As Stenotrophomonas são comumente encontradas no trato respiratório de pacientes com doenças pulmonares crônicas e também na rizosfera de plantas. Esse gênero apresenta resistência a diversos antibióticos, promove o crescimento de plantas e algumas espécies apresentam a capacidade de fixar o nitrogênio atmosférico. O Multi Locus Sequence Analysis (MLSA) é uma metodologia baseada em genes constitutivos para definição e alocação taxonômica de novas espécies. O objetivo geral do presente trabalho foi caracterizar taxonomicamente uma coleção ampla de Stenotrophomonas composta por isolados endófitos, linhagens-tipo e de referência. Para tanto, foi estabelecido um sistema de classificação e identificação de Stenotrophomonas por meio de MLSA. Foi possível através da metodologia de MLSA definir 9 novas espécies, detectar a presença de um novo gênero e estabelecer um sistema online de taxonomia para Stenotrophomonas. / The genus Stenotrophomonas is found in the respiratory treatment of patients with chronic pulmonary and also in the rizhosfera of plants. It presents resistance to several antibiotics, promotes the growth of plants and some species present the ability to fix atmospheric nitrogen. The Multi Locus Sequence Analysis (MLSA) is a methodology based on constitutive genes for definition and taxonomic allocation of new species. The general objective of the present work was to characterize a wide collection constituted by Stenotrophomonas from isolated endophytic, type and reference strains. In such a way, a system of classification and identification of Stenotrophomonas by means of MLSA was established. It was possible through the MLSA methodology to define 9 new species, to detect the presence of a new genus and to establish an online system for Stenotrophomonas taxonomy.

Quarenta e nove amostras EPEC típica (tEPEC) e atípica (aEPEC) pertencentes a diferentes sorotipos, isoladas de humanos e animais (cães, gatos, bovinos, ovinos, coelhos e sagüis) foram investigadas quanto ao perfil de virulência pela PCR e similaridade clonal por Multilocus Sequence Typing (MLST) e Pulsed-Field Gel Electrophoresis (PFGE). O objetivo deste estudo foi verificar se animais atuam como reservatório e fonte infecção de aEPEC para humanos. Os marcadores de virulência analisados revelaram que cepas aEPEC isoladas de animais possuem potencial para causar diarréia em humanos. As técnicas MLST e PFGE revelaram que amostras isoladas de animais e humanos compartilham relações clonais próximas ou idênticas. Estes resultados indicam que os animais estudados atuam como reservatório de aEPEC e representam fonte de infecção para humanos. Pelo fato de humanos, também atuarem como reservatório de aEPEC, ciclos de infecção cruzada animal-humano não podem ser descartados, pois a dinâmica de transmissão entre reservatórios de aEPEC não é muito bem compreendida. / Forty-nine typical and atypical EPEC strains belonging to different serotypes, isolated from humans, pets (cats and dogs), farm (bovines, sheep and rabbits) and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. Close clonal relationship between human and animal isolates was found with MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out, since the transmission dynamics between the reservoirs are not yet clearly understood.

Flagelinas de bactérias gram-negativas ativam o sistema imune inato resultando em efeitos adjuvantes para antígenos co-administrados. No entanto, não existem relatos sobre as propriedades adjuvantes de flagelinas de bactérias gram-positivas. O objetivo do projeto foi estudar os efeitos imunomoduladores da flagelina Hag de B. subtílis. Inicialmente foi purificada a Hag e subseqüentemente avaliada sua propriedade imunológica quando administrada junto com a ovalmbumina (OVA) pelas vias intra-nasal (i.n.) ou subcutânea (s.c.) em camundongos Balb/C e C57BL/6. Os resultados demonstraram as propriedades imunomoduladoras da Hag nas duas linhagens em relação à indução de anticorpos antígeno-específicos. Não foi possível demonstrar a ativação de linfócitos TCD4+ e CD8+ antígenos-específicos em animais Balb/C imunizados com OVA e Hag tanto pela via s.c. como pela i.n.. Resultados mais promissores do efeito adjuvante frente a células T foram encontrados em animais C57BL/6. Os resultados abrem perspectivas para o estudo do potencial da flagelina Hag de B. subtilis como adjuvante vacinal. / Flagellin of gram negatives bacteria actives the innate immune system resulting in adjuvants effects against coadministrated antigens. However, there is not information about immunological properties of flagellins produced by gram positive bacteria. This study aimed the evaluation of immunomodulator effects of Hag flagellin from B. subtilis. Vaccine formulations based in purified Hag and Ovalbumine protein (OVA) were administrated intranasal (i.n.) and subcutaneously (s.c.) in Balb/C and C57BL/6 mice and then the population of B and T lymphocytes were monitored for production of antibodies and citocines and/or surface markers expression. Results showed the immunomodulatory properties of Hag in both mice lineages regarding the induction of antigen specific antibodies when immunized with OVA and Hag by i.n. and s.c. via. Most promising results concerning the adjuvant effects observed on T cells activation were found in C57BL/6 mice. The results obtained open future perspectives for the study of the potential use of B. subtilis Hag flagellin as adjuvant in vaccines.

