Elias Gebru

Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin.

The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC(24 h))/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC(24 h)/MIC (AUC(24 h)/MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC(24 h)/MIC ratios of 40-60. AUC(24 h)/MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.

EstA protein, a novel virulence factor of Streptococcus pneumoniae, induces nitric oxide and pro-inflammatory cytokine production in RAW 264.7 macrophages through NF-κB/MAPK

In the present study we characterized the molecular mechanism by which esterase A (EstA) protein, a novel virulence factor of Streptococcus pneumoniae induces inflammation. Stimulation of RAW 264.7 macrophages with purified EstA protein induced the expression of inducible nitrogen oxide synthase (iNOS) mRNA and nitrogen oxide (NO) production in a concentration-dependent manner. Inhibitors of iNOS, NF-kappaB, p38 and ERK 1/2 MAPK pathways significantly decreased (50-78%) EstA-induced NO production. Similarly, EstA induced TNF-alpha, IL-1 beta and IL-6 mRNA expression in RAW 264.7 macrophages in a dose-dependent manner, and pre-treatment of the cell cultures with specific NF-kappaB, p38 and ERK 1/2 MAPK pathway inhibitors significantly decreased EstA-induced TNF-alpha, IL-1 beta and IL-6 protein production. Furthermore, immunoblot analysis revealed the degradation of the inhibitory kappa B (IKB-alpha) in response to EstA stimulation. Taken together, our data suggests that EstA protein is a novel inducer of NO and pro-inflammatory cytokines by activating the NF-kappaB, p38 and ERK 1/2 MAPK pathways during inflammatory responses. Future studies on the upstream protein kinases of the MAPK/NF-kappaB pathways and the kinetics of cytokine production will provide further details into the mechanism of EstA-induced inflammatory response.

Mechanism of macrophage activation induced by β-glucan produced from Paenibacillus polymyxa JB115

Beta-glucans are heterogeneous groups of glucose polymers found in the cell walls of fungi, plants and some bacteria. Our previous report showed that a novel beta-1,3/1,6-glucan produced from Paenibacillus (P.) polymyxa JB115 can induce nitric oxide (NO) production in RAW264.7 cells. In the present study, the beta-glucan significantly increased luciferase activity in cells transfected with NFkappaB or AP1, but not STAT1, reporter vector DNA, which contain their binding promoter site. All specific NFkappaB and MAPKs pathway inhibitors (pyrrolidine dithiocarbamate, AG490, U0126, SB203580 and SP600125) remarkably attenuated NO production induced by the beta-glucan. Furthermore, Western blot analysis revealed that the stimulation of Raw264.7 cells by beta-glucan induced phosphorylation of IkappaB and the consequent translocation of NFkappaB into the nucleus. Meanwhile, phosphorylation of ERK1/2, JNK/SAPK and p38 MAPKs in cytoplasm were also confirmed. All these results indicated that beta-glucan from P. polymyxa JB115 activates macrophages through MAPKs and NFkappaB signaling pathway.

Mycoplasma hyopneumoniae induces pro-inflammatory cytokine and nitric oxide production through NFκB and MAPK pathways in RAW264.7 cells

In the present study, we confirmed the ability of M. hyopneumoniae to induce the secretion of large amount of proinflammatory cytokine and nitric oxide (NO) in murine macrophage RAW 264.7 cells. Moreover, M. hyopneumoniae-induced activation of the MAPK and NF-кB pathways by phosphorylation of ERK1/2, p38 and JNK/SAPK and by dissociation of IκB from NF-κB. Translocation of transcription factor NF-κB and its binding was confirmed through western blot and electromobility shift assay. From these results, we further hypothesized that these signal proteins were involved in M. hyopneumoniae-induced proinflammatory cytokines and NO productions in macrophages. Hence, we utilized specific blockers of MAPK and NF-κB to investigate the signaling pathway involvement in cytokine and NO production through pharmacological approaches. The results demonstrated significant inhibition of TNF-α, IL-1β, IL-6 and NO by MAPK inhibitors. NF-κB inhibitor PDTC significantly inhibited IL-1β and NO production. These findings contribute to the understanding of the mechanisms of immune reactivity and may ultimately prove useful in the development of new therapeutic strategies. In summary, we found critical evidence for the involvement of NF-κB and MAPK signaling pathways in the upregulation of proinflammatory cytokine and NO induced by M. hyopneumoniae.

