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8 virus replication
S202
Antimicrobial effects and mechanisms P801 Eradication of induced mouse fibrosarcoma with combination of verotoxin1 and monophosphoryl lipid A H. Hosain Zadegan, M. Sattari, Z. Mohammad Hasan, A. Allameh (Khorram Abad, Tehran, IR) Objectives: Because of disadvantages of routine cancer therapies such as chemotherapy and radiotherapy, four essential approaches of different microbial metabolites have been used recently in treatment of cancers. These are enterotoxins, bacterial proteins, recombinant toxins, and combination therapies. Antitumour effects of verotoxin1 have known from several years ago, that produced from some kinds of Escherichia coli. Our purpose was to study the synergistic cytotoxicity of verotoxin1 and monophosphoryl lipidA (MPL) a nontoxic derivative of lipopolysaccharide on induced fibrosarcoma of Balb/c. Methods: For determining treatment doses of Verotoxin1 and MPL on animals, lethal dose 50 percent of them calculated with Reed & Munch method. Then intradermal tumours (mean diameter of 0/7−1 centimeter) induced with WEHI-164 cells on back of animals, and divided in four groups. Group 1 used as control, and groups 2, 3, 4 injected with MPL, verotoxin1, and combinations of MPL and verotoxin1 respectively. After treatment, eradication or decrease in volume of tumours monitored daily and compared in 4 groups. Results: verotoxin1 like standard vero cell line indicated cytotoxicity on WEHI-164 line in In vitro experiments. Simultaneous treatment of tumours with verotoxin1 & MPL eradicated malignant fibrosarcoms of balb/c. Animals of control group died after severe weight loss in comparison. Conclusion: Our results indicate that verotoxin1 have synergistic effects with MPL in eradication of malignant fibrosarcomas of Balb/c. And after further studies, it can be used for treatment of different tumours. Application of tow microbial metabolite as a treatment of tumours proposed and studies firstly in this study. On the other hand results indicate that tow factors have synergy in pathogenesis of verotoxigenic E. coli infections. P802 Effect of Berberis aetnensis C. Presl extract on tissue transglutaminase expression in primary astroglial cell cultures exposed to glutamate A. Campisi, A. Speciale, R. Acquaviva, G. Raciti, I. Barbagallo, S. Puglisi, L. Iauk (Catania, IT) Berberis aetnensis C. Presl (Berberidaceae) is a bushy spiny shrub presents on Mount Etna (Sicily). It contains several alkaloids showing several pharmacological properties (febrifugal, hypotensive, immunostimulating, antinflammatory). It is well known that excitotoxicity is a common feature in various neurological disorders. The excitotoxicity, increasing the intracellular calcium levels and activating specific genes, results in synthesis of several enzymes involved in cellular stress response, including tissue transglutaminase (TG-2). This is a multifunctional protein implicated in numerous cellular processes, as well as cellular differentiation, signal transduction, cell survival, and apoptosis. In previous researches, we demonstrated that the alterations of cellular redox state, evoked by glutamate in primary astrocyte cultures, are associated with the increase of TG-2 expression, and its effects were reduced by the antioxidant treatments. In this study, we investigated the protective effects of B. aetnensis extract in glutamate-evoked TG-2 up-regulation in primary rat astroglial cell cultures. Primary rat astroglial cell cultures were prepared from newborn albino cerebral cortex (1–2-day-old Wistar strain), maintained in cultures for two weeks, and exposed to 500 mM of glutamate for 24 hrs. Some cultures were also treated with the methanolic extract of plant roots at different concentrations and then exposed to the neurotransmitter. The exposure of the astroglial cell cultures to glutamate caused a dosedependent depletion of the glutathione levels, increased the reactive
17th ECCMID / 25th ICC, Posters oxygen species (ROS) production and induced DNA fragmentation. The treatment of the cells with B. aetnensis extracts recovered the oxidative status and reduced the glutamate-increased of TG-2 expression. These data suggest that B. aetnensis, preventing TG-2 up-regulation and reducing the oxidative stress induced by glutamate in primary astroglial cell cultures, may represent a new strategies in the neuropathological conditions associated to excitotoxicity. P803 Different antimicrobial activity of beta defensins and colonic biopsy extracts against aerobic and anaerobic bacterial strains of the gastrointestinal tract S. Nuding, L. Zabel, K. Fellermann, J. Wehkamp, H.A.G. Mueller, E.F. Stange (Stuttgart, Goppingen, DE) Objectives: The human gastrointestinal tract harbours a variety of aerobic and anaerobic