Bacterial infections are becoming progressively more difficult to treat due to antibiotic resistance and the decreasing rate at which new antibiotics are brought to market. The Turos laboratory has attempted to tackle this problem for the last 15 years with the discovery of N-thiolated β-lactams leading to the design of polyacrylate nanoparticles to deliver these and other drugs. Chapter 1 discusses many reported instances of utilizing different types of polymeric nanoparticles to deliver antibiotics. Poly(lactic-co-glycolic acid) (PLGA), poly(alkyl cyanoacrylate) (PACA), poly(styrene-co-butylacrylate), and chitosan are the main polymers used to make nanoparticles. Chapter 2 describes the synthesis, antibacterial activity, and mechanism of action of N-acyl ciprofloxacins, which have demonstrated in vitro bioactivity against Staphyloccocus aureus, methicillin-resistant Staphylococcus aureus, Bacillus anthracis, Enterococcus faecalis, Bartonella, and E. coli. Antimicrobial activity was found to diminish with increasingly lipophilic acyl groups of the derivatives. The N-acyl ciprofloxacins were found to utilize the same mechanism of action as the parent drug, ciprofloxacin, however, several exhibited lower mutation frequencies. Chapter 3 discusses the use of the N-acyl ciprofloxacins as probes for experimentation on the poly(vinyl benzoate) nanoparticles. These compounds were incorporated into poly(vinyl benzoate) nanoparticles, also designed in the Turos laboratory, to determine the effects of the lipophilic acyl groups on drug loading and drug release. N-acyl ciprofloxacins with higher lipophilicities (predicted logP values) experienced higher drug loading than the less lipophilic counterparts. However, the nanoparticles were unable to release large amounts of entrapped drug. N-acyl ciprofloxacins with increased hydrophilicity were found to either not be incorporated at all, or in incredibly small amounts. Drug release studies of these drugs indicate that possible the hydrophobic compounds that were associated with the nanoparticles were done so via adsorption onto the surface resulting in a burst release of drug. The work is concluded in Chapter 4, followed by experimental procedures and spectral data in Chapters 5 and 6.

Pas de résumé français

Dissertation (M.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 71-84).

Made available in DSpace on 2016-03-28T12:47:36Z (GMT). No. of bitstreams: 2 andrea_cabral_ioc_mest_2013.pdf: 830806 bytes, checksum: f948411de69b8059eee07de1790cd7c0 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / As bartoneloses são doenças mundialmente dispersas causadas por bactérias gram negativas do gênero Bartonella. Com mais de 24 espécies reconhecidas, B. bacilliformis, B. henselae e B. quintana são os principais e mais comuns agentes causadores de doença em humanos. Na literatura existem relatos de casos isolados e alguns estudos de prevalência sorológica sobre a doença, a maioria realizados em pacientes adultos, com escassa informação sobre a sua apresentação e a epidemiologia nas crianças. A proposta desse estudo retrospectivo foi analisar uma série casos de bartonelose em pacientes abaixo de 16 anos, no estado do Rio de Janeiro, durante o período de 2006 a 2012, a partir dos dados secundários obtidos no banco de dados do Laboratório de Referência Nacional para Rickettsioses, Laboratório de Hantavirose e Rickettsioses do Instituto Oswaldo Cruz, Rio de Janeiro. Dos 36 confirmados por análise sorológica utilizando teste de imunofluorescência comercial, com títulos de corte de 64, e/ ou por reação em cadeia da polimerase, 19 casos (52,7%) foram do sexo feminino, com uma variação por faixa etária de zero a 16 anos, 19 (52,7%) tinham entre 11 e 16 anos de idade A maioria dos casos - 22 casos (61,1%) -, foi procedente do município do Rio de Janeiro, com mais cinco (13,8%) e três (8,3%) em pacientes residentes nos municípios de Duque de Caxias e Nova Iguaçu. A informação sobre contato com gato estava disponível em apenas sete (19,4%). As manifestações clínicas observadas neste estudo foram semelhantes às descritas na literatura, na qual foi possível identificar doença da