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Microorganisms, that evolve to acquire resistance to environmental amoeba, are likely to become human pathogens due to the physiological similarities of free-living amoebae to human macrophages. In this sense free-living amoebae can be regarded as nurseries of pathogenic microorganisms. Due to the widespread distribution of amoebae in the environment and their resistance to current disinfection procedures, they may constitute important reservoirs of pathogenic microorganisms. Striking examples are Legionella pneumophila and several mycobacteria including Mycobacterium tuberculosis. This work represents the first attempt to detect and characterize amoebae and their associated microorganisms in Portugal.Detection, isolation and in vitro culture of amoebae from several environmental sources, including lagoon and estuarine water and sediments, were performed according to previously developed methods. Identification of the isolated amoebae was done by optical microscopy based on morphological criteria and by DNA sequence analysis of PCR products amplified with one of the following two sets of primers: EUK and ITS. Transmission electron microscopy (TEM) studies and PCR/sequencing approaches were used to detect and identify amoeba-associated microorganisms (AAMs). Bacterial 16S region was amplified with the primers 616V/630R. TEM studies were performed according to standard procedures. In short, samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.Several species of amoeba (Acanthamoeba lenticulata, A. polyphaga, A. rhyzodes, Platyamoeba oblongata, Saccamoeba limax, Vannella simplex, Vannella sp. and other unidentified amoebae) were found and AAMs were detected both by TEM and PCR/sequencing. The bacteria species Variovorax paradoxus, previously found in association with Saccamoeba and Arcella, were isolated and identified by the PCR/sequencing approach, which also allowed the detection of an unidentified species with about 85% identity with Marivirga tractuosa, a species not yet associated with amoebae. These results point to the existence of AAMs in the environments subjected to this study and the need to evaluate their pathogenic potential. This is an important issue particularly in environments related to human recreational, nutritional and public health activities.

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

Helicobacter pylori is a pathogenic spiral-shaped, microaerophilic, gram-negative bacterium, that inhabits the human stomach. Infection is usually acquired during childhood and always elicits an acute immune response that is, however, inefficient in bacteria clearance. Therefore, in the absence of effective treatment, infection and gastritis (non ulcer dyspepsia, NUD) persist throughout the patient’s life. Depending on its severity and pattern, in about 15% of infected adults, this silent destruction of the gastric mucosa may further progress to peptic ulcer disease (PUD) (gastric and duodenal ulcers, GU and DU respectively) and/or gastric cancer. Infection with H. pylori is also the major cause for the development of paediatric PUD, a rare event that may occur shortly after infection. In addition to the still undisclosed genetic susceptibility of these young patients, the virulence of the implicated H. pylori strain plays a crucial role in the paediatric PUD pathogenesis. Recently, we proved by in vitro infection assays that, compared with paediatric NUD-associated isolates, a group of paediatric ulcerogenic-strains present a greater ability to induce a marked decrease in the gastric cells viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment. Moreover, we showed that their enhanced virulence result from a synergy between the ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp562 virulence factors. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility.We are now characterizing/comparing the cellular and flagella morphologies of H. pylori strains isolated from Portuguese children, associated with DU, GU or NUD, belonging to the vast and multiethnic collection of the Instituto Nacional de Saúde Dr. Ricardo Jorge (Portugal). For that, bacteria were grown in H. pylori selective medium (Biogerm, Maia, Portugal) at 37ºC in a microaerobic environment (Anoxomat®, MART Microbiology BV, Drachten, The Netherlands) for 24 h. For Leifson staining analysis, a drop of each bacterial suspension (in PBS) was spread in cleaned microscope slides, stained with the Leifson dye solution until a golden film developed on the dye surface and a precipitate appeared throughout the sample, and analysed by optical microscopy. For Transmission-Electronic-Microscopy (TEM) studies bacterial pellets were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.Corroborating the better swimming abilities of the PUD strains, as previously shown by motility assays, optical microscopy analysis of Leifson stained slides demonstrated marked differences in the morphology of the studied strains (Figure 1). The H. pylori strain associated with DU (Hp 1152/04) seem longer than all the others and, in contrast, that associated with GU (Hp 499/02) is the shortest one and presents a, more pronounced, spiral morphology. Moreover, our preliminary data on TEM analysis indicate the presence of more abundant and apparently more organized flagella in the GU-associated strain Hp 499/02, in contrast to the NUD control strain, Hp 655/99 (Figure 2).Work supported by Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

