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The Journal of Antimicrobial... Oct 2016Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are...
BACKGROUND
Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are thought to be important factors in disease severity and duration. Macrolides including azithromycin are often used to treat the above diseases, but exhibit variable levels of efficacy. Inhaled corticosteroids are also readily used in treatment, but may lack specificity. Ideally, new treatment alternatives should suppress unwanted inflammation, but spare beneficial antiviral immunity.
METHODS
In the present study, we screened 225 novel macrolides and tested them for enhanced antiviral activity against rhinovirus, as well as anti-inflammatory activity and activity against Gram-positive and Gram-negative bacteria. Primary bronchial epithelial cells were grown from 10 asthmatic individuals and the effects of macrolides on rhinovirus replication were also examined. Another 30 structurally similar macrolides were also examined.
RESULTS
The oleandomycin derivative Mac5, compared with azithromycin, showed superior induction (up to 5-fold, EC50 = 5-11 μM) of rhinovirus-induced type I IFNβ, type III IFNλ1 and type III IFNλ2/3 mRNA and the IFN-stimulated genes viperin and MxA, yet had no effect on IL-6 and IL-8 mRNA. Mac5 also suppressed rhinovirus replication at 48 h, proving antiviral activity. Mac5 showed antibacterial activity against Gram-positive Streptococcus pneumoniae; however, it did not have any antibacterial properties compared with azithromycin when used against Gram-negative Escherichia coli (as a model organism) and also the respiratory pathogens Pseudomonas aeruginosa and non-typeable Haemophilus influenzae. Further non-toxic Mac5 derivatives were identified with various anti-inflammatory, antiviral and antibacterial activities.
CONCLUSIONS
The data support the idea that macrolides have antiviral properties through a mechanism that is yet to be ascertained. We also provide evidence that macrolides can be developed with anti-inflammatory, antibacterial and antiviral activity and show surprising versatility depending on the clinical need.
Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Antiviral Agents; Asthma; Bronchi; Cells, Cultured; Drug Discovery; Drug Evaluation, Preclinical; Epithelial Cells; Escherichia coli; Gram-Negative Bacteria; Gram-Positive Bacteria; Haemophilus influenzae; Humans; Interferon-beta; Interferons; Interleukin-6; Interleukin-8; Macrolides; Myxovirus Resistance Proteins; Oxidoreductases Acting on CH-CH Group Donors; Proteins; Pseudomonas aeruginosa; Rhinovirus; Virus Replication
PubMed: 27494903
DOI: 10.1093/jac/dkw222 -
New Zealand Veterinary Journal Nov 2016To test the non-inferiority of a novel combination intramammary product containing penicillin and cloxacillin to a reference intramammary product containing...
AIM
To test the non-inferiority of a novel combination intramammary product containing penicillin and cloxacillin to a reference intramammary product containing oxytetracycline, oleandomycin, neomycin and prednisolone with regard to bacteriological cure and clinical cure.
METHODS
Clinical cases of mastitis were sourced from 30 spring-calving dairy farms in the Southland region of New Zealand. Affected quarters were infused three times at 24 hourly intervals with either the novel combination product containing 1 g penicillin and 200 mg cloxacillin, or a reference product containing 200 mg oxytetracycline, 100 mg oleandomycin, 100 mg neomycin and 5 mg prednisolone. Cows were enrolled when a farmer detected a case of clinical mastitis. Milk samples were collected for microbiological culture immediately before treatment (Day 0) and on Days 9, 16 and 23. Bacteriological cure was compared for 187 and 178 quarters treated with the reference and novel product, respectively, and clinical cure was compared for 235 and 223 quarters, respectively. Non-inferiority was assessed by calculating the difference in cure rates between the two products and constructing a 95% CI around the difference, using the variance inflation factor to account for herd level clustering. The non-inferiority margin was 20% for both bacteriological and clinical cure. Generalising estimating equation models were used to determine predictor variables.
RESULTS
The bacteriological cure percentage, adjusted to account for herd-level clustering, was 8.5 (95% CI=-1.7-21.8)% higher for quarters treated with the novel than the reference product. The adjusted clinical cure percentage was 0.3 (95% CI=-11.2-12.0)% higher for clinical quarters treated with the novel than the reference product. Bacterial species was the only covariate for bacteriological cure (p=0.003), and quarter score at enrolment (indicating udder inflammation) was the only covariate for clinical cure (p=0.032) in the multivariable models.
