PGMICRO PROGRAMA DE PÓS-GRADUAÇÃO EM MICROBIOLOGIA (PGMICRO) INSTITUTO DE BIOLOGIA Phone: Not available
Dissertations/Thesis

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2023
Dissertations
1
  • Felipe Rangel do Vale Lima
  • Functional redundancy of microbial communities in diverse terrestrial ecosystems.

  • Advisor : PEDRO MILET MEIRELLES
  • COMMITTEE MEMBERS :
  • RONALDO BASTOS FRANCINI FILHO
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • PEDRO MILET MEIRELLES
  • Data: Jan 20, 2023


  • Show Abstract
  • Functional redundancy is a mechanism that guarantees the maintenance of ecological processes that are crucial to the functionality of the ecosystem, even with the loss of species. Although widely studied in animals and plants, little is understood about the functional redundancy in microbial communities despite the importance of these communities for the maintenance of numerous ecosystem services. Rare microorganisms, microbial taxa with relative abundance below 0.01%, play a key role in maintaining ecosystem services, but are generally neglected in studies of functional redundancy in microbial communities. In the present study, we collected 6794 metagenomic samples, obtained from 73 habitats distributed in 9 ecosystems across the planet, from which we calculated the functional redundancy in microbial communities from different ecosystems. To test the relationship between relative abundance and richness of the rare biosphere of the microorganism community and the functional redundancy indices found, we performed a simple linear regression considering significant the analyzes whose P value <0.05. Our findings demonstrate that functional redundancy differs considerably between the studied ecosystems. Data from this study reveal that the functional redundancy of microbial communities is apparently strongly influenced by the lifestyle of microorganisms and the environments in which they are inserted. The analysis carried out revealed that microbial communities with a high richness of rare microorganisms generally have reduced functional redundancy which leads us to believe that rare microorganisms can be important in performing highly specialized and distinct functions in each environment and their extinction can trigger cascading effects reducing the functionality of ecosystems.

2
  • LUCAS BARBOSA DE AMORIM CONCEIÇÃO
  • Identification and Characterization of Endogenous Viral Elements in Genomes of Bombus spp.

  • Advisor : ERIC ROBERTO GUIMARAES ROCHA AGUIAR
  • COMMITTEE MEMBERS :
  • RODRIGO ARAÚJO LIMA RODRIGUES
  • ERIC ROBERTO GUIMARAES ROCHA AGUIAR
  • LUIZ EDUARDO VIEIRA DEL BEM
  • Data: Feb 27, 2023


  • Show Abstract
  • Bee populations are extremely important for the maintenance of pollination-dependent populations of plants, which reflects on a market cap of 24 billion dollars, supporting global food production with more than US$ 200 bilhões.  The genus Bombus stands out by comprising efficient pollinator species in commercial crops and their natural habitats. Bee populations have been manifesting a concerning decline due to climate change, the use of pesticides and parasites, especially viruses. Therefore, it is necessary to investigate immunological mechanisms that act on these organisms' immune defenses, decreasing pathogens' influence on the populations. Non-retroviral endogenous elements (EVEs) may be transcribed and processed into small RNAs potentially helping on the antiviral immune response in insect species.  Interfering RNAs (RNAi) constitute the main antiviral response path in Arthropoda. The diversity and transcriptional activity of EVEs has already been proven in dipterans, but there is little data about EVEs in bees. The goal of this study is to identify and characterize EVEs in, publicly available, Bombus spp. Genomes. Firstly, the most recent genomes of 30 Bombus species were obtained from Genbank. After a prediction of Open Reading Frames (ORFs) and subsequent sequence similarity analysis against viral sequence databases, using the programs getorf and DIAMOND, respectively, the selected sequences were submitted to manual curation to eliminate repetitions, retrovirus-like sequences, transposons, DNA virus or other non-viral organisms, only mantaining putative non-retroviral RNA EVEs.. The curated sequences were processed again on NCBI, using both blastx and blastn online versions, to guarantee a better robustnessof the results, and aligned among one another for later inferences on the evolutionary history of these EVEs. It is possible to affirm that the EVEs presented similarities, partially, with four known viral families: Partitiviridae, Phasmaviridae, Totiviridae and Virgaviridae; whereas most of them were similar to non-classified viruses Altogether, each species presented between 1-20 EVEs, excluding B. superbus and B. waltoni, which did not manifest any EVEs; and B. terrestris, B. polaris and B. vosnesenskii, which had more robust results, with 32, 27 and 25 respectively. After aligning with one another, the sequences manifested, on the whole, many cases of similarity between different species, which may suggest an old and common evolutionary origin, endorsed by the data obtained from blastn. In future studies, the transcriptional activity of identified EVEs and the possible small-RNA production that might influence on bee’s antiviral response will be tested.

3
  • GRACIETE SOARES LIBORIO VERÍSSIMO
  • Genomic and phenotypic analyzes of the surface polysaccharide poly-N-acetylglucosamine (PNAG) in bacteria of the genus Leptospira.

  • Advisor : PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • COMMITTEE MEMBERS :
  • FLÁVIA FIGUEIRA ABURJAILE
  • ANGELA SILVA BARBOSA
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • Data: Jun 2, 2023


  • Show Abstract
  • Poly-β-1,6-N-acetyl-glucosamine (PNAG) is a surface polysaccharide widely expressed and present in the biofilm matrix of different bacterial species. PNAG is a virulence factor, involved in protection against innate host defenses, antibiotic therapy, and environmental stress conditions. Pathogenic leptospires are the etiological agents of leptospirosis, an anthropozoonosis with worldwide distribution that impacts human and animal health. Leptospira forms biofilms in the environment and in the kidneys of the reservoir Rattus norvegicus. However, information about the composition of the exopolymer matrix of Leptospira biofilms, as well as the proteins involved in PNAG expression, are poorly characterized. In this work we evaluated PNAG expression by Leptospira through immunofluorescence (IF), biochemical, and genomic approaches. Through IF, we demonstrated that PNAG is expressed on the cell surface of Leptospira with different degrees of pathogenicity and is present in the exopolymer matrix of the biofilm. Biochemical treatment with sodium metaperiodate cleaved N-acetylglucosamine-β- (1,6) bonds, confirming the presence of PNAG. Through comparative genomic analysis, we identified proteins with similarity to glycosyltransferases and deacetylases related to PNAG expression. In this study, we demonstrated the presence of PNAG in Leptospira in the planktonic phenotype and in the biofilm matrix. Our study expands knowledge about Leptospira biofilm composition and contributes to the understanding of possible Leptospira survival mechanisms in the environment and in the host-pathogen relationship.