Integration of Pharmacokinetic and Pharmacodynamic Indices of Orbifloxacin in Beagle Dogs after a Single Intravenous and Intramuscular Administration

ABSTRACT The pharmacokinetics (PK) and pharmacodynamics (PD) of orbifloxacin were studied in beagle dogs after intravenous (i.v.) and intramuscular (i.m.) administration at a dose of 2.5 mg/kg body weight. An absolute bioavailability of 100.1% ± 4.76%, a terminal half-life of 4.23 ± 0.2 h and 3.95 ± 0.15 h after i.v. and i.m. administration, a steady-state volume of distribution of 1.61 ± 0.13 liters/kg, and clearance of 0.31 ± 0.03 liters/h/kg were observed. Orbifloxacin showed rapid, concentration-dependent killing against the Escherichia coli, Staphylococcus aureus, Staphylococcus intermedius, and Proteus mirabilis clinical isolates. Computations based on PK-PD analysis indicated that the recommended dose is unlikely to be clinically effective against some strains like S. intermedius. Therefore, a higher dose of orbifloxacin would be worthy of consideration for treatment of certain bacterial infections in dogs.

Mutant prevention concentration and phenotypic and molecular basis of fluoroquinolone resistance in clinical isolates and in vitro-selected mutants of Escherichia coli from dogs

The antibacterial activity, selection of Escherichia coli (E. coli) mutants and mechanisms of fluoroquinolone resistance were investigated by integrating the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC) and in vitro dynamic model approaches. Difloxacin and orbifloxacin, for which the above information has been scarce, were used. A range of area under curve over a 24h interval (AUC(24h))/MIC ratios and selected E. coli strains were investigated using the dynamic models. Continuous incubation for three days in the presence of difloxacin or orbifloxacin resulted in losses in E. coli susceptibility. An AUC(24h)/MIC (AUC(24h)/MPC)-dependent fluoroquinolone activity and selection of E. coli mutants was confirmed. Maximum losses in susceptibility occurred at AUC(24h)/MIC ratios of 54 (orbifloxacin) and 57.3 (difloxacin). AUC(24h)/MIC ratios of 169.8 (orbifloxacin) and 199.5 (difloxacin) were estimated to be protective against the selection of E. coli mutants, and the corresponding ratios based on AUC(24h)/MPC predictions were 34 (orbifloxacin) and 36.3 (difloxacin). When integrating our in vitro data with pharmacokinetic data in dogs, the conventional clinical doses of both drugs were found to be inadequate to attain the above protective values for 90% of the mutant subpopulation (AUC(24h)/MPC(90)). Both target mutations, esp. at codon 83 (Ser to Leu) of gyrA, and overexpression of efflux pumps contributed to resistance development, with mutants also showing decreased susceptibility to enrofloxacin and marbofloxacin. Additional studies would determine the role of mutations found outside the QRDR, at codon 24 of gyrA, and at codon 116 of parC, and establish the significance of these observations in vivo.

Inflammatory responses to Mycoplasma hyopneumoniae in murine alveolar macrophage cell lines

