Formulação de um biofilme para controlo da listéria em queijos

Dissertação de Mestrado, Tecnologia e Segurança Alimentar, 12 Fevereiro de 2016, Universidade dos Açores.

Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts

Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut.

Secretory IgA (SIgA) serves as the first line of defense in protecting the intestinal epithelium from enteric toxins and pathogenic microorganisms. Through a process known as immune exclusion, SIgA promotes the clearance of antigens and pathogenic microorganisms from the intestinal lumen by blocking their access to epithelial receptors, entrapping them in mucus, and facilitating their removal by peristaltic and mucociliary activities. In addition, SIgA functions in mucosal immunity and intestinal homeostasis through mechanisms that have only recently been revealed. In just the past several years, SIgA has been identified as having the capacity to directly quench bacterial virulence factors, influence composition of the intestinal microbiota by Fab-dependent and Fab-independent mechanisms, promote retro-transport of antigens across the intestinal epithelium to dendritic cell subsets in gut-associated lymphoid tissue, and, finally, to downregulate proinflammatory responses normally associated with the uptake of highly pathogenic bacteria and potentially allergenic antigens. This review summarizes the intrinsic biological activities now associated with SIgA and their relationships with immunity and intestinal homeostasis.

Ecotoxicological Assessment of Antifouling Biocides in the Sediments of Cochin Estuarine System

The pollutants discharged into the estuaries are originate from two main sources-industrial and sewage. The former may be toxic which includes heavy metals, residues from antifouling paint particles and pesticides, while large discharges of sewage will contain pathogenic microorganisms. The contamination is enough to destroy the amenities of the waterfront, and the toxic substances may completely destroy the marine life and damage to birds, fishes and other marine organisms. Antifouling biocides are a type of chemical used in marine structure to prevent biofouling. These antifouling biocides gradually leach from the ships and other marine structures into water and finally settled in sediments. Once a saturation adsorption is reached they desorbed into overlying water and causes threat to marine organisms. Previous reports explained the imposex and shell thickening in bivalves owing to the effect of biocides. So bivalves are used as indicator organisms to understand the status of pollution. The nervous system is one of the best body part to understand the effect of toxicant. Acetylcholine esterase enzyme which is the main neurotransmitter in nervous was used to understand the effect of pollutants. Present study uses Acetylcholine esterase enzyme as pollution monitoring indicator

Influence of a Salt Water Regulator on the Survival Response of Salmonella Paratyphi in Vembanadu Lake: India

Contamination of environmental water by pathogenic microorganisms and subsequent infections originated from such sources during different contact and non- contact recreational activities are a major public health problem worldwide particularly in developing countries. The main pathogen frequently associated with enteric infection in developing countries are Salmonella enterica serovar typhi and paratyphi. Although the natural habitat of Salmonella is the gastrointestinal tract of animals, it find its way into natural water through faecal contamination and are frequently identified from various aquatic environments (Baudart et al., 2000; Dionisio et al., 2000; Martinez -Urtaza et al., 2004., Abhirosh et al., 2008). Typhoid fever caused by S. enterica serotype typhi and paratyphi are a common infectious disease occurring in all the parts of the world with its highest endemicity in certain parts of Asia, Africa, Latin America and in the Indian subcontinent with an estimated incidence of 33 million cases each year with significant morbidity and mortality (Threlfall, 2002). In most cases the disease is transmitted by polluted water (Girard et al., 2006) because of the poor hygienic conditions, inadequate clean water supplies and sewage treatment facilities. However in developed countries the disease is mainly associated with food (Bell et al., 2002) especially shellfish (Heinitz et al., 2000

Characterization of Bacteriocins from Bacillus licheniformis strain BTHT8 and Bacillus subtilis strain BTFK101 isolated from marine sediment

Pathogenic microorganisms such as Bacillus cereus, Listeria Monocytogenes and Staphylococcus sp have caused serious diseases, and consequently contributed to considerable economic loss in the food and agricultural industries. Antibiotics have been practically used to treat these pathogens since penicillin G was discovered more than half a century ago. Many different types of antibiotics have been discovered or synthesized to control pathogenic microorganisms. Repetitive use and misuse of antibiotics by the agricultural and pharmaceutical industries have caused the emergence of multidrug-resistant microorganisms, even to the strongest antibiotics currently available; therefore, the rapid development of more effective antimicrobial compounds is required to keep pace with demand. Bacteria were isolated from marine water and sediment samples collected from various locations off the coast of Cochin and salt pans of Tuticorin using pour plate technique. One hundred and twelve isolates were obtained. Seventeen isolates exhibiting antimicrobial activity were segregated after primary screening. The secondary screening which was aimed at selection of bacteria that produce proteinaceous inhibitory compounds, helped to select five strains viz. BTFK101, BTHT8, BTKM4, BTEK16 and BTSB22. The five isolates inhibited the growth of six Gram positive test organisms viz. B. cereus, B. circulans, B. coagulans, B. pumilus, Staphylococcus aureus and Clostridium perfringens. After quantitative estimation of the bacteriocin production, the two strains BTFK101 and BTHT8 were selected for further study.