microorganisms. To prevent colonisation and invasion of bacteria, the intestinal mucosa synthesizes antimicrobial peptides such as the cationic defensins. Based on RT-PCR and western blot, we determined a high induction of beta defensins in ulcerative colitis, whereas beta defensins in Crohn’s disease were less induced. To elucidate possible functional consequences for the intestinal barrier, we investigated the antimicrobial activity of defensins and cationic protein extracts, taken from the colon of patients with Crohn’s disease, ulcerative colitis or controls, against bacteria of the intestinal flora. Methods: To quantitate the antimicrobial activity, we established a flow cytometric assay with the membrane potential sensitive dye bis-(1,3dibutylbarbituric acid) trimethine oxonol. Depolarisation of the bacterial cell membrane caused by antimicrobial peptides leads to an uptake of the dye followed by an increasing green fluorescence. We tested the antibacterial activity of the constitutive human beta defensin HBD-1, the inducible defensin HBD-3 and cationic biopsy extracts of 22 patients with colonic Crohn’s disease, 32 with ulcerative colitis and 13 controls against ATCC strains of Escherichia coli and Staphylococcus aureus and a clinical isolate of Bacteroides vulgatus. Results: In populations of the aerobic strains E. coli and S. aureus as well as the anaerobic strain B. vulgatus 80% of the bacteria were killed by 2.5−5 mg HBD-3/mL. In contrast, HBD-1 in concentrations up to 15 mg/mL, which kill E. coli and S. aureus, exerted no bactericidal effect against B. vulgatus. The antimicrobial activity of cationic biopsy extracts was significantly diminished in extracts of patients with Crohn’s disease against E. coli and B. vulgatus compared to ulcerative colitis. The activity in extracts of Crohn’s disease against S. aureus was also reduced, but the differences versus controls and ulcerative colitis were less pronounced. Conclusion: The viability of B. vulgatus is not influenced by the constitutive HBD-1, this is in concordance with its augmented presence in the normal intestinal flora. The inducible defensin HBD-3 is a potent antimicrobial peptide against all 3 strains tested. The lower antimicrobial activity in extracts of Crohn’s disease corresponds well to the deficient beta defensin induction. The impact of other antimicrobial peptides must still be clarified. P804 Inhibitory effect of Melaleuca alternifolia (tea tree oil) on influenza A/PR/8 virus replication R. Timpanaro, A. Garozzo, B. Bisignano, A. Stivala, P.M. Furneri, G. Tempera, A. Castro (Catania, IT) Objectives: The Melaleuca alternifolia (Tea Tree) is a coniferous tree found in tropical regions, whose needles contain an essential oil that is used in medical and cosmetic products. The Tea Tree Oil (TTO) consists of about 50% oxygenated monoterpenes and 50% terpene hydrocarbons, terpinen-4-ol is the main active component. TTO has a wide spectrum of antimicrobial activity against bacteria, yeasts and fungi. The antimicrobial activity has been principally attributed to terpinen-4ol. The aim of this study was to investigate the antiviral activity and the mechanism of action of Tea Tree Oil and its components, terpinen-4-ol, g-terpinene, p-cymene, a-terpinene, terpinolene and a-terpineol, against Influenza A/PR/8 virus subtype H1N1 in MDCK cells.
Pathogenesis of Gram-negative infections Methods: The inhibitory effect was studied by measuring hemoagglutinin units (HAU) and 50% cytopathic effect (CPE50). In order to study the mode of action of the TTO, we carried out a series of experiments, including virucidal assay, pre-treatment assay, inhibition of attachment assay and time of addition assay. Results: The TTO, the terpinen-4-ol, the terpinolene, the a-terpineol were found to have an inhibitory effect on influenza virus replication at doses below the cytotoxic dose. No of the tested compounds showed virucidal activity nor any protective action for the MDCK cells. The effect of compound on different steps of the replicative cycle of influenza virus in MDCK cells was studied by adding compound at various times after infection (0, 1, 2, 4, 6 and 9 h). Viral replication, assessed as HAU/mL and CPE50 24 h after infection, revealed that this was significantly inhibited only if TTO was added within 2 h of infection, indicating an interference with an early step of the viral replicative cycle of Influenza virus. The influence of the compound on the virus adsorption step, studied by the infective centre assays, indicated that TTO did not interfere with cellular attachment of virus. Conclusion: These data suggest that TTO interferes with an early events of Influenza virus replication, after viral adsorption. Further studies are necessary to understand the precise mode of action of this compound.
