Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt / Sequenciamento e resistência antimicrobiana múltipla de Pseudomonas fluorescens isoladas de tilápia-do-nilo no Egito

Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128/400) of apparently healthy (9%; 36/400) and diseased (23%; 92/400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).

Pseudomonas fluorescens é uma das principais causas de doenças septicêmicas em peixes de água doce, causando graves perdas econômicas e diminuindo a eficiência da fazenda. Assim, esta pesquisa teve como objetivo investigar a ocorrência de P. fluorescens em peixes de tilápia-do-nilo (O. niloticus) no Egito, sequenciamento do gene 16S rDNA e suscetibilidade antimicrobiana. Cepas de P. fluorescens foram detectadas em 32% (128/400) de peixes tilápia-do-nilo aparentemente saudáveis ​​(9%; 36/400) e doentes (23%; 92/400). A maior prevalência foi observada nas brânquias dos peixes, 31,3%, seguida pelo intestino 26,9%, fígado 24,2% e rins 17,6%. Os resultados da PCR para o gene 16SrDNA de P. fluorescens mostraram o gene 16SrDNA em 30% dos isolados examinados. Além disso, a homogeneidade e uma forte relação entre cepas de P. fluorescens foi confirmada usando sequências de 16SrDNA. Além da responsabilidade do gene 16SrDNA na virulência de P. fluorescens. Os resultados dos testes de suscetibilidade antimicrobiana revelaram que todas as cepas foram resistentes à piperacilina (100%), seguida pela ceftazidima (29,7%) e cefepima (25,8%). As cepas de P. fluorescens foram altamente sensíveis à cefotaxima (74,2%), seguida pela ceftriaxona e levofloxacina (70,3% cada). Curiosamente, 29,7% das cepas de P. fluorescens eram multirresistentes a antimicrobianos (MAR).

Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt

Abstract Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128/400) of apparently healthy (9%; 36/400) and diseased (23%; 92/400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).

Resumo Pseudomonas fluorescens é uma das principais causas de doenças septicêmicas em peixes de água doce, causando graves perdas econômicas e diminuindo a eficiência da fazenda. Assim, esta pesquisa teve como objetivo investigar a ocorrência de P. fluorescens em peixes de tilápia-do-nilo (O. niloticus) no Egito, sequenciamento do gene 16S rDNA e suscetibilidade antimicrobiana. Cepas de P. fluorescens foram detectadas em 32% (128/400) de peixes tilápia-do-nilo aparentemente saudáveis (9%; 36/400) e doentes (23%; 92/400). A maior prevalência foi observada nas brânquias dos peixes, 31,3%, seguida pelo intestino 26,9%, fígado 24,2% e rins 17,6%. Os resultados da PCR para o gene 16SrDNA de P. fluorescens mostraram o gene 16SrDNA em 30% dos isolados examinados. Além disso, a homogeneidade e uma forte relação entre cepas de P. fluorescens foi confirmada usando sequências de 16SrDNA. Além da responsabilidade do gene 16SrDNA na virulência de P. fluorescens. Os resultados dos testes de suscetibilidade antimicrobiana revelaram que todas as cepas foram resistentes à piperacilina (100%), seguida pela ceftazidima (29,7%) e cefepima (25,8%). As cepas de P. fluorescens foram altamente sensíveis à cefotaxima (74,2%), seguida pela ceftriaxona e levofloxacina (70,3% cada). Curiosamente, 29,7% das cepas de P. fluorescens eram multirresistentes a antimicrobianos (MAR).

Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi

Background & objectives: Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (B426) produce mosquitocidal biosurfactant, surfactin and di-rhamnolipid. The objective of the study was to carry out a small-scale field evaluation of the two biosurfactants to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected sites in Goa, India. Methods: Surfactin (VCRC B471) and di-rhamnolipid (VCRC B426) were formulated as aqueous suspensions (5% AS), and were applied at the dosages of 34, 51 and 68 mL/m2 and 27, 41 and 54 mL/m2 respectively. Two experiments were carried out with the two formulations. Results: Surfactin (VCRC B471) formulation was effective at all the dosages and there was sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and up to 15 days in experiment 2. No pupae were found in the treated sites throughout the study. Di-rhamnolipid (VCRC B426) formulation was also found to reduce the immature density in the treated sites up to 14 days in experiment 1 and up to 15 days in experiment 2. Interpretation & conclusion: For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, 27mL/m2 . The formulations are to be applied fortnightly for effective control of Anopheles. The application dosage determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria

