The opportunistic fungal pathogen Scedosporium prolificans: Carbohydrate epitopes of its glycoproteins

Isolated from the mycelium, of Scedosporium prolificans were complex glycoproteins (RMP-Sp), with three structurally related components (HPSEC). RMP-Sp contained 35% protein and 62% carbohydrate with Rha, Ara, Man, Gal, Glc, and GlcNH(2) in a 18:1:24:8:6:5 molar ratio. Methylation analysis showed mainly nonreducing end- of Galp (13%), nonreducing end- (9%),2-O-(13%), and 3-O-subst. Rhap (7%), nonreducing end-(11%), 2-O-(10%), 3-O-(14%), and 2,6-di-O-subst. Manp units (13%). Mild reductive P-elimination of RMP-Sp gave alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-Manp-(1-->2)-D-Man-ol, with Man-ol substituted at O-6 with beta-D-Galp units, a related pentasaccharide lacking beta-D-Galp units, and beta-D-Galp-(1-->6)-[alpha-D-Manp-(1-->2)]-D-Man-ol in a 16:3:1 w/w ratio. Traces of Man-ol and Rha-ol were detected. ESI-MS showed HexHex-o1 and HCX(3-6)Hex-ol components. Three rhamnosyl units were peeled off successively from the penta- and hexasaccharide by ESI-MS-MS. The carbohydrate epitopes of RMP-Sp differ from those of the glycoprotein of Pseudallescheria boydii, a related opportunistic pathogen. (C) 2007 Elsevier B.V. All rights reserved.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Role of cis-cis muconic acid in the catalysis of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Chlorocatechol 1,2-dioxygenase (1,2-CCD) is a non-heme iron protein involved in the intradiol cleavage of aromatic compounds that are recalcitrant to biodegradation. In particular, 1,2-CCD catalyzes the conversion of catechol and its halogenated derivatives to cis-cis muconic acid. In this study we describe a series of experiments concerning the interaction of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp1,2-CCD) with cis-cis muconic acid. We used single-injection ITC to show that the reaction product inhibits enzyme kinetics. DSC and EPR measurements probed whether this was accomplished by a direct binding of the product to the enzyme active site. DSC shows that cis-cis muconic acid affects the thermal unfolding of the protein and allowed us to estimate a binding constant. Furthermore, EPR spectra of the Fe(III) center demonstrate that, upon product binding, a significant decrease in resonance intensity is observed, indicating that cis-cis muconic acid binds directly to the active site. Based on the increasing interest for understanding dioxygenases mechanism of action and, moreover, how to control such process, our data indicate that the product of the reaction does play a relevant role in the catalysis and should therefore be taken into account when one thinks about ways of regulating enzyme activity. (C) 2010 Elsevier B.V. All rights reserved.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.

A new member of the ribbon-helix-helix transcription factor superfamily from the plant pathogen Xanthomonas axonopodis pv. citri

XACb0070 is an uncharacterized protein coded by the two large plasmids isolated from Xanthomonas axonopodis pv. cirri, the agent of citrus canker and responsible for important economical losses in citrus world production. XACb0070 presents sequence homology only with other hypothetical proteins belonging to plant pathogens, none of which have their structure determined. The NMR-derived solution structure reveals this protein is a homodimer in which each monomer presents two domains with different structural and dynamic properties: a folded N-terminal domain with beta alpha alpha topology which mediates dimerization and a long disordered C-terminal tail. The folded domain shows high structural similarity to the ribbon-helix-helix transcriptional repressors, a family of DNA-binding proteins of conserved 3D fold but low sequence homology: indeed XACb0070 binds DNA. Primary sequence and fold comparison of XACb0070 with other proteins of the ribbon-helix-helix family together with examination of the genes in the vicinity of xacb0070 suggest the protein might be the component of a toxin-antitoxin system. (C) 2010 Elsevier Inc. All rights reserved.

