A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far

Infraspecific variability in the essential oil composition of Lychnophora ericoides

Variations in the composition of the leaf essential oils of wild Lychnophora ericoides, with and without scent, which were grown in three sampling sites, were examined by GC-MS. Results were submitted to principal component and cluster analysis which allowed for two groups of essential oils to be distinguished with regard to scent: cluster I with specimens exhibiting an aromatic scent and containing a high percentage of α-bisabolol (34 ± 23%) and o-cymene (8.4 ± 6.9%); cluster II with specimens without any scent and characterised by a high percentage of caryophyllene oxide (11 ± 9%) and δ-elemene (5.7 ± 6.9%). The two types of oil coexist in all the populations under study, although patterns of aromatic samples deriving from Caldas Novas' State Park reveal chemical differences in relation to the samples from Brasília's National Park and Santo Antônio do Descoberto. Such chemical variations clearly suggest, apart from a geographical influence, genetic differences between individuals in the populations. The high content of α-bisabolol supports the folk medicinal use of arnica as anti-inflammatory

Design of a lipid nanovesicle system encapsulating bacteriophages integrated in a multiple emulsion formulation: a proof-of-concept

Development of a biotechnological process for the inhalational administration of a bacteriophage was pursued, using strategies of nanoencapsulation within lipid nanovesicles. As a proof-of-concept for the nanoencapsulation strategy, a bacteriophage with broad lytic spectrum was entrapped within W/O/W multiple nanoemulsions. Physicochemical characterization of the optimized bacteriophage-encasing nanovesicles encompassed determination of particle hydrodynamic size, size distribution and particle charge via DLS, surface morphology via CRYO-SEM, and thermal analysis via DSC, whereas antimicrobial activity of the nanoemulsions produced was assessed in vitro using several bacterial strains. The optimized nanosystems showed no phase separation and encompassed nanovesicles with an average size of ca. 114 nm and an average Zeta Potential of ca. -13 mV, which were maintained stable over a storage timeframe of ca. 3 months.(undefined

Design of a lipid nanovesicle system encasing bacteriophages for inhalational therapy : a proof-of-concept

Inflammatory diseases that occur in the pharynx and involving both the adenoids and tonsils are important not only for being very frequent, but also because they often require minor surgery for their resolution. These structures have immunological functions leading to production of antibodies, and work in the local immunity of the pharynx and protection of the entire body. The most common etiologic agent of sore throats is Streptococcus pyogenes, an important pathogen of the beta-hemolytic group A which causes streptococcal pharyngitis. The emergence of antibiotic-resistant bacterial strains and the poor penetration of chemical antibiotics in bacterial biofilms raise the need for safe and effective options of antimicrobial treatment. The application of bacteriophages (or cocktails therefrom) has been proposed as an alternative (or complement) to conventional chemical antibiotics, allowing the release of natural predators of bacteria directly on these biofilms. The major advantage of bacteriophage-based antibiotherapy relative to its conventional chemical counterpart is that bacteriophages replicate at the site of infection, being available in abundance where they are needed the most. When compared with chemical antibiotics, bacteriophages have other important advantages: (i) strong tissue permeability, (ii) bacteriophage concentration remains high at the focus of infection, continuously increasing with bacterial (host) presence, (iii) elimination of the focus of infection occurs only after eradication of the host bacterium, (iv) bacteriophages are fully compatible with antibiotics and may act synergistically, (v) they are specific against the target bacteria, (vi) have a superior ability to penetrate bacterial biofilms, inducing production of enzymes that hydrolyze the biofilm polymeric matrix, (vii) although bacteria can develop resistance to bacteriophages, isolation of new lytic bacteriophages is much simpler and cheaper than developing a new chemical antibiotic. In this research effort, development of a biotechnological process for the inhalational administration of a bacteriophage cocktail (endotoxin free) was pursued, using strategies of nanoencapsulation within lipid nanovesicles (as forms of protection for the bacteriophage against the immune system) to treat infectious pathologies such as pharyngo-tonsillitis caused by Streptococcus pyogenes. This method of targeting may have a high potential for the treatment of bacterial infections of the respiratory tract, since inhalation therapy is considered to be favorable to certain respiratory infections because the aerosol is delivered directly at the site of infection, accelerating the action of bacterial predators. Additionally, a smaller amount of bioactive substance is needed, thus preventing or reducing possible side effects. As a proof of concept for the nanoencapsulation strategy, and since there is not yet available a strictly lytic bacteriophage cocktail for Streptococcus pyogenes, a well-defined and characterized bacteriophage was utilized, viz. bacteriophage T4. Water-in-oil-in-water (W/O/W) multiple emulsions are nanosystems in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase. Due to their compartimentalized internal structure, multiple emulsions present important advantages over simple O/W emulsions for encapsulation of biomolecules, such as the ability to carry both polar and non-polar molecules, and a better control over releasing of therapeutic molecules. T4 bacteriophage was entrapped within W/O/W multiple nanoemulsions, aiming at mimicking the multifunctional design of biology, optimized with several lipid matrices, poloxamers and stabilizing layer compositions. Physicochemical characterization of the optimized bacteriophage-encasing nanovesicle formulations encompassed determination of particle size, size distribution and particle charge, via Zeta potential analysis, surface morphology via CRYO-SEM, and thermal analysis via DSC, whereas antimicrobial activity of the nanoemulsions produced were evaluated via the “spot-test” using appropriate bacterial cultures