arranhadura do gato (DAG), febre de origem prolongada e forma hepatoesplênica. Assim, das fichas com dados disponíveis, foi possível verificar que 30 de 32 (94%) e 25 de 29 (90%) dos pacientes apresentaram febre e linfadenopatia, respectivamente. Hepatoesplenomegalia foi identificada em quatro dos 16 pacientes, cujas fichas tinham a informação disponível. Dor abdominal, observada em 33% dos pacientes, associada com náuseas, vômitos, hepatoesplenomegalia e diarreia, em concordância com relatos de casos descritos na literatura, apontam para a dificuldade do diagnóstico diferencial com doenças comuns nas faixas etárias mais baixas, chamando a atenção para as gastroenterites Apesar da restrição de um estudo retrospectivo e da inexistência de uma ficha epidemiológica específica para bartoneloses - as informações foram obtidas da ficha epidemiológica para febre maculosa-, os resultados deste estudo confirmam a necessidade de inclusão das bartoneloses no diagnóstico diferencial de doenças febris associadas com adenomegalia e do estabelecimento desta zoonose como doença de notificação compulsória no Brasil / Bartonelloses are globally dispersed diseases caused by gram-negative bacteria from Bartonella genus. With over 24 recognized species, B. bacilliformis, B. henselae and B. Quintana are the main and most common disease-causing agents in humans. In the literature, there are reports of isolated cases and some seroprevalence studies about the disease, most performed in adult patients, with limited information about its presentation and epidemiology in children. The purpose of this retrospective study was to analyze a case series of bartonellosis in patients aged less than 16 years, in the State of Rio de Janeiro, during the period 2006-2012. We assessed secondary data from the National Reference Laboratory for Rickettsiosis, Laboratory of Hantaviroses and Rickettsioses of Oswaldo Cruz Institute, Rio de Janeiro. Nineteen cases (52,7%), from 36 confirmed by serological analysis using commercial immunofluorescence test, with antibody cut off titers of 64 , and / or polymerase chain reaction, were female, with an age variation from zero to 16 years old, 19 (52.7%) were between 11 and 16 years old. Most of cases - 22 (61.1%) – were found in the city of Rio de Janeiro, with another five (13.8%) and three (8.3%) in patients residing in the cities of Duque de Caxias and Nova Iguaçu. Information about contact with cats was available in just seven (19,4%). Clinical manifestations observed in this study were similar to those described in the literature, in which it was possible to identify cat scratch disease (CSD), fever of unknown origin, and hepatosplenic form. Thus, from the records with available data, it was possible to find that 30 of 32 (94%) and 25 of 29 (90%) of the patients had fever and lymphadenopathy, respectively. Hepatosplenomegaly was identified in four of the 16 patients whose records had information available. Abdominal pain, occurring in 33% of patients, associated with nausea, vomiting, diarrhea and hepatosplenomegaly, in accordance with reported cases in the literature point to the difficulty of the differential diagnosis of common diseases in the lower age groups, drawing attention to gastroenteritis. Despite the restrictions of a retrospective study and the absence of a specific epidemiological record for bartonella - Information has been obtained from epidemiological record for spotted fever -, the results of this study confirm the need for inclusion of bartonellosis in the differential diagnosis of febrile illnesses associated with lymph node enlargement and the establishment of this zoonosis as a reportable disease in Brazil

