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Brazilian purpuric fever is a febrile hemorrhagic pediatric disease caused by Haemophilus influenzae biogroup aegyptius , a bacterium which was formerly associated with only self-limited purulent conjunctivitis. Here, we present draft genomes of strains from five Brazilian purpuric fever cases and one conjunctivitis case.

Levofloxacin is a synthetic broad-spectrum antibacterial agent for oral or intravenous administration. Chemically, levofloxacin is the levorotatory isomer (L-isomer) of racemate ofloxacin, a fluoroquinolone antibacterial agent. Quinolone derivatives rapidly and specifically inhibit the synthesis of bacterial DNA. Levofloxacin hasin vitroactivity against a broad range of aerobic and anaerobic Gram-positive and Gram-negative bacteria. However, formulation of combined poloxamers thermoregulated (as Pluronic® F127) and levofloxacin for use in multiresistant bacterial treatment were poorly described in the current literature. Thus, the aim of the present work is to characterize poloxamers for levofloxacin controlled release and their use in the treatment of multidrug bacterial resistance. Micelles were produced in colloidal dispersions, with a diameter between 5 and 100 nm, which form spontaneously from amphiphilic molecules under certain conditions as concentration and temperature. Encapsulation of levofloxacin into nanospheres showed efficiency and enhancement of antimicrobial activity againstEscherichia coli,Pseudomonas aeruginosa, andKlebsiella pneumoniaewhen compared with only levofloxacin. Furthermore, all formulations were not cytotoxic for NIH/3T3 cell lineage. In conclusion, poloxamers combined with levofloxacin have shown promising results, better than alone, decreasing the minimal inhibitory concentration of the studied bacterial multiresistance strains. In the future, this new formulation will be used after being tested in animal models in patients with resistant bacterial strains.

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.

Orientadores: Marcelo Lancellotti, Luciana Maria de Hollanda Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A Febre Purpúrica Brasileira (BPF) é uma doença infantil sistêmica caracterizada por conjuntivite, febre, púrpura e sepse. Seu agente etiológico é o Haemophilus influenzae biotipo aegyptius (Hae), uma bactéria anteriormente somente relacionada a casos de conjuntivite purulenta. Os fatores de virulência desse patógeno ainda não são totalmente conhecidos. Assim, o presente estudo teve por objetivo aprofundar o conhecimento sobre esta bactéria. Para tal, nove linhagens de Hae, das quais sete são causadoras de BPF, foram comparadas quanto ao seu genoma, à quantificação da expressão de possíveis genes de virulência e ao perfil de lipídios em infecções in vitro e in vivo. Linhagens causadoras de BPF apresentaram genes ausentes nas demais linhagens Hae estudadas, tais como crgA e hadA. Hae não foi competente ao processo de transformação e análises dos genomas de Hae revelaram a ausência de genes relacionados à competência deste processo no gênero Haemophilus, o que justifica a incapacidade de manipulação genética nesta bactéria. Os genes hadA e tabA foram expressos de forma distinta em linhagens de Hae nos hospedeiros in vitro e in vivo, não sendo possível inferir seu papel na virulência de Hae. Enquanto que o gene las parece ser modulado negativamente em contato com o hospedeiro. Apenas linhagens causadoras de BPF foram recuperadas de ratos Sprague-Dawley, o que sugere a presença de determinantes específicos de virulência que auxiliam na sobrevivência no hospedeiro nestas linhagens. Dados de espectrometria de massas dos lipídios totais mostraram modificações em Hae após a passagem in vivo. O conjunto de resultados mostra que, apesar de semelhantes entre si quanto ao genoma, as linhagens analisadas de Hae associadas a BPF não apresentam característica clonal. Tais linhagens apresentam fatores de virulência específicos possivelmente correlacionados à ocorrência de BPF, os quais são candidatos promissores para estudos futuros sobre a doença Abstract: Brazilian Purpuric Fever (BPF) is a fulminant pediatric disease characterized by conjunctivitis, fever, purpura and sepsis. The illness is caused by Haemophilus influenzae biotype aegyptius (Hae), a bacterium which was formerly associated with only self-limited purulent conjunctivitis. The virulence factors of Hae are not yet fully known. Therefore, this study proposes a better understanding of this pathogen. Nine strains of Hae, of which seven are associated with BPF, were compared for their genomes, expression of candidate genes for virulence and characterization of Hae's lipids in in vitro and in vivo infections assays. Hae associated with BPF presented genes that are absent in other Hae strains studied, such as crgA and hadA. Hae was not competent to transformation process and analysis of Hae genomes revealed the absence of genes related to competence in Haemophilus, which could explain the inability to genetically manipulate this bacterium. hadA e tabA genes showed distinct profiles of expression in Hae in hosts in vitro and in vivo, therefore it was not possible to infer their role in Hae¿s virulence. While las gene seems to be modulated negatively after interation with the host. Only Hae strains associated with BPF were recovered from infected Sprague-Dawley rats, suggesting that these strains acquired specific virulence determinants which assist in survival in the host. Mass spectrometry data of total lipids suggest that Hae suffers modifications after passing through the animal model. The findings showed that the strains analyzed associated with BPF not exhibit clonal characteristic, despite the similarities in their genomes. These strains have specific virulence factors possibly correlated with the occurrence of BPF and are promising candidates for future studies on the disease Doutorado Bioquímica Doutora em Biologia Funcional e Molecular FAPESP 2011/01319-5; 2012/15046-3 CNPQ 141710/2011-0

