Proteolytic and lipolytic potential of Pseudomonas spp. from goat and bovine raw milk / Potencial proteolítico e lipolítico de espécies de Pseudomonas do leite cru caprino e bovino

Pseudomonas, the main genus of gram-negative microorganisms isolated from milk, is psychrotrophic, biofilm-forming, and thermo-resistant deteriorating enzyme producers. The aim of this study was to quantify Pseudomonas spp. in goat's and cow's milk produced in the Paraná state, Brazil, to evaluate the deteriorating activity of the isolates at mesophilic and psychrotrophic conditions and to identify, at the species level, the isolates with alkaline metalloprotease (aprX gene) production potential. Microbiological, biochemical and molecular methods were used for isolating, confirming and identifying of isolates. The mean counts were 1.6 (±6.3)x104 and 0.89(±3)x102 CFU/mL for goat and bovine milk samples, respectively, immediately after milking. Of the Pseudomonas colonies isolated from goat milk (n=60), 91.7% showed proteolytic potential when incubated at 35°C/48 h and 80% at 7°C/10 days, and lipolytic potential was observed in 95% of the isolates incubated in mesophilic and 78.3% at refrigeration conditions. From the isolates of bovine milk (n=20), 35% showed proteolytic activity only when incubated at 35°C/48 h, and lipolytic potential was observed in 25% of the isolates incubated at 7°C/10d and 35°C/48h. It was observed that 83.3% and 25% of the isolates genetically confirmed as Pseudomonas spp. of goat and bovine milk showed the potential for alkaline metalloprotease production, with the species P. azotoformans, P. koreensis, P. gessardii, P. monteilii and P. lurida being the most frequent in goat milk and P. aeruginosa the only species identified in cow milk.(AU)

Pseudomonas é o principal gênero de micro-organismos Gram negativos isolados do leite, são psicrotróficos, formadores de biofilmes e produtores de enzimas deteriorantes termodúricas. O objetivo do presente trabalho foi quantificar Pseudomonas spp. no leite de cabras e vacas produzido no estado do Paraná, Brasil, avaliar a atividade deteriorante em temperatura mesofílica e psicrotrófica e identificar, em nível de espécie, os isolados com potencial de produção de metaloprotease alcalina (geneaprX). Foram utilizados métodos microbiológicos, bioquímicos e moleculares para isolamento, confirmação e identificação dos isolados. As contagens médias foram de 1,6 (±6,3) x 104 e 0,9 (±3) x 102 UFC/mL para as amostras de leite caprino e bovino, respectivamente. Dos isolados de Pseudomonas do leite de cabra (n=60), 91,7% demonstraram potencial proteolítico quando incubadas a 35°C/48h e 80% a 7°C/10dias e lipolíticos em 95% dos isolados incubados em mesofilia e em 78,3% dos incubados em temperatura de refrigeração. Dos isolados do leite bovino (n=20), foi verificada atividade proteolítica de 35% apenas quando incubadas a 35°C/48h e lipolítica em 25% dos isolados incubados a 7°C/10d e 35°C/48h. Foi observado que 83,3% e 25% dos isolados confirmados geneticamente como Pseudomonas spp. do leite caprino e bovino, respectivamente, apresentaram o potencial de produção de metaloprotease alcalina, sendo as espécies P. azotoformans, P. koreensis, P. gessardii, P. monteilii e P. lurida as mais frequentes no leite de cabras e P. aeruginosa a única identificada do leite de vacas.(AU)

Bacteria in combination with fertilizers promote root and shoot growth of maize in saline-sodic soil

Salinity is the leading abiotic stress hampering maize (Zea mays L.) growth throughout the world, especially in Pakistan. During salinity stress, the endogenous ethylene level in plants increases, which retards proper root growth and consequent shoot growth of the plants. However, certain bacteria contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which converts 1-aminocyclopropane-1-carboxylic acid (an immediate precursor of ethylene biosynthesis in higher plants) into ammonia and α-ketobutyrate instead of ethylene. In the present study, two Pseudomonas bacterial strains containing ACC-deaminase were tested separately and in combinations with mineral fertilizers to determine their potential to minimize/undo the effects of salinity on maize plants grown under saline-sodic field conditions. The data recorded at 30, 50 and 70 days after sowing revealed that both the Pseudomonas bacterial strains improved root and shoot length, root and shoot fresh weight, and root and shoot dry weight up to 34, 43, 35, 71, 55 and 68%, respectively, when applied without chemical fertilizers: these parameter were enhanced up to 108, 95, 100, 131, 100 and 198%, respectively, when the strains were applied along with chemical fertilizers. It can be concluded that ACC-deaminase Pseudomonas bacterial strains applied alone and in conjunction with mineral fertilizers improved the root and shoot growth of maize seedlings grown in saline-sodic soil.

