Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

This study identified Gram-positive bacteria in three sub-tropical marine fish species: Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warned, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warned were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body. E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.

Weakening of the Gram-negative bacterial outer membrane - A tool for increasing microbiological safety

Gram-negative bacteria are harmful in various surroundings. In the food industy their metabolites are potential cause of spoilage and this group also includes many severe or potential pathogens, such as Salmonella. Due to their ability to produce biofilms Gram-negative bacteria also cause problems in many industrial processes as well as in clinical surroundings. Control of Gram-negative bacteria is hampered by the outer membrane (OM) in the outermost layer of the cells. This layer is an intrinsic barrier for many hydrophobic agents and macromolecules. Permeabilizers are compounds that weaken OM and can thus increase the activity of antimicrobials by facililating entry of hydrophobic compounds and macromolecules into the cell where they can reach their target sites and inhibit or destroy cellular functions. The work described in this thesis shows that lactic acid acts as a permeabilizer and destabilizes the OM of Gram-negative bacteria. In addition, organic acids present in berriers, i.e. malic, sorbic and benzoic acid, were shown to weaken the OM of Gram-negative bacteria. Organic acids can poteniate the antimicrobial activity of other compounds. Microbial colonic degradation products of plant-derived phenolic compounds (3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3-phenylpropionic acid and 3-hydroxyphenylpropionic acid) efficiently destabilized OM of Salmonella. The studies increase our understanding of the mechanism of action of the classical chelator, ethylenediaminetetra-acetic acid (EDTA). In addition, the results indicate that the biocidic activity of benzalkonium chloride against Pseudomonas can be increased by combined use with polyethylenimine (PEI). In addition to PEI, several other potential permeabilizers, such as succimer, were shown to destabilize the OM of Gram-negative bacteria. Furthermore, combination of the results obtained from various permeability assays (e.g. uptake of a hydrophobic probe, sensitization to hydrophobic antibiotics and detergents, release of lipopolysaccharide (LPS) and LPS-specific fatty acids) with atomic force microscopy (AFM) image results increases our knowledge of the action of permeabilizers.

Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

Highly Luminescent Mn-Doped ZnS Nanocrystals: Gram-Scale Synthesis

Following growth doping technique highly luminescent (quantum yield >50%) Mn-doped ZnS nanocrystals are synthesized via colloidal synthetictechnique. The dopant emission has been optimized with varying reaction parameters and found the ratio of Zn and S as well as the percentage of introduced dopant in the reaction mixture are key factors for controlling the intensity. The method is simple, hassle free, and can be scalable to gram level without hindering the quality of nanocrystals. These nanocrystals retain their emission during various ligand exchange processes and aqueous dispersion.

A study of the purification and properties of tryptophan synthetase of Bengal gram (Cicer arietinum)

Active preparations of tryptophan synthetase were obtained from Bengal gram (Cicer arietinum) by the following procedure: (1) precipitation of inactive materials by manganous sulfate, (2) Adsorption of impurities on Alumina Cγ, (3) Adsorption of tryptophan synthetase on tricalcium phosphate gel, removal of inert protein from the gel by treatment with phosphate buffer (pH 7.2), and selective elution of the enzyme by 0.15 M phosphate buffer pH 7.2 containing 10% ammonium sulfate and 10−3 M serine. A 220-fold purification of the enzyme with 44% recovery of the activity was achieved. The pH optimum, effect of temperature, and substrate concentration and other properties of the purified enzyme have been studied in detail. Only the Image -isomer of serine takes part in the reaction. The Km values for indole, Image -serine, and Image -serine were calculated to be 0.66, 4.1, and 8.6 × 10−4 M, respectively. A kinetic study of the inhibition of tryptophan synthetase by indole-propionic acid has shown that it is of a competitive type. It has been demonstrated for the first time that 4-nitro-salicylaldehyde can replace pyridoxal phosphate as a coenzyme for the tryptophan synthetase reaction.

Suite of tools for statistical N-gram language modeling for pattern mining in whole genome sequences

Genome sequences contain a number of patterns that have biomedical significance. Repetitive sequences of various kinds are a primary component of most of the genomic sequence patterns. We extended the suffix-array based Biological Language Modeling Toolkit to compute n-gram frequencies as well as n-gram language-model based perplexity in windows over the whole genome sequence to find biologically relevant patterns. We present the suite of tools and their application for analysis on whole human genome sequence.

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci

The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 x 10(-6)m Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.