4
  • ALICE MARIA ABREU GUSMAO SOARES
  • Monitoring antibody production in patients infected with SARS-CoV-2 and vaccinated for SARS-CoV-2, in Salvador, Bahia, Brazil

  • Advisor : GUBIO SOARES CAMPOS
  • COMMITTEE MEMBERS :
  • ELISANGELA DE JESUS CAMPOS
  • GUBIO SOARES CAMPOS
  • LUANA LEANDRO GOIS
  • Data: Jul 10, 2023


  • Show Abstract
  • SARS-COV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is the virus that causesCOVID-19 (Coronavirus Disease 2019). The reach and negative impact of SARS-CoV-2across the world has placed this pandemic among the most notorious on record. Because it isa global emergency, as well as the growing number of cases and deaths caused and thecharacteristics of SARS-CoV-2, such as the high transmissibility between individuals and thevariability of clinical manifestations associated with the infection, make laboratory diagnosisa tool very important in the fight against the pandemic. Epidemiological screening for SARS-CoV-2 aims to correctly identify patients exposed to the virus to better understand theprevalence, epidemiology and dynamics of the disease. There is a lot to understand about theinteraction between SARS-CoV-2 and its host. With this, the search for a betterunderstanding of the immunological aspects of SARS-CoV-2 has increased. Thus, understanding how the antibody response works during and after natural infection is essentialto better understand the disease, as well as evaluating the immune response induced by thevaccine to understand how the antibody response works during and after infection. In thissense, a long-term follow-up study is important to understand, monitor and investigate IgGantibody levels. This study aimed to evaluate the level of antibodies to SARS-CoV-2 ingroups of infected patients (previously tested by qRT-PCR with detectable results for SARS-CoV2) and subsequently vaccinated in Salvador, Bahia, Brazil. For this, serologicalcollections were carried out from 245 individuals, where six analyzes of the presence ofantibodies of the IgG class were carried out through the enzyme-linked immunoabsorptiontest (ELISA) in the serum samples of these individuals over approximately 1 year, at differenttimes in the Virology Laboratory of the Federal University of Bahia - UFBA. In this work, itis learned that primary vaccination induced a significant increase in SARS-CoV-2 specificantibodies, which was further intensified by the booster dose. However, in individualsrecovered from SARS-CoV2, there was no further increase in antibody levels after thesecond vaccine dose. Notably, IgG levels were similar in individuals who were not infectedwith SARS-CoV-2 and in individuals who recovered from SARS-CoV-2 after a booster dose.

5
  • TACILA ANDER OLIVEIRA ALMEIDA
  • BIOTECHNOLOGICAL POTENTIAL OF FRESHWATER MITOSPORY FUNGI

  • Advisor : ADRIANA OLIVEIRA MEDEIROS
  • COMMITTEE MEMBERS :
  • ADRIANA OLIVEIRA MEDEIROS
  • RICARDO BIZOGNE SOUTO
  • VERA LUCIA COSTA VALE
  • Data: Aug 31, 2023


  • Show Abstract
  • : Fungi that occupy freshwater environments have been reported to produce metabolites with wide chemical diversity and a wide range of biological activities. Among the groups of fungi that occur in these environments are the mitosporic fungi, which consist of asexual states, mainly of ascomycetes and some basidiomycetes. Aquatic hyphomycetes are mitosporic fungi recognized mainly for their role in the decomposition of allochthonous leaf organic matter in lotic environments and for their ability to sporulate below the surface. The main objective of this work is to evaluate the biotechnological potential of freshwater mitosporic fungi. Chapter 1 is an integrative review on the metabolic production of aquatic hyphomycetes and its biotechnological applications. The results showed this group as important producers of several metabolites with applications in several sectors of industry, agriculture and health, mainly enzymes associated with the breakdown of lignocellulolytic compounds and with antimicrobial potential. Chapter 2 is an experimental work that aimed to evaluate the antimicrobial activity of four species of mitosporic fungi isolated in different streams associated with decomposing plant material. The fungi were grown in different culture media and their crude extracts were tested against nine bacterial and fungal pathogens using the broth microdilution technique. The fungi F. curvula, A. penicillioides and P. submersus strain 133057 inhibited most bacterial pathogens, but none showed antifungal activity. Strains of the same species collected from different environments and extracts from cultures in different culture media showed differences in terms of bioactivity, which are important aspects to be considered in the bioprospecting of antimicrobial activity

6
  • THAYRA GOMES DOS SANTOS
  • Genomic analysis of Lactiplantibacillus plantarum in the identification of probiotic properties

     

  • Advisor : RODRIGO DIAS DE OLIVEIRA CARVALHO
  • COMMITTEE MEMBERS :
  • RODRIGO DIAS DE OLIVEIRA CARVALHO
  • SANDEEP TIWARI
  • WANDERSON MARQUES DA SILVA
  • Data: Sep 25, 2023


  • Show Abstract
  • Probiotics are microorganisms with beneficial properties for the health of the host who consumes them. These can be applied both in industry and in the promotion of human health, such as for the treatment of inflammatory bowel diseases. Currently, it is possible to find several studies on lactobacilli, especially Lactiplantibacillus plantarum, however, due to its great genetic diversity, the species is still little explored. In this sense, the isolation and genomic characterization of new Lb. plantarum of Brazilian biodiversity could allow the discovery of exclusive protective mechanisms and presents potential for the development of alternative therapies to combat inflammation. As a result, a growing line of research, probiogenomics, has provided a more comprehensive and assertive understanding of the molecular bases of these probiotics. In this context, through the application of bioinformatics analyses, this work aimed to taxonomically and functionally characterize the complete genomes of six strains isolated in Brazil. In this way, the taxonomy of the six lineages was confirmed using the Average Nucleotide Identity by BLAST (ANIb), using the genome of the Lb lineage as a reference. plantarum SK151 from the GENBANK database (NCBI). In contrast, a multilocus phylogenetic analysis was performed using 220 complete Lb genomes. plantarum from the aforementioned database, to investigate evolutionary relationships with other probiotic lineages of the same species. Our results suggest high genetic proximity to probiotics already marketed, or that have demonstrated anti-inflammatory effects in clinical trials or preclinical studies of inflammatory diseases. Next, the analysis of antimicrobial peptides in the genomes of Brazilian isolates identified several plantaricin genes, suggesting antimicrobial potential. The presence of the gadB gene was also identified in the six lineages, which may be involved in the production of gamma-aminobutyric acid through the action of the enzyme glutamate decarboxylase, suggesting an important role in the regulation of the intestine-brain axis. Through SNP analyzes and molecular modeling, the lpl4 lineage showed a high degree of genetic and structural conservation of this enzyme when compared to the homologous protein considered functional in the Taj-Apis362 lineage. Furthermore, pangenomic analysis among all strains revealed the presence of exclusive genes in the lpl4 strain, namely, five hypothetical proteins and a sulfotransferase, involved in the synthesis of vitamin B6. To analyze the safety of the strains, the presence of antibiotic resistance genes was investigated, where only one marker was observed in a region of genomic plasticity on the chromosome of the E6 strain. Therefore, it is possible to conclude that the strains evaluated have probiotic properties with therapeutic potential for inflammatory diseases that affect the intestine-brain axis, especially lpl4, and can be considered safe for human and animal use. However, more studies are needed to investigate its therapeutic potential.

7
  • LUIZ RICARDO FRASER SILVA
  • A comparative evaluation of automated platforms for assembly and analysis of SARS-CoV-2 genomes

  • Advisor : LUIS GUSTAVO CARVALHO PACHECO
  • COMMITTEE MEMBERS :
  • BRENA MOTA MOITINHO SANT'ANNA
  • GUBIO SOARES CAMPOS
  • LUIS GUSTAVO CARVALHO PACHECO
  • Data: Oct 5, 2023