Abstract AIM: To investigate the mechanism by which Mycoplasma hyopneumoniae induces inflammatory responses in murine alveolar macrophage (MH-S) cells. METHODS: A pathogenic strain of M. hyopneumoniae cultured in modified Friis medium was used to investigate the inflammatory response in MH-S cell lines. The effect of stimulation by M. hyopneumoniae on the production of nitric oxide (NO) and cytokines in MH-S cells and inhibition of their production, using specific inhibitors of signalling pathways, was investigated using the Griess reaction and ELISA respectively. A Western blot assay was used to confirm activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Nuclear translocation of NF-κB was further confirmed using transient transfection and luciferase gene reporter assay. RESULTS: The results revealed dose-dependent production of NO in MH-S cells stimulated by M. hyopneumoniae. Increased concentrations of the cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 were also observed (p<0.05). Using immunoblot analysis, involvement of three MAPK pathways, extracellular signal-regulated kinase I/II (ERK1/2), p38 and Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) was confirmed. Specific inhibitors of signal pathways also demonstrated their effect on the NO and cytokine responses of MH-S cells. Degradation and phosphorylation of inhibitory kappa B (IκB)-alpha was observed, while the luciferase gene reporter assays revealed activation of NF-κB after stimulation by M. hyopneumoniae. Inhibition of NF-κB by pyrrolidine dithiocarbamate decreased M. hyopneumoniae-induced production of NO and IL-1β (p<0.05), whereas no inhibitory effect was observed on concentrations of TNF-α, and IL-6. CONCLUSION: These findings indicate that M. hyopneumoniae induces NO and pro-inflammatory cytokines, and NF-κB and the three MAPK pathways are involved in the process.

Evaluation of Anti-Quorum Sensing Activity of 97 Indigenous Plant Extracts From Korea through Bioreporter Bacterial Strains Chromobacterium violaceum and Pseudomonas aeruginosa

Quorum sensing (QS) is a recently discovered chemical communication system that enhances survival of bacteria, as a group allowing resident bacteria to assume specialized roles vital for intra- and inter bacterial gene regulation, and for keeping bacterial colonies intact. Furthermore, with the continuing emergence and spread of multidrug-resistant bacteria, antipathogenic strategy to combat bacterial infections through the interruption of quorum sensing controlled virulence factors had been shown to receive increased attention. With this prospect in the current study, we attempt to screen anti-quorum sensing activity of 97 indigenous plant extracts from Korea, through biomonitor bacterial strains, Chromobacterium violaceum (CV12472) and Pseudomonas aeruginosa (PAO1). Standard disc-diffusion assays were used to detect anti-quorum sensing activity (ring of colorless, but viable cells around the disk), of the plant extracts for CV12472. A special swarm media that allow swarming motility growth of POA1 was used to conduct inhibition of swarming motility assay. Minimum Inhibitory Concentration (MIC) test for the 97 plant extracts against bioreporter strains (CV12472 and PAO1) revealed antibacterial activity of three plant extracts (Potentilla cryptotaeniae, Viburnum carlesii and Prunus armeniaca var. ansu). Out of the 97 plant extracts, significant inhibition of pigment production were detected by six plant extracts in CV12472, while 16 plant extracts had shown inhibition of swarming motility in POA1. In conclusion, a total of 18 plant extracts were screened for their anti-quorum sensing activity by the two bioreporter strains. Of the 18 plant extracts, four had shown anti-quorum activities in both bioreporter strains.

Immunomodulatory activities and subacute toxicity of a novel β-glucan from Paenibacillus polymyxa JB115 in rats

Subacute toxicity and immunopharmacological activities of β-glucan from P. polymyxa JB115 was evaluated in a 28-day feeding study in rats. The white blood cell count, red blood cell count, hematocrit, hemoglobin, thrombocytes (THR) and thrombocytocrit were significantly higher in male fed with β-glucan than control rats and the insignificant lower eosinophil count, mean corpuscular volume, mean cell hemoglobin and uninfected THR (uTHR) levels were observed in male whereas no marked changes in female rats. No other significant differences in serum chemistry and liver, kidney, and spleen weights were observed. The pathological changes and other abnormal indicators were not detected in urine. Female rats fed with diet supplemented with 0.01% β-glucan also showed marked increase in the percentage of blood cytotoxic T-lymphocytes compared to that of the control group while not significant differences in the percentage of blood B-lymphocytes. No adverse effects on general condition and behavior, growth, feed and water consumption and feed conversion efficiency were found. The results suggest that consumption of the novel β-1, 3/1, 6-glucan from P. polymyxa JB115 was not associated with any obvious toxic effects in rats, indicating its safety as a potential immunostimulant or as an adjuvant of some animal vaccines.