Carrageenan from red algae: an application in the development of inhalable tuberculosis therapy targeting the macrophages.

Macrophages have unique surface receptors that might recognize preferentially several moieties present on the surface of infecting organisms, including in the bacterial cell wall. Benefiting from a similar composition regarding the referred moieties, polysaccharides might be good candidates to compose the matrix of drug carriers aimed at macrophage targeting, as they can use the same recognition pathways of the infecting organisms. Carrageenan (CRG), a polysaccharide extracted from red edible seaweed, is an interesting possibility for the approach of directly targeting alveolar macrophages, as its composition is reported to be recognized by several macrophage lectin receptors. Inhalable starch/CRG microparticles were successfully produced, effectively associating isoniazid (96%) and rifabutin (74%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.3 and 3.9 µm. It was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells for the drug-loaded formulation. Starch/CRG microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. This work provides indications on the potential of the starch/CRG carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases. Additionally, if tuberculosis is focused, these microparticles can be used as inhalable drug carriers. Graphical abstractz.

Inhalable Fucoidan Microparticles Combining Two Antitubercular Drugs with Potential Application in Pulmonary Tuberculosis Therapy.

The pulmonary delivery of antitubercular drugs is a promising approach to treat lung tuberculosis. This strategy not only allows targeting the infected organ instantly, it can also reduce the systemic adverse effects of the antibiotics. In light of that, this work aimed at producing fucoidan-based inhalable microparticles that are able to associate a combination of two first-line antitubercular drugs in a single formulation. Fucoidan is a polysaccharide composed of chemical units that have been reported to be specifically recognised by alveolar macrophages (the hosts of Mycobacterium). Inhalable fucoidan microparticles were successfully produced, effectively associating isoniazid (97%) and rifabutin (95%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.6⁻3.9 µm. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells at the highest tested concentration (1 mg/mL). Fucoidan microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. Furthermore, drug-loaded microparticles effectively inhibited mycobacterial growth in vitro. Thus, the produced fucoidan microparticles are considered to hold potential as pulmonary delivery systems for the treatment of tuberculosis.

Antioxidant, Anti-inflammatory and Anti-hyperglycaemic Activities of Essential Oils from Thymbra capitata, Thymus albicans, Thymus caespititius, Thymus carnosus, Thymus lotocephalus and Thymus mastichina from Portugal.

The in viro antioxidant, anti-inflammatory, anti-hyperglycaemic, and anti-acetylcholinesterase activities of the essential oils (EOs) isolated from six Lamiaceae species (Thymbra capitata,. Thymus albicans, Th. caespititius, Th. carnosus, Th. lotocephalus and Th. mastichina) grown in Portugal, were evaluated. Th. caespititius and T. capitata carvacrol/thymol-rich EOs showed the best capacity for preventing lipid peroxidation, and scavenging the 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulphonid acid) (ABTS) and peroxyl free radicals, as well as for inhibiting lipoxygenase and a-amylase. However, Th. caespititius and Th. lotocephalus 1,8-cineole and linalool rich EOs were the best inhibitors of a-glucosidase. T capitata, Th. lotocephalus and Th. albicans EOs were the: most active for inhibiting acetyleholinesterase. Th. caespititius and Th. mastichina EOs were the main scavengers of nitric oxide (NO) radicals. The comparison between the present data with a survey of the existing literature on the in vitro biological activities of the essential oils isolated from the same species from Portuguese origin, using other.methodologies, showed some differences. For instance the use of two oxidizable substrates (egg yolk andlecithin liposomes) led to distinct results mainly for those samples with relatively low activity. In addition, the EOs capacity for scavenging peroxyl radicals-was also influenced by the presence of cyclodextrins, as a synergism seemed to occur between EOs and those carbohydrates.

Volatile-oils composition, and bioactivity of the essential oils of Plectranthus barbatus, P. neochilus, and P. ornatus grown in Portugal.

Volatile-oils chemical composition and bioactivity of the essentail oils from Plectranthus barbatus, P. neochilus, and P. ornatus (Lamiaceae) were assessed. Aerial parts from these three related Plectranthus species were collected from cultivated plants grown in Portugal, during vegetative and flowering phases. Volatiles, isolated by distillation-extraction, were analyzed by GC and GC/MS. Monoterpene hydrocarbons (12-74%) and sesquiterpene hydrocarbons (4-45%) constituted the main fractions in all volatiles. α-Pinene (3; 12-67%), oct-1-en-3-ol (6; traces-28%), ß-pinene (7; 0.1-22%), and ß-caryophyllene (50; 7-12%) dominated P. barbatus volatiles. P. neochilus major volatile components were α-terpenyl acetate (41; traces-48%), α-thujone (2; 2-28%), ß-caryophyllene (50; 2-28%), ß-pinene (7; 1-25%), and α-pinene (3; 1-19%). Oct-1-en-3-ol (6; 13-31%), ß-pinene (7; 11-24%), α-pinene (3; 11-19%), and ß-caryophyllene (50; traces-11%) were the main constituents from P. ornatus volatiles. These chemical compositions were rather different from those previously found for specimens harvested in Africa and Brazil. Moreover, the volatiles from the flowers are herewith reported for the first time. Essential oils, isolated by hydrodistillation from leaves and stems, showed a yellowish color and unpleasant odor, with yields ranging from 0.08% to 0.84% (v/dry weight). Antioxidant and antimicrobial activities of the essential oils were evaluated by DPPH· and TBARS assays, and agar disc-diffusion method, respectively. Results showed low or moderate antioxidant capacity and significant antimicrobial activity against Gram-positive bacteria.