  • Show Abstract
  • The COVID-19 pandemic, caused by the SARS-CoV-2 virus (Severe Acute Respiratory Syndrome Coronavirus 2), began in the province of Wuhan in China and quickly spread around the globe, generating socioeconomic and public health impacts. The discovery of the viral agent causing the disease was only possible thanks to new nucleic acid sequencing technologies, collectively known as NGS (Next Generation Sequencing). Currently, these same technologies are being used for genomic surveillance of the virus, including the identification of mutations and determination of new variants. In 2022, the World Health Organization issued a global genomic surveillance strategy for potentially pandemic and epidemic pathogens (2022-2032). In order to contribute to this strategy and improve access to viral detection tools, the main objective of this work is to implement, compare, and validate analysis protocols using only raw NGS data (FASTQ files) for the diagnosis and variant calling of SARS-CoV-2. For this purpose, a total of 15 new SARS-CoV-2 sequencing samples obtained by Ion AmpliSeq targeted methodology (n=10) or metatranscriptomic approach (n=5) were used as the sample universe. These FASTQ files were subjected to assembly using the automated assemblers: PATRIC, BV-BRC, ID-seq, CoronaSPAdes, and Genome Detective. Variant calling was performed using the platforms GISAID, Genome Detective, Nextclade, and BV-BRC, which have an integrated approach to assembly and annotation. The analyses were carried out at two different time points to identify possible changes in the results after platform updates. The generated results were compared, among other aspects, in terms of the ability to call SARS-CoV-2 variants, the average time spent on analysis, identified viral ORFs (Open Reading Frames), and the percentage of G+C content in the assembled genome. Initially, the BV-BRC platform proved to be capable of correctly determining the variant, with the highest number of results generated in the shortest time. ID-seq and Genome Detective were able to perform genome assembly, and from the generated FASTA files, variant calling was performed using the Nextclade platform and Genome Detective itself for annotation. After updates, BV-BRC, despite still producing the highest number of results in the shortest time, showed lower specificity in variant calling. However, Genome Detective presented an integrated approach to assembly and annotation with a greater ability to determine the variant. The assembler PATRIC and the annotation platform GISAID were not able to generate satisfactory results for the evaluated samples.

8
  • JULIA DE MELO JUREMA GUIMARÃES
  • Effect of pH on survival and biofilm formation by Leptospira

  • Advisor : PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • COMMITTEE MEMBERS :
  • LUCIANA DOS SANTOS MEDEIROS
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • RODRIGO DIAS DE OLIVEIRA CARVALHO
  • Data: Dec 7, 2023


  • Show Abstract
  • Leptospires are Gram negative bacteria with a wide physical-chemical agents sensibility profile, previous works revealed that Leptospira may survive for months in water and soil. Therefore, leptospires remain as a relevant human and veterinary health issue. Leptopsira form biofilms in vitro, in vivo and on the environment. However, there is no literature to our knowledge describing if continuous exposition to unfavorable temperatures and pHs may or not alter biofilm formation in Leptospira. The aim of this work is to evaluate pH and temperature stress action in biofilm formation and cell survival. Leptospiral biofilm biomass cultivated at 30°C with pHs 5.0; 6.5; 7.5 e 8.5 was quantified using crystal violet stain method. We generated growth curves of planktonic Leptospira at 30 °C with pH 5.0; 7.5 and 8.5. Preliminary results show that pH 5.0 and elevate temperatures cultivation conditions inhibit biofilm formation in Leptospira. The present results alongside our future results may enlighten if physycal-chemical stress exposition interfere in Leptospira biofilm formation

2022
Dissertations
1
  • Rodrigo Alex Henriquez Arancibia
  • THE DYNAMICS OF TRYPANOSOMA CRUZI PERSISTENCE: AN ECOLOGICAL-EVOLUTIONARY APPROACH

  • Advisor : LEANDRO MARTINS DE FREITAS
  • COMMITTEE MEMBERS :
  • BRUNO LOPES BASTOS
  • LEANDRO MARTINS DE FREITAS
  • LUCAS MIRANDA MARQUES
  • Data: Feb 21, 2022


  • Show Abstract
  • Chagas disease is a neglected tropical disease endemic to the American continent with Trypanosoma cruzi as its etiologic agent. This disease has a major impact on public health, affecting about 6 -8 million people around the world. Currently, there are limitations in its treatment and prevention, such as the lack of vaccines and widely effective drugs. The parasite typically presents a complex life cycle involving the triatomine insect vector and the mammalian host, in addition to other means of transmission such as oral and congenital. Chagas disease in its clinical course has two phases: the acute phase characterized by a strong immune response, reduction of parasitemia, but without the elimination of the infection leading to chronification and the chronic phase, predominantly asymptomatic in a smaller percentage, manifesting cardiac and/or gastrointestinal syndromes. years after infection. However, the causes that lead to the manifestation or not of symptoms of the chronic phase are not fully understood. We propose to investigate the dynamics and mechanisms underlying chronification. The Trypanosoma cruzi has virulence factors that help it evade the immune system and maintain it in the host. Considering the long process of molecular interaction as a selective pressure agent, we suggest that the efficiency of some of these virulence mechanisms, with those carried out by proteins of parasitic surface, is due to a certain similarity with human proteins due to a molecular evolutionary convergence. we search for evidence of this hypothesis; we carry out analyzes of similarity between Trypanosoma cruzi surface proteins and the immune system and human signal transduction. Also, the events that occurred during the acute phase seem to have a decisive impact on the later development of the disease, as in the definition of the host's state of resistance and susceptibility. We then present a mathematical model that proposes to investigate the scenarios in the parasite-system dynamics that end the states of susceptibility and resistance in infection by Trypanosoma cruzi. It simulates relevant characteristics of the dynamics of the acute phase of Chagas disease.

2
  • Emília Turlande Sêneca Ribeiro dos Santos
  • PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF Escherichia coli ISOLATED FROM FREE-RANGE POULTRY PRODUCTS INFORMALLY SOLD IN A TROPICAL CLIMATE REGION

  • Advisor : MELISSA HANZEN PINNA VALENTIM
  • COMMITTEE MEMBERS :
  • CARLOS PASQUALIN CAVALHEIRO
  • MATEUS MATIUZZI DA COSTA
  • MELISSA HANZEN PINNA VALENTIM
  • Data: Jun 20, 2022


  • Show Abstract
  • Foodborne Diseases are public health events with immediate compulsory notification and often associated with the consumption of animal products contaminated by bacteria. Escherichia coli is the main pathogen involved in foodborne outbreaks in Brazil. In recent years, free-range poultry products have shown greater visibility in the market. However, may there is negligence in terms of health surveillance for the marketing of these products, which provides health risk due to the greater possibility of spreading pathogenic microorganisms, including those resistant to antimicrobials. The aim was to investigate the presence of Escherichia coli in free-range poultry products sold informally and evaluate antimicrobials resistant E. coli circulation. Therefore, 38 free-range egg pools and 26 free-range chicken carcasses from informal marketing in Salvador-Bahia were analyzed. 30 isolates were obtained and identified as Escherichia coli by MALDI-TOF, with contamination rate of 92.3% (24/26 carcasses) of meat samples and 15.8% (6/38 pools) of egg samples. The isolates were submitted to the VITEK®2 System to determine the susceptibility pattern to clinically relevant classes of antimicrobials, such as penicillins, cephalosporins and carbapenems of the β-lactam group, aminoglycosides and quinolones. A total index of 46.7% (14/30) of resistance to at least one of tested antimicrobials was identified, with 35.7% (5/14) characterized as multidrug-resistants because they presented resistance to three or more classes of antimicrobials. The prevalence of resistance was higher for Ciprofloxacin with 33.3% (10/30), Ceftriaxone with 26.7% (8/30) and Ampicillin /Sulbactam with 20% (6/30). 8/30 (26.7%) presented phenotype for Extended-Spectrum Beta-Lactamase (ESBL) producing. In molecular characterization by PCR, 60% (18/30) of E. coli isolates were characterized as DEC group, with a higher prevalence for the atypical EAEC [38.9% (7/18)] and DAEC [22, 0% (4/18)], in addition to characterization of hybrid strains. Of the 30 isolates, 19 (63.3%) presented at least one β-lactam resistance gene, with a higher frequency observed for the blaCTX-M 1 and blaTEM genes, with 63% (12/19) and 47% (9/19), respectively, followed by blaOXA-1 and blaSHV with 10.5% (2/19) each. The detection of diarrheagenic, multidrug-resistant and/or ESBL-producing E.coli in free-range poultry products sold in a country for tourist purposes confirms the potential for these microorganisms to be transmitted through production animals, hence contributing to the occurrence of Foodborne Diseases and the global emergence of antimicrobial resistance.