P805 Essential oil composition and antibacterial effects of Ziziphora clinopodioides M. Chitsaz, M. Barton, M. Bazargan, M. Kamalinejad (Tehran, IR; Adelaide, AU) Background: The plant of Ziziphora clinopodioides (LAM), which is habitant of Iran, has been used in Iranian Traditional Medicine for treatment of some infectious conditions. In this survey, its antibacterial effects were examined against some bacterial species. Methods: Hydro-alcoholic extract of plant obtained by solvent (methanol 70%) and essential oil obtained by hydro-distillation procedure. The methanolic extract and essential oil were tested against Staphylococcus aureus (ATCC25923), Streptococcus pyogenes (PTCC1470), Escherichia coli (ATCC25922), Salmonella typhimurium (PTCC1609), Klebsiella pneumoniae (PTCC1053), and Pseudomonas aeruginosa (ATCC27853). Standard agar diffusion method was used for antimicrobial assay of methanolic extract and essential oil. MIC and MBC were determined by using macrobroth dilution method. Compounds of essential oil separated and identified by GC and GC/MS analysis. Results: The growth of Gram-positive organisms (S. aureus and S. pyogenes) were inhibited by methanolic extract at a concentration of 25 mg/well, but it did not inhibit any of the tested Gram-negative species. Essential oil halted the growth of all tested Gram-positive and Gramnegative organisms, with highest effect on S. typhimurium (with MIC and MBC of 225 micg/mL). Gas chromatographic analysis revealed 22 different compounds in essential oil which five of them comprise more than 73% of oil and pulegon is the highest one. Conclusion: The results suggest that Ziziphora clinopodioides has potent antibacterial effects on some Gram-positive and Gram-negative bacterial species especially on Salmonella typhimurium.
Pathogenesis of Gram-negative infections P806 Assessment of tryptophan metabolism and cytokine profile in cerebrospinal fluid samples from patients with bacterial meningitis L.G. Coutinho, C.L. Bellac, D. Grandgirard, M. Wittwer, R.S. Coimbra, S. Christen, L.F. Agnez-Lima, L.A. Marinho, S.L. Leib (Berne, CH) Objectives: Activation of the kynurenine (KYN) pathway (KP) has been observed in experimental bacterial meningitis (BM). Here we assessed the association of chemo-/cytokine levels with the concentration of KP metabolites in CSF and plasma samples from patients with BM.
S203 Methods: CSF samples were collected from 22 hospitalised patients. Nine patients were diagnosed with bacterial meningitis, 6 patients with viral meningitis and 7 patients with non-infectious neurological disorders. Microsphere-based multiplex assays (Lincoplex® , Linco Research Inc., St Charles, MA, USA) was used to assess the concentrations of 14 chemo-/cytokines separately in CSF and serum. The CSF and serum concentration of metabolites from the kynurenine pathway was assessed by high pressure liquid chromatography. Results: The concentration of TNF-a, IL-6, IL-1b, IFN-g, IL-10, IL-1 receptor antagonist, MIP-1a, MIP-1b, MCP-1 and G-CSF were 100-fold higher in CSF from patients with BM compared to the two other groups. In all CSF samples the concentration of IL-2, IL-12(p70), IL-4 and GMCSF was below the detection limit. In plasma samples the concentrations of IL-6, IL-10, IL-1 receptor antagonist, MCP-1 and G-CSF were significantly increased in patients with BM. The concentrations of the KP metabolites kynurenine, anthranilic acid and kynurenic acid were 10-fold higher in CSF of patients with BM compared to the other two groups. In contrast to what was found in CSF, the concentrations of KP metabolites in the plasma were not significantly different between the three groups. Tryptophan levels in plasma samples were higher than in CSF samples and were significantly decreased in patients with BM. Conclusion: BM is associated with increased levels of pro-inflammatory cytokines and KP metabolites. This increase in KP metabolites is most likely due to activation of KP by IFN-g and TNF-alpha. Based on the comparison of tryptophan and KP metabolite concentrations between plasma and CSF samples we conclude that the activation of the tryptophan pathway upon BM occurs within the brain. P807 Characterisation of the soluble domain of nitrite reductase from Neisseria meningitidis strains P. Stefanelli, G. Colotti, A. Neri, M.L. Salucci, R. Miccoli, L. Di Leandro, R. Ippoliti (Rome, L’Aquila, IT) Objectives: The objective of the study was to characterise the molecular and biochemical properties of the soluble domain of the AniA protein of N. meningitidis serogroup B MC58, and thereafter evaluate genomic conservation and protein expression in a panel of N. meningitidis clinical isolates. Methods: The soluble domain of the aniA gene of N. meningitidis MC58 was cloned and expressed in E. coli following standard procedures. Nitrite consumption was evaluated by the Griess method at different time points. For the pH dependence, the