Mass spectrometric investigation of the DNA-binding properties of an anthracycline with two trisaccharide chains

Cosmomycin D (CosD) is an anthracycline that has two trisaccharide chains linked to its ring system. Gel electrophoresis showed that CosD formed stable complexes with plasmid DNA under conditions where daunorubicin (Dn) and doxorubicin (Dx) dissociated to some extent during the experiments. The footprint and stability of CosD complexed with 10- and 16 trier DNA was investigated using several applications of electrospray ionisation mass spectrometry (ESI-MS). ESI-MS binding profiles showed that fewer CosD molecules bound to the sequences than Dn or Dx. In agreement with this, ESI-MS analysis of nuclease digestion products of the complexes showed that CosD protected the DNA to a greater extent than Dn or Dx. In tandem MS experiments, all CosD-DNA complexes were more stable than Dn- and Dx-DNA complexes. These results Support that CosD binds more tightly to DNA and exerts a larger footprint than ESI-MS investigations of the binding properties of CosD Could be carried out rapidly and using only small amounts of sample. (C) 2008 Elsevier Inc. All rights reserved.

DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells

Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene. We assessed the induction of apoptosis (Sub-G(1)) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay). Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay. Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.

Rates of antimicrobial resistance in latin america (2004-2007) and in vitro activity of the glycylcycline tigecycline and of other antibiotics

As a part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.), Gram-positive and Gram-negative bacterial isolates were collected from 33 centers in Latin America (centers in Argentina, Brazil, Chile, Colombia, Guatemala, Honduras, Jamaica, Mexico, Panama, Puerto Rico, and Venezuela) from January 2004 to September 2007. Argentina and Mexico were the greatest contributors of isolates to this study. Susceptibilities were determined according to Clinical Laboratory Standards Institute guidelines. Resistance levels were high for most key organisms across Latin America: 48.3% of Staphylococcus aureus isolates were methicillin-resistant while 21.4% of Acinetobacter spp. isolates were imipenem-resistant. Extended-spectrum β-lactamase were reported in 36.7% of Klebsiella pneumoniae and 20.8% of E. coli isolates. Tigecycline was the most active agent against Gram-positive isolates. Tigecycline was also highly active against all Gram-negative organisms, with the exception of Pseuodomonas aeruginosa, against which piperacillin-tazobactam was the most active agent tested (79.3% of isolates susceptible). The in vitro activity of tigecycline against both Gram-positive and Gram-negative isolates indicates that it may be an useful tool for the treatment of nosocomial infections, even those caused by organisms that are resistant to other antibacterial agents.

Time- and concentration-dependent cytotoxicity of antibiotics used in endodontic therapy

OBJECTIVE: New drugs have to be assessed in endodontic therapy due to the presence of microorganisms resistant to therapeutic procedures. Thus, this study evaluated the time- and concentration-dependent cytotoxicity of different antibiotics used in endodontic therapy. MATERIAL AND METHODS: Human gingival fibroblasts were treated and divided into the following experimental groups: Group I - control; Group II - ciprofoxacin hydrochloride; Group III - clyndamicin hydrochloride; and Group IV - metronidazole. Each drug was used at concentrations of 5, 50, 150, and 300 mg/L for 24, 48, 72, and 96 h. Cytotoxicity was evaluated by the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and spectrophotometric reading of ELISA plates. The results were analyzed by BioEstat 4.0 software using Kruskal-Wallis and Dunn's tests at a signifcance level of 5%. Cell viability was assessed for the different concentrations and times. RESULTS: All drugs presented dose-dependent cytotoxicity. Concentrations of 5 and 50 mgjL produced viable fibroblasts at all experimental times in all groups. CONCLUSIONS: Cell viability at 24 h was greater than in the other experimental times. Comparison between the same concentrations of antibiotics at different times showed that metronidazole presented the highest cell viability at 72 and 96 h compared to the other antibiotics, whereas clyndamicin hydrochloride showed higher cell viability at 72 h than ciprofoxacin hydrochloride.

The effect of laparoscopy access and antibiotics on the outcome of severe bacterial peritonitis in rats

Introduction: Treatment of severe bacterial peritonitis, especially by videolaparoscopy, is still a matter of investigation. The aim of the present study was to evaluate the effect of videolaparoscopy and laparotomy access with or without antibiotics on the outcome of severe bacterial peritonitis in rats. Materials and Methods: Sixty-four male Wistar rats were equally assigned to 8 groups: Sham surgery (SHAM), SHAM+antibiotics (SHAM+AB), cecal ligation and puncture (CLP), CLP+AB, CLP+videolaparoscopy (VLAP), CLP+laparotomy (LAP), VLAP+AB, and LAP+AB. All treated animals were submitted to an evaluation of bacteremia, white cell counts, and cytokine determinations: interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha). The groups treated with antibiotics received gentamicin and metronidazole. Survival was monitored over a period of 7 days. Results: Peritonitis induced by CLP was severe, with IL-1, IL-6, and TNF-alpha levels and lethality being significantly higher compared to the SHAM group. The IL-6 levels in the VLAP group were significantly higher compared to the CLP and VLAP+AB groups, and the TNF-alpha levels in the VLAP and LAP+AB groups were significantly higher compared to the LAP group. The survival time was significantly higher in the CLP+AB and VLAP+AB groups, when compared to the CLP group. There was no significant difference in bacteremia and lethality rates between the resources employed for treatment of peritonitis. Conclusions: Although the use of laparoscopic access itself exacerbates the inflammatory response, the combination with antibiotics minimizes this effect and increases the survival time. However, all of the resources used for treating severe peritonitis, when applied alone or in combination, have an equivalent influence on bacteremia and lethality rates.