Astrocytomas, or glioblastomas, are aggressive malignancies of the central nervous system which has poor prognosis, even when submitted to surgery, radiation and chemotherapy. Established permanent cell lines are valuable tools for the study of the glioma pathology, diagnostic and treatment. In this work, were analyzed the adhesion, induction of apoptosis and chemokines’ expression of the bacterial pathogen Neisseria meningitidis in the astrocytome cell line NG97. The analysis of the adhesion and morphological alteration by optical microscopy showed significant alterations mediated by meningococci infection in NG97 cells. Scanning-electron microscopy assays demonstrated a decrease in the number of microvilli on the cell surface as well as the meningococcal adhesion to the cells matrix when compared with the non-infected NG97 cells. Cells infected with different meningococci strains revealed activation of caspase-3, an apoptotic route. Also, the induction of apoptosis in NG97 cells infected was demonstrated by an increased pro- inflammatory chemokines expression, such as TNFα. These data suggest that N. meningitidis is a useful tool in the war against cancer.

Background H. influenzae is a natural competent bacterium that can uptake DNA from the environment and recombine into bacterial genome. The outbreaks of Brazilian purpuric fever, heavily polluted areas of a different H. influenzae biogroup - aegyptius - as well as gene transference between Neisseria meningitis make the transformation process an important evolutionary factor. This work studied the horizontal transference of the ompP2 gene from a multiresistant strain of H. influenzae 07 (NTHi), under the influence of graphene oxide nanoparticles in order to mimic an atmosphere rich in suspended particles and this way verify if the CFU transformants number was increased. Material and methods In this article the gene ompP2 was transformed into different strains of H. influenzae mediated or not by graphene oxide nanoparticles in suspension, followed by the adhesion tests in Hec-1B (human endometrium adenocarcinoma) and A549 (pulmonary epithelial carcinoma) cells lines. The transformation frequency and the adhesion capacity were determined in all the mutants to which the ompP2 gene was transferred and compared to their wild type strains. Results The nanoparticles increased the transformation ratio of one particular strain isolated from a pneumonia case. The adhesion patterns to A549 and Hec1b cell lines of these mutated bacteria has their capacity increased when compared to the wild type. Conclusions Graphene oxide nanoparticles aid the transformation process, helping to increase the number of CFUs, and the mutants generated with the ompP2 gene from a H. influenzae resistant strain not only present a chloramphenicol resistance but also have an increased adherence patterns in A549 and Hec1B cell lines.