experiments were performed using different buffers 20 mM acetate buffer in the range 4.5−5.5, 20 mM Hepes buffer in the range 5.5−6.6, and 20 mM phosphate buffer in the range 6.5−7.5. Direct Nitrogen Oxide production was recorded by a specific electrode (WPI, UK). A total of 11 N. meningitidis strains, 7 disease-associated and 4 carriers, were cultured with 5% CO2 at 37ºC on Thayer-Martin plates. The aniA sequences were analyzed with the BLAST programme. The antisera was prepared with 1.5 mg of purified protein to immunise New Zealand rabbits, Charles River and used to perform western-blot analysis. Results: The biochemical results show an ordered mechanism for the catalysis, since the binding of nitrite needed to induce electron transfer from the type I to the type II Cu-site. Furthermore sAniA shows a dependence of the catalytic activity upon acidification. Sequence analysis of the coding region of the gene in 11 N. meningitidis strains showed that, notwithstanding a high degree of gene sequence similarity among them, two gene variants were found due to insertions or deletions. In 1 disease associated strain a 9-residue insertion was located close to the catalytic site. No amplification was obtained in 2 carried strains due to the presence of a gene for a transposase protein. The AniA was expressed in all the strains, except for two with no amplification reaction. Conclusion: These preliminary results describe the basic biochemical properties of the sAniA from N. meningitidis MC58, in particular its pH dependence. The conservation of the gene among the majority of the examined strains suggests that preservation of its integrity is important for bacterial survival. However, further investigation will be performed
Antimicrobial effects and mechanisms P801 Eradication of induced mouse fibrosarcoma with combination of verotoxin1 and monophosphoryl lipid A H. Hosain Zadegan, M. Sattari, Z. Mohammad Hasan, A. Allameh (Khorram Abad, Tehran, IR) Objectives: Because of disadvantages of routine cancer therapies such as chemotherapy and radiotherapy, four essential approaches of different microbial metabolites have been used recently in treatment of cancers. These are enterotoxins, bacterial proteins, recombinant toxins, and combination therapies. Antitumour effects of verotoxin1 have known from several years ago, that produced from some kinds of Escherichia coli. Our purpose was to study the synergistic cytotoxicity of verotoxin1 and monophosphoryl lipidA (MPL) a nontoxic derivative of lipopolysaccharide on induced fibrosarcoma of Balb/c. Methods: For determining treatment doses of Verotoxin1 and MPL on animals, lethal dose 50 percent of them calculated with Reed & Munch method. Then intradermal tumours (mean diameter of 0/7−1 centimeter) induced with WEHI-164 cells on back of animals, and divided in four groups. Group 1 used as control, and groups 2, 3, 4 injected with MPL, verotoxin1, and combinations of MPL and verotoxin1 respectively. After treatment, eradication or decrease in volume of tumours monitored daily and compared in 4 groups. Results: verotoxin1 like standard vero cell line indicated cytotoxicity on WEHI-164 line in In vitro experiments. Simultaneous treatment of tumours with verotoxin1 & MPL eradicated malignant fibrosarcoms of balb/c. Animals of control group died after severe weight loss in comparison. Conclusion: Our results indicate that verotoxin1 have synergistic effects with MPL in eradication of malignant fibrosarcomas of Balb/c. And after further studies, it can be used for treatment of different tumours. Application of tow microbial metabolite as a treatment of tumours proposed and studies firstly in this study. On the other hand results indicate that tow factors have synergy in pathogenesis of verotoxigenic E. coli infections. P802 Effect of Berberis aetnensis C. Presl extract on tissue transglutaminase expression in primary astroglial cell cultures exposed to glutamate A. Campisi, A. Speciale, R. Acquaviva, G. Raciti, I. Barbagallo, S. Puglisi, L. Iauk (Catania, IT) Berberis aetnensis C. Presl (Berberidaceae) is a bushy spiny shrub presents on Mount Etna (Sicily). It contains several alkaloids showing several pharmacological properties (febrifugal, hypotensive, immunostimulating, antinflammatory). It is well known that excitotoxicity is a common feature in various neurological disorders. The excitotoxicity, increasing the intracellular calcium levels and activating specific genes, results in synthesis of several enzymes involved in cellular stress response, including tissue transglutaminase (TG-2). This is a multifunctional protein implicated in numerous cellular processes, as well as cellular differentiation, signal transduction, cell survival, and apoptosis. In previous researches, we demonstrated that the alterations of cellular redox state, evoked by glutamate in primary astrocyte cultures, are associated with the increase of TG-2 expression, and its effects were reduced by the antioxidant treatments. In this study, we investigated the protective effects of B. aetnensis extract in glutamate-evoked TG-2 up-regulation in primary rat astroglial cell cultures. Primary rat astroglial cell cultures were prepared from newborn albino cerebral cortex (1–2-day-old Wistar strain), maintained in cultures for two weeks, and exposed to 500 mM of glutamate for 24 hrs. Some cultures were also treated with the methanolic extract of plant roots at different concentrations and then exposed to the neurotransmitter. The exposure of the astroglial cell cultures to glutamate caused a dosedependent depletion of the glutathione levels, increased the reactive