CONCLUSION
The novel combination product was demonstrated to be non-inferior to the reference product with regards to both bacteriological cure and clinical cure.
CLINICAL RELEVANCE
Clinicians treating mastitis now have access to this novel combination intramammary product, and demonstration of its non-inferiority compared to the existing reference product will provide options for treatment approaches. The novel product contains fewer antimicrobials; which are of a narrower spectrum of activity.
Topics: Animals; Anti-Infective Agents; Cattle; Cloxacillin; Drug Therapy, Combination; Female; Injections; Mammary Glands, Animal; Mastitis, Bovine; Neomycin; Oleandomycin; Oxytetracycline; Penicillins; Prednisolone; Treatment Outcome
PubMed: 27430313
DOI: 10.1080/00480169.2016.1210044 -
Enzyme and Microbial Technology May 2016The present study highlights the microbial synthesis of silver and gold nanoparticles by Sporosarcina koreensis DC4 strain, in an efficient way. The synthesized...
The present study highlights the microbial synthesis of silver and gold nanoparticles by Sporosarcina koreensis DC4 strain, in an efficient way. The synthesized nanoparticles were characterized by ultraviolet-visible spectrophotometry, which displayed maximum absorbance at 424nm and 531nm for silver and gold nanoparticles, respectively. The spherical shape of nanoparticles was characterized by field emission transmission electron microscopy. The energy dispersive X-ray spectroscopy and elemental mapping were displayed the purity and maximum elemental distribution of silver and gold elements in the respective nanoproducts. The X-ray diffraction spectroscopy results demonstrate the crystalline nature of synthesized nanoparticles. The particle size analysis demonstrate the nanoparticles distribution with respect to intensity, volume and number of nanoparticles. For biological applications, the silver nanoparticles have been explored in terms of MIC and MBC against pathogenic microorganisms such as Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, Bacillus anthracis, Bacillus cereus and Staphylococcus aureus. Moreover, the silver nanoparticles in combination with commercial antibiotics, such as vancomycin, rifampicin, oleandomycin, penicillin G, novobiocin, and lincomycin have been explored for the enhancement of antibacterial activity and the obtained results showed that 3μg concentration of silver nanoparticles sufficiently enhance the antimicrobial efficacy of commercial antibiotics against pathogenic microorganism. Furthermore, the silver nanoparticles potential has been reconnoitered for the biofilm inhibition by S. aureus, Pseudomonas aeruginosa and E. coli and the results revealed sufficient activity at 6μg concentration. In addition, gold nanoparticles have been applied for catalytic activity, for the reduction of 4-nitrophenol to 4-aminophenol using sodium borohydride and positive results were attained.
Topics: Anti-Infective Agents; Gold; Metal Nanoparticles; Microbial Sensitivity Tests; Nanotechnology; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Silver; Sporosarcina
PubMed: 26992796
DOI: 10.1016/j.enzmictec.2016.02.005 -
Biochimica Et Biophysica Acta Mar 2016OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not...
Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis.
BACKGROUND
OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism.
METHODS
The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent.
RESULTS
Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates.
CONCLUSIONS
Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP.
GENERAL SIGNIFICANCE
Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.
Topics: Anti-Bacterial Agents; Catalytic Domain; Clotrimazole; Crystallography; Cytochrome P-450 Enzyme System; Oleandomycin; Oxidoreductases; Protein Structure, Secondary; Streptomyces antibioticus; Structure-Activity Relationship
PubMed: 26475642
DOI: 10.1016/j.bbagen.2015.10.009 -
Journal of Separation Science Nov 2015A simple extraction technique has been developed for seven macrolide antibiotics in milk. The procedure involves a modified quick, easy, cheap, effective, rugged, and...
Innovative mixture of salts in the quick, easy, cheap, effective, rugged, and safe method for the extraction of residual macrolides in milk followed by analysis with liquid chromatography and tandem mass spectrometry.