Percepção dos receptores sanguíneos quanto ao processo transfusional / Blood transfusion receivers' perception of the transfusion process / Percepción de transfusión de sangre en el caso de los receptores

Pesquisa qualitativa, exploratória descritiva, que objetivou conhecer a percepção dos receptores sanguíneos quanto ao processo transfusional. A pesquisa foi realizada em uma unidade de hemoterapia de um município da região sul do Brasil e os dados foram analisados por meio do Discurso do Sujeito Coletivo. Foram entrevistados, por meio de instrumento semiestruturado, onze pacientes, homens e mulheres entre 30 e 95 anos, em recuperação pós-cirúrgica de cirurgia cardíaca, submetidos à transfusão sanguínea. Emergiram quatro Ideias Centrais: Perda e reposição sanguínea; Preservação da vida; Reconhecimento do processo transfusional e Segurança transfusional. A percepção sobre a mudança que os pós-transfundidos começam a vivenciar a partir do processo transfusional traz à tona uma ressignificação da própria vida. Este estudo mostrou que os pacientes transfundidos percebem o processo transfusional como uma alternativa de sobrevivência e, mesmo tendo conhecimento sobre o processo e seus significados, permanecem receios e angústias que podem ser minimizados pela equipe multiprofissional.

Qualitative research, descriptive exploratory, aimed to know the perception of blood transfusion recipients as to the process. The research was carried out at a blood bank in a city in southern Brazil, and the data were analyzed using the Collective Subject Discourse. Were interviewed using a semistructured instrument, eleven patients, men and women between 30 and 95 years, post-surgical recovery of cardiac surgery, underwent blood transfusion. Four central ideas emerged: loss and blood replacement; Preservation of life; Recognition of the transfusion process; and transfusion safety. The perception about the change that post-transfusion begin to live from the transfusion process raises a reframing of life itself. This study showed that transfused patients perceive the transfusion process as a means of survival, and even having knowledge about the process and their meanings, there is the permanence of fears and anxieties that can be minimized by the multidisciplinary team.

Investigación cualitativa, de tipo exploratorio descriptivo, con el objetivo de conocer la percepción de los receptores de transfusiones de sangre en cuanto al proceso. La investigación se llevó a cabo en un banco de sangre en una ciudad en el sur de Brasil, y los datos fueron analizados utilizando el Discurso del Sujeto Colectivo. Fueron entrevistados mediante un instrumento semi-estructurado, once pacientes, hombres y mujeres de entre 30 y 95 años de recuperación post-quirúrgica de la cirugía cardíaca, se sometió a una transfusión de sangre. Cuatro ideas centrales surgieron: la pérdida y reemplazo de sangre; La preservación de la vida; Reconocimiento del proceso de transfusión; y seguridad de las transfusiones. La percepción sobre el cambio que después de la transfusión comenzar a vivir desde el proceso de transfusión plantea una reformulación de la vida misma. Este estudio mostró que los pacientes transfundidos perciben el proceso de transfusión como un medio de supervivencia, e incluso tener conocimiento sobre el proceso y sus significados, no es la permanencia de los temores y ansiedades que pueden minimizarse por el equipo multidisciplinario.