17th ECCMID / 25th ICC, Posters oxygen species (ROS) production and induced DNA fragmentation. The treatment of the cells with B. aetnensis extracts recovered the oxidative status and reduced the glutamate-increased of TG-2 expression. These data suggest that B. aetnensis, preventing TG-2 up-regulation and reducing the oxidative stress induced by glutamate in primary astroglial cell cultures, may represent a new strategies in the neuropathological conditions associated to excitotoxicity. P803 Different antimicrobial activity of beta defensins and colonic biopsy extracts against aerobic and anaerobic bacterial strains of the gastrointestinal tract S. Nuding, L. Zabel, K. Fellermann, J. Wehkamp, H.A.G. Mueller, E.F. Stange (Stuttgart, Goppingen, DE) Objectives: The human gastrointestinal tract harbours a variety of aerobic and anaerobic microorganisms. To prevent colonisation and invasion of bacteria, the intestinal mucosa synthesizes antimicrobial peptides such as the cationic defensins. Based on RT-PCR and western blot, we determined a high induction of beta defensins in ulcerative colitis, whereas beta defensins in Crohn’s disease were less induced. To elucidate possible functional consequences for the intestinal barrier, we investigated the antimicrobial activity of defensins and cationic protein extracts, taken from the colon of patients with Crohn’s disease, ulcerative colitis or controls, against bacteria of the intestinal flora. Methods: To quantitate the antimicrobial activity, we established a flow cytometric assay with the membrane potential sensitive dye bis-(1,3dibutylbarbituric acid) trimethine oxonol. Depolarisation of the bacterial cell membrane caused by antimicrobial peptides leads to an uptake of the dye followed by an increasing green fluorescence. We tested the antibacterial activity of the constitutive human beta defensin HBD-1, the inducible defensin HBD-3 and cationic biopsy extracts of 22 patients with colonic Crohn’s disease, 32 with ulcerative colitis and 13 controls against ATCC strains of Escherichia coli and Staphylococcus aureus and a clinical isolate of Bacteroides vulgatus. Results: In populations of the aerobic strains E. coli and S. aureus as well as the anaerobic strain B. vulgatus 80% of the bacteria were killed by 2.5−5 mg HBD-3/mL. In contrast, HBD-1 in concentrations up to 15 mg/mL, which kill E. coli and S. aureus, exerted no bactericidal effect against B. vulgatus. The antimicrobial activity of cationic biopsy extracts was significantly diminished in extracts of patients with Crohn’s disease against E. coli and B. vulgatus compared to ulcerative colitis. The activity in extracts of Crohn’s disease against S. aureus was also reduced, but the differences versus controls and ulcerative colitis were less pronounced. Conclusion: The viability of B. vulgatus is not influenced by the constitutive HBD-1, this is in concordance with its augmented presence in the normal intestinal flora. The inducible defensin HBD-3 is a potent antimicrobial peptide against all 3 strains tested. The lower antimicrobial activity in extracts of Crohn’s disease corresponds well to the deficient beta defensin induction. The impact of other antimicrobial peptides must still be clarified. P804 Inhibitory effect of Melaleuca alternifolia (tea tree oil) on influenza A/PR/8 virus replication R. Timpanaro, A. Garozzo, B. Bisignano, A. Stivala, P.M. Furneri, G. Tempera, A. Castro (Catania, IT) Objectives: The Melaleuca alternifolia (Tea Tree) is a coniferous tree found in tropical regions, whose needles contain an essential oil that is used in medical and cosmetic products. The Tea Tree Oil (TTO) consists of about 50% oxygenated monoterpenes and 50% terpene hydrocarbons, terpinen-4-ol is the main active component. TTO has a wide spectrum of antimicrobial activity against bacteria, yeasts and fungi. The antimicrobial activity has been principally attributed to terpinen-4ol. The aim of this study was to investigate the antiviral activity and the mechanism of action of Tea Tree Oil and its components, terpinen-4-ol, g-terpinene, p-cymene, a-terpinene, terpinolene and a-terpineol, against Influenza A/PR/8 virus subtype H1N1 in MDCK cells.
