Synthesis and total H-1- and C-13-NMR assignment of cephem derivatives for use in ADEPT approaches

We report the synthesis and total NMR characterization of 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[[[(4''-nitrophenoxy)carbonyl]oxy]-methyl]-8-oxo-7[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-dioxide (5), a new cephalosporin derivative. This compound can be used as the carrier of a wide range of drugs containing an amino group. The preparation of the intermediate product, 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[methyl-4-(6-methoxyquinolin-8-ylamino) pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]-diphenylmethyl ester-5-dioxide (6), as well as the synthesis of the antimalarial primaquine prodrug 5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid-3-[methyl-4-(6-methoxyquinolin-8-ylamino) pentylcarbamate]-8-oxo-7-[(2-thienyloxoacetyl)amino]-5-dioxide (7) are also described, together with their total H-1- and C-13-NMR assignments.

Efficacy of a 7-day course of furazolidone, levofloxacin, and lansoprazole after failed Helicobacter pylori eradication

Background: Increasing resistance to clarithromycin and nitroimidazole is the main cause of failure in the Helicobacter pylori eradication. The ideal retreatment regimen remains unclear, especially in developing countries, where the infection presents high prevalence and resistance to antibiotics. The study aimed at determining the efficacy, compliance and adverse effects of a regimen that included furazolidone, levofloxacin and lansoprazole in patients with persistent Helicobacter pylori infection, who had failed to respond to at least one prior eradication treatment regimen. Methods: This study included 48 patients with peptic ulcer disease. Helicobacter pylori infection was confirmed by a rapid urease test and histological examination of samples obtained from the antrum and corpus during endoscopy. The eradication therapy consisted of a 7-day twice daily oral administration of lansoprazole 30 mg, furazolidone 200 mg and levofloxacin 250 mg. Therapeutic success was confirmed by a negative rapid urease test, histological examination and 14C- urea breath test, performed 12 weeks after treatment completion. The Chi-square method was used for comparisons among eradication rates, previous treatments and previous furazolidone use. Results: Only one of the 48 patients failed to take all medications, which was due to adverse effects (vomiting). Per-protocol and intention-to-treat eradication rates were 89% (95% CI-89%-99%) and 88% (88-92%), respectively. Mild and moderate adverse effects were reported by 41 patients (85%). For patients with one previous treatment failure, the eradication rate was 100%. Compared to furazolidone-nave patients, eradication rates were lower in those who had failed prior furazolidone-containing regimen(s) (74% vs. 100%, p = 0.002). Conclusion: An empiric salvage-regimen including levofloxacin, furazolidone and lansoprazole is very effective in the eradication of Helicobacter pylori, particularly in patients that have failed one prior eradication therapy.

Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion

Background: The supraceliac aortic cross-clamping can be an option to save patients with hipovolemic shock due to abdominal trauma. However, this maneuver is associated with ischemia/reperfusion (I/R) injury strongly related to oxidative stress and reduction of nitric oxide bioavailability. Moreover, several studies demonstrated impairment in relaxation after I/R, but the time course of I/R necessary to induce vascular dysfunction is still controversial. We investigated whether 60 minutes of ischemia followed by 30 minutes of reperfusion do not change the relaxation of visceral arteries nor the plasma and renal levels of malondialdehyde (MDA) and nitrite plus nitrate (NOx). Methods: Male mongrel dogs (n = 27) were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (supraceliac aortic cross-clamping for 60 minutes, n = 9), and I/R (60 minutes of ischemia followed by reperfusion for 30 minutes, n = 9). Relaxation of visceral arteries (celiac trunk, renal and superior mesenteric arteries) was studied in organ chambers. MDA and NOx concentrations were determined using a commercially available kit and an ozone-based chemiluminescence assay, respectively. Results: Both acetylcholine and calcium ionophore caused relaxation in endothelium-intact rings and no statistical differences were observed among the three groups. Sodium nitroprusside promoted relaxation in endothelium-denuded rings, and there were no inter-group statistical differences. Both plasma and renal concentrations of MDA and NOx showed no significant difference among the groups. Conclusion: Supraceliac aortic cross-clamping for 60 minutes alone and followed by 30 minutes of reperfusion did not impair relaxation of canine visceral arteries nor evoke biochemical alterations in plasma or renal tissue.