Modelamiento in silica de la liasa organomercurial (MerB) de Pseudamanas fluarescens / In silico modeling of Pseudomonas fluoresceins organomercurial lyase (MerB) / Modelagem in silica da liase organomercurial (MerB) de Pseudamanas fluarescens

Resumen El modelamiento ¡n silíco ha sido de gran contribución en los procesos proteómicos, desarrollando estructuras de las secuencias proteicas ya existentes, que por motivos de altos costos y las diferentes tecnologías necesarias para el desarrollo de estas metodologías, se encuentran deficientes en el número de modelamientos de proteínas disponibles. Entre aquellas secuencias con carencia de estructura proteica se encuentra la proteína liasa organomercurial (MerB) de Pseudomonas /luorescens, importante en la resistencia al mercurio. En el presente artículo se analizó tanto estructural como funcionalmente la proteína MerB en Pseudomonas jluorescens, utilizando la herramienta de la química estructural "modelamiento por homología" mediante plataformas bioinformáticas, con el fin de obtener un modelo que represente la estructura 3D más precisa y que capturen las mejores variantes estructurales entre todas las posibles conformaciones de las proteínas en la familia. En este trabajo, se desarrolló un método comparativo de la secuencia estudiada con las reportadas en las bases de datos para las proteínas MerB del género Pseudomonas. Se propone un modelo tridimensional para la enzima (MerB) en P. jluorescens, mediante el modelamiento por homología, se muestra la caracterización en la estructura secundaria, terciaria, la caracterización del dominio catalítico y los motivos estructurales presentes.

Abstract In silico modeling has made a great contribution to proteomic processes, developing structures of the already existing protein sequences, which for reasons of high costs and the different technologies necessary for the development of these methodologies, are deficient in the number of models of available proteins. Among those sequences lacking protein structure is the organomercurial lyase (MerB) protein from Pseudomonas fluoresceins, important in mercury resistance. In this article, the MerB protein in Pseudomonas fluorescens was analyzed both structurally and functionally, using the structural chemistry tool "homology modeling" using bioinformatic platforms, in order to obtain a model that represents the most accurate 3D structure and that captures the best structural variants among all the possible conformations of the proteins in the family. In this work, a comparative method of the sequence studied with those reported in the databases for MerB proteins of the genus Pseudomonas was developed. A three-dimensional model for the enzyme (MerB) in P. fluorescens is proposed, through homology modeling, the characterization at the secondary and tertiary structure level, the characterization of the catalytic domain and the structural motifs present is shown.

Resumo A modelagem in silico tem dado um grande contributo para os processos proteómicos, desenvolvendo estruturas de sequências de proteínas já existentes, as quais, pelos elevados custos e pelas diferentes tecnologias necessárias ao desenvolvimento destas metodologias, são deficientes no número de modelos de proteínas disponíveis. Entre as sequências sem estrutura protéica está a proteína organomercurial liase (MerB) de Pseudomonas fluorescens, importante na resistência ao mercúrio. Neste artigo, a proteína MerB em Pseudomonas fluorescens foi analisada estrutural e funcionalmente, usando a ferramenta de química estrutural "modelagem de homologia" usando plataformas de bioinformática, a fim de obter um modelo que represente a estrutura 3D mais precisa e que capture as melhores variantes estruturais. entre todas as conformações possíveis das proteínas da família. Neste trabalho, foi desenvolvido um método comparativo da sequência estudada com aqueles relatados em bancos de dados para proteínas MerB do gênero Pseudomonas. Um modelo tridimensional para a enzima (MerB) em P. fluorescens é proposto, através de modelagem por homologia, a caracterização em nível de estrutura secundária e terciária, a caracterização do domínio catalítico e os motivos estruturais presentes são mostradas.

Actinomyces y síndrome de Kartagener: Reporte de caso y revisión de la literatura / Actinomyces and Kartagener syndrome: Case report and literature review

RESUMEN El síndrome de Kartagener, el cual hace parte del subgrupo de las discinesias ciliares primarias predispone a infecciones respiratorias recurrentes del tracto respiratorio por Haemophilus influenzae, Staphylococcus aureus y Streptococcus pneumoniae. Se describe a continuación el caso de un paciente con diagnóstico de síndrome de Kartagener en quien se documentó colonización por Pseudomonas fluorescens y neumonía con empiema asociado por Actinomyces spp, una asociación poco frecuente en la literatura.