Pathogenesis of Gram-negative infections Methods: The inhibitory effect was studied by measuring hemoagglutinin units (HAU) and 50% cytopathic effect (CPE50). In order to study the mode of action of the TTO, we carried out a series of experiments, including virucidal assay, pre-treatment assay, inhibition of attachment assay and time of addition assay. Results: The TTO, the terpinen-4-ol, the terpinolene, the a-terpineol were found to have an inhibitory effect on influenza virus replication at doses below the cytotoxic dose. No of the tested compounds showed virucidal activity nor any protective action for the MDCK cells. The effect of compound on different steps of the replicative cycle of influenza virus in MDCK cells was studied by adding compound at various times after infection (0, 1, 2, 4, 6 and 9 h). Viral replication, assessed as HAU/mL and CPE50 24 h after infection, revealed that this was significantly inhibited only if TTO was added within 2 h of infection, indicating an interference with an early step of the viral replicative cycle of Influenza virus. The influence of the compound on the virus adsorption step, studied by the infective centre assays, indicated that TTO did not interfere with cellular attachment of virus. Conclusion: These data suggest that TTO interferes with an early events of Influenza virus replication, after viral adsorption. Further studies are necessary to understand the precise mode of action of this compound.
P805 Essential oil composition and antibacterial effects of Ziziphora clinopodioides M. Chitsaz, M. Barton, M. Bazargan, M. Kamalinejad (Tehran, IR; Adelaide, AU) Background: The plant of Ziziphora clinopodioides (LAM), which is habitant of Iran, has been used in Iranian Traditional Medicine for treatment of some infectious conditions. In this survey, its antibacterial effects were examined against some bacterial species. Methods: Hydro-alcoholic extract of plant obtained by solvent (methanol 70%) and essential oil obtained by hydro-distillation procedure. The methanolic extract and essential oil were tested against Staphylococcus aureus (ATCC25923), Streptococcus pyogenes (PTCC1470), Escherichia coli (ATCC25922), Salmonella typhimurium (PTCC1609), Klebsiella pneumoniae (PTCC1053), and Pseudomonas aeruginosa (ATCC27853). Standard agar diffusion method was used for antimicrobial assay of methanolic extract and essential oil. MIC and MBC were determined by using macrobroth dilution method. Compounds of essential oil separated and identified by GC and GC/MS analysis. Results: The growth of Gram-positive organisms (S. aureus and S. pyogenes) were inhibited by methanolic extract at a concentration of 25 mg/well, but it did not inhibit any of the tested Gram-negative species. Essential oil halted the growth of all tested Gram-positive and Gramnegative organisms, with highest effect on S. typhimurium (with MIC and MBC of 225 micg/mL). Gas chromatographic analysis revealed 22 different compounds in essential oil which five of them comprise more than 73% of oil and pulegon is the highest one. Conclusion: The results suggest that Ziziphora clinopodioides has potent antibacterial effects on some Gram-positive and Gram-negative bacterial species especially on Salmonella typhimurium.