Wettability of Aqueous Rhamnolipids Solutions Produced by Pseudomonas aeruginosa LBI

The wetting behavior of rhamnolipids produced by Pseudomonas aeruginosa LBI strain grown on waste oil substrate and sodium dodecyl sulfate (SDS) on glass, polyethylene terephthalate (PET), poly(vinyl chloride) (PVC), poly(epsilon-caprolactone) (PCL) and polymer blend (PVC-PCL) was investigated by the measuring contact angle of sessile drops, to determine the wetting characteristics of rhamnolipids. The comparison of the wetting profiles showed that at low SDS and rhamnolipid concentrations, the contact angle increased and when the concentration of the surfactant increased further, the contact angle decreased. The blend surface (PVC-PCL) showed better wettability than the homopolymers themselves and the blend changed the surface hydrophobicity of the polymer, making it more hydrophilic. The rhamnolipids produced by the LBI strain exhibited superior wetting abilities than the chemical surfactant SDS one. This is the first work that evaluates the wetting properties of rhamnolipids on polymer blends.

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography-mass spectrophotometry (MS) and by high-performance liquid chromatography-MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l(-1). Under these conditions, the surface tension of the culture decreased to 30 mN m(-1), and the critical micelle concentration was 26.5 mg l(-1). It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.

A Broad-spectrum mer Operon in a Multi-drug Resistant Strain of the Fish Pathogen, Aeromonas salmonicida.

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

Caracterização de metalo-beta-lactamases produzidas por amostras de pseudomonas aeruginosa isoladas em dois hospitais de Porto Alegre

Introdução: P. aeruginosa é o principal agente causador de infecção hospitalar sendo a primeira causa de pneumonia nosocomial em hospitais brasileiros. Os carbapenêmicos são geralmente o tratamento empírico de escolha para infecções graves causadas por esta bactéria. Entretanto, seu uso tem sido limitado pelas elevadas taxas de resistência entre os isolados de P. aeruginosa principalmente os produtores de metalo-β-lactamases (Mβla). Objetivos: Caracterizar e avaliar a produção de metalo-β-lactamase em amostras de Pseudomonas aeruginosa resistentes a ceftazidima e/ou imipenem em dois hospitais universitários de Porto Alegre. Métodos: O método de disco difusão padronizado pelo NCCLS foi utilizado para avaliar o perfil de susceptibilidade aos antimicrobianos. Um teste de aproximação de discos utilizando ceftazidima com ácido 2-mercaptopropiônico foi utilizado para triagem de amostras produtoras de Mβla. A fita de Etest combinada imipenem com EDTA também foi utilizada como teste fenotípico. Os resultados destes dois testes foram comparados à PCR para pesquisa dos genes blaSPM-1, blaIMP-1 e blaVIM-2 .Os isolados produtores de Mβla foram submetidos a tipagem molecular pela técnica de macrorestrição de DNA seguida de pulsed-field gel electrophoresis (PFGE). O perfil de hidrólise para o imipenem foi avaliado nas amostras produtoras de Mβla através da variação de absorção medida a 298 nm. Resultados: Dos 92 isolados clínicos analisados, 33 foram positivos no teste de aproximação de discos. Destes, 18 foram produtores de SPM-1 e 5 de IMP-1. Todas as amostras produtoras de SPM-1 apresentaram razão de IP/IPI na fita combinada ≥ 8. Os 18 isolados de SPM-1 foram classificados como um único padrão de PFGE que foi o predominante. Seis isolados pertenciam a um segundo padrão de PFGE, sendo que cinco destes eram IMP-1. O perfil de hidrólise mostrou que os isolados produtores de SPM-1 degradam mais efetivamente o imipenem quando comparado aos produtores de IMP-1 e aos não produtores de Mβla. Conclusões: Há uma alta prevalência de Mβla entre isolados de P. aeruginosa resistentes a imipenem e/ou ceftazidima. O gene SPM-1 é o elemento genético mais prevalente entre as amostras de P. aeruginosa Mβla positivas e a disseminação clonal têm contribuído para os elevados níveis de resistência aos carbapenêmicos entre os isolados de P. aeruginosa nos hospitais deste estudo.

