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.

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...

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.

Production of polyhydroxyalkanoate [PHAs] and copolymer [P[HB-co-HV]] by soil bacterial isolates in batch and two-stage batch cultures

NINETY TWO local bacterial isolates, from the rhizosphere and soil around the root system of bean [Viciafaba] grown in Kalubia Governorate in Egypt, were bio-prospected for polyhydroxyalkanoate [PHA] accumulation. Three isolates accumulated >/=20% of PHAs, they were identified as Pseudomonas flu orescens S48, Bacillus megaterium 7A and B. megaterium UBFI9. The tested isolates gave the maximum PHAs content on basal medium containing glucose and ammonium sulfate at C/N ratio of 30/1 after 72 hr at 30°C using shake flask culture technique. Two-stage batch were implemented with varying loading levels of nitrogen and phosphorus, inoculated with washed cells. Nitrogen omission of 70% led to increase the PHAs content by 19%, 3% and 8.5% using washed cells of Ps. fluorescens S48, B. megaterium UBF 19 and Bacillus megaterium 7A, respectively comparing with batch production on the same medium after 72 hr. The Copolymer poly[hydroxybutyrate-co-hydroxyvalerate] [P [HB-co-HV]] content level was increased when valerie/glucose [V/G] was 0.19 mol.mo[-1] after 96 hr being 25.97% and 20.11% by Ps. fluorescens S48 and B. megaterium UBFI9, respectively and reached 23.73% by B. megaterium 7A at propionic/glucose [PIG] of 0.5 mol.mol[-1]. The corresponding highest values of valeric content of copolymer at V/G 3.08 mol.mol[-1] were 63%, 49% and 45%, respectively, comparing with other V/G ratios by using GC analysis . Replacing glucose with 2% corn oil or 1% soybean oil increased the PHAs content of Ps. fluorescens S48 cells to 54.21% and 52.12%, respectively, after 72

The proteolytic activity of Pseudomonas fluorescens 07A isolated from milk is not regulated by quorum sensing signals

The proteolytic activity of Pseudomonas fluorescens 07A was investigated, and was optimal on tryptone-calcium medium. N-acyl-homoserine lactones (AHLs) were not detected on supernatants of late-exponential and stationary-phase culture broths. Synthetic AHLs or bacterial cell extracts added to the medium did not influence growth or proteolytic activity suggesting that quorum sensing might not regulate protease production in this strain.

Supervivencia de Pseudomonas fluorescens en suelos con diferente contenido de materia orgánica / Pseudomonas fluorescens survival in soils with different contents of organic matter

Pseudomonas fluorescens es una bacteria PGPR (plant growth promoting rhizobacteria), heterótrofa, capaz de combatir fitopatógenos edáficos. Su supervivencia podría estar favorecida por el elevado contenido de materia orgánica del suelo (MOS). Para probarlo, se inocularon, en condiciones de laboratorio, tres cepas de P. fluorescens: UP61, C7R12, y P190 (nativa de Balcarce, Buenos Aires) en suelos rizosféricos de tomate representativos de diferentes zonas de Argentina: suelo Argiudol (Balcarce, y Zavalla, Santa Fe) y suelo Torrifluvens (Cipolletti, Río Negro) (MOS %: 7,2; 4,3 y 2,6 respectivamente). Los resultados indicaron que la supervivencia de P. fluorescens en los suelos Argiudoles fue similar; aunque las pendientes de las curvas de supervivencia en el suelo de Zavalla fueron menores que las observadas en el suelo de Balcarce. La producción de CO2 fue superior en el suelo de Balcarce que en el suelo de Zavalla (4,3 y 2,8 mmol.g-1suelo), esta diferencia podría ser explicada por la existencia de una mayor presión competitiva por parte de la microflora nativa. La supervivencia en el suelo Torrifluvens resultó mínima, lo que sería atribuible a su elevada conductividad eléctrica más que al menor contenido de MOS. La cepa UP61 presentó en todos los casos la mejor supervivencia.