The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics

Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the P-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins. (C) 2009 Elsevier Inc. All rights reserved.

Characterisation of anthracyclines from a cosmomycin D-producing species of Streptomyces by collisionally-activated dissociation and ion mobility mass spectrometry

Cultures of cosmomycin D-producing Streptomyces olindensis ICB20 that were propagated for many generations underwent mutations that resulted in production of a range of related anthracyclines by the bacteria. The anthracyclines that retained the two trisaccharide chains of the parent compound were separated by HPLC. Exact mass determination of these compounds revealed that they differed from cosmomycin D (CosD) in that they contained one to three fewer oxygen atoms (loss of hydroxyl groups). Some of the anthracyclines that were separated by HPLC had the same mass. The location from which the hydroxyl groups had been lost relative to CosD (on the aglycone and/or on the sugar residues) was probed by collisionally-activated dissociation using an electrospray ionisation linear quadrupole ion trap mass spectrometer. The presence of anthracyclines with the same mass, but different structure, was confirmed using an electrospray ionisation travelling wave ion mobility mass spectrometer.

Economic impact of treatment for surgical site infections in cases of total knee arthroplasty in a tertiary public hospital in Brazil

The aim of this study was to estimate the additional cost of treatment of a group of nosocomial infections in a tertiary public hospital. A retrospective observational cohort study was conducted by means of analyzing the medical records of 34 patients with infection after total knee arthroplasty, diagnosed in 2006 and 2007, who met the criteria for nosocomial infection according to the Centers for Disease Control and Prevention. To estimate the direct costs of treatment for these patients, the following data were gathered: length of hospital stay, laboratory tests, imaging examinations, and surgical procedures performed. Their costs were estimated from the minimum values according to the Brazilian Medical Association. The estimated cost of the antibiotics used was also obtained. The total length of stay in the ward was 976 days, at a cost of US$ 18,994.63, and, in the intensive care unit, it was 34 days at a cost of US$ 5,031.37. Forty-two debridement procedures were performed, at a cost of US$ 5,798.06, and 1965 tests (laboratory and imaging) were also performed, at a cost of US$ 15,359.25. US$ 20,845.01 was spent on antibiotics and US$ 1,735.16 on vacuum assisted closure therapy, microsurgical flaps, implant removal, spacer use, and surgical revision. The total additional cost of these cases of hospital infection in 2006 and 2007 was of US$ 91,843.75. Based on that, we demonstrate that the high cost of treatment for hospital infections emphasizes the importance of taking measures to prevent and control hospital infection.

Doença granulomatosa crônica: diagnóstico no primeiro episódio infeccioso

OBJETIVO: Relatar caso ilustrativo de doença granulomatosa crônica cujo diagnóstico ocorreu durante o aparecimento do primeiro episódio infeccioso, colaborando com a iniciativa do Brazilian Group for Immunodeficiency para a sensibilização do pediatra geral em relação ao diagnóstico precoce das imunodeficiências primárias, o que está associado a melhor qualidade de vida e maior sobrevida desses indivíduos. DESCRIÇÃO DE CASO: Paciente do sexo masculino, 39 dias de vida, admitido em pronto-socorro pediátrico por febre alta há cinco dias e irritabilidade. No dia seguinte, observou-se abscesso cervical, isolando-se Staphylococcus aureus comunitário. Durante a internação, ocorreram outros abscessos superficiais e em cadeias ganglionares profundas, além de resposta lenta aos antimicrobianos. Solicitou-se investigação para imunodeficiências, que confirmou a hipótese de doença granulomatosa crônica por quantificação dos ânions superóxido e teste de redução do nitrobluetetrazolio. Paciente foi encaminhado a serviço especializado, no qual identificou-se doador de medula óssea compatível, realizando-se o transplante seis meses após o diagnóstico. Quatro meses após o transplante, ocorreu normalização do burst oxidativo, indicando sucesso. COMENTÁRIOS: O paciente mostrou apresentação típica da doença, o que permitiu seu diagnóstico por pediatras gerais já na primeira infecção, tendo como consequência o acompanhamento por especialistas em imunodeficiências primárias, a introdução da profilaxia antimicrobiana e a procura bem sucedida de doador de medula HLA-compatível.