A simple extraction technique has been developed for seven macrolide antibiotics in milk. The procedure involves a modified quick, easy, cheap, effective, rugged, and safe method based on acetonitrile extraction, followed by the addition of a mixture of salts (sodium sulfate, sodium chloride, and potassium carbonate) not yet reported in literature. The method was validated for tylosin and was selective, free of matrix effect, and linear in the range of 0.78-18.75 ng/mL in the final extract, corresponding to 0.125-3 times the maximum residue limit. The limit of detection, limit of quantification, decision limit, and detection capability were, respectively, 0.84, 2.79, 58.4, and 71.7 μg/kg. The overall average recovery at 25, 50, and 75 μg/kg ranged from 89-97%. Repeatability and intermediate precision expressed by relative standard deviations were below 10.5 and 12%, respectively. The extension of the validation for spiramycin, throleandomycin, oleandomycin, roxithromycin, erythromycin, and clarithromycin is under consideration since the procedure proved to be able to efficiently extract all studied macrolides, with recoveries from 74-104% at 50 μg/kg for tylosin, erythromycin, spiramycin, and oleandomycin and 20 μg/kg for throleandomycin, roxithromycin, and clarithromycin.
Topics: Animals; Chromatography, Liquid; Limit of Detection; Macrolides; Milk; Reproducibility of Results; Salts; Tandem Mass Spectrometry
PubMed: 26340418
DOI: 10.1002/jssc.201500373 -
Drug Metabolism and Disposition: the... Jul 2015Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive...
Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats.
Topics: Alanine Transaminase; Animals; Anti-Bacterial Agents; Antifungal Agents; Buthionine Sulfoximine; Carbamazepine; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP3A; Enzyme Induction; Enzyme Inhibitors; Glutathione; Hydroxylation; Ketoconazole; Liver; Male; Microsomes, Liver; Rats; Rats, Inbred F344; Troleandomycin
PubMed: 25870103
DOI: 10.1124/dmd.115.063370 -
International Journal of Nanomedicine 2015In the present study, the strain Brevibacterium frigoritolerans DC2 was explored for the efficient and extracellular synthesis of silver nanoparticles. These...
In the present study, the strain Brevibacterium frigoritolerans DC2 was explored for the efficient and extracellular synthesis of silver nanoparticles. These biosynthesized silver nanoparticles were characterized by ultraviolet-visible spectrophotometry, which detected the formation of silver nanoparticles in the reaction mixture and showed a maximum absorbance at 420 nm. In addition, field emission transmission electron microscopy revealed the spherical shape of the nanoparticles. The dynamic light scattering results indicated the average particle size of the product was 97 nm with a 0.191 polydispersity index. Furthermore, the product was analyzed by energy dispersive X-ray spectroscopy, X-ray diffraction, and elemental mapping, which displayed the presence of elemental silver in the product. Moreover, on a medical platform, the product was checked against pathogenic microorganisms including Vibrio parahaemolyticus, Salmonella enterica, Bacillus anthracis, Bacillus cereus, Escherichia coli, and Candida albicans. The nanoparticles demonstrated antimicrobial activity against all of these pathogenic microorganisms. Additionally, the silver nanoparticles were evaluated for their combined effects with the commercial antibiotics lincomycin, oleandomycin, vancomycin, novobiocin, penicillin G, and rifampicin against these pathogenic microorganisms. These results indicated that the combination of antibiotics with biosynthesized silver nanoparticles enhanced the antimicrobial effects of antibiotics. Therefore, the current study is a demonstration of an efficient biological synthesis of silver nanoparticles by B. frigoritolerans DC2 and its effect on the enhancement of the antimicrobial efficacy of well-known commercial antibiotics.
Topics: Anti-Infective Agents; Bacteria; Biotechnology; Brevibacterium; Candida; Metal Nanoparticles; Particle Size; Silver
PubMed: 25848272
DOI: 10.2147/IJN.S72313 -
Xenobiotica; the Fate of Foreign... 20151. Precision-cut liver slices are one of the in vitro models used in studies concerning xenobiotic metabolism. Sparse information on this field is actually available...
