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).

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Eficiencia de solubilización de fósforo de aislados nativos guatemaltecos de Pseudomonas fluorescens / Phosphorus solubilization efficiency of native Guatemalan isolates of Pseudomonas fluorescens

El fósforo (P) es un elemento esencial en la producción agrícola, pero debido a su compleja dinámica en el suelo, solo una pequeña cantidad es aprovechable para las plantas, ya que la mayoría del P se encuentra en formas insolubles, especialmente, en suelos Andisoles de origen volcánico. Los microorganismos con capacidad solubilizadora de fósforo (MSF) son una alternativa para transformar el P a formas solubles y aprovechables por las plantas; además de brindar múltiples beneficios ambientales. Este trabajo identificó y evaluó in vitro, aislados nativos de Pseudomonas fluorescens Mingula, obtenidos de regiones guatemaltecas con suelos Andisoles que limitan la producción agrícola por la alta fijación de P. Se realizaron cultivos in vitro de la bacteria en medio National Botanical Research Instituteís phosphate growth (NBRIP), con fosfato tricálcico Ca3(PO4)2 como fuente de P insoluble y se midió el índice de solubilización de fósforo (ISF). Un total de 35 aislados de P. fluorescensfueron identificados y confirmados por PCR específico. El análisis de relaciones genéticas con el marcador AFLP, mostró dos grupos: el grupo A incluyó a los aislados con ISF mayores a 1.75, mientras el grupo B incluyó a aquellos con ISF menor a 1.75. La comparación de ISF entre los aislados y departamentos, demostró diferencia estadísticamente significativa (p < .001), con el aislado Pf_33 como más eficiente. Debido al potencial de solubilización de los aislados nativos del grupo genético A (ISF > 1.75), estos se recomiendan para futuras investigaciones que determinen su respuesta a condiciones de campo y estrategias para el desarrollo de biofertilizantes.

Phosphorus (P) is an essential element in agricultural production, but due to its complex dynamics in the soil, only a tiny amount is usable by plants. This is because most P is in insoluble forms, especially in volcanic Andisol soils. Microorganisms with phosphorus solubilizing capacity (MSF) are an alternative for transforming P into soluble forms usable by plants and providing multiple environmental benefits. This research identified and evaluated in vitro native isolates of Pseudomonas fluorescens Mingula, obtained from Guatemalan regions with Andisol soils that limit agricultural production due to high P fixation. In vitro cultures of the bacteria were grown on the National Botanical Research Instituteís phosphate medium (NBRIP), with tricalcium phosphate Ca3(PO4)2 as a source of insoluble P, and We measured the phosphorus solubilization index (PSI). We identified and confirmed a total of 35 isolates of P. fluorescens by specific PCR. Using the AFLP marker, genetic relationship analysis showed two groups: group A included isolates with PSI greater than 1.75, while group B included those with FSI less than 1.75. Comparing of PSI between isolates and departments showed statistically significant dif-ferences (p < 0.001), respectively, with the Pf_33 isolate as the most efficient. Because of the high solubilization potential of the native isolates of genetic group A (FSI > 1.75), We recommend future research to determine their response to field conditions and strategies for biofertilizer development.

Effectiveness of indigenous fluorescent pseudomonads in suppressing Rhizoctonia solani root rot disease and promoting plant growth in chilli seedlings

Aims@#This study aimed to isolate and evaluate the indigenous fluorescent Pseudomonas spp. with bio-control potential against Rhizoctonia solani and promoting growth in chilli seedlings. @*Methodology@#A total of 120 fluorescent bacterial were isolated from the healthy chilli rhizosphere soil from the seven major chilli cultivation localities in Terengganu, Malaysia. Only 115 Gram negative fluorescent isolates were further invitro screened for antagonistic activities against R. solani and plant growth-promoting properties. The 50 most effective fluorescent Pseudomonads antagonist against R. solani with minimum percentage inhibition of radial growth (PIRG) of 65% were selected. Hierarchical cluster analysis was further conducted with two dendrograms derived from SPSS Statistic 20 to facilitate the comparison between these 50 isolates for antagonistic and growth-promoting properties. A total of 40 fluorescent isolates within the most potential cluster were further selected and identified using 16S rRNA sequencing. Thirty four fluorescent isolates were identified as Pseudomonas spp. and six isolates as Burkholderia spp. The top 13 ranked fluorescent Pseudomonas spp. from the scoring index were evaluated for seed germination and vigor index in chilli seedlings. There was no significant difference in germination rate between fluorescent Pseudomonas inoculated with control. However, vigor index of chilli seeds pre-inoculated with fluorescent P. putida (B5C1), P. aeruginosa (B3C56) and P. putida (B5C7) were significantly increased with 4684.9, 4657.3 and 4401.0 over control (P ≤ 0.05).@*Conclusion, significance and impact of study@#These selected fluorescent isolates: P. putida (B5C1), P. aeruginosa (B3C56) and P. putida (B5C7) have the potential to be developed as biofungicide against R. solani and as growthpromoter in chilli production system.