3
  • Sabrina Ferreira de Santana
  • Use of metatranscriptomics for pathogen detection

  • Advisor : ERIC ROBERTO GUIMARAES ROCHA AGUIAR
  • COMMITTEE MEMBERS :
  • PAULA LUIZE CAMARGOS FONSECA
  • ERIC ROBERTO GUIMARAES ROCHA AGUIAR
  • LUIS GUSTAVO CARVALHO PACHECO
  • Data: Jun 30, 2022


  • Show Abstract
  • There are several pathogens that constitute constant threats to both human public health and agriculture, leading to great social and economic impacts. Currently, viruses are more prominent due to their rapid mutational rate and spread, being influenced by the environment and being associated with a wide range of hosts, infecting from higher mammals to unicellular organisms. The most recent example related to human public health is SARS-CoV-2, which has a rapid spread associated with the absence of population immunity, leading to the pandemic scenario. There are also viruses associated with other microorganisms, such as fungi. Many fungi have a great impact on agriculture, such as Ceratocystis which infects several plants of agricultural importance such as mango and eucalyptus, impacting productivity and leading to death. Thus, monitoring becomes an important strategy for epidemiological assessment of infectious agents and associated microorganisms that can impact the outcome of infections, causing both hypovirulence and hypervirulence. New computational tools can help in the development of more efficient actions in the monitoring and development of disease management strategies. Robust methods assist in the evaluation of complex data allowing rapid monitoring of threats. With this in mind, the objective of this project was to evaluate and apply metatranscriptomics as a tool to identify pathogens in samples derived from different organisms. To this end, data from patients positive for COVID-19 in the State of Bahia and the public library of the fungus Ceratocystis cacaofunesta were analyzed. In both cases, part of the bioinformatics analysis was carried out through the Galaxy online platform and the taxonomic classification was carried out with the Kaiju and Kraken tools. For Ceratocystis, the BLAST program was also used to search for similarity, HMMER and InterProScan to find the conserved domains, AliView to edit the sequences, MAFFT to perform the global alignment, CIPRES to search for the best model and build the tree. phylogenetics and, finally, iTol to visualize the phylogenetic tree. After analysis, for samples derived from humans known to be infected with SARS-CoV-2, there were mainly bacteria present in the respiratory tract of these individuals. Thus, the data were compared with the literature in order to assess which microorganisms belong to the respiratory tract and identify which of the species found have potential or have already been described acting as opportunistic pathogens co-circulating with SARS-CoV-2. As for the fungus Ceratocystis, several viral sequences were found, including those belonging to the families Hypoviridae, Mymonaviridae, Narnaviridae and Partitiviridae. Conserved domains associated with the sequences were also observed, in addition to having confirmed the family-level designation of the new viruses through phylogenetic analysis. In short, metagenomic analyzes associated with bioinformatics have been shown to be essential for a better understanding and monitoring of pathogens and our work reinforced the potential of Next Generation RNAs Sequencing in this process.

4
  • TATIALE DE OLIVEIRA RODRIGUES
  • PRODUCTION OF RECOMBINANT PROTEIN Pr55gag FOR USE IN THE DIAGNOSIS OF EQUINE INFECTIOUS ANEMIA

  • Advisor : SILVIA INES SARDI
  • COMMITTEE MEMBERS :
  • CAMILA FONSECA LOPES BRANDAO
  • DELLANE MARTINS TIGRE
  • SILVIA INES SARDI
  • Data: Jul 6, 2022


  • Show Abstract
  • Equine Infectious Anemia is caused by a Lentivirus that affects equids all over the world and clinical manifestation ranges from an asymptomatic to a lethal disease. There is no reliable vaccine, so the disease control and management, as recommended by the sanitary authorities includes testing animals before relocation and euthanasia for the positive ones. Due to the gravity of the disease and the fact that the diagnosis settles the destiny of animals’ lives, it is crucial that diagnostic be the most accurate as possible. The golden standard diagnosis method is Agarose Gel Immunodiffusion (AGID) with virion core protein p26, obtained from eukaryotic cell culture, as antigen. As the technology has improved, it is possible to produce viral proteins using the recombinant DNA and use them as antigen to develop more effective tests. The precursor protein Pr55gag shares antigenicity with the structural proteins of EIAV into which it is cleaved, therefore it should promote greater sensitivity to the diagnostic test. This study aimed to produce the Pr55gag protein in a prokaryotic system to avaluate its potential as an antigen in serological diagnosis. Thus, EIAV gag gene was inserted in plasmid pET_TEV that was cloned into Escherichia coli C41 (DE3) linage. This expression system was used to express the protein inducted with IPTG. The protein was purified on a histidine affinity column, underwent dialysis in PBS to remove excess salts and imidazole and was concentrated with a VivaSpin® system of centrifugation. The recombinant protein obtained from this methodology was used as an antigen in a Western blotting, in which 56 equine sera provided by the State Agricultural Defense Agency of Bahia (ADAB) and previously submitted to AGID were tested. Kappa coefficient of 0.82 indicates an almost perfect agreement between the results of both tests. In addition to being faster to perform, it is suggested that Western blotting with the recombinant protein is more sensitive, as it was able to identify more positive animals (23/56) than IDGA (19/56). Preliminary results suggest that the recombinant protein Pr55gag, obtained quickly, efficiently, with low cost and satisfactory yield, can be used as an antigen in diagnostic tests for Equine Infectious Anemia providing a more sensitive test by detecting a higher number of positive animals.

5
  • Carolina Ferreira Amorim
  • Activity of photoactivated porphyrins on Candida spp. 

  • Advisor : VASCO ARISTON DE CARVALHO AZEVEDO
  • COMMITTEE MEMBERS :
  • Aristóteles Góes-Neto
  • SAMIRA ABDALLAH HANNA
  • VASCO ARISTON DE CARVALHO AZEVEDO
  • Data: Jul 28, 2022


  • Show Abstract
  • Candida spp. is the main fungal genus associated to infections in humans, and its treatment has become a challenge due to the production of biofilm and its resistance/multi-resistance profile to conventional antifungals. Antimicrobial photodynamic therapy stands out as a treatment characterized by a broad spectrum of antimicrobial action, being able to induce oxidative stress in different microorganisms, and porphyrins are photosensitizers with high selectivity to pathogens. Thus, this work aimed to analyze the photoinactivation of different species of Candida by two cationic (4-H2TMeP+ and 3-H2TMeP+) and one anionic (4-H2TPSP-) porphyrins. Microdilution assays were performed to determine the MIC100, with subsequent determination of MFC100, verification of the action on biofilm, determination of the oxidative species involved through the use of scavengers, potentiation with iodide, and analysis using the atomic force microscopy (AFM). Cationic porphyrins were significantly efficient in inactivating Candida albicans and non-albicans species with 100% growth inhibition and fungicidal activity (MFC100/MIC100 < 4). The cationic porphyrins were able to interfere in Candida spp biofilm formation. The photooxidative mechanism activated by 3-H2TMeP+ in Candida spp. involved mainly the production of singlet oxygen. The photooxidant effect in these yeasts was potentiated with the addition of potassium iodide. In the AFM, 3-H2TMeP+ was able to reduce C. parapsilosis adhesion to the surface. Thus, it is concluded that the use of porphyrin photosensitizers has significant potential in the development of an alternative and/or complementary treatment for infections caused by different Candida species.