Made available in DSpace on 2016-06-02T19:22:03Z (GMT). No. of bitstreams: 1 2193.pdf: 1048976 bytes, checksum: 7425ebc2647d9f4d59a0843f3908283e (MD5) Previous issue date: 2008-10-12 / Universidade Federal de Minas Gerais / Many studies have demonstrated that infection by Bartonella vinsonni subspecies berkhoffii may cause cardiac arrhythmia, myocarditis and infectious endocarditis in humans. There is strong evidence that Bartonella vinsonni is an important cardiac pathogen in both humans and dogs The Bartonella sp has been associated with several human diseases, being responsible for a number of zoonoses. The aim of this work was to identify and correlate the Bartonella vinsonni subsp. berkhoffii with the cardiac disease by using specific oligonucleotides as Bh16SF and Bh16SR, which amplify a 185-bp fragment of 16S rRNA. The PCR technique was used for detection of this bacterium in human leukocytes. The amplification products were analyzed in 1.5% agarose gel in order to confirm the 185-bp fragment, which in turn indicates the presence of the bacterium. Thirty eight (39%) out of the 96 humans with cardiopathies as well as 16 (15%) out of the 104 humans from the control group showed positive PCR amplification for Bartonella vinsonni subsp. berkhoffii, thus suggesting a positive correlation of this subspecies with the cardiopathies. Sequencing of the amplified product confirmed the presence of Bartonella vinsonni subspecies berkhoffii. / Estudos comprovam a existência de casos de arritmia cardíaca, miocardite e endocardite infecciosa causados por infecção pela bactéria Bartonella vinsonni subespécie berkhoffii em humanos. Há uma grande evidência que a espécie da Bartonella vinsonni é um patógeno cardíaco importante em humanos e cães. A Bartonella sp está associada a uma variedade de doenças humanas, sendo considerado uma zoonose. O objetivo deste trabalho foi identificar e correlacionar a bactéria Bartonella vinsonni subespécie berkhoffii com a patologia cardíaca utilizando oligonucleotídeos específicos Bh16SF e Bh16SR, que amplificam um segmento de 185 pb para o gene 16SrRNA e averiguar, por PCR, a presença de bactérias em leucócitos humanos. Os produtos de amplificação foram analisados em gel de agarose 1,5% para confirmação da amplificação do fragmento de DNA de 185 pb que indica a presença da bactéria. Trinta e oito (39%) dos 96 humanos cardiopatas e dezesseis (15%) dos 104 humanos do grupo controle apresentaram amplificação positiva por PCR para a bactéria Bartonella vinsonni subespécie berkhoffii, sugerindo uma correlação entre a bactéria e a cardiopatia. Análise por sequenciamento do material amplificado confirmou a presença da Bartonella vinsonni subespécie berkhoffii. Estes achados confirmam a existência de uma correlação entre a presença da bactéria Bartonella vinsonni subespécie berkhoffii e problemas cardíacos em humanos.

Submitted by Luiz Ricardo Gonçalves null (tatinho_tva@hotmail.com) on 2016-07-27T18:25:27Z No. of bitstreams: 1 dissertação LRG.pdf: 2432897 bytes, checksum: 0ce4cc1f4bd2a278c07c6602aa1453d0 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-07-29T13:34:29Z (GMT) No. of bitstreams: 1 golcalves_lr_me_jabo.pdf: 2432897 bytes, checksum: 0ce4cc1f4bd2a278c07c6602aa1453d0 (MD5) / Made available in DSpace on 2016-07-29T13:34:29Z (GMT). No. of bitstreams: 1 golcalves_lr_me_jabo.pdf: 2432897 bytes, checksum: 0ce4cc1f4bd2a278c07c6602aa1453d0 (MD5) Previous issue date: 2016-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Patógenos transmitidos por vetores artrópodes são mundialmente importantes, podendo causar enfermidades tanto no homen quanto nos animais. Recentemente, diversos estudos têm sido realizados a fim de elucidar o papel dos animais selvagens na epidemiologia desses patógenos. A identificação desses microrganismos, assim como dos seus vetores e hospedeiros, permite mapear áreas de ocorrência desses patógenos, auxiliando os órgãos competentes na elaboração de medidas de controle. Sendo assim, o presente estudo teve como objetivo detectar e caracterizar, por métodos moleculares, micoplasmas hemotróficos e bartonelas em amostras de baço de roedores selvagens e sinantrópicos distribuídos em cinco biomas brasileiros. Para tal, foram analisadas 500 amostras de roedores pertencentes a 51 espécies distribuídas em 13 estados. Dentre as 457 amostras de DNA positivas nos ensaios de PCR baseados nos genes Interphotoreceptor retinoid binding protein [IRBP] ou Glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 100 (21,9%) mostraram-se positivas para Mycoplasma spp. por meio da cPCR baseada no gene 16S rRNA. A análise filogenética de 24 sequências do