Quantification of psychrotrophic bacteria and molecular identification of Pseudomonas fluorescens in refrigerated raw milk / Quantificação de bactérias psicrotróficas e identificação molecular de Pseudomonas fluorescens em leite cru refrigerado

In this study, we investigated the contamination of refrigerated raw milk produced in the western region of Paraná, southern Brazil, with psychrotrophic microorganisms, aiming to assay the proteolytic activity of the isolates and to identify Pseudomonas fluorescens, the main proteolytic species associated with the spoilage of milk products. Raw milk samples from 50 dairy farms were submitted to the counting of psychrotrophic microorganisms, being the microbiota characterized by its mesophilic behavior and proteolytic capacity, besides molecular identification of P. fluorescens. Of the samples evaluated, 94% had psychrotrophic counts ranging from 3 to 7.1 log CFU mL-1, and 48.5% of these showed mesophilic behavior. Of the isolates, 48.0% had proteolytic activity in at least one evaluated temperature (21 and 30°C), and 39.3% had proteolytic activity in both temperatures. Among the 61 isolates submitted to molecular identification by polymerase chain reaction (PCR), 86.8% contained the expression of the 16S gene characteristic for P. fluorescens. In this study, we demonstrated that P. fluorescens is the most prevalent psychrotrophic bacteria species in raw refrigerated milk and their proteolytic ability poses high risks to the dairy industry.(AU)

No presente estudo, investigamos a contaminação do leite cru refrigerado produzido na região oeste do Paraná, sul do Brasil, com micro-organismos psicrotróficos, visando testar a atividade proteolítica dos isolados e identificar Pseudomonas fluorescens, a principal espécie proteolítica associada à deterioração de produtos lácteos. Amostras de leite cru de 50 fazendas leiteiras foram submetidas à contagem de micro-organismos psicrotróficos, caracterizando-se a microbiota por seu comportamento mesofílico e sua capacidade proteolítica, além de identificação molecular de P. fluorescens. Entre as amostras avaliadas, 94% apresentaram contagem psicrotrófica variando de 3 a 7,1 log UFC mL-1 e 48,5% destas apresentaram comportamento mesofílico. Entre os isolados, 48,0% apresentaram atividade proteolítica em pelo menos uma das temperaturas testadas (21 e 30°C) e 39,3% apresentaram atividade proteolítica em ambas as temperaturas. Entre os 61 isolados sub-metidos à identificação molecular por reação em cadeia da polimerase (PCR), 86,8% continham expressão do gene 16S característico de P. fluorescens. Neste estudo, demonstramos que P. fluorescens é a espécie de bactérias psicrotróficas mais prevalente em leite refrigerado cru e sua capacidade proteolítica promove elevados riscos de deterioração para a indústria de laticínios.(AU)

Anti-Bacterial Effect of Lactobacillus rhamnosus Cell-Free Supernatant Possessing Lysozyme Activity Against Pathogenic Bacteria