Adsorção de atrazina, desetilatrazina e hidroxiatrazina em latossolo vermelho escuro sob cerrado e sob plantio direto no Distrito Federal

The environmental fate of a pesticide depends on many factors, among them the soil characteristics as mineralogy, soil chemistry and organic matter content. This study aims to verify how the adaptation of chemical and mineralogical properties of a dark red latosol from the Brazilian savanna under the continuous use of no till practice can affect its affinity to atrazine and two metabolites (deethylatrazine and hydroxyatrazine). Therefore, beyond the characterization of the cited parameters, batch studies were performed with both no till and native vegetation soils. The comparison between them showed that the changes in some soil properties due to agriculture use of it were enough to change significantly its affinity for the studied compounds. Atrazine and deethylatrazine showed significant affinity to the high organic matter content horizon, while deeper horizons with smaller amounts of organic matter atrazine sorption was neglectible, deethylatrazine was present, but in smaller amounts.        O destino de um agrotóxico no meio ambiente depende de vários fatores, entre estes as alguns atributos do solo como mineralogia, composição química e conteúdo de matéria orgânica. Este estudo teve como objetivos verificar de que forma a adaptação de algumas das características de um Latossolo Vermelho Escuro do cerrado ao uso continuado do plantio direto (PD) influencia a retenção da atrazina e dois de seus principiais metabólitos (desetilatrazina e hidroxiatrazina). Desta forma, além da caracterização do solo, desenvolveram-se estudos da interação do solo com a atrazina, tanto com solos utilizados para PD, quanto solos de mata nativa nunca usados na prática agrícola. A comparação entre ambos evidenciou que as mudanças ocasionadas pelo solo agrícola, embora pequenas, foram suficientes para causar ligeira modificação na afinidade deste pelos compostos estudados. Atrazina e desetilatrazina apresentaram maior afinidade com os horizontes mais ricos em matéria orgânica, enquanto que os horizontes mais profundos, mais pobres em matéria orgânica, a adsorção da atrazina foi desprazível, desetilatrazina esta presente, porém em quantidades pequenas

Development of an inhalational therapeutical system based on bacteriophages to treat pharyngo-tonsillitis : a nanoencapsulation approach