Necessidades e preocupações dos pais em diferentes etapas do ciclo vital / Parents' needs and concerns at different stages of the life cycle / Necesidades y preocupaciones de los padres en diferentes etapas del ciclo vital

Objetivo: identificar as necessidades e as preocupações prioritárias, manifestadas pelos pais no desempenho do seu papel, em três etapas do ciclo vital: adolescência, idade produtiva e idade madura. Metodologia: estudo exploratório com abordagem qualitativa, desenvolvido com quatorze pais residentes em um município no extremo sul do Brasil. Os dados foram coletados entre maio e agosto de 2011, por meio de entrevista em profundidade. Através da técnica da análise textual discursiva e da matriz construída com base na teoria bioecológica de Bronfenbrenner, foram construídas três categorias: Necessidades/preocupações do pai, geradas em sua relação com o mundo do trabalho; Necessidades/preocupações que emergem da relação de cuidado com os filhos e Preocupações dos pais com relação ao futuro dos filhos. Conclusão: identificou-se que a preocupação com o futuro dos filhos foi apontada por pais de todas as faixas-etárias investigadas. .

Objective: this study aimed to identify priority needs and concerns expressed by fathers in the performance of their role in three stages of the life cycle: adolescence, productive age, and mature age. Methodology: this is an exploratory study with a qualitative approach, conducted with fourteen fathers residing in a municipality in the extreme south of Brazil. The data were collected between May and August 2011 by means of the in-depth interview. Through the technique of written discourse analysis and the array built upon Bronfenbrenner's bioecological theory, we obtained three categories: fathers' needs/concerns, generated in their relationship with the world of work; needs/concerns that emerged from the relationship of care with the children; and fathers' concerns about the future of the children. Conclusions: we identified that the concern with the future of the children was pointed out by fathers of all age groups investigated. .

Objetivo: identificar las necesidades y preocupaciones prioritarias, manifestadas por los padres en el desempeño de su función, en tres etapas del ciclo de vida: adolescencia, edad productiva y edad madura. Metodología: estudio exploratorio con abordaje cualitativo, desarrollado con catorce padres residentes en un municipio en el extremo sur de Brasil. Los datos fueran colectados entre mayo y agosto de 2011, a través de entrevistas en profundidad. A través de la técnica de análisis textual y discursiva e de la matriz construida basada en la teoria bioecologica de Bronfenbrenner, fueran construidas tres categorías: Necesidades/ preocupaciones de lo padre, generado en suya relación con el mundo de lo trabajo; Necesidades/preocupaciones que emergen de la relación de cuidado con hijos e preocupaciones de los padres con lo futuro de los hijos. Conclusión: Se identifico que la preocupación con el futuro de los hijos fue apuntado por los padres de todas las edades averiguadas. .

Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria.

Background & objectives: The production of carbapenemases is an important mechanism responsible for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase producers is essential. A prospective study was undertaken to detect metallo-β-lactamases (MBLs) and AmpC β-lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for detection of carbapenemases and MBLs. Methods: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. lwoffi i) and 140 Pseudomonas spp. (103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby- Bauer disc diffusion method. Modifi ed Hodge test, EDTA disk synergy (EDS) test and AmpC disk test were used for the detection of carbapenemases, MBLs and AmpC β-lactamases, respectively. Results: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffi i, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC β-lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9 (28.1%) found positive for carbapenemases by modifi ed Hodge test. Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC β-lactamase producers, 3 (6.5%) MBL producers, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test. One P. aeruginosa was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase. Interpretation & conclusions: MBL production is an important mechanism of carbapenem resistance among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection of MBLs than modifi ed Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance. AmpC β-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations / Vírus influenza e bactéria proteolítica co-infectantes em trato respiratório de indivíduos com manifestações respiratórias

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09 percent) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64 percent) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13 percent) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51 percent) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 ºC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

O papel da bactéria proteolítica na exacerbação do vírus influenza tem sido demonstrado em hospedeiros naturais como porcos e humanos. Foram coletadas 407 amostras do trato respiratório de indivíduos apresentando manifestações clínicas, durante a estação da influenza (2003-2005) na cidade de São Paulo. Este trabalho teve como objetivo avaliar a incidência de determinadas bactérias que junto com vírus co-infectarem o trato respiratório humano. Testes bacteriológicos, e virológicos como imunofluorescência (IF), RT/PCR e hemaglutinação (HA) foram usados nas investigações viral e bacteriana. Pelo teste de IF foram selecionadas trinta e sete (9,09 por cento) amostras positivas para o vírus influenza. A presença do vírus influenza foi confirmada pela técnica de RT/PCR. Pelos testes bacteriológicos e do agar caseina, verificou-se que 18 (48,64 por cento) dos indivíduos foram infectados com bactérias proteolíticas tais como Staphylococcus spp., Streptococcus spp. e Pseudomonas spp. Destas amostras, 13 (35,13 por cento) foram co-infectadas com vírus influenza tipo A, e 5 (13,51 por cento) com influenza tipo B. No experimento in vitro com influenza e S. aureus, detectou-se aumento do título hemaglutinante deste vírus, após contacto de 30 min a 25 ºC. Os resultados obtidos revelaram a ocorrência de co-infecção com bactéria proteolítica e vírus influenza nos indivíduos avaliados. Estes achados corroboram com a investigação do sinergismo, entre bactéria e vírus, que poderia ser capaz de potencializar infecção respiratória, aumentando os riscos aos hospedeiros.