2021
Dissertations
1
  • ICARO SANTOS LOPES
  • Identification and classification of CRISPR arrays found in Xanthomonas

  • Advisor : PEDRO MILET MEIRELLES
  • COMMITTEE MEMBERS :
  • BRENA MOTA MOITINHO SANT'ANNA
  • MILTON RICARDO DE ABREU ROQUE
  • PEDRO MILET MEIRELLES
  • THIAGO LUIZ DE PAULA CASTRO
  • VIVIANE GRAZIELLE DA SILVA
  • Data: Feb 22, 2021


  • Show Abstract
  • One of the decade’s most important discoveries in biotechnology is a defense mechanism found in prokaryotes that concedes adaptative immunity to its owner. This system called CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) consists in genomic integration of some sequences from bacteriophages, creating cellular “memory”. At this point, there are few studies about CRISPR’s role in prokaryotes’ interactions with the environment and organisms other than viruses. There is also little knowledge about how bacteria acquire this mechanism and its prevalence in species of economical interest, like phytopathogens. With this work, we intend to identify and classify CRISPR sequences found in the Xanthomonas genus, describing how this system affects interactions between the bacteria, their host plants and environmental viruses. In addition, we verify the hypothesis concerning these sequences’ origins in each Xanthomonas species, considering environmental influences and inheritance from a common ancestor. 239 complete Xanthomonas genomes were analyzed and CRISPR sequences were found in 102 of them. The final results imply that both environmental conditions and the common ancestor play a part to determine the presence, absence and characterization of CRISPR sequences in Xanthomonas. Therefore, it’s possible that both hypotheses are partially correct. Vast diversity observed in the spacers across the genomes indicates an intense network of environmental interactions to which bacteria are subordinated in order to carry out their phytopathogenic role.

2
  • Susanna Rita Oliveira Schumacher
  • Comparative genomics study of Serratia marcescens using a phytopathogenic strain isolated in Bahia

  • Advisor : THIAGO LUIZ DE PAULA CASTRO
  • COMMITTEE MEMBERS :
  • SAMIRA ABDALLAH HANNA
  • SANDEEP TIWARI
  • THIAGO LUIZ DE PAULA CASTRO
  • Data: Dec 6, 2021


  • Show Abstract
  • Serratia marcescens is a ubiquitous species with a complex behavior in the environment, acting as an opportunistic pathogen in animals and plants, endosymbiont through endophytism and also found in free-living form in soil, water and food. For a microorganism-host relationship to be established, bacteria must be able to colonize, adapt, and adhere to internal host tissues. To this end, bacteria have developed several secretion systems, which play an important role in adhesion, motility, competition with other microorganisms, and the secretion of virulence factors. In this work we describe the genome sequence, assembly and annotation of the phytopathogenic isolate S. marcescens BP2 followed by comparative analyses of seven S. marcescens genomes, being those of opportunistic human pathogens, two endophytes, one phytopathogenic and two free-living, deposited in the NCBI database. The aim of this study was to understand the phylogenetic relationships and to identify genes and secretion systems correlated with the diverse lifestyle of this group of bacteria. Phylogenomic and ANI analyses, revealed the occurrence of two different groups in the taxonomic tree: one composed of phytopathogenic and free-living and one of endophytic and opportunistic pathogens. Additionally, the search for orthologous gene clusters demonstrated that all strains share a total of 3862 clusters, while the same groups present in the phylogenomic analyses, share a greater amount of clusters among themselves. The search for structural components of the secretion systems was performed using the TXSScan tool and it was possible to find the presence of type I, V and VI systems in all isolates. Moreover, we could observe the presence of several virulence factors in pathogenic strains and also in endophytic strains, suggesting that these genes may contribute to the competitiveness and adaptability of this bacterium in the environment. Finally, the presentation of a complete genome of phytopathogenic S. marcescens may help in future pangenomic studies of this species.

2020
Dissertations
1
  • TAMIRES PASCOAL DOS SANTOS
  • INFLUENCE OF HUMIC ACIDS IN THE AQUATIC HYPHOMYCET COMMUNITY COMBINED WITH NUTRIENT CONCENTRATIONS AND pH VALUES

  • Advisor : ADRIANA OLIVEIRA MEDEIROS
  • COMMITTEE MEMBERS :
  • ADRIANA OLIVEIRA MEDEIROS
  • PATRICIA OLIVEIRA FIUZA
  • PAULA BENEVIDES DE MORAIS
  • Data: Aug 7, 2020


  • Show Abstract
  • Aquatic hyphomycete fungi are primary responsible for the process of decomposition in the allochthonous organic matter in headwater streams. Black waters streams have low leaf decomposition rate, fungal biomass and reproductive diversity of aquatic hyphomycetes. We hypothesize that the rates of fungal decomposition and colonization are low due to the low concentration of nutrients, acid pH and higher concentration of humic acid in these streams. In a microcosms conditions we tested a complete factorial design (3 [humic acid levels: 5 and 24mgL-1] x 2 [nutrient levels: 0.05mgL-1 nitrogen and 0.07mgL-1 phosphorus and 0.5mgL- 1 of nitrogen and 0.7 mgL-1 of phosphorus x 2 [two pH ranges: 4 and 7.8] + 3 [controls: river water] x 3 replicates). Senescent leaves of Miconia albicans were incubated in fine mesh bags for 15 days in 3 clear water streams for colonization by aquatic hyphomycetes. In the laboratory, a set of 10 discs from the pre-colonized leaves, for each incubation stream, was distributed in each treatment. Every three days the nutrient solution containing the suspension spores was changed and preserved in formalin (4%) to determine the richness, density and reproduction rate of the aquatic hyphomycetes. We identified 15 species of aquatic hyphomycetes associated with leaf litter in breakdown process. Flagelospora curvula, Lunullopora curvula and Triscelophorus monosporus were the most abundance organism in conidia production. Nitrogen was the main factor that drive fungal biomass, fungal sporulation density and richness, but not leaf litter breakdown. Statistical models suggest that higher concentrations of humic acid, low nitrogen availability and acidic pH negatively affect the leaf decomposition process and alter the aquatic hyphomycete community. Although, our study was carried out in vitro, new hypotheses about the decomposition process in black waters of tropical Brazilian savannah may note future investigations in this unique ecosystem, mainly to understand the interactions between humic substances and aquatic hyphomycetes.

2
  • TIAGO SOUSA DA SILVA
  • Molecular characterization of Mycobacterium tuberculosis complex strains isolated from a reference center for tuberculosis research

  • Advisor : MELISSA HANZEN PINNA VALENTIM
  • COMMITTEE MEMBERS :
  • MELISSA HANZEN PINNA VALENTIM
  • JOICE NEVES REIS PEDREIRA
  • SORAIA MACHADO CORDEIRO
  • Data: Nov 12, 2020


  • Show Abstract
  • Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis Complex (CMTB) mycobacteria, a disease that primarily affects the lungs. The use of tools based on molecular biology has allowed advances in the field of human, animal and environmental medicine, bringing many benefits in the diagnosis of infectious diseases, significantly reducing the time from diagnosis to the institution of treatment combined with this advance, techniques such as the spoligotyping associated with classical epidemiology has enabled the identification of transmission chains and can be used to assess the efficiency of disease control programs in different parts of the world. This study aims to investigate the genetic diversity of MTB complex strains isolated from patients at a reference center for tuberculosis investigation. This study included bacterial isolates belonging to the MTB complex, originating from sputum samples sent to the Bacteriology Laboratory of the Jose Silveira Foundation for mycobacteriological diagnosis, with decontamination by the NALC-NaOH method, during the period from 2016 to 2018. The isolates were genotyped by spoligotyping technique and the results were analyzed visually and entered in binary format and compared with the standards described in the SITVIT database in order to identify the corresponding Spoligo International Type (SIT). The 46 samples with complete genetic profile in hybridization were classified into 26 SIT. The LAM line was the most frequent 43.1% (22/51). Regarding the antimicrobial sensitivity of the 150 isolates genotyped as M.tuberculosis, 143/150 (95.3%) showed sensitivity in vitro to all drugs tested while 7/150 (4.7%) showed resistance to at least one anti-drug -ALSO. Genotyping helped in the discovery of epidemiologically related cases, it was possible to observe three regions with a higher concentration of cases in the neighborhoods. We believe that knowledge about the characteristics of these patients will contribute to the development of policies aimed at improving the diagnosis and treatment of patients. Therefore, this study will greatly contribute to the provision of laboratory indicators for the control of tuberculosis and will serve in decision making by Brazilian municipalities.