gene 16S rRNA mostrou a presença de dez diferentes clusters, todos agrupados dentro do grupo Mycoplasma haemofelis. O posicionamento filogenético associado com a baixa porcentagem de identidade entre as sequências amplificadas no presente estudo e aquelas previamente depositadas no Genbank sugere a circulação de novas espécies de hemoplasmas em roedores no Brasil. Por meio da qPCR, 117 (25,6%) amostras mostraram-se positivas para Bartonella sp. A análise de diversidade das sequências do gene gltA mostrou a presença de 15 haplótipos. A relação filogenética entre as sequências do gene gltA mostrou que a espécie de Bartonella detectada em roedores no Brasil é intimamente relacionada com Bartonella sp. denominada como grupo filogenético A detectada em roedores da Família Cricetidae na América do Norte, provavelmente formando uma única espécie ou um complexo de espécies. Por outro lado, as sequências amplificadas por meio de ensaios de cPCR adicionais, baseados nos genes ftsZ, groEL e pap-31, mostraram-se filogeneticamente próximas ao complexo Bartonella vinsonii. Por fim, as sequências de Bartonella amplificadas no presente estudo formaram um grupo monofilético e amplamente difundidas em sete diferentes espécies de roedores amostrados em três biomas. Tais achados sugerem uma provável dominância desse genótipo entre os roedores silvestres no Brasil. Copositividade para hemoplasmas e Bartonella sp. foi observada em 41 (9%) dos roedores amostrados. O presente estudo demonstrou, por meio de métodos moleculares, a ocorrência de micoplasmas hemotróficos e Bartonella sp. em roedores selvagens e sinantrópicos no Brasil. / Arthropod-borne pathogens are wordwide important, causing diseases in humans and animals. Recently, several studies have been performed in order to elucidate the role of wild animals in the epidemiology of these pathogens. The identification of these microorganisms, as well as their vectors and hosts, allow to map the areas of occurrence of these pathogens, helping competent agencies in the development of control measures. Therefore, the present study aimed to detect and characterize, using molecular methods, hemotrophic mycoplasmas and Bartonella in wild and sinantropic rodent spleen samples distributed in five Brazilian biomes. For this purpose, 500 rodent samples belonging to 51 species distributed in 13 states were analyzed. Among 457 DNA samples positive to PCR assays based on the Interphotoreceptor retinoid binding protein [IRBP] or Glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) genes, 100 (21.9%) were positive to Mycoplasma spp. by cPCR based on 16S rRNA gene. The phylogenetic analysis of 24 sequences of the 16S rRNA gene showed the presence of ten different clusters, which grouped within the Mycoplasma haemofelis group. The phylogenetic position associated with the low percentage of identity between the sequences amplified in the present study and those previously deposited in Genbank suggest the circulation of new hemoplasmas species in rodents in Brazil. Additionally, 117 (25.6%) samples were positive to Bartonella sp. by qPCR. The diversity analysis of the gltA gene sequences showed the presence of 15 haplotypes. The phylogenetic relationships between the sequences of gltA gene showed that Bartonella species detected in rodents in Brazil is closely related to Bartonella sp. namely phylogenetic group A detected in the rodents belonging to Cricetidae family in North America, probably constituting only one species, or a complex of species. On the other hand, the amplified sequences by additional cPCR assays based on ftsZ, groEL and pap-31 genes were phylogenetically positioned to Bartonella vinsonii complex. Finally, the Bartonella sequences amplified in the present study formed a monophyletic and widespread group in seven different species of rodents sampled in three biomes. These findings suggest a probable dominance of this genotype among wild rodents in Brazil. Co-positivity was observed in 41 (9%) of rodents sampled. The presente study demonstrated using molecular methods, the occurrence of hemotrophic mycoplasmas and Bartonella sp. in wild and synanthropic rodents in Brazil.

