Electron beam irradiation dose dependently damages the bacillus spore coat and spore membrane.

Effective control of spore-forming bacilli begs suitable physical or chemical methods. While many spore inactivation techniques have been proven effective, electron beam (EB) irradiation has been frequently chosen to eradicate Bacillus spores. Despite its widespread use, there are limited data evaluating the effects of EB irradiation on Bacillus spores. To study this, B. atrophaeus spores were purified, suspended in sterile, distilled water, and irradiated with EB (up to 20 kGy). Irradiated spores were found (1) to contain structural damage as observed by electron microscopy, (2) to have spilled cytoplasmic contents as measured by spectroscopy, (3) to have reduced membrane integrity as determined by fluorescence cytometry, and (4) to have fragmented genomic DNA as measured by gel electrophoresis, all in a dose-dependent manner. Additionally, cytometry data reveal decreased spore size, increased surface alterations, and increased uptake of propidium iodide, with increasing EB dose, suggesting spore coat alterations with membrane damage, prior to loss of spore viability. The present study suggests that EB irradiation of spores in water results in substantial structural damage of the spore coat and inner membrane, and that, along with DNA fragmentation, results in dose-dependent spore inactivation.

Lyotropic liquid crystal as a real-time detector of microbial immune complexes.

AIMS: To design a simple method for the detection of microbe-immune complexes exploiting the optical and elastic properties of a biocompatible liquid crystalline material. METHODS AND RESULTS: Aqueous solution of disodium cromoglycate (DSCG), a lyotropic chromonic liquid crystal (LCLC), was aligned in a glass cell so as to be optically dark in polarized light. Immune complexes of at least three to four organisms altered the DSCG alignment such that polarized light was subsequently transmitted to reveal the presence of pathogens as optically bright regions around the immune complexes. CONCLUSIONS: This work describes the first method to detect viable micro-organisms in real time using LCLC. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique provides a powerful tool for the detection of microbes in minutes, exploiting the optical and elastic properties of LC.

Real-time microbe detection based on director distortions around growing immune complexes in lyotropic chromonic liquid crystals.

We describe director distortions in the nematic liquid crystal (LC) caused by a spherical particle with tangential surface orientation of the director and show that light transmittance through the distorted region is a steep function of the particle's size. The effect allows us to propose a real-time microbial sensor based on a nontoxic lyotropic chromonic LC (LCLC) that detects and amplifies the presence of immune complexes. A cassette is filled with LCLC, antibody, and antigen-bearing particles. Small and isolated particles cause no macroscopic distortions of the LCLC. Upon antibody-antigen binding, the growing immune complexes distort the director and cause detectable optical transmittance between crossed polarizers.

Tetracycline delivery from fibrin controls peritoneal infection without measurable systemic antibiotic.

The addition of antibiotics to an adhesive haemostat results in an ideal system for the treatment of a localized infectious disease. Fibrin sealant (FS) is a biocompatible, resorbable, adherent haemostat that can deliver antibiotics. Previous use of fibrin to deliver antibiotics resulted in rapid release and limited bioactivity. We have reported previously that poorly soluble antibiotics significantly retard release from FS, resulting in extended delivery in vitro, and overcome antibiotic-resistant infection. We now report that localized antibiotic delivery from FS controls peritoneal infection without measurable systemic antibiotic. Rats and mice were implanted with preformed FS discs containing tetracycline free-base to evaluate control of peritoneal sepsis and to measure serum tetracycline levels. Infection was initiated with Staphylococcus aureus. Morbidity and mortality were evaluated for 14 days. Serum was isolated from jugular vein blood with subsequent evaluation for antimicrobial activity. Mice prophylactically treated with FS-tetracycline (FS-TET) 500 mg/kg 2 days before infection cleared the S. aureus infection, resulting in 100% survival. Mice treated with FS-TET 500 mg/kg 7 days before infection survived. Mice treated with FS-TET 1750 mg/kg 35 days before infection also survived. Rats treated with FS-TET 500 mg/kg had undetectable serum tetracycline levels, whereas in vitro release of tetracycline from FS-TET pellets in rat serum was readily detected. We conclude that fibrin is an excellent vehicle for extended delivery of low solubility tetracycline. Tetracycline delivered from FS is an appropriate chemotherapy for S. aureus peritonitis. FS-TET controls localized infection without a measurable concentration of systemic tetracycline.