Preliminary report of NAD[+]-dependent amino acid dehydrogenase producing bacteria isolated from soil

Amino acid dehydrogenases [L-amino acid: oxidoreductase deaminating; EC 1.4.1.X] are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD[+], NADP[+] or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria [PKU], maple syrup urine disease [MSUD], homocystinuria [HCY] and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteria. The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed. In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD[+]-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat /Km [s-1.mM -1] values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83. This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1.

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.

Prevalence of extended -spectrum beta -lactamases in enterobacteriaceae, Pseudomonas and stenotrophomonas as determined by VITEK2 and Etest syptems in Kuwait teaching hospital

To determine the prevalence of extended-spectrum beta-lactamase [ESBL]-producing members of the Enterobacteriaceae using VITEK 2 and E test systems. A total of 3,592 consecutive gram-negative isolates [single isolate per patient] of the family of Enterobacteriaceae and Pseudomonas adjudged to be clinically relevant to the patient's infection were studied for ESBL production over a period of 1 year at Mubarak Al-Kabeer Hospital, Kuwait. Two methods were used: the automated VITEK 2 system and E test ESBL, a manually manipulated plastic strip containing various gradients of beta-lactam antibiotics. These tests and interpretative criteria for the results were performed according to the manufacturer's instructions. Of the 3,592 bacterial isolates, 264 [7.5%] and 185 [5.2%] were positive for ESBL production by the VITEK 2 and E test, respectively. All the ESBL-producing Pseudomonas aeruginosa identified by VITEK 2 gave indeterminate results by E test. Prevalent ESBL producers, identified by the VITEK 2 versus E test, respectively, were: Citrobacter spp. [15 vs. 3.2%], K. pneumoniae [12.2 vs. 11.4%], Enterobacter spp. [12 vs. 3%], E. coli [6.5 vs. 5.6%], P. aeruginosa [6.5 vs. 0%] and Morganella spp. [2 vs. 1%]. The most common infection associated with ESBL-producing pathogens was urinary tract infection [68.2%], followed by wound infection [14.4%] and bloodstream infection [6.1%]. The result of this study showed a relatively high prevalence of clinically significant ESBL producers among the Enterobacteriaceae and Pseudomonas spp. at our teaching hospital. The VITEK 2 identified a higher prevalence of ESBL strains than the E test

Metabolism of 3-chlorobenzoate by a Pseudomonas (diff) spp.

Pseudomonas (diff) spp. was isolated from a complex petrochemical sludge, using benzoate as the sole source of carbon. The organism could metabolize 3-chlorobenzoate, releasing approximately 30% of organically bound chloride. 3-Chlorodihydrodihydroxybenzoate and 3-chlorocatechol were confirmed as pathway intermediates by mass spectral and HPLC analysis. About 3-fold higher levels of catechol 1,2-oxygenase were detected in cells grown on 3-chlorobenzoate as compared to that of benzoate. 3-Chlorocatechol inhibited the catechol 1,2-oxygenase activity, when used as assay substrate. A 15-fold purified catechol 1,2-oxygenase had a Km of 0.37 mumole and Vmax of 2.3 with 3-chlorocatechol. Catechol gave Km of 0.2 mumole and Vmax of 40, suggesting that 3-chlorocatechol is not metabolised further and hence blocks the metabolic pathway for 3-chlorobenzoate degradation. In contrast catechol 1,2-oxygenase was not inhibited by 4-chlorocatechol and probably is an intermediate for the total/complete degradation of 3-chlorobenzoate (approx. 30%).