Pseudomonas fluorescens are plant growth promoting rhizobacteria (PGPR). The survival of this inoculated heterotrophic bacterium may be affected by soil organic matter content (SOM). To confirm this hypothesis, three strains of P. fluorescens: UP61, C7R12 y P190 (native of Balcarce, Buenos Aires) were inoculated, in laboratory conditions, into three argentine rhizospheric soils: two Argiudolls (Balcarce, and Zavalla, Santa Fe) and a Torrifluvens (Cipolletti, Río Negro) with different SOM: 7,2; 4,3; and 2,6%, respectibily. The results indicated that the all three isolates survival in general was not different. The slopes of the regression curves in Zavalla soil were very similars, while in the Balcarce soil the strains behaviour were very different. CO2 production was superior in the Balcarce than the Zavalla soil. These results suggest that the situations that affected the survival in the Balcarce soil may be associated with the presence of a larger number of functional microflora compared with Zavalla soil. The survival in the Cipolletti soil was the lowest; independently of the protective effect of the SOM in relation with the capability of survival of the inoculated bacteria, the scarcity of survival in this soil, specially after the great fall observed, is not attributable to the low SOM content, it might be related with its high electric conductivity. The UP61 had the best survival rate in all soils.

Role of bacteria in the epizootic ulcerative syndrome (EUS) of fishes.

Bacteriological examination of certain water bodies and fishes carrying EUS was carried out. As a whole, 17 species of bacteria were isolated from the investigated water bodies and EUS affected fishes. The species of bacteria isolated from fishes are common to those isolated from water. Experimental infection trials conducted suggested that Aeromonas hydrophila in association with Pseudomonas fluorescens, may be playing the role of primary aetiological agent in producing EUS in fishes.

Identificación y caracterización de bacilos gramnegativos no fermentados aislados en el medio hospitalario / Identification and characterization of nonfermenting gramnegative bacili isolated from a hospital environment

Se estudio un total de 251 cepas aisladas en el medio hospitalario con diagnóstico presuntivo de bacilos gramnegativos no fermentadores, enviadas por los Laboratorios de Infecciones Nosocomiales de los Centros Provinciales de Higiene y Epidemiología de Ciudad de La Habana, Santiago de Cuba, Holguín, Guantánamo, Camagüey e Isla de la Juventud desde septiembre de 1992 hasta junio de 1993. Se empleó un esquema inicial para corroborar el diagnóstico primario de estas cepas, los medios de producción de pigmentos King A y King B para la identificación de Pseudomonas aeruginosas y pruebas bioquímicas claves para el diagnóstico microbiológico de otros no fermentadores, se utilizó la piocinotipia y la serotipia para la caracterización posterior de la especie aeruginosa. Resultaron confirmadas 238 cepas y de ellas, el 88,23 por ciento correspondió a la especie aeruginosa cuyos piocinotipo y serotipo predominantes fueron al 10a y 011, respectivamente. Otros bacilos encontrados con frecuencia fueron Pseudomonas cepacia (25 por ciento); Pseudomonas fluorescens (17,8 por ciento) y Acinetobacter calcoaceticus var. anitratus 17,8 por ciento

Competiçäo entre Pseudomonas fluorescens modificado geneticamente e a estirpe selvagem em microcosmos de solos subtropicais / Competition between a genetically modified pseudomonas fluorescens and its parent in subtropical soil microcosms

A capacidade de adaptaçäo ecológica da estirpe Pseudomonas fluorescens (Br12), modificada pela inserçäo cromossomal do transposon Tn5::cryIVB, em relaçäo à estirpe senvagem correspondente (Br15) e à microbiota indígena, foi avaliada em dois solos subtropicais com diferentes texturas (fina e grossa), plantados com milho. Ambas estirpes apresentaram crescimento similar quando cultivadas separadamente em meio líquido LB à 28ºC. Quando as estirpes foram cultivadas, misturadas nas mesmas condiçöes, a estirpe Br12 apresentou crescimento desvantajoso em relaçäo à estirpe Br5. Ambas as populaçöes das estirpes introduzidas, em inóculos mistos, resultaram em percentuais gradativamente menores das populaçöes de bactétias heterotróficas totais e de pseudomonadaceas fluorescentes totais, com pequena desvantagem para a estirpe modificada, quando comparada com sua parental selvagem. Entretanto, em solos argilosos a 25ºC, ambas as estirpes, Br12 e Br5, mantiveram proporçöes estáveis durante todo o período experimental em relaçäo às densidades de pseudomonadaceas fluorescentes totais. O efeito da modificaçäo ecológica foi aparentemente menor do que a influência dos efeitos ambientais sobre as populaçöes das estirpes introduzidas em comunidades microbianas indígenas de solo.