Proteomic analysis shows that individual Listeria monocytogenes strains use different strategies in response to gastric stress.

Ingestion of contaminated dairy products, in particular soft cheese, is one of the major routes of infection by the human pathogen Listeria monocytogenes. During cheese processing, this foodborne pathogen is exposed to sublethal acid and osmotic stress conditions, which may induce tolerance responses and influence subsequent survival in the gastric tract. The aim of the current study was to evaluate the impact on a L. monocytogenes cheese isolate (serotype 4b) and two cheese dairy isolates (T8, serotype 4b, isolated from vat; and A9, serotype 1/2b or 3b, isolated from shelf stand) of exposure to sublethal conditions of pH and salt (5.5 and 3.5% [w/v] NaCl) in a cheese-simulated medium and further challenge with gastric stress. The bacterial cells exposed to pH 7.0 and no added salt were considered non-adapted. Via two-dimensional gel electrophoresis (2-DE), the proteomes of cheese-simulated medium and gastric challenged Listeria cells were compared. All L. monocytogenes isolates were able to survive the high acidity of gastric fluid (pH 2.5), and no significant differences were observed between adapted and non-adapted cells. However, the analysis of the intracellular proteome profiles revealed a significant intra-strain variation in the protein arsenal used to respond to the adaptation in the cheese-based medium and to the gastric stress. In cheese-based medium, the three strains produced different stress proteins. All three strains showed a higher abundance of carbohydrate proteins, but there was no overlap between them. Exposure to the gastric fluid induced the production of a group of proteins in T8 adapted and non-adapted cells that had not been detected previously in the cheese-based proteome. No such response was shown by A9 and C882 strains. Taken together, this study evidences the proteome tools used by adapted and non-adapted cells to cope with the hostile microenvironment of the stomach.

Antimicrobial activity, cytotoxicity and intracellular growth inhibition of Portuguese Thymus essential oils

Thyme essential oils are well recognized by their excellent biological activities and the antimicrobial activity of Portuguese thyme essential oils has been investigated with promising results, particularly against food borne pathogens. In this study the potential antimicrobial activity of the essential oils of five species of Thymus (Lamiaceae), namely Th. caespititius Brot., Th. camphoratus Hoffmanns. & Link, Th. capitellatus Hoffmanns. & Link., Th. carnosus Boiss. and Th. zygis L. was evaluated against Candida albicans, Haemophilus influenza, Helicobacter pylori, Listeria monocytogenes, Salmonella enterica and Streptococcus pneumoniae. H. pylori strains were the most susceptible bacteria, particularly to the essential oils of Th. caespititius (Planalto Central), Th. zygis (Rebordãos) and Th. caespititius (Pico) which minimum inhibitory concentration (MIC) values ranged from 0.05 to 0.08 mg.mL-1. Th. caespititius essential oil from Planalto Central or its main component, carvacrol significantly (p<0.05) inhibited the intracellular growth of H. pylori, and showed no citotoxicity to the gastric cell line. Our results suggest the potential of this essential oil and its main component as a promising tool as anti-Helicobacter agent potentiating the eradication of this important gastroduodenal pathogen.

Characterization and activity studies of highly heavy metal resistant sulphate-reducing bacteria to be used in acid mine drainage decontamination.

Biological treatment with sulphate-reducing bacteria (SRB) has been considered as the most promising alternative for acid mine drainage (AMD) decontamination. Normally, these wastewaters contain high concentrations of sulphate and heavy metals, so the search for SRB highly resistant to metals is extremely important for the development of a bioremediation technology. A SRB consortium resistant to high concentrations of heavy metals (Fe, Cu and Zn), similar to those typically present in AMD, was obtained among several environmental samples, from a wastewater treatment plant. The phylogenetic analysis of the dsr gene sequence revealed that this consortium contains species of SRB affiliated to Desulfovibrio desulfuricans and Desulfobulbus rhabdoformis. The results show that the presence of usually lethal concentrations of Fe (400mg/L), Zn (150 mg/L) and Cu (80 mg/L) is not toxic for the sulphate-reducing bacteria present in this sample. As a consequence, a very good efficiency in terms of sulphate reduction and metals removal was obtained. Both ethanol and lactate can be used by this inoculum as carbon source. With the other samples tested sulphate reduction was inhibited by the presence of copper and zinc. This highly metal resistant consortium will be used to inoculate a bioreactor to carry out AMD decontamination.

Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L. monocytogenes but is absent from many field isolates.

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.

Composition of the essential oils of Thymus and Origanum species from Algeria and their antioxidant and antimicrobial activities.

The composition of the essential oils of Origanum and Thymus species restricted to Algeria and the North Africa region was determined. Antioxidant and antibacterial activities of the isolated essential oils were also determined. The oils of oregano plants were strongly characterized by p-cymene (16.8-24.9%), gamma-terpinene (16.8-24.9%), thymol (8.4-36.0%), and carvacrol (1.1-29.7%), a thymol chemotype for Origanum floribundum and a alpha-terpineol chemotype for Thymus numidicus being described for the first time. The strains of Listeria monocytogenes tested were relatively resistant to the action of essential oils of either Origanum or Thymus species. All essential oils possessed antioxidant activity, but this was dependent on the specific chemical composition and the method employed to determine such activity.