Recently, there has been a growing demand for natural preservatives because of increased consumer interest in health. In this study, we produced Lactobacillus rhamnosus cell-free supernatant (LCFS) and evaluated and compared its antimicrobial activity with existing natural preservatives against pathogenic microorganisms and in chicken breast meat contaminated with Escherichia coli and Staphylococcus aureus. Lactobacillus rhamnosus cell-free supernatant possessed 30 units of lysozyme activity and contained 18,835 mg/L of lactic acid, 2,051 mg/L of citric acid and 5,060 mg/L of acetic acid. Additionally, LCFS inhibited the growth of fourteen pathogenic bacteria, S. aureus, Bacillus cereus, Listeria monocytogenes, Vibrio parahaemolyticus, Listeria innocua, S. epidermidis, L. ivanovii, E. coli, Pseudomonas aeruginosa, Shigella sonnei, Shi. flexneri, Proteus vulgaris, Pseudomonas fluorescens, and Klebsiella pneumoniae. The antibacterial activity of LCFS was stronger than that of egg white lysozyme (EWL), Durafresh (DF) and grapefruit seed extract (GSE). Additionally, LCFS maintained its antimicrobial activity after heat treatment at 50℃~95℃ and at pH values of 3~9. Moreover, LCFS inhibited the growth of E. coli and S. aureus in chicken breast meat. In conclusion, it is expected that LCFS, which contains both lysozyme and three organic acids, will be useful as a good natural preservative in the food industry.

Predictive modeling of Pseudomonas fluorescens growth under different temperature and pH values

Abstract Meat is one of the most perishable foods owing to its nutrient availability, high water activity, and pH around 5.6. These properties are highly conducive for microbial growth. Fresh meat, when exposed to oxygen, is subjected to the action of aerobic psychrotrophic, proteolytic, and lipolytic spoilage microorganisms, such as Pseudomonas spp. The spoilage results in the appearance of slime and off-flavor in food. In order to predict the growth of Pseudomonas fluorescens in fresh meat at different pH values, stored under refrigeration, and temperature abuse, microbial mathematical modeling was applied. The primary Baranyi and Roberts and the modified Gompertz models were fitted to the experimental data to obtain the growth parameters. The Ratkowsky extended model was used to determine the effect of pH and temperature on the growth parameter µmax. The program DMFit 3.0 was used for model adjustment and fitting. The experimental data showed good fit for both the models tested, and the primary and secondary models based on the Baranyi and Roberts models showed better validation. Thus, these models can be applied to predict the growth of P. fluorescens under the conditions tested.

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.

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.

Expression and response surface optimization of the recovery and purification of recombinant D-galactose dehydrogenase from Pseudomonas fluorescens.

The enzyme D-galactose dehydrogenase (GalDH) has been used in diagnostic kits to screen blood serum of neonates for galactosemia. It is also a significant tool for the measurement of β-D-galactose, α-D-galactose and lactose as well. In this study, response surface methodology (RSM) was used to identify the suitable conditions for recovery of recombinant GalDH from Pseudomonas fluorescens in aqueous two-phase systems (ATPS). The identified GalDH gene was amplified by PCR and confirmed by further cloning and sequencing. E. coli BL-21 (DE3) containing the GalDH gene on a plasmid (pET28aGDH) was used to express and purify the recombinant enzyme. The polyethylene glycol (PEG) and ammonium sulfate concentrations and pH value were selected as variables to analyze purification of GalDH. To build mathematical models, RSM with a central composite design was applied based on the conditions for the highest separation. The recombinant GalDH enzyme was expressed after induction with IPTG. It showed NAD+-dependent dehydrogenase activity towards D-Galactose. According to the RSM modeling, an optimal ATPS was composed of PEG-2000 14.0% (w/w) and ammonium sulfate 12.0% (w/w) at pH 7.5. Under these conditions, GalDH preferentially concentrated in the top PEG-rich phase. The enzyme activity, purification factor (PF) and recovery (R) were 1400 U/ml, 60.0% and 270.0%, respectively. The PEG and salt concentrations were found to have significant effect on the recovery of enzyme. Briefly, our data showed that RSM could be an appropriate tool to define the best ATPS for recombinant P. fluorescens GalDH recovery.

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.

