the filamentous fungi tested showed more than 90% inhibition of viability by CMT-3. In contrast, AMB showed fungicidal activity against all yeasts tested. However, most of the filamentous fungi (16 of 19) showed less than 50% inhibition of viability by AMB, indicating that AMB is fungistatic against most of these filamentous fungi. To begin to identify the sites in fungal cells affected by CMT-3, C. albicans and a Penicillium sp. were incubated with the compound at 35°C, and then the fluorescence of CMT-3 was observed by confocal laser scanning electron microscopy. CMT-3 appeared to have widespread intracellular distribution throughout C. albicans and the Penicillium sp. The mechanisms of the antifungal activity of CMT-3 are now being explored.

Human epithelial cell processing of carbon and gold nanoparticles

This paper describes some early cellular and intracellular interactions of human polarised lung and colon epithelial cells (representative of two portals of entry, inhalation and ingestion), following exposure to specific carbon and gold engineered nanoparticles in vitro. Cells were incubated with functionalised and non-functionalised carbon nanotube-derived nanoloops (∼28?60 nm diameter), or gold nanoparticles (2 nm and 10 nm Au-core) which were either non-functionalised, or functionalised with biological proteins or ssDNA and analysed using viability staining, transmission electron microscopy (TEM) and field emission scanning (FESEM) electron microscopy. Even with such diverse nanoparticles and functionalisations, we found that the surface properties and size of the nanoparticles determined their cellular binding, incorporation and/or cytotoxicity. However the cells responded to the different types of nanoparticles using various intracellular routes which differed with the cell type, but all of the nanoparticles ultimately were consolidated into aggregates and transported to the basal cell surface. Nanoparticles that were completely covered with biological macromolecules (i.e., recombinant gClq-R protein, non-immune IgGk, monoclonal antibody to gClq-R, or ssDNA) did not cause ultrastructural damage or changes in the cell monolayers. Monoclonal antibody (mAb)-functionalised carbon nanoloops and ssDNA 100% covered Au-nanodots were incorporated and transported within the colon cells using different cellular pathways than those used by the lung cells. Citrate-capped Au-nanoparticles (2 nm and 10 nm) and 20% DNA covered Au-nanoparticles passed into the colon and lung cells through small holes in the apical cell membrane, which could possibly be produced by lipid peroxidation. Serious forms of cell damage were observed with citrate capped 2 nm and 10 nm Au-nanoparticles (i.e., nuclear localisation (2 nm-Au); intracellular membrane damage (10 nm-Au)). Vital staining used to identify cellular necrosis following nanoparticle exposure, was sometimes misleading showing cell necrosis statistics similar to normal controls, when TEM analysis revealed intracellular and organellar damage in identically treated cells.

The effects of low-frequency ultrasound on Staphylococcus epidermidis.

Abstract. Low frequency ultrasound (LFUS) significantly enhances skin permeability to a variety of drugs; however, its bacterial effects have not been well studied. Staphylococcus epidermidis organisms were grown and standardized to 105 cfu/ml 24 h prior to investigation and suspended in normal saline. LFUS was applied with two probes immersed in the bacterial suspensions over a range of suspension volumes, intensities, and exposure times. The suspension temperature was measured, and a sample was removed, streaked onto blood agar plates, and incubated at 37°C for 24 h. Quantitative bacterial counts were then obtained. LFUS resulted in significant reductions in bacterial counts that correlated with fluid temperature. Probe size and ultrasound intensity appeared to affect bacterial counts, but were also correlated with temperature. Bacterial growth was minimal with temperatures exceeding 45°C. While LFUS can reduce bacterial counts, these conditions have the potential to cause burns in humans.

The Exosporium of B.cereus Contains a Binding Site for gC1qR/p33: Implication in Spore Attachment and/or Entry

B. cereus, is a member of a genus of aerobic, gram-positive, spore-forming rod-like bacilli, which includes the deadly, B. anthracis. Preliminary experiments have shown that gC1qR binds to B. cereus spores that have been attached to microtiter plates. The present studies were therefore undertaken, to examine if cell surface gC1qR plays a role in B. cereus spore attachment and/or entry. Monolayers of human colon carcinoma (Caco-2) and lung cells were grown to confluency on 6 mm coverslips in shell vials with gentle swirling in a shaker incubator. Then, 2 microl of a suspension of strain SB460 B. cereus spores (3x10(8)/ml, in sterile water), were added and incubated (1-4 h; 36 degrees C) in the presence or absence of anti-gC1qR mAb-carbon nanoloops. Examination of these cells by EM revealed that: (1) When B. cereus endospores contacted the apical Caco-2 cell surface, or lung cells, gC1qR was simultaneously detectable, indicating upregulation of the molecule. (2) In areas showing spore contact with the cell surface, gC1qR expression was often adjacent to the spores in association with microvilli (Caco-2 cells) or cytoskeletal projections (lung cells). (3) Furthermore, the exosporia of the activated and germinating spores were often decorated with mAb-nanoloops. These observations were further corroborated by experiments in which B.cereus spores were readily taken up by monocytes and neutrophils, and this uptake was partially inhibited by mAb 60.11, which recognizes the C1q binding site on gC1qR. Taken together, the data suggest a role, for gC1qR at least in the initial stages of spore attachment and/or entry.

High resolution FESEM and TEM reveal bacterial spore attachment.

Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.

Biological calcium absorption edge imaging using monochromatic synchrotron radiation

Soft X-ray contact absorption edge images of unfixed, unstained biological specimens were made using monochromatic synchrotron radiation. X-ray contact replicas of unfixed, hydrated biological specimens at the nitrogen absorption edge and above and below the CaLIII absorption edge were compared to comparative conventional morphological and elemental high-resolution imaging methods (scanning and transmission electron microscopy, TEM-histochemistry and TEM-X-ray microanalysis). Soft X-ray absorption edge images made above the calcium absorption edge clearly revealed morphological detail and identified regions ladened with calcium as verified by TEM histochemistry of identical spores. Similarly, nitrogen absorption edge images identified residual nitrogenous material in the spore resuspension medium, and non-viable spores with nitrogen loss due to protoplast disaggregation.