Amostras de sangue total e suabes de orofaringe e conjuntiva foram coletadas de 57 felídeos Neotropicais (1 Leopardus geoffroyi, 17 L. wiedii, 22 L. tigrinus, 14 L. pardalis e 3 Puma yagouaroundi) mantidos em cativeiro no Refúgio Bela Vista, Foz do Iguaçu. Dados clínicos, hemograma e histórico dos animais foram disponibilizados. Materiais clínicos obtidos a partir dos suabes de orofaringe e conjuntiva foram submetidos ao cultivo para Mycoplasma spp em meio específico. DNA do sangue e suabes foram extraídos por meio de um kit comercial e pelo método de fervura, respectivamente. DNA extraído de amostras de sangue foram submetidos à PCR para detecção de Mycoplasma haemofelis (Mhf), Candidatus M. haemominutum (CMhm), DNA proviral do vírus da leucemia felina (FeLV) e Bartonella spp. DNA extraído dos suabes foram submetidos à PCR para detecção de Mollicutes e M. felis. Foi realizada uma análise de associação entre alterações clínicas e a infecção por Bartonella spp e um estudo de fator de risco da infecção por esse microrganismo. Apenas 1 (1,75%) animal foi positivo a reação para Mhf e nenhum foi positivo a reação para CMhm. Dois (3,5%) animais foram positivos a reação para FeLV e 6 (10,52%) foram positivos para Bartonella spp. Não houve co-infecção entre os agentes pesquisados nas amostras de sangue. Foram obtidos 5 (8,77%) isolados de Mycoplasma spp da orofaringe e nenhum de conjuntiva. DNA de Mollicutes foi detectado em 53 (93%) e 27 (47,36%) amostras de orofaringe e conjuntiva, respectivamente. Nenhuma amostra apresentou resultado positivo na detecção de DNA alvo de M. felis. Não houve associação entre as alterações hematológicas (anemia, desidratação, leucocitose, leucopenia, histórico de anemia) e a infecção por Bartonella spp. Machos apresentam maior risco de adquirir bartonelose do que fêmeas. Este é o primeiro relato da presença de DNA proviral de FeLV em L. tigrinus e L. pardalis no sul do aís, de DNA de B. henselae em L. tigrinus, L. pardalis, L. geoffroyii e P. yagouaroundi, e de um estudo de fator de risco associado a infecção por Bartonella spp em felídeos neotropicais. / Total blood samples and oropharinx and conjunctival swabs were collected from 57 neotropical felids (1 Leopardus geoffroyi, 17 L. wiedii, 22 L. tigrinus, 14 L. pardalis and 3 Puma yagouaroundi) maintained in captivity at Refúgio Bela Vista, Foz do Iguaçu. Clinical data, hemogram and clinical history of these animals were available. Clinical materials obtained from oropharinx and conjunctiva were cultured in specific media for Mycoplasma spp isolation. DNA of blood and swabs were extracted using a commercially available kit and a boiling method, respectively. DNA samples from swabs were submitted to a PCR for the detection of Mollicutes and M. felis. DNA samples from blood were submitted to a PCR for detection of Mycoplasma haemofelis (Mhf), Candidatus M. haemominutum (CMhm), feline leukemia virus (FeLV) proviral DNA , and Bartonella spp. The association between hematological alterations and bartonella infection was evaluated and a risk factor analysis was performed. Only 1 (1.75%) animal was positive for Mhf reaction, whereas all animals were negative for CMhm detection. Two (3.5%) animals were positives for FeLV and 6 (10.52%) animals were positive for Bartonella spp, by PCR. Co-infections among these agents were not observed. Five (8.77%) mycoplasma isolates were obtained from oropharinx samples and none was obtained from conjunctival samples. Mollicutes DNA was detected in 53 (93%) and 27 (47.36%) samples from oropharinx and conjunctiva, respectively. All samples were negative for M. felis detection. Hematological alterations (anemia, dehydration, leukocytosis, leucopenia, history of anemia) were not associated to Bartonella spp infection. Males are more likely to be infected than females. This is the first report of FeLV proviral DNA in L. tigrinus and L. pardalis in Southern Brazil, of B. henselae DNA in L. trigrinus, L. pardalis, L. geoffroyi and P. yagouaroundi, and the first study of risk factors for Bartonella spp infection in neotropical felids.