ABSTRACT Kartagener syndrome, which is part of the subgroup of the primary ciliary dyskinesias, predisposes to recurrent respiratory tract infections due to Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae. The case of a patient with a diagnosis of Kartagener syndrome in whom colonization by Pseudomonas fluorescens and pneumonia complicated with empyema by Actinomyces spp is a rare association in the literature, which is described below.

Antagonismo de rizobacterias sobre hongos fitopatógenos, y su actividad microbiana con potencial biofertilizante, bioestimulante y biocontrolador / Antagonism of rhizobacteria on phytopathogenic fungi, and their microbial activity with biofertilizer, biostimulant and biocontroller potential

RESUMEN Las rizobacterias forman parte de la gran cantidad de microorganismos que actúan como agentes de biocontrol, produciendo metabolitos que inducen resistencia sistémica en las plantas que inhiben el crecimiento de patógenos. El objetivo de esta investigación fue evaluar la capacidad de diez rizobacterias de los géneros Rhizobium, Bradyrhizobium, Sinorhizobium, Ochrobactrum y Pseudomonas para producir ácido cianhídrico (HCN), sideróforos y ácido indol-acético (AIA), disolver fosfato, fijar nitrógeno e inhibir el crecimiento de fitopatógenos. Se realizaron todas las pruebas fisiológicas y bioquímicas correspondientes, así como la prueba de antagonismo in vitro contra los fitopatógenos Fusarium oxysporum, Colletotrichum gloeosporioides y Rhizoctonia solani. Cinco cepas produjeron una mayor cantidad de AIA en relación a las otras en presencia de triptófano, la cepa ES1 (Ochrobactrum sp.) produjo HCN, el 50 % de las cepas evaluadas liberaron sideróforos, el 60 % disolvió fósforo, y todas resultaron positivas para la fijación de nitrógeno. Nueve cepas inhibieron el crecimiento de F. oxysporum entre 40 % y 65 %, la cepa Alf (Pseudomonas fluorescens) inhibió además el crecimiento de C. gloeosporioides en un 22 %, y ninguna inhibió el crecimiento de R. solani. Los rizobios evaluados y la cepa de Pseudomonas fluorescens podrían ejercer efectos beneficiosos sobre las plantas a través de mecanismos directos e indirectos, o una combinación de ambos, lo que las convierte en una opción sostenible para la producción de cultivos.

ABSTRACT Rhizobacteria are part of the large number of microorganisms that act as biocontrol agents, producing metabolites that induce systemic resistance in plants and inhibit the growth of pathogens. The objective of this research was to evaluate the capacity of ten rhizobacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Ochrobactrum and Pseudomonas to produce hydrogen cyanide (HCN), siderophores and indole acetic acid (IAA), dissolve phosphate, fix nitrogen and inhibit the growth of phytopathogens. All the corresponding physiological and biochemical tests were carried out, in addition to an in vitro antagonism test against the phytopathogens Fusarium oxysporum, Colletotrichum gloeosporioides and Rhizoctonia solani. Five strains produced a greater amount of IAA with respect to the others in the presence of tryptophan, the strain ES1 (Ochrobactrum sp.) produced HCN, 50% of the evaluated strains released siderophores, 60% solubilized phosphorus and all were positive for nitrogen fixation. Nine strains inhibited the growth of F. oxysporum by 40% to 65%. The Alf strain (Pseudomonas fluorescens) inhibited the growth of C. gloeosporioides by 22% while none inhibited the growth of R. solani. The rhizobia tested and the Pseudomonas fluorescens strain may have favorable effects on plants through direct and indirect mechanisms, or a combination of both, making them a sustainable option for crop production.