3
  • ARTUR FILIPE CANCIO RAMOS DOS SANTOS

  • In silico IDENTIFICATION OF TRANSCRIPTIONAL REGULATORY WAYS IN BIOFILMS OF Leptospira biflexa

  • Advisor : VASCO ARISTON DE CARVALHO AZEVEDO
  • COMMITTEE MEMBERS :
  • Aristóteles Góes-Neto
  • PABLO IVAN PEREIRA RAMOS
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • PEDRO MILET MEIRELLES
  • SANDEEP TIWARI
  • VASCO ARISTON DE CARVALHO AZEVEDO
  • Data: Nov 18, 2020


  • Show Abstract
  • Bacteria of the genus Leptospira comprehend 66 genomic species including pathogenic, intermediate and saprophytic groups. Pathogenic leptospires are the etiologic agent of leptospirosis, a disease of public health impact worldwide. Biofilms improve survival of microorganisms in hostile environments and are related to various medical conditions. Leptospira forms biofilms in vitro and in vivo. Nevertheless, the regulatory mechanisms of biofilm formation in Leptospira are poorly known. We aimed in this study to identify transcriptional regulators involved in biofilm formation in Leptospira biflexa and describe the co-regulation networks of these regulators. First, we collected transcriptional regulators predicted for Leptospira biflexa in the P2TF database. Than, a similarity search was conducted using BLASTp of those regulators against previous data from a Leptospira biflexa transcriptome analysis of biofilm versus planktonic cells, in two time points: 48h (mature) and 120 h (late). After identifying regulatory genes in Leptospira biflexa, we checked for their expression levels. We found 138 transcriptional regulators predicted for this species, comprising sigma factors, one-component systems, response regulators and other DNA-binding proteins. From those, 38 presented were identified  as participating in biofilm regulation. Leptospira genomes are poorly known, with half of it composed of hypothetical proteins. We believe our results will pave the way for an initial comprehension of the regulatory mechanisms in Leptospira biofilm.

4
  • LARISSA AMORIM TOURINHO DE VASCONCELOS
  • GENOMIC ANALYSIS OF SIGNALING MEDIATED BY c-di-GMP IN Leptospira biflexa BIOFILM 

  • Advisor : PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • COMMITTEE MEMBERS :
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • CRISTIANE RODRIGUES GUZZO CARVALHO
  • PABLO IVAN PEREIRA RAMOS
  • Data: Nov 27, 2020


  • Show Abstract
  • Leptospira form biofilms in vitro and in natural environments. Biofilm formation is driven by signaling systems and, amongst signaling molecules, cyclic di-GMP (c-di-GMP) is an important second messenger involved in biofilm formation. Studies in cell signaling in Leptospira biofilm are scarce and little is known about the c-di-GMP signaling pathway during biofilm formation. In this work, we performed a wide genomic study on the c-di- GMP signaling system in Leptospira biflexa. We executed similarity searches and accessed databases to identify c-di-GMP-related genes, predicted enzymatic and c-di-GMP-binding functions, and investigated the expression of the identified genes based on the L. biflexa biofilms transcriptome dataset. We identified 22 genes encoding putative diguanylate cyclases (DGCs), phosphodiesterases (PDEs) and hybrid proteins. From those, 16 genes were potentially functional. Two genes encoding the DGC LEPBI_I2876 and the PDE LEPBI_I0099 presented expression levels that suggested they modulated c-di-GMP concentration during biofilm development. We also detected nine putative effector genes, including a pnp ortholog that is more expressed in mature biofilms and four genes harbouring potentially functional PilZ domains. We also showed that csrA, bolA, hfq and fliA, known c- di-GMP regulators, were expressed in L. biflexa biofilms. Furthermore, we found five TetR family genes that were more expressed during biofilm dispersal in L. biflexa biofilm, and we suggested these genes could induce biofilm dispersal by repressing c-di-GMP signaling. Additionally, we proposed a coordinated action between Pnp, FliA and the DGC LEPBI_I2876 during biofilm development in L. biflexa. Finally, we were able to suggest a biological pathway for c-di-GMP signaling in L. biflexa. The present work, conducted in the saprophytic L. biflexa, pointed out components of a c-di-GMP signaling pathway that is in its early stages of study in Leptospira.

5
  • TALITA PEREIRA GOMES
  • INVESTIGATION OF THE ROLE OF EXTRACELLULAR DNA IN Leptospira BIOFILM

  • Advisor : PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • COMMITTEE MEMBERS :
  • ELIANE DE OLIVEIRA FERREIRA
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • VITOR ANTONIO FORTUNA
  • Data: Dec 11, 2020


  • Show Abstract
  • Extracellular DNA is an essential component for bacterial biofilms. Bacteria of the genus Leptospira form biofilms in vitro and in natural environment. In this study we performed a structural and architectural analysis of Leptospira biflexa biofilm, with regard to the role of extracellular DNA. We digested mature biofilms (48h-growth) using DNase I enzyme, followed by spectrophotometry analysis of biomass and analysis of structural parameters by confocal laser scanning microscopy. The destruction of extracellular DNA by DNase caused biofilm disruption. Comparing biofilms treated with DNase and non-treated (EMJH control), biomass was reduced from 0,8 to 0,4 (p= 1,89E-02). Considering the fluorescence of extracellular DNA, the following parameters were significantly reduced: biomass (from 2,0 μm3/μm2 to 0,05 μm3/μm2; p= 3,42E-04), surface area (from 422462,1 μm2 to 21158,6 μm2; p= 3,02E-04), thickness average (4,5 μm to 0,09 μm; p= 2,71E-05), roughness coefficient (0,4 to 1,9; p= 6,97E- 07) and suface/biovolume ratio (6,8 μm2/μm3 to 18,1 μm2/μm3; p= 2,24E-04). Our results demonstrated an important structural change when the biofilm was treated with the DNase enzyme. We concluded that extracellular DNA is essential for the three- dimensional architecture of Leptospira biflexa biofilm.