Inflammatory diseases that occur in the pharynx and involving both the adenoids and tonsils are important not only for being very frequent, but also because they often require minor surgery for their resolution. These structures have immunological functions leading to production of antibodies, and work in the local immunity of the pharynx and protection of the entire body. The most common etiologic agent of sore throats is Streptococcus pyogenes, an important pathogen of the beta-hemolytic group A which causes streptococcal pharyngitis. The emergence of antibiotic-resistant bacterial strains and the poor penetration of chemical antibiotics in bacterial biofilms raise the need for safe and effective options of antimicrobial treatment. The application of bacteriophages (or cocktails therefrom) has been proposed as an alternative (or complement) to conventional chemical antibiotics, allowing the release of natural predators of bacteria directly on these biofilms. The major advantage of bacteriophage-based antibiotherapy relative to its conventional chemical counterpart is that bacteriophages replicate at the site of infection, being available in abundance where they are needed the most. When compared with chemical antibiotics, bacteriophages have other important advantages: (i) strong tissue permeability, (ii) bacteriophage concentration remains high at the focus of infection, continuously increasing with bacterial (host) presence, (iii) elimination of the focus of infection occurs only after eradication of the host bacterium, (iv) bacteriophages are fully compatible with antibiotics and may act synergistically, (v) they are specific against the target bacteria, (vi) have a superior ability to penetrate bacterial biofilms, inducing production of enzymes that hydrolyze the biofilm polymeric matrix, (vii) although bacteria can develop resistance to bacteriophages, isolation of new lytic bacteriophages is much simpler and cheaper than developing a new chemical antibiotic. In this research effort, development of a biotechnological process for the inhalational administration of a bacteriophage cocktail (endotoxin free) was pursued, using strategies of nanoencapsulation within lipid nanovesicles (as forms of protection for the bacteriophage against the immune system) to treat infectious pathologies such as pharyngo-tonsillitis caused by Streptococcus pyogenes. This method of targeting may have a high potential for the treatment of bacterial infections of the respiratory tract, since inhalation therapy is considered to be favorable to certain respiratory infections because the aerosol is delivered directly at the site of infection, accelerating the action of bacterial predators. Additionally, a smaller amount of bioactive substance is needed, thus preventing or reducing possible side effects. As a proof of concept for the nanoencapsulation strategy, and since there is not yet available a strictly lytic bacteriophage cocktail for Streptococcus pyogenes, a well-defined and characterized bacteriophage was utilized, viz. bacteriophage T4. Water-in-oil-in-water (W/O/W) multiple emulsions are nanosystems in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase. Due to their compartimentalized internal structure, multiple emulsions present important advantages over simple O/W emulsions for encapsulation of biomolecules, such as the ability to carry both polar and non-polar molecules, and a better control over releasing of therapeutic molecules. T4 bacteriophage was entrapped within W/O/W multiple nanoemulsions, aiming at mimicking the multifunctional design of biology, optimized with several lipid matrices, poloxamers and stabilizing layer compositions. Physicochemical characterization of the optimized bacteriophageencasing nanovesicle formulations encompassed determination of particle size, size distribution and particle charge, via Zeta potential analysis, surface morphology via CRYO-SEM, and thermal analysis via DSC

Safety issues concerning phage therapy for veterinary applications

Phages have been proposed as natural antimicrobial agents to fight bacterial infections in animals. This is supported by several scientific evidences of the efficacy of phage therapy for veterinary applications. However, there are important safety issues that should be taken into consideration when developing a phage product for veterinary applications. For example it is of utmost importance to guarantee that phages are non‐temperate and do not encode bacterial toxins. The phage administration strategy and timing should also be adequate in order to reduce the development of phage‐resistant mutants. The present work describes the main strategies used to ensure a safe phage product for veterinary application, based on the results obtained on the scope of the European Project Phagevet‐P (Veterinary Phage Therapies as Alternatives to Antibiotics in Poultry Production FP6‐2003‐Food‐2‐A:007224)