Productivity Enhancement Of Coriandrum Sativum Using Plant Biologicals

Objective: The present study was designed to investigate phytopromotional effects of Sebacina vermifera on economically and medicinally important aromatic plant - Coriandrum sativum (coriander). Methods: Phytopromotional effects of Sebacina vermifera were evaluated on coriander, under greenhouse and field conditions. The evaluations were carried out with reference to emergence, growth promotion and quantitative as well as the qualitative composition of essential oil. Beside this the overall effects were comparatively assessed with the effects of (a) Phosphate solubilizing bacteria (Pseudomonas fluorescens) (b) Nitrogen-fixing bacteria (Azotobacter chroococcum) on coriander using same parameters. Results: Mycorrhizal fungus (Sebacina vermifera) was observed with the most significant effect in all aspects viz. emergence, growth promotion and quantitative as well as the qualitative composition of essential oil. Conclusion: Based upon the observations, Sebacina vermifera is highly recommended as a potential biological agent that could be applied for phytopromotional effects and economic cultivation of aromatic plants.

Ocular infection by a psychrophile: Pseudomonas fluorescens

Accurate identification of infectious pathogens is essential for appropriate management of ocular infections. Routine laboratory protocols typically support bacterial growth at 37°C. We report a case, wherein we serendipitously isolated Pseudomonas fluorescens – an organism that prefers lower temperatures for optimal growth (psychrophilic) in the environment – from eviscerated contents of an eye with total corneal melt. This case highlights the need for being vigilant for organisms with different temperature sensitivities in culture media than that found in routine protocols.

Response of arbuscular mycorrhizal fungi along with Trichoderma viride and Pseudomonas fluorescens on the growth, biochemical attributes and vase life of Chrysanthemum indicum

Aim: The present study aimed to investigate the effects of two dominant arbuscular mycorrhizal (AM) fungi along with Trichoderma viride and Pseudomonas fluorescens on the growth and vase life of Chrysanthemum indicum ('Garden Mum'- Kathleen Dark Red). Methodology: An experiment was conducted to evaluate the effect of G. mosseae (G) and A. laevis (A) along with T. viride (T) and Pseudomonas fluorescens (P) on the growth and vase life of C. indicum L. under polyhouse conditions. The experiment was laid in a randomized block design with five replicates. Results: AM fungi along with other bioinoculants showed maximum root colonization leading to increased water absorption and various important nutrients, especially phosphorous, thereby enhancing the growth and different biochemical attributes. For the vase life experiment, bioinoculants treated plants showed better result with minimum peroxidase activity, thereby delaying flower senescence. Interpretation: AMF inoculation should be recommended at nursery level as biofertilizers are cost effective and also a substitute for chemical fertilizers

16s rrna gene analysis for characterization of pseudomonas spp. isolated from groundnut rhizosphere

The present study assessed 16S rRNA gene sequence analysis method as a tool for identification of fluorescent pseudomonads phenotypically difficult to identify. Using 16S rRNA gene sequencing the isolates were identified to the genus level to the species level. In this study, phylogenetic tree was constructed using complete sequence within the 16S rRNA gene. Distance tree was constructed to find out genetic similarity between the organisms. Although the phylogenetic analysis is not decisive, it is consistent with other observations, especially the capacities of the strain as a biocontrol agent. The results suggest that the JS16, JS7, JS31 and JS52 and strains are high homology with Pseudomonas fluorescens, P. Plecogloccida and P. Putida.

Compatibility of Azospirillum brasilense and Pseudomonas fluorescens in growth promotion of groundnut ( Arachis hypogea L)

ABSTRACT We attempted to study the compatibility among plant beneficial bacteria in the culture level by growing them near in the nutrient agar plates. Among all the bacteria tested, Rhizobium was found to inhibit the growth of other bacteria. From the compatible group of PGPR, we have selected one biofertilizer (Azospirillum brasilense strain TNAU) and one biocontrol agent (Pseudomonas fluorescens strain PF1) for further studies in the pot culture. We have also developed a bioformulation which is talc powder based, for individual bacteria and mixed culture. This formulation was used as seed treatment, soil application, seedling root dip and foliar spray in groundnut crop in vitro germination conditions. A. brasilense was found to enhance the tap root growth and P. fluorescens, the lateral root growth. The other growth parameters like shoot growth, number of leaves were enhanced by the combination of both of the bacteria than their individual formulations. Among the method of application tested in our study, soil application was found to be the best in yielding better results of plant growth promotion.