1. Precision-cut liver slices are one of the in vitro models used in studies concerning xenobiotic metabolism. Sparse information on this field is actually available for cattle and other veterinary species. 2. The aim of the current work was to study the effect of dexamethasone (DEX) on the gene expression and function of CYP3A23 (in rat), CYP3A28 (in cattle) and the transcriptional factors involved in their regulation. 3. DEX (at 100 µM) up-regulated CYP3A23 mRNA (3.2-fold, p = 0.028) in rat liver slices after 12 h culture, whereas the gene expression profiles of transcriptional factors involved in CYP3A regulation were unaffected. A CYP3A-dependent enzyme activity (triacetyl-oleandomycin N-demethylase) increased 3.4-fold (p < 0.05) in rat liver slices cultured in the presence of DEX. 4. The protocol used for rat liver slices was used as reference to study the expression of a CYP3A isoenzyme in cattle liver slices. Oppositely, DEX did neither affect the gene expression profile of CYP3A28 nor the CYP3A activity tested in cattle liver slices. 5. The data reported here are a further contribution to demonstrate the usefulness of liver slices as an in vitro tool for studies on the expression and function of xenobiotic metabolizing enzymes in cattle and in other ruminant species.
Topics: Animals; Cattle; Cytochrome P-450 CYP3A; Dexamethasone; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Isoenzymes; L-Lactate Dehydrogenase; Liver; Male; Mitochondria, Liver; Rats, Wistar; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Tissue Survival
PubMed: 25630049
DOI: 10.3109/00498254.2014.1002122 -
Chemico-biological Interactions Dec 2014Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently,...
Neferine is a bisbenzylisoquinoline alkaloid isolated from the seed embryos of Nelumbonucifera Gaertn (Lotus) with various potent pharmacological effects. Recently, neferine has attracted attention for its anti-tumor activities. Our study explored its metabolism and cytotoxicity mechanism. Approaches using chemical inhibitors and recombinant human enzymes to characterize the involved enzymes and kinetic studies indicated that the demethylation of neferine by cytochrome P450 (CYP) 2D6 and CYP3A4 fitted a biphasic kinetic profile. Glutathione (GSH) was used as a trapping agent to identify reactive metabolites of neferine, and four novel GSH conjugates were detected with [M+H](+) ions at m/z 902.4, 916.2, 916.1, and 930.4. Based on its structure containing para-methylene phenol and results from a product ion scan, GSH tends to conjugate with C9' after undergoing oxidative metabolism to form the binding site predominated by CYP3A4. Furthermore, the addition of recombinant human GSTA1, GSTT1, and GSTP1 had little effect on the production of the GSH conjugates. In a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay, combined with the GSH modulators l-buthionine sulfoximine or N-acetyl-l-cysteine, neferine treatment of MDCK-hCYP3A4 and HepG2 cells revealed that CYP3A4 expression and cellular GSH content could cause an EC50 shift. Metabolic activation mediated by CYP3A4 and GSH depletion significantly enhanced neferine-induced cytotoxicity.
Topics: Acetylcysteine; Animals; Antineoplastic Agents; Benzylisoquinolines; Buthionine Sulfoximine; Cytochrome P-450 CYP2C8; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dogs; Glutathione; Hep G2 Cells; Humans; Indolequinones; Ketoconazole; Kinetics; Madin Darby Canine Kidney Cells; Microsomes, Liver; Troleandomycin
PubMed: 25451576
DOI: 10.1016/j.cbi.2014.10.014 -
Food Chemistry Mar 2015Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, prevention and growth promotion. In this work, monoclonal antibodies...
Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, prevention and growth promotion. In this work, monoclonal antibodies (Mabs) against erythromycin were produced and used to develop a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of erythromycin in milk. A novel carboxyphenyl derivative of erythromycin (ERO-CMO) was synthesized and conjugated with bovine serum (BSA) for use as the immunogen or ovalbumin (OVA) as the coating antigen. Four hybridoma cell lines were isolated, which produced Mabs that competed with erythromycin. The 6C1 and 5B2 Mabs had IC50 values for erythromycin of 14.40 and 0.94 μg L(-)(1), respectively. These Mabs demonstrated high cross-reactivity to the macrolides containing 14-membered rings, but not to oleandomycin. No cross-reactivity was observed for 12 macrolides that contained 15 or 16-membered lactone rings or for 2 pleuromutilins. The ciELISA developed using the 5B2 Mab afforded recovery values that ranged from 76.9% to 85.7% with only a 10-fold sample dilution prior to analysis.
Topics: Animals; Anti-Bacterial Agents; Antibodies, Monoclonal; Antibody Specificity; Cattle; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Erythromycin; Female; Food Analysis; Haptens; Inhibitory Concentration 50; Macrolides; Mice; Mice, Inbred BALB C; Milk; Ovalbumin
PubMed: 25308648
DOI: 10.1016/j.foodchem.2014.08.104