Subverting bacterial resistance using high dose, low solubility antibiotics in fibrin.

Antibiotics (ABs) delivered from fibrin were evaluated for control of multi-drug resistant (MDR) Staphylococcus aureus. ABs having low aqueous solubility (< or = 1 mg/ml) were encapsulated by fibrin (composed of fibrinogen, thrombin, Factor XIIIa and calcium chloride) and examined. Electron microscopy revealed fibrin-caged, tetracycline crystals that were 0.26 to 2.8 microns in size and bound within the reticular matrix. Antibiograms documented that S. aureus ATCC 27659 was resistant to erythromycin (ERY), penicillin G (PEN), streptomycin (STR), sulfamethoxazole-trimethoprim (SXT) and tetracycline (TET). However, low solubility formulations of STR (10 mg/ml) or SXT (0.5 mg/ml), delivered from fibrin and evaluated by the agar disk diffusion assay, produced zones of growth inhibition after 18-24 h at 37 degrees C in vitro, indicating renewed susceptibility of S. aureus ATCC 27659 to these ABs. ERY, PEN and TET were unable to overcome resistance at concentrations up to 10 mg/ml. In vivo, intraperitoneal (i.p.) injection of 150 mg/kg STR delivered from fibrin resulted in 100% survival of rats with MDR S. aureus peritonitis as compared with control rats receiving i.p. STR (150 mg/kg) in 0.9% saline. The results demonstrate that some low solubility ABs delivered from fibrin are efficacious in controlling infection mediated by MDR S. aureus.

Highlights from the poster sessions.

Scientific papers relating to the bioactivity of experimental and clinical therapeutics, drug delivery devices, microbial sensitivity to drugs, microbial sensitivity to combination therapies, and mechanisms of drug resistance were reported at several poster presentations. There were 134 such posters presented at six sessions scheduled during the four-day ASM meeting. Several notable papers are summarized in this report.

Toxicity of two carbamide peroxide products used in nightguard vital bleaching.

This study evaluated the effects of two generic classes of 10% carbamide-peroxide (CP) oxygenating agents (currently under clinical assessment for nightguard vital tooth bleaching) for lethality and genetic mutation after oral administration to mice, and for cellular cytotoxicity to mouse fibroblasts in vitro. The single dose LD50 values for a non-carbopol-containing CP (Gly-Oxide) and a carbopol-containing CP (Proxigel) in mice were found to be 143.8 mg/kg and 87.2 mg/kg, respectively. Genotoxicity, as measured by the Mouse Micronucleus test for mutagenicity, was negative for both 10% CP agents in comparison with positive and negative controls. Cytotoxicity as measured in the L929 fibroblast lysis assay resulted in 50% killing of L929 fibroblasts at 0.62 for the non-carbopol-containing CP and 1.88 mmol/L for the carbopol-containing CP. Both 10% CP agents were compared with seven widely-used dental products in the L929 fibroblast lysis assay and found to be no more toxic than these products.

Identification and characterization of rat intestinal lamina propria cells: consequences of microbial colonization.

Germ-free (GF) animals exhibit an abnormally diminished, cell-mediated immune response which can be rapidly normalized by bacterial colonization of the intestine. This conventionalization suggests that the development and/or regulation of the immune system is dependent upon intestinal bacteria or their products. Here we consider the ontogeny of gut-associated lymphoid tissue (GALT) immunocytes by isolating and characterizing the intestinal lamina propria cells (LPC) of GF rats responding to bacterial colonization or an irrelevant protein antigen, and compared to LPC of specific pathogen-free (SPF) rats which were conventionalized (CV) from birth. Isolation of cells was accomplished by successive EDTA washings of small intestine to remove the epithelium, and enzymatic digestion of the tissue generating single-cell suspensions. Resulting cell suspensions were characterized by monoclonal antibodies directed against leukocyte epitopes using flow cytometry. Functional characterization was measured by the tritiated thymidine proliferation assay with concanavalin A (Con A) and lipopolysaccharide (LPS) as co-stimulators. Germ-free and SPF rats had fewer total LPC than CV rats. Antibody staining revealed that GF rats had fewer total leukocytes than CV and SPF rats, and that CV rats had a greater percentage of T-cells and cells positive for the C3 receptor than GF rats. Co-stimulation of LPC with mitogens only increased proliferation of cells from CV rats compared to GF and SPF rats. In addition, spleen cells from CV rats demonstrated significantly enhanced proliferative responses compared to spleen cells from GF rat and were more analogous to spleen cells from SPF rats in their ability to proliferate in vitro, with and without mitogens. We conclude that T-cells and CD35-positive (C3BR+) cells are recruited and/or proliferate in response to intestinal bacteria and/or their products, and that this results in the induction of immune competency.