Helicobacter pylori is a human pathogen involved in gastritis and gastric cancer whose mode of transmission remains unknown. Association of H. pylori with humans is thought to date from remote antiquity and the bacterium has apparently evolved together with the human host. A few studies have shown the presence of H. pylori in aquatic environments, which might provide a route of transmission of the bacteria to humans. A recent study has also disclosed the association of the bacteria with Acantamoeba castellanii. Amoeba are known to harbor and promote the persistence of several human pathogens in the environment, representing a significant source of contamination in community and hospital acquired infections.

Amoebae graze on bacteria from the environment. In some cases, bacteria acquire resistance to the destructive action of amoebae and survive inside the host. The resistance of microorganisms to amoebae is often accompanied by resistance to the physiologically related human macrophage and displays increased pathogenic potential to humans. The association of bacteria with amoebae, that are ubiquitous in the environment, protects the bacteria from common disinfection measures. This leads to the creation of reservoirs of pathogenic microorganisms that are difficult to eradicate. This process occurs widely in the environment, but is of special concern in hospitals, where many established and emerging pathogens involved in nosocomial disease, such as Legionella pneumophila, are increasingly being identified in association with amoebae. One particularly important amoeba-associated microorganism (AAM) is the newly recognized giant mimivirus, a member of the Nucleo-Cytoplasmic Large Deoxyribovirus (NCLDV) group. The virus has been isolated from hospital sources in France and, together with other members of the NCLDV group, is known from metagenomic studies to be widespread in the Oceans, representing one of the main groups of marine viruses. Our project aims to detect amoeba and potentially pathogenic AAMs with particular relevance to the NCLDVs in the environment and in hospital facilities. In the present work we have developed methods for detection, isolation and in vitro culture of amoebae from several environmental sources including hospital dust, sea water and estuarine sediments.

****, F. Morgado***** and R. Marschang*******Centro de Estudos do Ambiente e do Mar (CESAM) – Faculdade de Ciencias da Universidadede Lisboa, Campo Grande, 1749-016, Lisboa, Portugal** Centro Hospitalar de Lisboa Central - HCC, Anatomia Patologica, R. da Beneficencia 8, 1069-166 Lisboa, Portugal*** Faculdade de Engenharia, Universidade Catolica Portuguesa, Lisboa, Portugal**** Laboratorio de Optica, Lasers e Sistemas, Faculdade de Ciencias da Universidade de Lisboa, 1749-016, Lisboa, Portugal***** CESAM, Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, Portugal****** Institut fur Umwelt und Tierhygiene, Hohenheim University, Garbenstr. 30, 70599 Stuttgart, GermanyHeterotermic vertebrates are hosts of a variety of iridoviruses, many with recognized economical importance for aquaculture and with a role in the decline of amphibian populations, and others with no known disease associations. These include members of the Lymphocystisvirus, Magalocystisvirus and Ranavirus genera [1].Erythrocytic infections of reptiles, amphibians and fish with large icosahedral iridovirus-like viruseswere detected in the beginning of the XXth century by light microscopy, since they produce large cytoplasmic inclusions in the infected cells (Figure 1) [2, 3]. Our work has confirmed the viral nature of the inclusions (Figure 2) and identified 14 new host species worldwide [4, 5]. Our ultrastructural results detected significant morphological variations among the new viruses that suggest that it is a widespread and diverse group of potential related viruses [5]. Recent data on the sequence of a conserved region of the DNA polymerase gene from our work in