Synthesis and evaluation of cyclic cationic peptides as antimicrobial agents for use in plant protection

Aquesta tesi doctoral se centra en l'estudi de l'aplicació de pèptids antimicrobians en la lluita contra agents patògens de cultius de plantes d'interès econòmic.L'estratègia sintètica s'ha portat a terme utilitzant metodologies convencionals de síntesi de pèptids en fase sòlida com l'estratègia tridimensional ortogonal Fmoc/tBut/Allyl. Ha calgut fer la recerca de les condicions òptimes per a l'eliminació del grup Allyl i la ciclació. D'entre els pèptids cíclics de 4-10 aminoacids sintetitzats, el decapèptid c(Lys-Leu-Lys-Leu-Lys-Phe-Lys-Lys-Leu-Gln) ha resultat ésser el més efectiu i s'ha pres com a base per al disseny d'una quimioteca de 56 pèptids. Dels resultats obtinguts s'ha sintetitzat una segona quimioteca basada en l'estructura general c(X1-X2-X3-X4-Lys-Phe-Lys-Lys-Leu-Gln) determinada com la que posseix el millor perfil d'activitat. Els pèptids més efectius obtinguts constituixen els primers exemples de pèptids cíclics actius contra E. amylovora i poden ser considerats com a bons candidats pel desenvolupament d'agents antimicrobians efectius en protecció vegetal.

Eukaryotic ectosymbionts of Acari

The Acari is the most numerous and diverse group of the subphylum Chelicerata. With approximately 55 000 described species (and estimates of up to 1 million extant species), their adaptations for parasitism, phytophagy, mycophagy, saprophagy and predation rival other arthropods and challenge us with a wide variety of biological interactions. While a few studies have unravelled the nature of some endosymbiotic associations between mites or ticks and prokaryotes, almost nothing has been done yet regarding acarine eukaryotic ectosymbionts. Microbial ectosymbionts can benefit their hosts by providing nutrients, by aiding digestion, by enhancing communication, by assisting in mating and/or fertilization, by protecting their host against pathogenic microorganisms, against predation and so on. In this sketch, we introduce a number of described cases of fungal and protist ectosymbionts and discuss the role they might play in the life of their acarine hosts.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

Effects of phytophthora cinnamomi on heathland flora and fauna of the Eastern Otway Ranges

The plant pathogen, Phytophthora dnnamomi, is a cause of dieback disease observed in sclerophyll vegetation in Australia, The effects of P. dnnamomi on flora and fauna were studied at two locations in heathland vegetation near the coastal town of Anglesea, Victoria. The pathogen was isolated from soils beneath diseased heathland plants. The extent of diseased vegetation was assessed by the presence and absence of highly sensitive indicator species, Xanthorrhoea australis and hopogon ceratophyllus. The characteristics of heathland vegetation exhibiting dieback disease associated with the presence of P. dnnamomi were investigated. Plant species richness was similar between diseased and non-diseased areas however diseased areas were characterised by significant declines in the cover and frequency of susceptible species, increases in resistant species and increases in percent cover of open ground. Compared to non-diseased areas, diseased areas exhibited fewer shrub species and decreased shrub cover. The percentage cover and number of species of sedges, lilies and grasses were higher in diseased areas. Structural differences were significant between 0-0.6 m with decreased cover of vegetation in diseased areas. Differences in structure between diseased and non-diseased areas were not as great as expected due to increases in the cover of resistant species. A number of regenerating X australis were observed in post-disease areas. Cluster analysis of floristic data could clearly separate diseased and non-diseased trap stations. The population dynamics and habitat use of eight small mammal species present were compared in diseased and non-diseased areas using trapping and radio-tracking techniques. The number of small mammal species captured in post-disease areas was significantly lower than non-diseased areas. Mean captures of Antechinus stuartii and Rattus fiisdpes were significantly lower in diseased areas on Grid B. Mean captures of Rattus lutreolus were significantly lower in diseased areas on both study grids. Significant differences were not observed in every season over the two year study period. Radio tracking revealed more observations of Sminthopsis leucopus in non-diseased vegetation than in diseased. Cercartetus nanus was frequently observed to utilise the disease susceptible X. australis for nesting. At one location, the recovery of vegetation and small mammal communities in non-diseased and diseased vegetation after fuel reduction burning was monitored for three years post-fire. Return of plant species after fire in both disease classes were similar, reaching 75% of pre-fire richness after three years. Vegetation cover was slower to return after fire in diseased areas. Of the seven small mammal species captured pre-fire, five were regularly captured in the three years after fire. General linear model analysis revealed a significant influence of disease on capture rates for total small mammals before fire and a significant influence of fire on capture rates for total small mammals after fire. After three years, the influence of fire on capture rates was reduced no significant difference was detected between disease classes. Measurements of microclimate indicate that diseased, burnt heathland was likely to experience greater extremes of temperature and wind speed. Seeding of diseased heathland with X. australis resulted in the establishment of seedlings of this sensitive species. The reported distributions of the mamma] species in Victoria were analysed to determine which species were associated with the reported distribution of dieback disease. Twenty-two species have more than 20% of their known distribution in diseased areas. Five of these species, Pseudomys novaehollandiae, Pseudomys fumeust Pseudomys shortridgei, Potorous longipes and Petrogale pencillata are rare or endangered in Victoria. Four of the twenty-two species, Sminthopsis leucopus, Isoodon obesulus, Cercartetus nanus and Rottus lutreolus am observed in Victorian heathlands. Phytophthora cinnamomi changes both the structure and floristics of heathland vegetation in the eastern Qtway Ranges. Small mammals respond to these changes through decreased utilisation of diseased heathland. The pathogen threatens the diversity of species present and future research efforts should be directed towards limiting its spread and rehabilitating diseased areas.