Development and formulation of azospirillum lipoferum and pseudomonas fluorescens as effective biological agents for enhanced agro-productivity / Desenvolvimento e formulação de azospirillum lipoferum e pseudomonas fluorescens enquanto agentes biológicos eficazes para uma agro-produtividade melhorada

Biofertilizer is a group of beneficial microorganisms used for improving the productivity of soil by fixing atmospheric nitrogen or by solubilizing soil phosphorus. They also stimulate plant growth through synthesis of growth promoting substances. In this present study, Azospirillum lipoferum is grown in Nitrogen free Bromothymol blue (Nfb) medium and Pseudomonas fluorescens in King's B medium. Bioprocess condition was optimized for both of the culture and found that Pseudomonas fluorescens has shown highest growth at 300C in pH 8 after 72 hours of incubation where as Azospirillum lipoferum showed highest cell concentration at 310C in pH 7, with incubation period of 72 hours. The optimized culture is mixed with different formulations of powder and liquid carrier such as Saw dust, Rice husk, Date seed powder, Matka khad, Jiwamrit and Beejamrit respectively. Shelf life study for 0, 30, 60, 90 and 120 days by cell counting and spread plate method showed that shelf life of the biofertilizer produced from Powder and liquid carriers had high amount of viable microbial population up to 120 days storage. Among biofertilizer based bio inoculants, Saw dust showed maximum population of 77x109cfu/ml for Azospirillum lipoferum and 72 x 109 CFU/ml for Pseudomonas strain on 120th day and the liquid carrier Matka khad showed 85x109 cfu/ml for Azospirillum lipoferum and 78 x 109 CFU/ml for Pseudomonas fluorescens.

Biofertilizante é um grupo de microorganismos benéficos utilizados para melhorar a produtividade do solo através da fixação de azoto atmosférico ou por solubilização de fósforo no solo. Eles também estimulam o crescimento vegetal através de síntese de substâncias promotoras do crescimento. No presente estudo, Azospirillum lipoferum é cultivado em um meio de azul de bromotimol sem nitrogênio (Nfb) e Pseudomonas fluorescens num meio de King's B. A condição de bioprocesso foi optimizada para ambas as culturas e descobriram que Pseudomonas fluorescens mostraram maior crescimento a 300ºC em pH 8 após 72 horas de incubação, enquanto que Azospirillum lipoferum mostraram maior concentração de células a 310ºC em pH 7, com um período de incubação de 72 horas. A cultura optimizada é misturada com diferentes formulações de pó e veículo líquido tal como serragem, casca de arroz, pó de semente de tâmaras, Matka khad, Jiwamrit e Beejamrit respectivamente. O estudo do prazo de validade para 0, 30, 60, 90 e 120 dias por contagem celular e método de espalhamento em placa mostrou que o prazo de validade do biofertilizante produzido a partir do pó e veículos líquidos teve grande quantidade de população microbiana viável até 120 dias de armazenamento. Entre inoculantes biológicos de base biofertilizantes, a serragem mostrou população máxima de 77x109 CFU/ml para Azospirillum lipoferum e 72 x 109 CFU/ml para a estirpe Pseudomonas no 120º dia e um veículo líquido Matka khad mostrou 85x109 CFU/ml para Azospirillum lipoferum e 78x109 CFU/ml para Pseudomonas fluorescens.

PROMOCIÓN DEL CRECIMIENTO DE Baccharis macrantha (ASTERACEAE) CON BACTERIAS SOLUBILIZADORAS DE FOSFATOS ASOCIADAS A SU RIZOSFERA / Growth Promotion of Baccharis macrantha (Asteraceae) by Phosphate Solubilizing Rhizosphere Bacteria

El objetivo de esta investigación fue aislar y caracterizar bacterias solubilizadoras de fosfatos (BSF) asociadas a la rizosfera de Baccharis macrantha y Viburnum triphyllum, y evaluar su capacidad para solubilizar fosfatos en condiciones in vitro. Además se determinó el efecto de la inoculación de las cepas de BSF más eficientes sobre el crecimiento de B. macrantha. Las muestras de suelo rizosférico de B. macrantha y V. triphyllum fueron colectadas en los meses de mayo-período de lluvia y septiembre-período seco del 2012. Para la cuantificación de bacterias heterótrofas cultivables y BSF se empleó el método de recuento en placa en los medios Agar Tripticasa de Soya y Pikovskaya (PVK) respectivamente. La capacidad de solubilización de fosfatos de las cepas aisladas se estimó a partir del diámetro de los halos formados alrededor de las colonias en el medio de cultivo PVK después de 7 días de incubación a 28 °C. Los ensayos de inoculación en B. macrantha se realizaron con las BSF más eficientes. La inoculación de las BSF B. firmusy P. fluorescens de forma individual y como inoculante combinado mostro un efecto benéfico, incrementando significativamente el porcentaje de germinación de semillas, la altura de la plántula, la longitud de la raíz y el peso seco de B. macrantha. La inoculación de BSF podría ser considerada una estrategia para mejorar el crecimiento y establecimiento de B. macrantha en pastizales abandonados.