Evaluación de dos densidades de siembra y selección negativa como opciones de manejo del complejo mosca blanca begomovirus en el cultivo de tomate (Lycopersicum esculentum Mill) en dos regiones de Nicaragua

Con el objetivo de evaluar el efecto de dos densidades de siembra y selección negativa de plantas, como prácticas de Manejo Integrado de Plagas (MIP) para manejar el complejo mosca blanca (Bemisia tabaci Genn) – Begomovirus, en el cultivo de tomate Lycopersicun esculentum (Mill), se establecieron dos ensayos de campo. Uno en Santa Lucia departamento de Boaco, con semillero MIP y manejo del productor y otro, en el Centro Nacional de Investigación Agropecuaria (CNIA), Managua, con manejo convencional. En las dos localidades se hicieron recuentos de moscas blancas tanto en la fase de semillero como de campo. En las dos regiones, se estableció un diseño en bloques completo al azar, con cuatro parcelas y cuatro repeticiones, se usaron dos densidades con dos testigos. Para ambas localidades se muestreó el número de moscas blancas por planta y a éstos datos se les realizó un análisis de varianza con respecto al tiempo, para determinar el comportamiento de las poblaciones en los tratamientos y en las fechas de recuento. Para el CNIA se realizó selección negativa de plantas con síntomas virales, en los tratamientos tres y cuatro, desde los 23 días después del transplante (DDT); fecha en que aparecieron los primeros síntomas de virosis, hasta los 41 DDT cuando ya habían adquirido el 100% de incidencia. Para el cálculo de severidad viral se uso la escala de propuesta por REDCAHOR y modificada por Rojas en el 2000. Se evaluó el porcentaje de plantas con síntomas virales en las dos densidades, las cuales se compararon mediante una prueba de “t” de Student, también se evaluaron variables de rendimiento como racimos florales, flores y frutos por planta. Los resultados demuestran que en Santa Lucia Boaco, las poblaciones de mosca blanca en semillero, fueron mayores en el cultivo trampa (fríjol) con un promedio de 0.17 moscas por planta, que el encontrado en plantas de tomate con un promedio de 0.13 moscas por planta, donde no se alcanzo el umbral para aplicar químicos. Para el caso del CNIA Managua las poblaciones de mosca blanca por planta si alcanzaron el umbral de aplicación de químicos de 0.2 moscas por planta a los cuatro días después de la vi germinación (DDG), sin embargo no se aplicó. Para el caso de Santa Lucia Boaco las poblaciones de mosca blanca en campo, únicamente se encontró diferencia en cuanto a las fechas, pero para las densidades fue igual. Similares resultados se obtuvieron en el CNIA Managua donde las poblaciones de mosca blan ca fueron estadísticamente igual para las densidades. El comportamiento de la virosis en Santa Lucia Boaco tardó mas en alcanzar un 100%, el cual se logró a los 29 días después de aparecer los primeros síntomas, pero la enfermedad provocó mayor daño que en el CNIA Managua, donde se alcanzo un 100% de incidencia 18 días después de aparecer los primeros síntomas, pero en este caso la enfermedad no fue muy severa ya que algunas plantas produjeron alcanzándose un promedio de 0.8 frutos por planta, aunque en términos comerciales esto significa 100% de perdida. El aumento de densidades y selección negativa, no tuvo efecto sobre la incidencia y severidad de virosis en tomate.

Tratado de pazes, entre os serenissimos e poderosissimos Principes D. Carlos II, Rey Catholico, e D. Afonso VI, Rey de Portugal, feito, e concluso no Convento de Sancto Eloy da Cidade de Lisboa, aos 13 de fevereiro de 1668 : sendo mediator o serenissimo, e poderosissimo Principe Carlos II, Rey da Gram Bretanha

Analisa esse tratado que põe fim às guerras da Restauração de Portugal, mandando devolver a cada uma das partes as conquistas que à outra tiverem feito. Nas colônias, a paz passaria a vigorar um ano após a sua publicação, dado o tempo necessário para que a notícia chegasse à América e à Ásia.

Tratado definitivo de paz, e união entre os serenissimos, e potentissimos Principes D. Joseph I. Rey Fidelissimo de Portugal, e dos Algarves, Jorge III, Rey da Gram Bretanha, de huma parte, Luiz XV, Rey Christianissimo de França, e D. Carlos III, Rey Catholico de Hespanha, da outra parte assignado em Pariz a dez de fevereiro de mil setecentos sessenta e tres :como plenos poderes, e ratificaçoens dos quatro monarcas contratantes, ajustando-se os actos que se passarão no dia 9 de março do mesmo anno, em que as ditas ratificações forão trocadas na mesma Corte de Pariz

Na Europa Central, onde ocorreram as principais batalhas terrestres, chamou-se Guerra dos Sete Anos; na Inglaterra, que foi a principal vencedora, chamou-se A Grande Guerra do Império; Nos Estados Unidos e no Canadá, que passou a ser inglês ao final dos combates, chamou-se de Guerra Franco-Índia.