Pathogenesis of Gram-negative infections P806 Assessment of tryptophan metabolism and cytokine profile in cerebrospinal fluid samples from patients with bacterial meningitis L.G. Coutinho, C.L. Bellac, D. Grandgirard, M. Wittwer, R.S. Coimbra, S. Christen, L.F. Agnez-Lima, L.A. Marinho, S.L. Leib (Berne, CH) Objectives: Activation of the kynurenine (KYN) pathway (KP) has been observed in experimental bacterial meningitis (BM). Here we assessed the association of chemo-/cytokine levels with the concentration of KP metabolites in CSF and plasma samples from patients with BM.
S203 Methods: CSF samples were collected from 22 hospitalised patients. Nine patients were diagnosed with bacterial meningitis, 6 patients with viral meningitis and 7 patients with non-infectious neurological disorders. Microsphere-based multiplex assays (Lincoplex® , Linco Research Inc., St Charles, MA, USA) was used to assess the concentrations of 14 chemo-/cytokines separately in CSF and serum. The CSF and serum concentration of metabolites from the kynurenine pathway was assessed by high pressure liquid chromatography. Results: The concentration of TNF-a, IL-6, IL-1b, IFN-g, IL-10, IL-1 receptor antagonist, MIP-1a, MIP-1b, MCP-1 and G-CSF were 100-fold higher in CSF from patients with BM compared to the two other groups. In all CSF samples the concentration of IL-2, IL-12(p70), IL-4 and GMCSF was below the detection limit. In plasma samples the concentrations of IL-6, IL-10, IL-1 receptor antagonist, MCP-1 and G-CSF were significantly increased in patients with BM. The concentrations of the KP metabolites kynurenine, anthranilic acid and kynurenic acid were 10-fold higher in CSF of patients with BM compared to the other two groups. In contrast to what was found in CSF, the concentrations of KP metabolites in the plasma were not significantly different between the three groups. Tryptophan levels in plasma samples were higher than in CSF samples and were significantly decreased in patients with BM. Conclusion: BM is associated with increased levels of pro-inflammatory cytokines and KP metabolites. This increase in KP metabolites is most likely due to activation of KP by IFN-g and TNF-alpha. Based on the comparison of tryptophan and KP metabolite concentrations between plasma and CSF samples we conclude that the activation of the tryptophan pathway upon BM occurs within the brain. P807 Characterisation of the soluble domain of nitrite reductase from Neisseria meningitidis strains P. Stefanelli, G. Colotti, A. Neri, M.L. Salucci, R. Miccoli, L. Di Leandro, R. Ippoliti (Rome, L’Aquila, IT) Objectives: The objective of the study was to characterise the molecular and biochemical properties of the soluble domain of the AniA protein of N. meningitidis serogroup B MC58, and thereafter evaluate genomic conservation and protein expression in a panel of N. meningitidis clinical isolates. Methods: The soluble domain of the aniA gene of N. meningitidis MC58 was cloned and expressed in E. coli following standard procedures. Nitrite consumption was evaluated by the Griess method at different time points. For the pH dependence, the experiments were performed using different buffers 20 mM acetate buffer in the range 4.5−5.5, 20 mM Hepes buffer in the range 5.5−6.6, and 20 mM phosphate buffer in the range 6.5−7.5. Direct Nitrogen Oxide production was recorded by a specific electrode (WPI, UK). A total of 11 N. meningitidis strains, 7 disease-associated and 4 carriers, were cultured with 5% CO2 at 37ºC on Thayer-Martin plates. The aniA sequences were analyzed with the BLAST programme. The antisera was prepared with 1.5 mg of purified protein to immunise New Zealand rabbits, Charles River and used to perform western-blot analysis. Results: The biochemical results show an ordered mechanism for the catalysis, since the binding of nitrite needed to induce electron transfer from the type I to the type II Cu-site. Furthermore sAniA shows a dependence of the catalytic activity upon acidification. Sequence analysis of the coding region of the gene in 11 N. meningitidis strains showed that, notwithstanding a high degree of gene sequence similarity among them, two gene variants were found due to insertions or deletions. In 1 disease associated strain a 9-residue insertion was located close to the catalytic site. No amplification was obtained in 2 carried strains due to the presence of a gene for a transposase protein. The AniA was expressed in all the strains, except for two with no amplification reaction. Conclusion: These preliminary results describe the basic biochemical properties of the sAniA from N. meningitidis MC58, in particular its pH dependence. The conservation of the gene among the majority of the examined strains suggests that preservation of its integrity is important for bacterial survival. However, further investigation will be performed