Investigations in associated protozoa-bacterial infections of cyprinids from a fish farm situated on the Jijia river in N-E of Romania / Investigações sobre infecções protozoárias-bacterianas associadas a ciprinídeos da exploração piscícola no rio Jijia, situado no NE da Romênia

In autumn 2011 in cyprinid farms located in Iasi on the Jijia river, several infections with bacterial strains and macroscopical external cysts on the skin were diagnosedwhich developed as a result of the stress induced by biotic and abiotic factors. On the examination of the cyst contents the presence of numerous spores was observed, mostly of the Dermocystidium sp genusThe samples were taken from the common carp (Cyprinus carpio) and crucian carp (Carassius auratus gibelio) species from the fish farm as well as from the Jijia River. 35 fish were examined, all of them showing cysts, fragmentation of their dorsal fin and congestion of the gills. Histological examination of the skin showed a field of multiple dermal cysts with round light eosinophilic formations (14-16µm) containing a central refractable body similar to that reported for Dermocystidium sp. Gills samples were taken from the affected areas for the SEM examination with the purpose of evaluating not only aspects of normal morphology, but also aspects of some modifications of the affected areas as well as the presence of the etiologically incriminated bacteria Pseudomonas fluorescens. The isolates were identified through phenotypic methods. All the strains that showed mobility and oxidase-positivity were tested using API 20 NE strip. Consequently, they were taxonomically grouped into the species Pseudomonas fluorescens. The scanning electron microscope (SEM) was used for the first time in the characterization of the bacterial lesions produced by Pseudomonas strains on Cyprinus carpio and Carassius auratus gibelio gills. The diagnosis of septicemia with conditional pathogen species of Pseudomonas fluorescens was correlated with the results of the physico-chemical investigations of water and the data concerning the breeding conditions of the investigated livestock...

No outono de 2011, em fazendas de ciprinídeos localizadas em Iasi, no rio Jijia, diversas infecções bacterianas e cistos externos macroscópicos na pele se desenvolveram como resultado do estresse induzido por fatores bióticos e abióticos. No exame do conteúdo dos cistos, a presença de diversos esporos foi observada, a maioria do gênero Dermocystidium sp. As amostras foram colhidas das seguintes espécies: carpa comum (Cyprinus carpio) e carpa cruciana (Carassius auratus gibelio) de fazenda piscícola, além do rio Jijia. Assim sendo, 35 peixes foram examinados, todos demonstrando cistos, fragmentação da barbatana dorsal e congestão das guelras. O exame histológico da pele mostrou um campo de múltiplos cistos dérmicos com formações circulares claras eosinofílicas (14-16µm) contendo corpo central refratado similar ao relatado para Dermocystidium sp. Amostras de guelras foram retiradas das áreas afetadas para exame MEV, com o propósito de se avaliar não apenas os aspectos da morfologia normal, mas também os aspectos de algumas modificações das áreas afetadas, além da presença da bactéria etiologicamente incriminada: Pseudomonas fluorescens. Os isolados foram identificados por meio de métodos fenotípicos. Todas as amostras que mostraram mobilidade e positividade-oxidase foram testadas usando-se fita API 20 NE. Consequentemente, estas foram taxonomicamente agrupadas na espécie Pseudomonas fluorescens. O microscópio eletrônico de varredura (MEV) foi usado pela primeira vez na caracterização de lesões bacterianas produzidas por Pseudomonas nas guelras de Cyprinus carpio e Carassius auratus gibelio. O diagnóstico de septicemia com espécies condicionais de patogênico de Pseudomonas fluorescens foi correlacionado com os resultados das investigações físico-químicas da água e de dados sobre as condições de reprodução dos animais investigados...

Characterization and phylogenetic analysis of Magnaporthe spp. strains on various hosts in Iran