The objectives of this research was to isolate and characterize phosphate solubilizing bacteria (BSF) associated to the rhizosphere of Baccharis macrantha and Viburnum triphyllum, and to assess their ability to solubilize phosphate under conditions in vitro. Furthermore to determine the effect of inoculation of the strains BSF more efficient on the growth of B. macrantha. Rhizosphere soil samples of B. macrantha and V. triphyllum were collected in the months of May-rainy season and September-period dry the 2012. Trypticase Soya Agar and Pikovskaya (PVK) were used for quantification of culturable heterotrophic bacteria and BSF, respectively. The phosphate solubilizing capacity of the isolated strains was estimated from the diameter of the halo around the colonies formed in the culture medium PVK after 7 days incubation at 28 °C. Inoculation assays were performed with more efficient BSF in B. macrantha. Inoculation of BSF Bacillus firmus and Pseudomona fluorescens individually and as inoculant combined showed a beneficial effect, significantly increasing the percentage of seed germination, seedling height, root length and dry weight of B . macrantha. Inoculation the BSF could be considered a strategy to improve the growth and development of B. macrantha in abandoned pastures.

Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk

The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli. The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca+2. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca+2, on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.

Fabrication of an antimicrobial active packaging and its effect on the growth of Pseudomonas and aerobic mesophilic bacteria in chicken / Elaboración de un envase activo antimicrobiano y su efecto en el desarrollo de Pseudomonas y bacterias aerobias de pollo

Background: One of the aims of food packaging is to protect the product from environmental factors that can cause a reduction in quality. Surface growth of microorganism is one of the leading causes of food spoilage. One option is to use antimicrobial packaging to provide an increased margin of safety and quality. Objectives: The aim of this study was evaluate the effect of active packaging with eugenol on growth of Pseudomonas and aerobic mesophilic bacteria in fresh chicken pieces. Methods: Three batches of low-density polyethylene (LDPE) film, containing 0, 9.0 and 7.7, mg g-1 eugenol (control, AAF1 and AAF2, respectively), were extruded in a pilot-plant scale blown-extrusion machine. The films with eugenol lost 42.7% and 36.8% (AAF1 and AAF2, respectively) of eugenol during processing and absorbed UV-visible light at 300-261 nm. The kinetics of eugenol release from the AAF1 into the air at 5°C and 25ºC displayed Fick's behavior, and a diffusion coefficient of 10-8 cm2 s-1 was calculated. Results: Eugenol showed antimicrobial activity on in vitro, using paper discs with 1.74, 0.87 and 0.36 mg eugenol on 108 CFU mL-1 of Pseudomonas fluorescens in Muller-Hinton agar. Chicken thighs were wrapped in the AAF2 film, and the effects on the growth of Pseudomonas and aerobic mesophilic bacteria (AMB) were evaluated after storage for 5 d at 5°C. The AAF2 showed a moderately antimicrobial effect in reducing the growth of Pseudomonas (1.1 x 106 CFU g-1) relative to growth in the control film (6.0 x 106 CFU g-1) (P < 0.05). The film with eugenol was effective in reducing the growth of AMB (9.0 x 105 CFU g-1) relative to growth in the control film (1.7 x 106 CFU g-1) (P < 0.05). Conclusions: Despite the high losses of eugenol during the extrusion of the films, they showed an antimicrobial effect during contact with fresh chicken under commercial conditions. This study shows the potential use of eugenol for application in LDPE antimicrobial packaging film.