Carrion’s disease is a neglected tropical disease caused by Bartonella bacilliformis, a vector-borne pathogen restricted to the Andean valleys of Peru, Ecuador and Colombia. Carrion’s disease is a biphasic illness; in the acute phase the case-fatality rate can be as high as 88 %, related to high parasitemia, arriving to almost all erythrocytes, and secondary bacterial infections close related with the development of transient immunosuppression in the earlier illness phases. In addition, there are an undefined number of asymptomatic carriers that are reservoirs of the etiological agent of Carrion’s disease in endemic areas, they make take into account due to they are the perpetuators of this disease. The actual scenario of Carrion’s disease, in which the illness is arriving to new areas, due to the expansion of the vector’s distribution, suggests that now may be a crucial time to design a strategy focusing on its elimination.

Analysis of immune responses in Bartonella bacilliformis carriers are needed to understand acquisition of immunity to Carrion’s disease and may allow identifying biomarkers associated with bacterial infection and disease phases. Serum samples from 144 healthy subjects from 5 villages in the North of Peru collected in 2014 were analyzed. Four villages had a Carrion’s disease outbreak in 2013, and the other is a traditionally endemic area. Thirty cytokines, chemokines and growth factors were determined in sera by fluorescent bead-based quantitative suspension array technology, and analyzed in relation to available data on bacteremia quantified by RT-PCR, and IgM and IgG levels measured by ELISA against B. bacilliformis lysates. The presence of bacteremia was associated with low concentrations of HGF (p = 0.005), IL-15 (p = 0.002), IL-6 (p = 0.05), IP-10 (p = 0.008), MIG (p = 0.03) and MIP-1α (p = 0.03). In multi-marker analysis, the same and further TH1-related and pro-inflammatory biomarkers were inversely associated with infection, whereas angiogenic chemokines and IL-10 were positively associated. Only EGF and eotaxin showed a moderate positive correlation with bacteremia. IgM seropositivity, which reflects a recent acute infection, was associated with lower levels of eotaxin (p = 0.05), IL-6 (p = 0.001), and VEGF (p = 0.03). Only GM-CSF and IL-10 concentrations were positively associated with higher levels of IgM (p = 0.01 and p = 0.007). Additionally, IgG seropositivity and levels were associated with high levels of angiogenic markers VEGF (p = 0.047) and eotaxin (p = 0.006), respectively. Our findings suggest that B. bacilliformis infection causes immunosuppression, led in part by overproduction of IL-10. This immunosuppression probably contributes to the chronicity of asymptomatic infections favoring B. bacilliformis persistence in the host, allowing the subsequent transmission to the vector. In addition, angiogenic markers associated with bacteremia and IgG levels may be related to the induction of endothelial cell proliferation in cutaneous lesions during chronic infections, being possible candidate biomarkers of asymptomatic infections.

The Selection during Niche AdaPtation (SNAP) hypothesis aims to explain how the gene order in bacterial chromosomes can change as the result of bacteria adapting to a new environment. It starts with a duplication of a chromosomal segment that includes some genes providing a fitness advantage. The duplication of these genes is preserved by positive selection. However, the rest of the duplicated segment accumulates mutations, including deletions. This results in a rearranged gene order. In this work, we develop a method to identify SNAP in bacterial chromosomes. The method was tested in Salmonella and Bartonella genomes. First, each gene was assigned an orthologous group (OG). For each genus, single-copy panorthologs (SCPos), the OGs that were present in most of the genomes as one copy, were targeted. If these SCPos were present twice or more in a genome, they were used to build duplicated regions within said genome. The resulting regions were visualized and their possible compatibility with the SNAP hypothesis was discussed. Even though the method proved to be effective on Bartonella genomes, it was less efficient on Salmonella. In addition, no strong evidence of SNAP was detected in Salmonella genomes.