Use of phosphite as a control for Phytophthora cinnamomi in southeastern Victorian vegetation communities

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

Aspects of the interaction between Xanthorrhoea australis and Phytophthora cinnamomi in south-western Victoria, Australia

Diseases in natural ecosystems are often assumed to be less severe than those observed in domestic cropping systems due to the extensive biodiversity exhibited in wild vegetation communities. In Australia, it is this natural biodiversity that is now under threat from Phytophthora cinnamomi. The soilborne Oomycete causes severe decline of native vegetation communities in south-western Victoria, Australia, disrupting the ecological balance of native forest and heathland communities. While the effect of disease caused by P. cinnamomi on native vegetation communities in Victoria has been extensively investigated, little work has focused on the Anglesea healthlands in south-western Victoria. Nothing is known about the population structure of P. cinnamomi at Anglesea. This project was divided into two main components to investigate fundamental issues affecting the management of P. cinnamomi in the Anglesea heathlands. The first component examined the phenotypic characteristics of P. cinnamomi isolates sampled from the population at Anglesea, and compared these with isolates from other regions in Victoria, and also from Western Australia. The second component of the project investigated the effect of the fungicide phosphonate on the host response following infection by P. cinnamomi. Following soil sampling in the Anglesea heathlands, a collection of P, cinnamomi isolates was established. Morphological and physiological traits of each isolate were examined. All isolates were found to be of the A2 mating type. Variation was demonstrated among isolates in the following characteristics: radial growth rate on various nutrient media, sporangial production, and sporangial dimensions. Oogonial dimensions did not differ significantly between isolates. Morphological and physiological variation was rarely dependant on isolate origin. To examine the genetic diversity among isolates and to determine whether phenotypic variation observed was genetically based, Random Amplified Polymorphic DNA (RAPD) analyses were conducted. No significant variation was observed among isolates based on an analysis of molecular variance (AMQVA). The results are discussed in relation to population biology, and the effect of genetic variation on population structure and population dynamics. X australis, an arborescent monocotyledon indigenous to Australia, is highly susceptible to infection by P. cinnamomi. It forms an important component of the heathland vegetation community, providing habitat for native flora and fauna, A cell suspension culture system was developed to investigate the effect of the fungicide phosphonate on the host-pathogen interaction between X. australis and P. cinnamomi. This allowed the interaction between the host and the pathogen to be examined at a cellular level. Subsequently, histological studies using X. australis seedlings were undertaken to support the cellular study. Observations in the cell culture system correlated well with those in the plant. The anatomical structure of X australis roots was examined to assist in the interpretation of results of histopathological studies. The infection of single cells and roots of X. australis, and the effect of phosphonate on the interaction are described. Phosphonate application prior to inoculation with P. cinnamomi reduced the infection of cells in culture and of cells in planta. In particular, phosphonate was found to stimulate the production of phenolic material in roots of X australis seedlings and in cells in suspension cultures. In phosphonate-treated roots of X australis seedlings, the deposition of electron dense material, possibly lignin or cellulose, was observed following infection with P. cinnamomi. It is proposed that this is a significant consequence of the stimulation of plant defence pathways by the fungicide. Results of the study are discussed in terms of the implications of the findings on management of the Anglesea heathlands in Victoria, taking into account variation in pathogen morphology, pathogenicity and genotype. The mode of action of phosphonate in the plant is discussed in relation to plant physiology and biochemistry.

Effects of Phytophthora cinnamomi on heathland fauna and ecosystem function

Progression of disease caused by the plant pathogen Phytophthora cinnamomi was correlated to rainfall events and resulted in a loss of plant species diversity in heathland vegetation at Anglesea, Victoria. Lower captures of small mammals were recorded in diseased areas. Management of disease using the chemical phosphite was also evaluated.

Profiling secondary metabolites of plant roots inoculated with Phytophthora cinnamomi

The soil-borne pathogen, Phytophthora cinnamomi, continues to cause severe dieback in Australian native forest species and is of great international significance due to its global distribution. This research established a protocol to successfully identify phyto-chemicals associated with the defense response of plants challenged by the disease caused by Phytophthora cinnamomi.