Recent works has highlighted the contribution of microbes to animal function. In this regard, the microbial community associated with corals has become a growing field of research in order to understand how microbes contribute to the host organisms’ response to environmental changes. It has been shown that microbes associated with corals have important functions in the coral holobiont such as immunity and nutrient assimilation. However, corals are notoriously difficult to work with. To this end, the sea anemone Aiptasia is becoming a model organism for coral symbiosis. Given the importance of host-­microbiome interactions, the topic of this thesis is to assess microbial structure of Aiptasia, culture prominent bacterial members, and compare bacterial community structure to corals. Different molecular methods have been applied using 16S rRNA bacterial gene fragments to characterize the microbial composition of Aiptasia. 16S rRNA gene sequence derived from cultured bacteria was compared to 16S rRNA gene sequences retrieved from native Red Sea Aiptasia. Inter-­individual as well as methodological differences were found to account for variance in microbiome composition. However, all approaches showed a highly abundant microbial taxon belonging to the genus Alteromonas in all samples. The Alteromonas species was successfully isolated for further research targeting microbiome selection mechanisms in Aiptasia. Future investigations by using different molecular tools will help to define the functions and relationship between the Aiptasia and its complex microbiome.

Since the soils nutrient composition along with the associated biotic and abiotic factors direct the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies have been focused on sediment types with unique features characteristic of extreme environments. Red Sea lagoon ecosystems are environments with such unique features as they are highly saline. However, not much is known about the potential of their microbiomes to produce bioactive compounds. Here, we explored sediment types such as mangrove mud, microbial mat, and barren soil collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL) as sources for antibiotic bioprospecting. Our antibiotic bioprospecting process started with a metagenomic study that provides a more precise view of the microbial community inhabiting these sites and serves as a preliminary screen for potential antibiotics. Taking the outcomes of the metagenomic screening into account, the next step we established a library of culturable strains from the analyzed samples. We screened each strain from that library for antibiotic activity against four target strains (Staphylococcus aureus ATCC 25923, Escherichia coli dh5 α, Pseudomonas syringae pv. tomato dc3000 and Salmonella typhimurium dt2) and for the presence of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes known to support synthesis of secondary metabolites that act like antimicrobial agents. The metagenomics study showed a shift in dominant phyla consistent with a historical exposure to hydrocarbon contamination and that AKL unexpectedly displayed more contamination than RHL. This may be due to dominant phyla in AKL being consistent with early hydrocarbon exposure (when contamination levels are still high) and the dominant phyla in RHL being consistent with late hydrocarbon exposure (when contamination levels are lower as a result of an extended period of hydrocarbon degradation). Additionally, RHL samples showed a higher percentage of enzymes associated with antibiotic synthesis, PKS and NRPS. When considering sediment type, mangrove mud samples showed a higher percentage of enzymes associated with antibiotic synthesis than microbial mat samples. Taken together, RHL was shown to be the better location with an increased probability of successful antibiotic bioprospecting, while the best sediment type in RHL for this purpose is microbial mat. Moreover, the phylum Actinobacteria tends to be the common target for research when it comes to antibiotic bioprospecting. However this culture-independent metagenomic study suggests the tremendous potential of Proteobacteria, Bacteroidetes, Cyanobacteria and Firmicutes for this purpose. Taking into account that the metagenomic screen suggests other phyla beyond Actinobacteria for antibiotic bioprospecting, the culture-dependent experiments were not designed to target actinobacteria alone. A total of 251 bacterial strains were isolated from the three collected sediments. Phylogenetic characterization of 251 bacterial isolates, based on 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity of ≤ 98 % to other strain sequences in the NCBI database. We demonstrate that 52 of the 251 isolates exhibit the potential to produce an antimicrobial effect. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 52 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards all target strains. In conclusion, this study demonstrated the significant microbial diversity associated with Red Sea harbor/lagoon systems and their potential to produce antimicrobial compounds and novel secondary metabolites. To the best of our knowledge, this is the first study that has analyzed the microbiomes in Red Sea lagoons for antibiotic bioprospecting.

