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.

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.

Detecção de Pseudomonas viridiflava em sementes importadas de couve chinesa (Brassica rapa var. pekinensis)

A ocorrência de Pseudomonas viridiflava é descrita em sementes de couve chinesa (Brassica rapa var. pekinensis) importadas do Japão. do ponto de vista epidemiológico, a detecção dessa bactéria é de extrema importância. Embora já existam, em nosso país, relatos desse patógeno nas culturas de alface, alho, cebola, cenoura, feijão e mandioca, sua presença em sementes de couve chinesa pode se constituir num risco potencial para outras espécies de brássicas aqui cultivadas.

Avaliação, identificação e atividade enzimática de bactérias psicotróficas presentes no leite cru refrigerado

Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4°C, 10oC and 7°C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk

Recuperação e purificação de ramnolipídeos produzidos por pseudomonas aeruginosa P029-GVIIA utilizando melaço de cana como substrato

Biosurfactants are molecules produced by microorganisms mainly bacteria as Pseudomonas and Bacillus. Among the biosurfactants, rhamnolipids play an important role due to their tensoactive as well as emulsifying properties. Besides can be produced in a well consolidated way the production costs of biosurfactants are quite expansive mainly if downstream processing is goning to be considered. Actually, attention has been given to identification of biosurfactants as well as optimization of its fermentative processes including downstream ones. This work deals with the development of strategies to recovery and purification of rhamnolipids produced by Pseudomonas aeruginosa P029-GVIIA using sugar-cane molasses as substrate. Broth free of cells was used in order to investigate the best strategies to recovery and purification produced by this system. Between the studied acids (HCl and H2SO4) for the acid precipitation step, HCl was the best one as has been showed by the experimental design 24. Extraction has been carried out using petroleum ether and quantification has been done using the thioglycolic acid method. Adsorption studies were carried out with activated carbon in a batch mode using a 24 experimental design as well as combined with an hydrophobic resin Streamline Phenyl aiming to separate the produced biosurfactant. Biosurfactant partial identification was carried out using High Performance Liquid Chromatography (HPLC). Experiments in batch mode showed that adsorption has been controlled mainly by pH and temperature. It was observed a reduction of 41.4% for the liquid phase and the solid phase it was possible to adsorb up to 15 mg of rhamnolipd/g of activated carbon. The kinetics of adsorption has been well fitted to a pseudo-first order reaction with velocity constant (k1) of 1.93 x 10-2 min-1. Experiments in packed bed ranging concentration on eluent (acetone) has been shown the highest recovery factor of 98% when pure acetone has been used. The combined effect if using activated carbon with an hydrophobic resin Streamline Phenyl has been shown successful for the rhamnolipids purification. It has been possible to purify a fraction of the crude broth with 98% of purity when the eluted of activated carbon packed bed was used with pure acetone

Estudo da produção de biossurfactantes sintetizados por Pseudomonas aeruginosa AP029-GVIIA utilizando manipueira como fonte de carbono

This study evaluates the biosurfactants production from cassava wastewater, an agro industrial residue, to be used as carbon source. Using a factorial design 24-1 (half fraction), 10 tests were performed using Pseudomonas aeruginosa AP029/GVII-A in submerged batch cultivation in rotating incubator (shaker). The influence of factors (temperature, agitation, aeration ratio and concentration of cultivation medium) at two different levels for the synthesis of the biosurfactant. Samples were collected throughout the cultivation by 132 hours of fermentation were completed. The best outcome was intended by following production through substrate consumption, dry matter, reduction of surface tension (ring method) and emulsification index. The kinetics of microorganism was assessed for the carbon source used. The results showed that the cassava wastewater is a well assimilable substrate for the production of biotensoactive, reaching 91 % of consumption by the micro-organism under study. The growth temperature was found to be one of the leading factors in the synthesis of the metabolite, followed by aeration and also due to the agitation. The best results showed a 30 % reduction in surface tension (% RTS) for the environment, reaching values of 30 mN/m; 3.0 g /L of biomass and emulsifying index greater than 65 %. The metabolite synthesized still remained stable for different salt concentrations (1, 5 and 10 % w/ v) and alkaline pH (8-10).

Detection of SPM and IMP metallo-beta-lactamases in clinical specimens of Pseudomonas aeruginosa from a Brazilian public tertiary hospital

Phenotypic and genotypic SPM and IMP metallo-beta-lactamases (MBL) detection and also the determination of minimal inhibitory concentrations (MIC) to imipenem, meropenem and ceftazidime were evaluated in 47 multidrug-resistant Pseudomonas aeruginosa isolates from clinical specimens. Polymerase chain reaction detected 14 positive samples to either bla(SPM) or bla(IMP) genes, while the best phenotypic assay (ceftazidime substrate and mercaptopropionic acid inhibitor) detected 13 of these samples. Imipenem, meropenem and ceftazidime MICs were higher for MBL positive compared to MBL negative isolates. We describe here the SPM and IMP MBL findings in clinical specimens of P. aeruginosa from the University Hospital of Botucatu Medical School, São Paulo, Brazil, that reinforce local studies showing the high spreading of bla(SPM) and bla(IMP) genes among Brazilian clinical isolates.

Risk factors for recovery of Imipenem-or ceftazidime-resistant Pseudomonas aeruginosa among patients admitted to a teaching hospital in Brazil

background. The prevalence of resistance to imipenem and ceftazidime among Pseudomonas aeruginosa isolates is increasing worldwide.objective. Risk factors for nosocomial recovery ( defined as the finding of culture- positive isolates after hospital admission) of imipenemresistant P. aeruginosa ( IRPA) and ceftazidime- resistant P. aeruginosa ( CRPA) were determined.design. Two separate case- control studies were conducted. Control subjects were matched to case patients ( ratio, 2: 1) on the basis of admission to the same ward at the same time as the case patient. Variables investigated included demographic characteristics, comorbid conditions, and the classes of antimicrobials used.setting. The study was conducted in a 400- bed general teaching hospital in Campinas, Brazil that has 14,500 admissions per year. Case patients and control subjects were selected from persons who were admitted to the hospital during 1992 - 2002.results. IRPA and CRPA isolates were obtained from 108 and 55 patients, respectively. Statistically significant risk factors for acquisition of IRPA were previous admission to another hospital ( odds ratio [ OR], 4.21 [ 95% confidence interval {CI}, 1.40- 12.66];), hemodialysis Pp. 01 ( OR, 7.79 [ 95% CI, 1.59- 38.16];), and therapy with imipenem ( OR, 18.51 [ 95% CI, 6.30- 54.43];), amikacin ( OR, 3.22 Pp. 01 P !.001 [ 95% CI, 1.40- 7.41];), and/ or vancomycin ( OR, 2.48 [ 95% CI, 1.08- 5.64];). Risk factors for recovery of CRPA were Pp. 005 Pp. 03 previous admission to another hospital ( OR, 18.69 [ 95% CI, 2.00- 174.28];) and amikacin use ( OR, 3.69 [ 95% CI, 1.32- 10.35]; Pp. 01). Pp. 01conclusion. Our study suggests a definite role for several classes of antimicrobials as risk factors for recovery of IRPA but not for recovery of CRPA. Limiting the use of only imipenem and ceftazidime may not be a wise strategy to contain the spread of resistant P. aeruginosa strains.