Treatment of Osteomyelitis in the Feet of Diabetic Patients by Photodynamic Antimicrobial Chemotherapy

Objective: This article describes two inexpensive photodynamic antimicrobial chemotherapy (PACT) protocols to provide intensive local care on ulcerated feet of diabetic patients with osteomyelitis. Background Data: Patients with this condition generally have poor quality of life. The usual treatment consists of the administration of a cocktail of drugs including anti-inflammatories, promoters of blood circulation, and systemic antibiotics. However, depending on the conditions of the tissues, amputation may be required. Consequently, it is important to develop PACT protocols that can help avoid amputation. Materials and Methods: Two PACT protocols were applied to two diabetic patients with osteomyelitis. These protocols were based on several PACT sessions that consisted of: (1) local injection of mixtures of phenothiazines (2% in water) and Hypericum perforatum extract (10% in propylene glycol), and (2) illumination, lasting 10 min, applied to the lesion's interior and exterior using, respectively, an optical fiber and a non-coherent light source. The frequency of PACT was daily or every other day in the beginning, and weekly after tissue recovery begun. The patients were followed clinically and by radiographic testing. Results: Both PACT protocols helped cure these patients who were about to have amputation of their feet. Radiograms showed that bone had healed and that the bone's texture had improved. Conclusion: Here we have described efficient and affordable PACT protocols to treat osteomyelitis in the feet of diabetic patients. This treatment modality should be considered by vascular surgeons and by orthopedists to treat osteomyelitis that is resistant to conventional treatments.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the beta-lactam producer Penicillium chrysogenum

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of g-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA. (C) 2011 Elsevier Inc. All rights reserved.

Diversity of endophytic enterobacteria associated with different host plants

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these. isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P, stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.

Foundation and Perspectives of the Use of Plant Extracts as Performance Enhancers in Broilers

Feed is responsible for about 70% of broilers production costs, leading to an increasing number of studies on alternative dietary products that benefit bird performance and lower production costs. Since the 1950s, antimicrobial additives are the most frequently used performance enhancers in animal production and their positive results are observed even in high-challenge conditions. Since the 1990s, due to the ban of the use of some antibiotics as growth promoters and the growing trend of the public to consume natural products, plant extracts have been researched as alternatives to antibiotic growth promoters. The first study that evaluated the antibacterial activities of plant extracts was carried out in 1881; however, they started to be used as flavor enhancers only during the next decades. With the emergence of antibiotics in the 1950s, the use of plant extracts as antimicrobial agents almost disappeared. There are several studies in literature assessing the use of plant extracts, individually or in combination, as antimicrobials, antioxidants, or digestibility enhancers in animal feeds. Research results on the factors affecting their action, such as plant variety, harvest time, processing, extraction, as well as the technology employed to manufacture the commercial product and dietary inclusion levels show controversial results, warranting the need of further research and standardization for the effective use of plant extracts as performance enhancers, when added to animal feeds. This article aims at presenting plant extracts as alternatives to antibiotics, explaining their main modes of action as performance enhancers in broiler production.

Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell-free supernatants containing bacteriocins, added to 3-h-old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto-aggregation was strain-specific, and values ranged from 7 center dot 2% for ET35 to 12 center dot 1% for ET05. Various degrees of co-aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61 center dot 9-64 center dot 6%), Lact. fermentum (78 center dot 9%), Lact. delbrueckii (43 center dot 7%) and Ped. acidilactici (51 center dot 3%), which are higher than the one recorded for Lact. rhamnosus GG (53 center dot 3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain-dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco-2 cells was within the range reported for Lact. rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.

Evaluation of the role of environmental factors in the human gastrointestinal tract on the behaviour of probiotic cultures of Lactobacillus casei Shirota and Lactobacillus casei LC01 by the use of a semi-dynamic in vitro model

This study evaluated the influence of gastrointestinal environmental factors (pH, digestive enzymes, food components, medicaments) on the survival of Lactobacillus casei Shirota and Lactobacillus casei LC01, using a semi-dynamic in vitro model that simulates the transit of microorganisms through the human GIT. The strains were first exposed to different simulated gastric juices for different periods of time (0, 30, 60 and 120 min), and then to simulated intestinal fluids for zero, 120, 180 and 240 min, in a step-wise format. The number of viable cells was determined after each step. The influence of food residues (skim milk) in the fluids and resistance to medicaments commonly used for varied therapeutic purposes (analgesics, antiarrhythmics, antibiotics, antihistaminics, proton pump inhibitors, etc.) were also evaluated. Results indicated that survival of both cultures was pH and time dependent, and digestive enzymes had little influence. Milk components presented a protective effect, and medicaments, especially anti-inflammatory drugs, influenced markedly the viability of the probiotic cultures, indicating that the beneficial effects of the two probiotic cultures to health are dependent of environmental factors encountered in the human gastrointestinal tract.

Quantitative Determination of Gemifloxacin Mesylate in Tablets by Capillary Zone Electrophoresis and High Performance Liquid Chromatography

The aim of this study was to develop and validate selective and sensitive methods for quantitative determination of an antibacterial agent, gemifloxacin, in tablets by high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). The HPLC method was carried out on a LiChrospher (R) 100 RP-8e, 5 mu m (125 x 4 mm) column with a mobile phase composed of tetrahydrofuran-water (25:75, v/v) with 0.5 % of triethylamine and pH adjusted to 3.0 with orthophosphoric acid. The CZE method was performed using 50 mM sodium tetraborate buffer (pH 8.6). Samples were injected hydrodynamicaly (0.5 psi, 5 s) and the electrophoretic system was operated under normal polarity, at +20 kV and capillary temperature of 18 degrees C. A fused-silica capillary 40.2 cm (30 cm effective length) x 75 mu m i.d. was used. Both, HPLC and CZE could be interesting and efficient techniques to be applied for quality control in pharmaceutical industries.