Eicosanoid regulation of acute intestinal vascular permeability induced by intravenous peptidoglycan-polysaccharide polymers.

A model of rapidly developing, self-limited acute vascular permeability changes localized to the gut-associated lymphoid tissue (GALT) of the rat was used to study the role of prostaglandins (PGs), thromboxanes (Txs), and leukotrienes (LTs) in the in vivo regulation of early intestinal inflammatory events. Sprague-Dawley rats were pretreated with metabolites, enzyme inhibitors, or receptor antagonists of the arachidonic acid pathway before intravenous injection of sonicated peptidoglycan-polysaccharide polymers derived from group A streptococci (PG-APS, 5 micrograms rhamnose/g body weight). Rats were killed five minutes after PG-APS injection and were evaluated grossly for petechiae of the intestinal parenchyma and lymphoid aggregates. Indomethacin or dexamethasone increased intestinal injury by PG-APS by inducing mid-small bowel and cecal parenchymal hemorrhage. Indomethacin significantly diminished colonic lymphoid aggregate hemorrhage. PGE1, PGE2, and prostacyclin dramatically inhibited GALT hemorrhage; prostacyclin was the most potent with an effective dose of 0.1 microgram/kg. Dazmegrel, a specific Tx synthetase antagonist, significantly inhibited PG-APS-induced vascular permeability. Dazmegrel continued to diminish colonic lymphoid aggregated hemorrhage during concurrent treatment with indomethacin, which removed potential endogenous prostaglandin protection. Diethylcarbamazine, a lipoxygenase inhibitor, and FPL-55712, a LT receptor antagonist, inhibited the PG-APS-induced lesions, with FPL-55712 being more potent. LT blockade had a predominant effect on the intestinal parenchymal hemorrhage. We postulate that the normal homeostatic suppression of inflammation induced by phlogistic bacterial cell wall polymers is PG mediated, and that pathological responses are Tx and LT dependent.

Effects of eugenol on polymorphonuclear cell migration and chemiluminescence.

In this study, the effects of eugenol on human polymorphonuclear (PMN) cell migration and chemiluminescence were examined in vitro. Utilizing zymosan-activated serum or crude Bacteroides sonicate fractions as chemotractants, we found that eugenol inhibits PMN migration at 6.6 X 10(-2) to 6.6 X 10(-5) mol/L (P less than 0.05). Also, similar effects were observed in PMNs pre-incubated in eugenol. Regardless of concentration, eugenol was not found to induce chemotaxis of PMNs. An examination of PMN membrane activation through chemiluminescence gave results consistent with the chemotaxis data, demonstrating a decrease in light emission at concentrations as low as 6.6 X 10(-6) mol/L (P less than 0.05). In view of these data, the potential effect of eugenol on in vivo (sulcular or periapical) PMN function deserves further study.

Stimulation of human PMNs in vitro by a succinimide molecular complex of methylfurylbutyrolactone.

The immunopotentiating effects on human neutrophils of a new synthetic immune biological response modifer were studied. The compound is a succinimide crystalline molecular complex of methylfurylbutyrolactone (MFBL). The MFBL succinimide (MFBL-S) derivative was tested for its in vitro effects on polymorphonuclear leukocyte (PMN) functions. At microgram quantities, MFBL-S stimulated a twofold increase in: directed migration of PMNs; adherence to nylon; and uptake of E. coli lipopolysaccharide. The MFBL-S enhanced phagocytosis by PMNs of S. epidermidis and E. coli. Additionally, intracellular killing of S. epidermidis by MFBL-S treated PMNs was significantly increased at all doses studied, whereas killing of E. coli was only significantly different from controls at concentrations of 10 micrograms/ml.