Fatores de risco para aquisição de pseudomonas aeruginosa resistente a imipenem em paccientes hospitalizados

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Pesquisa de genes de metalo-beta-lactamases em pseudomonas aeruginosa isoladas em três hospitais universitários de Porto Alegre

Objetivos: Padronizar uma técnica molecular, Reação em Cadeia da Polimerase (PCR) para detectar os genes blaSPM-1 , blaVIM-2 e blaIMP-1 que conferem a Pseudomonas aeruginosa resistência a carbapenêmicos (imipenem e meropenem) através da produção das metalo-β-lactamases (MBL) SPM-1, VIM-2 e IMP-1. Comparar os resultados obtidos com a técnica de PCR com os obtidos através de uma técnica fenotípica para detecção desse mecanismo enzimático de resistência. Descrever a prevalência dos genes de MBL. Metodologia: Utilizando 48 amostras clínicas de P. aeruginosa resistentes a ceftazidima e/ou imipenem sendo 37 caracterizadas como produtoras de MBL através de uma técnica fenotípica (aproximação de disco) e 11 como não produtoras pela mesma técnica. Esses isolados foram testados com três primers específicos para os seguintes genes: blaSPM-1 , blaVIM-2 e blaIMP-1 Resultados e Discussão: Dos 37 isolados produtores de metalo-β-lactamase, 29 tiveram resultado positivo na PCR sendo 27 positivos para o gene blaSPM-1 e 2 positivos para o gene blaIMP-1 . Em nenhum isolado foi detectado o gene blaVIM-2 .Oito isolados caracterizados como produtores de MBL não tiveram nenhum produto amplificado com os três pares de primers. Os 11 isolados caracterizados como não produtores de MBL não tiveram produto amplificado. Assim, a sensibilidade da técnica molecular foi de 78,40% e a especificidade 100,0% considerando a técnica fenotípica de aproximação de disco como padrão ouro Conclusões: Foi possível a padronização da técnica de PCR para a detecção dos genes blaSPM-1 , blaVIM-2 e blaIMP-1 bem como foi possível a comparação entre a técnica de PCR e a técnica fenotípica, sendo que a metodologia molecular apresentou 100% de especificidade e 78,40% de sensibilidade. O gene de MBL mais prevalente foi blaSPM-1 encontrado em 27/29 amostras de P. aeruginosa resistentes ao imipenem. O gene blaIMP-1 foi detectado em apenas 2/29 amostras de P. aeruginosa resistentes ao imipenem. O gene blaVIM-2 e não foi detectado nas amostras de P.aeruginosa analisadas neste estudo.

Pseudomonas aeruginosa LBI production as an integrated process using the wastes from sunflower-oil refining as a substrate

Pseudomonas aeruginosa LBI produced surface active rhamnolipids when cultivated on waste from the sunflower-oil process under different conditions. These biosurfactants, which reduce the superficial and interfacial tensions between fluids, offer advantages over their chemical counterparts, especially because of their ecological acceptability. These molecules can be used in fields as diverse as chemical, pharmaceutical and petrochemical industries. In this work, we present the effect of C/N ratio on growth and production yield. The best production yields (Y-P/S) were achieved for C/N ratios (in g/g) of 8/1 (0.22) and 6.4/1 (0.23). The product concentration was very satisfactory (7.3 g/L) at C/N ratio of 8/1, especially when considering that the substrate was basically composed of wastes that would otherwise constitute an environmental disposal problem. (c) 2007 Elsevier Ltd. All rights reserved.

Isolation of Pseudomonas aeruginosa strains from dental office environments and units in Barretos, state of São Paulo, Brazil, and analysis of their susceptibility to antimicrobial drugs

Uma ampla variedade de patógenos oportunistas tem sido detectadas nos tubos de alimentação de água dos equipos odontológicos, particularmente no biofilme formado na superfície do tubo. Entre os patógenos oportunistas encontrados nos tubos de água, Pseudomonas aeruginosa é reconhecida como uma das principais causadoras de infecções nosocomiais. Foram coletadas 160 amostras de água e 200 amostras de fomites em quarenta clinicas odontológicas na cidade de Barretos, São Paulo, Brasil, durante o período de Janeiro a Julho de 2005. Setenta e seis cepas de P. aeruginosa, isoladas a partir dos fomites (5 cepas) e das amostras de água (71 cepas), foram analisadas quanto à susceptibilidade à seis drogas antimicrobianas freqüentemente utilizadas para o tratamento de infecções provocadas por P. aeruginosa. As principais suscetibilidades observadas foram para a ciprofloxacina, seguida pelo meropenem. A necessidade de um mecanismo efetivo para reduzir a contaminação bacteriana dentro dos tubos de alimentação de água dos equipos odontológicos foi enfatizada, e o risco da exposição ocupacional e infecção cruzada na prática odontológica, em especial quando causada por patógenos oportunistas como a P. aeruginosa foi realçado.