Populations of Magnaporthe, the causal agent of rice blast disease, are pathotypically and genetically diverse and therefore their interaction with different rice cultivars and also antagonistic microorganisms are very complicated. The objectives of the present study were to characterize phylogenetic relationships of 114 native Magnaporthe strains, isolated from rice and different weeds in the North region of Iran and to study their interaction with the fungal and bacterial antagonists. Phylogenetic studies [lineage structure, cluster analysis and gene flow] were performed using AFLP DNA fingerprinting. Antagonistic effects of the native fungal [Trichoderma harzianum] and bacterial [Bacillus subtilis and Pseudomonas fluorescens] against Magnaporthe strains were assayed at In vitro levels using factorial experiments based on completely randomized designs [CRD] and mean comparison tests. In total, 39 clonal lineages including 48 haplotypes were identified among the strains of M. grisea and designated here as A-Z. AFLP marker could finely differentiate the strains isolated from various hosts. The strains isolated from Setaria sp. were much close to those from rice [Oryza sativa L.]. Magnaporthe strains isolated from Digitaria sp. showed higher genetic variation than other strains. Genetic distances revealed by the AFLP markers could be finely differentiated M. grisea and M. salvinii. The rate of gene flow was an evidence of low gene transferring among Magnaporthe populations and the existence of a complex species for Magnaporthe strains. The fungal and bacterial antagonists showed different reactions against different Magnaporthe strains. These results confirmed high genetic diversity between the Magnaporthe strains which was also previously determined by the AFLP experiments. It was concluded that the Magnaporthe populations in Iran have a complex genetic diversity, and therefore, to achieve an efficient control of the different strains and pathotypes of Magnaporthe sp, it is necessary to use different bacterial and fungal biocontrol agents as a dynamic and integrated control system

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.

Survival of Escherichia coli o157: h7 co-cultured with different levels of Pseudomonas fluorescens and Lactobacillus plantarum on fresh beef

The purpose of this study was to investigate the effect of different levels of Pseudomonas fluorescens (10² and 10(6)log10 cfu/ml)and Lactobacillus plantarum (10² and 10(4)log10 cfu/ml)on the growth of Escherichia coli O157:H7 on beef loins. Beef loins inoculated with E. coli O157:H7 and P. fluorescens were aerobically stored for 7 days at 4 ºC, while those inoculated with E. coli O157:H7 and L. plantarum were vacuum packaged and stored for 8 weeks at 4 ºC. Aerobic Plate Counts (APC), E. coli O157:H7 and either P. fluorescens or L. plantarum counts were determined at different storage intervals. For the aerobically packaged beef loins, E. coli O157:H7 was detected throughout the 7 day storage period regardless of the P. fluorescens level in the inoculum. For the vacuum packaged beef loins, similar inoculum levels of E. coli O157:H7 and L. plantarum allowed E. coli O157:H7 to survive until week 5 of storage, while a higher inoculum level of L. plantarum inhibited E. coli O157:H7 from week 3. Once fresh beef has been contaminated with E. coli O157:H7, the level of P. fluorescens in the background flora does not inhibit its survival and growth. However, under vacuum storage, the application of L. plantarum as a biopreservative inhibits the survival of E. coli O157:H7 on beef. The higher the level of L. plantarum in the system, the earlier the onset of the inhibition. Farmers and abattoirs have to strengthen preventive strategies to eliminate contamination of beef carcasses with E. coli O157:H7.

Pathogenic variation in isolates of Pseudomonas causing the brown blotch of cultivated mushroom, Agaricus bisporus

Twenty seven bacterial isolates were isolated from superficial brown discolorations on the caps of cultivated Agaricus bisporus. After White Line Assay (WLA) and the assist of Biolog computer-identification system, isolates were divided into groups: (I) comprised ninteen bacterial isolates that positively responded to a Pseudomonas "reactans" reference strain (NCPPB1311) in WLA and were identified as Pseudomonas tolaasii, (II) comprised two isolates which were WLA+ towards the reference strain (JCM21583) of P. tolaasii and were proposed to be P. "reactans". The third group comprised six isolates, two of which weakly responded to the strain of P. tolaasii and were identified as P. gingeri whereas the other four were WLA- and identified as P. fluorescens (three isolates) and P. marginalis (one isolate). Isolates of P. tolaasii showed high aggressiveness compared with those of P. "reactans" in pathogenicity tests. Cubes of 1 cm³ of A. bisporus turned brown and decreased in size when were inoculated with 10 µl of P. tolaasii suspension containing 10(8) CFU ml-1, whereas a similar concentration of P. "reactans" caused only light browning. Fifty µl of the same concentration of P. tolaasii isolates gave typical brown blotch symptoms on fresh mushroom sporophores whereas the two P. "reactans" isolates caused superficial light discoloration only after inoculation with 100 µl of the same concentration. Mixture from both bacterial suspensions increased the brown areas formed on the pileus. This is the first pathogenicity report of P. tolasii and P. "reactans" isolated from cultivated A. bisporus in Egypt.

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.