A new class of synthetic biological response modifiers: the methylfurylbutyrolactones (Nafocare B).

A new class of synthetic biological response modifiers (BRMs) has been produced by combining a highly electrophilic reactant, 2-methyl-2, 5-dihydrofuran (a cyclic acetal of cis-3-acetyl acrolein), with L-ascorbic acid. The parent class of compounds can be referred to as methylfurylbutyrolactones (MFBL), previously termed Nafocare B. This parent molecule is amorphous, has a molecular weight of 252.7, and the chemical name [3,6] cyclohemiketal of 2-(5-methyl-2-furyl)-3-keto-L-butyrolactone. Two crystalline forms were produced by a reaction of the MFBL parent molecule with either succinic anhydride or succinimide, to create MFBL-SA (Nafocare B2) and MFBL-S (Nafocare B3) dimers, respectively. The structure of these compounds has been confirmed by modern methods of analytical chemistry, including x-ray crystallography. All three forms of the MFBLs showed negligible toxicity in single-dose acute LD-50s in mice. Also, the MFBLs did not demonstrate genotoxic activity at 800 mg/kg in the mouse micronucleus assay. The MFBLs are immunostimulatory in assays involving T- and B-lymphocytes, but not in immunoassays on macrophages derived from resident- or thioglycollate-elicited peritoneal exudate cells (PEC). Spleen cells from mice treated 4 days via the intraperitoneal, intravenous, or the oral routes responded significantly over controls to suboptimal stimulatory concentrations of polyclonal mitogens in the lymphocyte stimulation assay. The MFBLs were not mitogenic, since they did not increase DNA synthesis in resting spleen cells from MFBL-treated mice. Antibody production is also amplified by the MFBLs. Mice immunized with sheep erythrocytes, a T-cell-dependent antigen, and treated with MFBLs had a 200-800% increase in the numbers of antibody-producing splenic lymphocytes detected by the Jerne hemolytic plaque assay. Also, mice immunized with soluble bovine serum albumin (BSA), and treated with a MFBL, demonstrated at least a fourfold increase in IgG-specific antibodies to BSA, when compared with controls. To demonstrate effects of MFBLs on macrophages, we used the Fc receptor (FcR) surface marker and superoxide anion assays. Only at the highest in vitro dose of MFBL (16 micrograms/ml) was a significant increase in erythrocyte antibody rosette formation detected, using resident macrophages isolated from PEC. In the superoxide anion release assay neither resident- nor thioglycollate-elicited PECs, obtained from in vivo-treated mice or macrophages treated in vitro, showed increased production of superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of eugenol for mutagenicity by the mouse micronucleus test.

Mutagenicity of eugenol (2-methoxy-4-allylphenol) was evaluated by an in vivo eukaryotic assay in mice. A 50% lethal dose (LD50) for intraperitoneal (IP) delivery of eugenol was found to be 1109.6 mg/kg body weight (7.5% eugenol-in-saline). Oral (PO) delivery via stainless-steel, esophageal cannulation was not lethal to 14,794 mg/kg body weight (100%) eugenol. Based upon recommended procedure, 80 and 25% LD50 doses were administered IP in 250 microliter volumes. Undiluted eugenol was administered PO in 100 microliter volumes. Delivery of eugenol by both regimes to male mice induced anaphase mutations in polychromatic erythrocytes as measured by the bone marrow micronucleus test. IP delivery of both doses induced the formation of micronuclei to significant levels (P less than 0.001) compared to saline controls. PO delivery of eugenol induced a much reduced frequency of micronuclei when compared to the IP route. However, a significant increase in micronuclei was evident when this test population was compared to its control group (P less than 0.003). These results suggest that eugenol presents some mutagenic capacity in eukaryotic hosts and should be evaluated for further toxicological effects.