2019
Dissertations
1
  • CAMILA PIMENTEL SOBRINHO
  • Prevalence of enteropathogenic bacteria with zoonotic potential in urban rats in Salvador, Bahia

  • Advisor : FEDERICO COSTA
  • COMMITTEE MEMBERS :
  • FEDERICO COSTA
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • SORAIA MACHADO CORDEIRO
  • Data: Jan 17, 2019


  • Show Abstract
  • Urban rats (Rattus spp.) are considered natural reservoirs of several zoonotic pathogens, such as their enterobacteria that are responsible by several infectious diseases that affect the human kind. Studies evaluating the occurrence of enterobacteria, as well as the antimicrobial resistances, in these animals in urban communities of tropical countries are non-existent, therefore, this study aimed to determine the prevalence of enterobacteria with zoonotic potential in urban rats in Salvador, Brazil. Rats were trapped between April and June 2018. Fecal samples were obtained through rectal swab, thereafter cultivated in selective medium. For microbiological identification, biochemical proofs and serologic characterization were performed. Salmonella samples were submitted to Vitek® for species confirmation and susceptibility profile. Escherichia coli isolates were submitted to evaluations on resistance to ceftriaxone and ertapenem. Klebisiella pneumoniae strains were phenotypically evaluated for production of extended spectrum β-lactamases (ESBL). 72 rats were captured, 67 R. norvegicus and five R. rattus. Eight enterobacteria genera were isolated from rat samples: Escherichia 70.8 %, Citrobacter 38.9 %, Enterobacter 16.7 %, Klebsiella 16.7 %, Serratia 8.3 %, Proteus 4.2 %, Kluyvera 1.4 % and Salmonella 1.4 %. Salmonella was resistant to cefalotin, cefuroxime, axetil, amikacin and gentamicin. E. coli isolates were susceptible to ceftriaxone and ertapenem. Klebsiella pneumoniae strains presented negative phenotype for ESBL. Diarrheagenic E. coli (DEC) were isolated in 31.3 % of the 67 R. norvegicus. The pathotypes detected more frequently were shiga toxin E. coli (STEC) in 11.9 %, followed by atypical enteropathogenic E. coli (aEPEC) in 10.4 % and enteroinvasive E. coli (EIEC) in 4.5 %. From the five R. rattus, 40 % presented DEC. Shigella was not isolated from any of the rats analyzed. Our findings indicate that rats are host of several enterobacteria and contribute to increase the importance of rodents as potential sources of pathogenic agents for humans. Although the epidemiology of these microorganisms needs to be further investigated, as well as its impact in the health of residents who have contact with these animals

2
  • JULIANA DOS SANTOS DAHORA ALMEIDA
  • DOES BLACK WATER AFFECT THE DECOMPOSITION ACTIVITY OF AQUATIC HYPHOMYCETE?

  • Advisor : ADRIANA OLIVEIRA MEDEIROS
  • COMMITTEE MEMBERS :
  • ADRIANA OLIVEIRA MEDEIROS
  • MILTON RICARDO DE ABREU ROQUE
  • PAULA BENEVIDES DE MORAIS
  • Data: Mar 28, 2019


  • Show Abstract
  • Aquatic hyphomycetes (AH) are decomposer fungus of submersed organic matter. The objective of this work was to evaluate whether black waters affect the reproduction and activity of AH. For this matter, we executed two experiments. Experiment 1: leaves of Miconia albicans and Davilla angustifólia (Atlantic Forest); Clusia burlemaxxi and Vochysia acuminata (Brazilian savanna) were incubated in a low order stream of their respective biomes (the Brazilian savanna stream representing black waters and the Atlantic forest stream representing white waters). We did not find a significative difference in the decomposition rates between the black waters and white waters streams; however, sporulation rate and fungal biomass were higher in the white waters streams. Experiment 2: leaves of M. albicans e C. burlemaxxi were incubated in streams of their respective biomes, and after 15 days of incubation, half of the experiment was transplanted between the streams (litter bags from white waters streams were placed into black waters streams and vice versa). After the transplant the decomposition rates were slightly accelerated, but there was no significative difference between treatments (RM-ANOVA p>0,05). Twenty-one (21) AH species were identified associated to decomposing leaves. No structural change in the AH community was detected after the streams transplant. The sporulation rate and the fungal biomass were higher in the white waters streams. Therefore, we assume that black waters are unfavorable for the reproductive activity of AH.

3
  • CATHERINE BORGES MACÊDO
  • Sequence analysis of the zika virus detection in Aedes aegypti excreta post oral infection

  • Advisor : SILVIA INES SARDI
  • COMMITTEE MEMBERS :
  • SILVIA INES SARDI
  • GUBIO SOARES CAMPOS
  • ARTUR GOMES DIAS LIMA
  • Data: Apr 30, 2019


  • Show Abstract
  • Background & objectives: The zika virus (ZIKV) is transmitted by Aedes aegypti, which found favorable conditions in the Americas for its propagation. The resurgence of outbreaks occurring in cities, led us to investigate the possibility of the ZIKV being kept in nature through a new route of dissemination. The objective of this study was evaluate, after oral infection of Ae. aegypti with ZIKV, replication, viral excretion and infective capacity of the virus in the excreta.  Methods: Mosquitoes were infected with ZIKV and monitored sequentially to assess viral excretion by excreta, midgut, Malpighian tubules and salivary glands were dissected to verify the presence of the virus by RT-qPCR. We analyzed by virus isolation in cell culture the infecting capacity of the virus in excretas. Results: We identified the presence of ZIKV in the excreta between 1 to 3, 6 to 10, 14 and 15 days post infection (dpi). We verified the viral presence in the midgut, Malpighian tubules with 6 and 11 dpi, and salivary glands with 13 and 15 dpi. We confirmed by virus isolation in cell culture the excretion of the virus provably infectious. Interpretation & conclusion: The viral presence at 3 dpi is no longer related to a residual viral load since the total digestion of blood has already occurred. The detection of ZIKV present in the midgut, Malpighian tubules and later in the salivary glands suggests the a viral dissemination. Viral replication in C6/36 cells inoculated with the excreta shows that the virus remains viable. The results suggest a possible potential secondary pathway for propagation of ZIKV in nature.

2018
Dissertations
1
  • PRISCYLA DOS SANTOS RIBEIRO
  • Revisiting leptospiral growth through biofilms: a decrease in growth metabolism

  • Advisor : PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • COMMITTEE MEMBERS :
  • PAULA CARVALHAL LAGE VON BUETTNER RISTOW
  • ANGELA SILVA BARBOSA
  • LUCY SELDIN
  • Data: Nov 28, 2018


  • Show Abstract
  • Biofilms are the main way of life of microorganisms in nature. In this phenotype, bacteria
    have unique metabolic characteristics, including changes in growth such as cell size and
    generation time. Leptospires, whose pathogenic species cause leptospirosis, form biofilms
    in vitro and in natural environments. There is little information on the metabolism of
    leptospires in biofilms. In this work we analyzed physiological and genomic aspects of
    Leptospira growth in biofilms. We cultivated the saprophytic species Leptospira biflexa in
    biofilms (BIOF) and planktonic cells (PLANK) for seven days at 29ºC. We performed
    growth curves by counting bacteria every 12 h. For the cell viability test, we stained the
    biofilms with the Live / Dead reagent, with observation under a fluorescence microscope.
    We measured the cell length for morphological evaluation during the growth phases. We
    analyzed the differential expression of OmpL36 structural protein by Western blotting,
    comparing BIOF and PLANK. Finally, we performed analysis of growth gene homologies
    in the complete genome of L. biflexa and of the transcriptional expression of the identified
    genes (Bioproject PRJNA288909). BIOF and PLANK cells presented classic growth curves.
    The mean generation time (GT) was 4.9 h (± 0.32) for BIOF, two hours less than for PLANK
    (6.9 h ± 0.24). Despite the faster growth demonstrated by lower GT, the cell concentration
    of BIOF was approximately ten times lower than that of PLANK. The viability test showed
    that leptospires in the biofilm began to die earlier, with 24 h of incubation, during
    exponential phase, which may explain the lower concentration of cells observed for this
    phenotype. In addition, we observed negative regulation of 58% of growth genes in the
    biofilm. This change was more prominent in mature biofilm (48 h) when cells are in
    transition to stationary state. Cell length analyzes showed longer BIOF cells during
    stationary and decline phases. The OmpL36 protein was more expressed in the mature
    biofilm than in planktonic cells. Collectively, our data provide evidence of metabolic
    changes and growth limitation of Leptospira in biofilms.