Antecedentes: Uno de los principales objetivos del envasado de alimentos es protegerlo de factores que puedan afectar y causar una reducción en la calidad. El desarrollo de microorganismos en la superficie es uno de las causas principales del deterioro de los alimentos. Una opción es el empleo de envases con propiedades antimicrobianas. Objetivos: El objetivo del presente estudio fue evaluar el efecto de un envase antimicrobiano conteniendo eugenol en el desarrollo de Pseudomonas y bacterias mesofílicas aerobias (BMA) en piezas de pollo. Métodos: Tres lotes de película de polietileno de baja densidad (PEBD) conteniendo 0, 9.0 y 7.7 mg g-1 de eugenol (control, AAF1, AAF2, respectivamente) fueron obtenidas por extrusión-soplo utilizando un extrusor a nivel planta piloto. Se calculó la cinética de liberación del eugenol de la AAF1 hacia el aire a 5°C y 25°C. Se evaluó la capacidad antimicrobiana in vitro del eugenol sobre 108 UFC mL-1 de Pseudomona fluorescens utilizando discos de papel conteniendo 1.74, 0.87 y 0.36 mg de eugenol en agar Muller-Hinton. Las piezas de pollo fueron envueltas en la película AAF2 y almacenadas a 5°C evaluando a los 5 días el efecto de la película en el desarrollo de Pseudomonas y en BMA. Resultados: El eugenol mostró actividad antimicrobiana inhibiendo el crecimiento de P. fluorescens. Las películas conteniendo eugenol perdieron durante el proceso de extrusión 42.7% y 36.8% (AAF1 y AAF2 respectivamente) del total añadido mostrando un comportamiento fickiano con un coeficiente de difusión de 10-8 cm2 s-1. Las AAF2 mostraron un efecto moderado en la reducción del desarrollo de Pseudomonas (1.1 x 106 CFU g-1) comparadas con el control (6.0 x 106 CFU g-1) (P < 0.05). Las películas con eugenol (AAF2) fueron efectivas al reducir el desarrollo de las BMA (9.0 x 105 CFU g-1) comparadas con la película control (1.7 x 106 CFU g-1) (P < 0.05). Conclusiones: A pesar de las pérdidas del eugenol durante el proceso de extrusión para la obtención de las películas, estas mostraron un efecto antimicrobiano sobre las piezas de pollo. Por lo tanto, este estudio muestra el uso potencial del eugenol para la aplicación en envases antimicrobianos elaborados a base de PEBD.

Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk

Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.

Clinical significance of serum anti-microbial antibodies test in inflammatory bowel disease / 中华消化杂志

Objective To investigate the clinical significance of serum anti-Saccharomyces cerevisias antibody (ASCA),anti-outer membrane porin C (anti-OmpC),antibody to Pseudomonas fluorescens-associated sequence I2 (anti-I2 )and antibody to bacterial flagellin (anti-CBirl )in the diagnosis and treatment of inflammatory bowel disease (IBD).Methods From 2011 to 2013,87 patients with IBD were enrolled and divided into Crohn′s disease (CD)group (66 cases)and ulcerative colitis (UC)group (21 cases).A total of 62 age and gender matched healthy individuals were enrolled as the control group. Fasting blood samples (2 mL)of the subjects were collected.The expression of ASCA,anti-OmpC,anti-I2 and anti-Cbirl antibodies was detected with enzyme-linked immunosorbent assay (ELISA)kits.The diagnosis value of each antibody in IBD and the differential diagnostic value of in UC and CD were compared by receiver operating characteristic (ROC)curve.Results The area under the curve (AUC)of ASCA between IBD and the healthy control group,between CD group and UC group was 0.580 and 0.512, respectively;the accuracy in diagnosis was low.The AUC of anti-CBirl between IBD and the healthy control group was 0.617.There was no differential diagnosis significance of the other antibodies.The positive rate of ASCA in IBD group was 62.1 % (54/87),which was significantly higher than that in the control group (38.7%,24/62).The positive rates of anti-OmpC and anti-I2 in IBD group was significantly lower than those in the control group and the differences were statistically significant (both P 0.05).The specificity,sensitivity,positive predictive value (PPV)and negative predictive value (NPV)of ASCA in differential diagnosis of CD and UC was 52.4%,66.7%,81 .48% and 33.33%,respectively.The specificity and sensitivity of anti-OmpC,anti-I2 and anti-CBirl in differential diagnosis of CD and UC was 81 .0% to 100.0% and 9.1 % to 37.9%,respectively.The specificity,sensitivity,PPV and NPV of double-positive ASCA and anti-I2 in the diagnosis of CD was 57.1 %,86.4%,82.6% and 50.0%, respectively.The positive rate of ASCA and anti-I2 in CD group was significantly higher than that in UC group (84.8%(56/66)vs 57.1 % (12/21 );χ2 =5 .633,P =0.018 ).Conclusions Positive ASCA has some significance in the diagnosis of patients with IBD in our country.The detection of anti-I2 can help to diagnose ASCA negative CD.Because of low sensitivity and positive rate,anti-OmpC and anti-CBirl have limited value in the diagnosis of IBD and the differential diagnosis of UC and CD in our country.