Ácido clavulânico e cefamicina c: uma perspectiva da biossíntese, processos de isolamento e mecanismo de ação

The present article reviews different aspects of the chemistry of two widely used β-lactam antibiotics Clavulanic Acid and Cephamycin C. The article discusses important details of the biosynthesis of these compounds, their action mechanism and, principally, the methods employed in their isolation and purification, in accordance with the available literature. Despite the large quantity of available articles and patents concerning β-lactam antibiotics, those which describe the isolation and purification of Clavulanic Acid and Cephamycin C are rare. Overall, the intention of this article is to discuss the up-to-date scientific research related to the compounds under review.

Screening for antifungal activities of extracts of the brazilian seaweed genus Laurencia (Ceramiales, Rhodophyta)

The resistance of pathogens to commonly used antibiotics has enhanced morbidity and mortality and has triggered the search for new drugs. Several species of the red alga genus Laurencia are very interesting candidates as potential sources of natural products with pharmaceutical activity because they are known to produce a wide range of chemically interesting halogenated secondary metabolites. This is an initial report of the antifungal activities of the secondary metabolites of five species of Laurencia, collected in the state of Espírito Santo, against three strains of pathogenic fungi: Candida albicans (CA), Candida parapsilosis (CP), and Cryptococcus neoformans (CN). Minimum inhibitory concentrations (MIC) of the algal extracts were determined by serial dilution method in RPMI 1640 Medium in 96-well plates according to the NCCLS and microbial growth was determined by absorbance at 492nm. A result showing maintenance or reduction of the inoculum was defined as fungistatic, while fungicidal action was no observed growth in the 10 µL fungistatic samples subcultured in Sabouraud Agar. Our results indicate that apolar extracts of Laurencia species possess antifungal properties and encourage continued research to find new drugs for therapy of infectious diseases in these algae.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Perfil de prescrições e uso de antibióticos em infecções comunitárias

INTRODUÇÃO: O objetivo deste estudo foi conhecer os padrões de utilização de antibióticos no município de Sorocaba, avaliando o diagnóstico referido, a terapêutica empregada e sua utilização. MÉTODOS: Utilizou-se um instrumento de avaliação aplicado por 12 meses em usuários de antibióticos. Foram coletados dados de 403 usuários e referiram-se à: informações sociodemográficas e de saúde, diagnóstico e terapêutica. RESULTADOS: Encontrou-se grande utilização e a maior utilização prévia na faixa etária de 0-10 anos (p<0,05). As infecções com envolvimento pulmonar foram as mais citadas (p<0,05) e as penicilinas, os fármacos mais utilizados, presentes em 45,1% das prescrições. Tempo médio de terapêutica (8,9 dias) esteve abaixo do preconizado para otites. Em sinusites, 22% das prescrições não orientaram para o uso recomendado (10 dias). CONCLUSÕES: A inexistência e não utilização de protocolos terapêuticos têm resultado em grande diferença nos padrões de prescrição, levando a insucesso terapêutico e recidiva de infecções - situações frequentemente encontradas neste estudo.

Efeitos da enramicina e monensina sódica sobre a digestão de nutrientes em bovinos alimentados com dietas contendo alto nível de concentrados

Estudaram-se os efeitos da administração de enramicina e monensina sódica sobre a digestão total dos nutrientes da dieta e o consumo de matéria seca digestível em bovinos. Doze fêmeas bovinas não-gestantes e não-lactantes (675 ± 63 kg PV) foram distribuídas inteiramente ao acaso em três tratamentos (controle, enramicina e monensina) e alimentados com dieta contendo 60% de concentrados (milho, farelo de soja e minerais) e 40% de volumoso (cana-de-açúcar). A enramicina foi administrada na dose de 20 mg/animal/dia e a monensina na dose de 300 mg/animal/dia. O experimento teve duração total de 21 dias, de modo que os últimos dez dias foram utilizados para administração do marcador externo (15 g de óxido crômico/animal/dia) e os últimos cinco dias para a coleta de fezes e amostragem dos alimentos. Nenhum dos antibióticos alterou os consumos de matéria seca digestível e NDT e a digestibilidade de matéria seca, proteína bruta, extrato etéreo, fibra em detergente ácido, fibra em detergente neutro, amido, energia bruta e nutrientes digestíveis totais.

Development of a Multicommutated Flow System with Chemiluminometric Detection for Quantification of Gentamicin in Pharmaceuticals

A new flow procedure based on multicommutation with chemiluminometric detection was developed to quantify gentamicin sulphate in pharmaceutical formulations. This approach is based on gentamicin's ability to inhibit the chemiluminometric reaction between luminol and hypochlorite in alkaline medium, causing a decrease in the analytical signal. The inhibition of the analytical signal is proportional to the concentration of gentamicin sulphate, within a linear range of 1 to 4 mu g mL(-1) with a coefficient variation <3%. A sample throughput of 55 samples h(-1) was obtained. The developed method is sensitive, simple, with low reagent consumption, reproducible, and inexpensive, and when applied to the analysis of pharmaceutical formulations (eye drops and injections) it gave results with RSD between 1.10 and 4.40%.