2
  • JOSY DE LIMA GODOI
  • Phenotypic and molecular identification of E. coli strains from shrimp (Litopenaeus vannamei) obtained of informal trade in a tropical climate region

  • Advisor : MELISSA HANZEN PINNA VALENTIM
  • COMMITTEE MEMBERS :
  • MELISSA HANZEN PINNA VALENTIM
  • JOICE NEVES REIS PEDREIRA
  • JOSE ROBERTO PINHO DE ANDRADE LIMA
  • Data: Nov 29, 2018


  • Show Abstract
  • Many of the commercialization of shrimp in tropical countries is in natura and carried out through informal trade, with a precarious hygienic-sanitary condition, favoring the contamination of the food, mainly by E. coli and consequent risk of enteric infection for the population, through exposure to virulent ESBL-producing strains with relevance to public health. The objective of this study was to investigate virulence and resistance genes in E. coli isolates obtained from shrimp (Litopenaeus vannamei), marketed informally, through phenotypic and molecular identification. For this, 22 shrimp samples (500g, each) were collected. After maceration and serial dilution, the colimetric index was determined for total and thermotolerant coliforms and then the culture for isolation and phenotypic identification by the automated system (VITEK® 2), as well as the test for microbial resistance. As for the virulence and resistance genes, the polymerase chain reaction was carried out for molecular characterization. There were obtained 24 E. coli isolates, including 17 commensal strains (17/24 – 70,8%) and seven pathogenic strains (7/24 – 29,2%), including four EIEC isolates (4/7 – 57,1%), two STEC isolates (2/7 – 28,6%); an O157: H7 serotype EHEC (1/7 – 14,3%) and an STEC / ETEC hybrid (1/7 – 14,3%). The gene encoding ESBL CTX-M was identified in 25% of the isolates (one EHEC and five commensals). The identification of E. coli pathophyses with resistance profile in shrimps demonstrates gene circulation and relevance for public health since it contributes to the occurrence of infections resistant to the therapeutic approach, mainly in countries with tourist purposes, which receive people of different nationalities, which ratifies the importance of food safety for public health.

3
  • AMANDA OLIVEIRA DOS SANTOS MELO NASCIMENTO
  • Phenotype to genotype: Genome wide-based metabolic reconstruction of ligninolytic Klebsiella strains isolated from Caatinga biome

  • Advisor : THIAGO BRUCE RODRIGUES
  • COMMITTEE MEMBERS :
  • THIAGO BRUCE RODRIGUES
  • PEDRO MILET MEIRELLES
  • AMARO EMILIANO TRINDADE SILVA
  • Data: Nov 29, 2018


  • Show Abstract
  • Lignin is a three-dimensional amorphous macromolecule formed by binding, predominating
    β-aryl ether between coumaril, synapyl and coniferyl alcohols. Justified by the recalcitrance
    of lignin, the search for naturally biodegrading bacteria lignin has been the subject of studies
    because they are easy to grow and manipulate, but it does not have a complete understanding
    of how bacteria have the ability to degrade lignin. Aiming to identify and characterize the
    lignin degradation system of environmental prokaryotes, we used dependent and
    independent approaches to culture. In order to achieve this, in chapter 1, culture-dependent
    approaches allowed taxonomic identification and functional characterization of lineages of
    bacteria FP10-5.22 (Klebsiella variicola), P3TM1 (Klebsiella variicola) and FP10-5.23
    (Klebsiella oxytoca), which presented growth capacity in lignin as the sole carbon source
    (stationary phase in 24 hours) and up to 98% degradation of the methylene blue dye in 48
    hours. In chapter 2, the data referring to the sequencing of the complete genome of the
    ligninolytic line P1CD1 (K. variicola) are presented. The sequencing of the complete
    genome allowed the identification of the genetic repertoire involved in the degradation of
    lignin, as well as to establish the possible metabolic routes used by the lineage P1CD1 to
    degrade lignin. 251 genes related to transferase, dehydrogenase, lyase,
    transcriptional/regulatory, monooxygenase, dioxygenase, reductase, hydrolase, peroxidase,
    isomerase, oxidase, transport, ligase, hydroxylase, superoxide and redox proteins have been
    identified. The identified genetic repertoire suggests that lignin is degraded in two steps; the
    first step relates to the depolymerization of the primary lignin molecule (decoupling of the
    precursor alcohols interunit bonds) by the activity of the lignin-modifying enzymes (LMEs)
    and the auxiliary of lignin degradation enzymes of (LDAs) generating peripheral
    metabolites. The second stage is related to the decomposition of lignin-derived fragments
    through the activity of the enzymes present in the subsystems belonging to the metabolism
    of aromatic compounds, transforming the peripheral metabolites into intermediary lignin
    metabolites. The results of chapters 1 and 2 suggest that prokaryotes of Caatinga water and
    soil samples are a viable alternative for the optimization of the pre-treatment stage of
    lignocellulosic biomass, since they presented high ligninolytic potential after genomic and
    phenotypic analysis.

4
  • LARISSA MORAES DOS SANTOS FONSECA
  • Emerging infectious diseases research: Mayaro, Oropouche, and Rocio  virus
  • Advisor : GUBIO SOARES CAMPOS
  • COMMITTEE MEMBERS :
  • GUBIO SOARES CAMPOS
  • LUIS GUSTAVO CARVALHO PACHECO
  • ELISANGELA DE JESUS CAMPOS
  • Data: Nov 30, 2018


  • Show Abstract
  • Arbovirus (Arthropod-borne viruses) are viruses transmitted through the bite of hematophagous arthropods and, therefore, responsible for causing diseases known as arboviruses. With very similar and unspecific clinical symptoms, such as fever, headache, myalgia, arthralgia and muscle pain, arboviruses represent a serious global public health problem with relevant social and economic impacts. Among the Brazilian arboviruses, Chikungunya (CHIKV), Dengue (DENV), Yellow Fever (YFV), Mayaro (MAYV), Oropouche (OROV), Rocio (ROCV) and Zika (ZIKV) viruses are responsible for most cases of human arboviruses in Brazil. However, except for CHIKV, DENV, YFV, and ZIKV, there is no information about the circulation of these viruses in Bahia State. The emergence of arboviruses such as CHIKV and Zika ZIKV in urban centers of Brazil highlights the concern with the emergence of other arboviruses not previously found in other regions beyond the Amazon Region. The aim of this study was to investigate the presence of MAY, ORO and ROC viruses in serum, urine and saliva samples of patients living in the State of Bahia with clinical suspicion of dengue. These patients manifested a febrile symptom in addition to other symptoms such as arthralgia, headache, myalgia, vomiting and diarrhoea. A total of 554 samples from patients from a private hospital in Salvador, Brazil, were tested for DENV, CHIKV and ZIKV. RT-PCR and nested-PCR were performed for the detection of MAY, ORO and ROC viruses. No samples were positive for MAYV and ROCV and 18 samples were positive for OROV among them 6 of serum, 7 of saliva and 5 of urine. This was the first time OROV was identified in Brazil outside the Amazon region, in Bahia State, and in samples of saliva and urine. Sequencing of the gene fragment coding for the N and NSs proteins of the 7 positive samples was performed and phylogenetic analysis showed that the virus present in Bahia State was grouped in genotype I, which is being the most distributed in the Brazilian Amazon region. The detection of OROV in Bahia shows the need for an efficient surveillance system to control the spread of this virus in the population. In addition, this work suggests the use of saliva and urine samples as an alternative for the detection of OROV when serum samples are not available.

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