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-06T13:04:06Z No. of bitstreams: 1 arquivototal.pdf: 1188137 bytes, checksum: 010a2962d49aa738285e5f27b77c15b3 (MD5) / Made available in DSpace on 2017-09-06T13:04:06Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1188137 bytes, checksum: 010a2962d49aa738285e5f27b77c15b3 (MD5) Previous issue date: 2015-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The microbial communities play a fundamental role in the health of coral and their changes can lead to the onset of disease. Many diseases affect coral reefs in several parts of the world, however, little is known about the pathogenic agents and the factors that trigger the pathological process. In coastal reefs of Carapibus Beach of Conde (Paraiba state, Brazil), a tissue necrosis affects the zoanthid Palythoa caribaeorum. This study aimed to compare the diversity of culturable bacteria in the samples of healthy and necrotic tissue from the P. caribaeorum colony collected from the Carapibus reefs. The present work aimed also to analyze the production of proteolytic enzymes by isolated bacteria. The density of total heterotrophic bacteria in the necrotic tissue was higher than in healthy tissue of P. caribaeorum. Phylogenetic analysis of isolated bacteria based on the partial 16S rRNA gene sequences revealed that the majority of bacterial strains belonged to the Bacilli class of Firmicutes phylum (65.2%), followed by the Gamma Proteobacteria class (34.7%) of Proteobacteria phylum. The genus Bacillus was dominant among the strains of healthy tissue of P. caribaeroum, while among the bacteria of necrotic tissue the Bacillus and Vibrio were the most abundant. The higher number of strains belonged to the Vibrio genus were found in necrotic tissue (42.1%) in comparison with healthy tissue (9.6%). Among bacteria from healthy tissue were found also Staphylococcus sp. isolate and from necrotic tissue Marinobacter spp. and Alteromonas spp. isolates. Among the isolates from healthy tissue only Bacillus spp. showed proteolytic activity, while proteolytic isolates from necrotic tissue belonged to the genera: Bacillus, Vibrio, Alteromonas and Marinobacter. The data suggest that bacteria of the genus Vibrio and proteolytic bacteria may play a role in the development of tissue necrosis of zoanthid studied. / As comunidades microbianas desempenham um papel fundamental na saúde do coral, e suas alterações podem levar ao aparecimento de doenças. Muitas doenças afetam os corais dos recifes em várias partes do mundo, entretanto pouco se sabe sobre os agentes patogênicos e os fatores que desencadeiam o processo patológico. Nos recifes costeiros da praia de Carapibus, Conde (Paraiba, Brasil), uma doença necrosante afeta o zoantideo Palythoa caribaeorum. Em virtude disso, neste trabalho objetivou-se comparar a diversidade de bactérias cultiváveis nas amostras do tecido sadio e necrosado coletadas da colônia de P. caribaeorum dos recifes de Carapibus. Objetivou-se também analisar a produção de enzimas proteolíticas por bactérias isoladas. A densidade de bactérias heterotróficas totais no tecido necrosado foi maior que no tecido sadio. A análise filogenética dos isolados bacterianos realizada na base das sequencias parciais do gene RNAr 16S revelou que o maior número de isolados de bactérias pertenceram a classe Bacilli do filo Firmicutes (65,2%), seguida pela classe Gama-Proteobacteria (34,7%) do filo Proteobacteria. O gênero Bacillus foi predominante entre os isolados do tecido sadio de P. caribaeroum, enquanto entre as bactérias do tecido necrosado os gêneros Bacillus e Vibrio foram dominantes. Um número maior de isolados pertencentes ao gênero Vibrio foi encontrado no tecido necrosado (42,1%) em relação ao tecido sadio (9,6%) de P. caribaeorum. Foram encontradas também as bactérias Staphylococcus sp. no tecido sadio, Marinobacter spp. e Alteromonas spp. no tecido necrosado. Dentre os isolados do tecido sadio de P. caribaeorum, apenas o gênero Bacillus apresentou a atividade proteolítica, enquanto os isolados proteolíticos do tecido necrosado pertenceram aos gêneros: Bacillus, Vibrio, Alteromonas e Marinobacter. Os dados obtidos sugerem que as bactérias do gênero Vibrio e as bactérias proteolíticas podem desempenhar um papel no desenvolvimento da doença necrosante do zoantídeo estudado.

Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.