Screening of Variables Influencing the Clavulanic Acid Production by Streptomyces DAUFPE 3060 Strain

Clavulanic acid (CA) is a beta-lactam antibiotic, which has a potent beta-lactamase inhibiting activity. The influence of five variables, namely pH (6.0, 6.4, and 6.8), temperature (28A degrees C, 30A degrees C, and 32A degrees C), agitation intensity (150, 200, and 250 rpm), glycerol concentration (5.0, 7.5, and 10 g/L) and soybean flour concentration (5.0, 12.5, and 20 g/L), on CA production by a new isolate of Streptomyces (DAUFPE 3060) was investigated in 250-mL Erlenmeyer flasks using a fractional factorial design. Temperature and soybean flour concentration were shown to be the two variables that exerted the most important effects on the production of CA at 95% confidence level. The highest CA concentration (494 mg/L) was obtained after 48 h at 150 rpm, 32A degrees C, pH 6.0, 5.0 g/L glycerol, and 20 g/L soybean flour concentrations. Under these conditions, the yields of biomass and product on consumed substrate were 0.26 g(X)/g(S) and 64.3 mg(P)/g(S), respectively. Fermentations performed in 3.0-L bench-scale fermenter allowed increasing the CA production by about 60%.

Stability of clavulanic acid under variable pH, ionic strength and temperature conditions. A new kinetic approach

Clavulanic acid (CA) is a beta-lactam antibiotic that alone exhibits only weak antibacterial activity, but is a potent inhibitor of beta-lactamases enzymes. For this reason it is used as a therapeutic in conjunction with penicillins and cephalosporins. However, it is a well-known fact that it is unstable not only during its production phase, but also during downstream processing. Therefore, the main objective of this study was the evaluation of CA long-term stability under different conditions of pH and temperature, in the presence of variable levels of different salts, so as to suggest the best conditions to perform its simultaneous production and recovery by two-phase polymer/salt liquid-liquid extractive fermentation. To this purpose, the CA stability was investigated at different values of pH (4.0-8.0) and temperature (20-45 degrees C), and the best conditions were met at a pH 6.0-7.2 and 20 degrees C. Its stability was also investigated at 30 degrees C in the presence of NaCl, Na(2)SO(4), CaCl(2) and MgSO(4) at concentrations of 0.1 and 0.5 M in Mcllvaine buffer (pH 6.5). All salts led to increased CA instability with respect to the buffer alone, and this effect decreased in following sequence: Na(2)SO(4) > MgSO(4) > CaCl(2) > NaCl. Kinetic and thermodynamic parameters of CA degradation were calculated adopting a new model that took into consideration the equilibrium between the active and a reversibly inactivated form of CA after long-time degradation. (C) 2009 Elsevier B.V. All rights reserved.

Identification of Staphylococcus aureus Carrying the mecA Gene in Ready-to-Eat Food Products Sold in Brazil

Since Staphylococcus aureus can cause several types of diseases, the development of antibiotic resistance poses an even greater threat to public health. S. aureus is known to possess the adaptive capability to promptly respond to antibiotics, making it resistant and increasingly difficult to treat; methicillin-resistant strains of S. aureus are a major concern with regard to this species. Previous studies reported the identification of methicillin-resistant S. aureus in food, demonstrating that this can represent a source of S. aureus which may carry the mecA gene. Fifty-seven S. aureus isolates, previously obtained from different types of food, were screened by polymerase chain reaction with specific primers for the mecA gene, which mediates methicillin resistance. Five (9%) isolates showed the presence of mecA gene, demonstrating that food may contain microorganisms possessing resistance genes. This study emphasizes the need to include food as a possible source of S. aureus carrying mecA gene and the need to monitor these products. Moreover, this is the first report of the presence of mecA genes in S. aureus isolated from ready-to-eat food in Brazil and Latin America.

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: nonselective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L acidophilus La05, L casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 degrees C or 42 degrees C, anaerobiosis; L acidophilus: MRS agar with bile salts, 37 degrees C or 42 degrees C, aerobiosis; L casei: MRS agar with lithium chloride and sodium propionate, 37 degrees C or 42 degrees C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 degrees C or 42 degrees C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 degrees C, anaerobiosis, L acidophilus: LC medium, 42 degrees C, aerobiosis or anaerobiosis and L casei: LP-MRS, 42 degrees C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P <= 0.05). (C) 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.