Semi-scale production of PHAs from waste frying oil by Pseudomonas fluorescens S48

The present study aimed at developing a strategy to improve the volumetric production of PHAs by Pseudomonas fluorescens S48 using waste frying oil (WFO) as the sole carbon source. For this purpose, several cultivations were set up to steadily improve nutrients supply to attain high cell density and high biopolymer productivity. The production of PHAs was examined in a 14 L bioreactor as one-stage batch, two-stage batch, and high-cell-density fed-batch cultures. The highest value of polymer content in one-stage bioreactor was obtained after 60 h (33.7%). Whereas, the two-stage batch culture increased the polymer content to 50.1% after 54 h. High-cell-density (0.64 g/L) at continuous feeding rate 0.55 mL/l/h of WFO recorded the highest polymer content after 54 h (55.34%). Semi-scale application (10 L working volume) increased the polymer content in one-stage batch, two-stage batch and high cell density fed-batch cultures by about 12.3%, 5.8% and 11.3%, respectively, as compared with that obtained in 2 L fermentation culture. Six different methods for biopolymer extraction were done to investigate their efficiency for optimum polymer recovery. The maximum efficiency of solvent recovery of PHA was attained by chloroform-hypochlorite dispersion extraction. Gas chromatography (GC) analysis of biopolymer produced by Pseudomonas fluorescens S48 indicated that it solely composed of 3-hydrobutyric acid (98.7%). A bioplastic film was prepared from the obtained PHB. The isolate studied shares the same identical sequence, which is nearly the complete 16S rRNA gene. The identity of this sequence to the closest pseudomonads strains is about 98-99%. It was probably closely related to support another meaningful parsiomony analysis and construction of a phylogenetic tree. The isolate is so close to Egyptian strain named EG 639838.

A Case of Pseudomonas fluorescens Infection after Dacryocystorhinostomy and Silicone Tube Intubation

PURPOSE: To report a case of Pseudomonas fluorescens infection following endoscopic dacryocystorhinostomy and silicone tube intubation in a healthy patient who was using steroid nasal spray. In addition, a literature review is conducted. CASE SUMMARY: A 72-year-old female patient came to our clinic with tearing and hyperemia in the right eye. Ten months prior, she had undergone endoscopic dacryocystorhinostomy and silicone tube intubation due to nasolacrimal duct obstruction in the right eye. Six months after the first operation, dacryocystorhinostomy revision with silicone tube exchange was performed due to obstruction of the nasal bony orifice. In addition, the patient was using a steroid nasal spray. On slit lamp examination, conjunctival injection, marked inflammation and punctal edema around the tube were observed. The silicone tube was removed and the tube cultured. Pseudomonas fluorescens was isolated from the tube contents. The patients was treated with topical 0.3% gatifloxacin 4 times a day, methylol cephalexin lysinate 1000 mg 3 times a day and the nasal spray was discontinued. Two weeks later, all symptoms were resolved after treatment with antibiotic treatment. CONCLUSIONS: A case of Pseudomonas fluorescens canaliculitis which occurred in healthy patient who was using steroid nasal spray is presented with a literature review. Pseudomonas fluorescens canaliculitis can be treated by using proper antibiotics.

Characterization and lytic activity of Pseudomonas fluorescens phages from sewage

Pseudomonas fluorescens phages from sewage were tested against P. fluorescens isolates of soil and sewage. The phages were characterized as to host range, morphology, structural proteins and genome fingerprint. Of the seven phages isolated, one was found to be abundant in sewage (5.9×10(7) pfu/mL), having broad host range, and distinct protein and DNA profile when compared to the other six phages. DNA restriction and protein profiles of the phages and their morphology indicate the diversity in the sewage environment. None of the isolates from the rhizosphere regions of various cultivated soils were susceptible to phages isolated from sewage.