A deficiência da proteína C5 do sistema complemento humano é rara com 38 casos relatados na literatura e freqüentemente associada a severas infecções provocadas por bactérias Neisseria. O objetivo do trabalho é caracterizar imunológica e geneticamente esta deficiência encontrada pela primeira vez em brasileiros. Por imunodifusão dupla obtivemos níveis expressivos de C3, C4, C6, C7, C8, C9, Fator B, Fator H e Fator I em todos os membros desta família, a proteína C5 não foi detectada no soro de três irmãos: II:9, II:4 e II:5. Por ELISA a concentração de C5 nestes indivíduos foi (0,9; 1,0; 1,3 µg/ml, 45- 190 µg/ml). Soros destes probandos não apresentam atividade hemolítica mediada pelo sistema complemento. O cDNA de C5 dos indivíduos I:1, I:2, II:4 e II:9 apresenta a deleção do éxon 30. Causada pela substituição de GAG4028 por GAA4028 no último nucleotídeo deste éxon que leva a um erro no splicing. Este defeito provavelmente produz uma proteína incompleta e destinada à degradação. / The deficiency of the C5 component of the complement system is rare with 38 described cases in the literature. This deficiency is frequently associated with severe infections, especially caused by Neisseria. Our objective is to characterize immunologically and genetically this deficiency, the first of its type described in the Brazilian population.We noted that C3, C4, C6, C7, C8, C9, Factor B, Factor H and Factor I have expressive levels in all the individuals sera of this family. C5 was absent in individuals II:4, II:5 and II:9. By ELISA a C5 concentration in this individuals were 0,9; 1,0; 1,3 µg/ml (normal: 45 - 190 µg/ml). Their serum doesn´t present hemolytic activity mediated by complement system. The C5 cDNA from individuals I:1, I:2, II:4 and II:9 has éxon 30 deleted. Caused by the substitution of GAG4028 for a GAA4028 in the last codon of exon 30. This defect was responsible for the deficiency of C5 in this family and this deletion would probably produce an unstable protein destined for degradation.

Methylobacterium mesophilicum SR1.6/6 é uma bactéria endofítica isolada de ramo de citros previamente esterilizado superficialmente. Esta bactéria possui a capacidade de associar-se com uma ampla variedade de espécies e tecidos de plantas, principalmente nas raízes, mediado pela formação de biofilme e superfícies do hipocótilo de plântulas in vitro. Este trabalho teve como objetivo o desenvolvimento e a aplicação de um modelo para o estudo in vitro da associação entre esta bactéria e plântulas de soja. Metodologias de genômica e transcriptômica foram aplicadas para a obtenção do draft genômico desta bactéria e de um amplo perfil de sequências expressas, obtidas em dois tratamentos distintos; i) células de biofilme células bacterianas aderidas às raízes das plântulas removidas por sonicação, e ii) células planctônicas células bacterianas em suspensão (i.e. interagindo somente com exsudados radiculares). Os dados genômicos obtidos por 454-pirosequenciamento resultaram em uma cobertura de 37 vezes o tamanho do genoma e geraram 242 contigs. Entre estes, 187 contigs grandes representaram 96% do genoma (tamanho estimado de 6.8 Mb), com um conteúdo GC de 69.5%. Considerando a análise da expressão gênica, um procedimento para monitorar a aderência das células bacterianas às raízes das plântulas foi realizado por meio de microscopia eletrônica de varredura de amostras de raízes coletadas durante o experimento (i.e. 24, 48 e 72h após a inoculação). As células bacterianas obtidas em cada tratamento foram inicialmente submetidas à extração de RNA total, seguida de um processo de enriquecimento de mRNA e sequenciamento por meio da tecnologia de 454-pirosequenciamento (RNA-Seq). O amplo perfil de expressão gênica obtido foi mapeado no draft genômico, resultando em um total de 1.930 clusters gênicos. Posteriormente, estes clusters foram filtrados de acordo com sua abundância e ocorrência diferencial em cada tratamento, resultando em 280 genes diferencialmente expressos. Funções relacionadas ao metabolismo de etanol/metanol, divisão celular, resposta ao estresse oxidativo, produção de sideróforos, biossíntese de peptidoglicanos e hopanóides foram induzidas nas células bacterianas aderidas às raízes das plântulas, enquanto que genes relacionados ao metabolismo essencial das células foram observados principalmente nos tratamentos controle e planctônico. Estes dados fornecem uma base para estudos relacionados a mecanismos moduladores da interação bactériaplanta, distinguindo significativamente os tratamentos biofilme e planctônico, mostrando assim que o contato físico é essencial para o sucesso da interação em estudo. Por fim, estas análises permitiram uma ampla visualização de perfis de expressão gênica desta bactéria, utilizando o draft genômico primeiramente obtido como base para o estudo desta interação com a planta hospedeira. Estudos futuros podem ser desenvolvidos visando caracterizar os mecanismos adaptativos desta bactéria, como seu metabolismo metilotrófico e outros metabolismos específicos, os quais podem dar suporte ao comportamento endofítico deste organismo. / Methylobacterium mesophilicum strain SR1.6/6 is an endophytic bacterium, which has originally been isolated from surface-sterilized healthy citrus branch. This bacterium is able to associate with a range of plant species, rather in the roots, mediated by a biofilm structure, and in hypocotyl surfaces of in vitro seedlings. The aim of the present study was the development and application of a model to study in vitro the association between this bacterium and soybean seedlings. Genomic and transcriptomic approaches were applied resulting a draft of this bacterium genome and a broad profile of mapped mRNA sequences obtained in two different treatments; i) biofilm cells root adhered bacterial cells were removed by sonication, and ii) planktonic cells bacteria cells in suspension (i.e. interacting only with root exudates). Genomic data, obtained by 454-pyrosequencing have had an average depth of 37-fold coverage of the genome and yielded 242 contigs. Among these, 187 large contigs represented 96% of the genome sequence (estimate size of 6.8 Mb) with a GC content of 69.5%. Concerning the gene expression survey, the process to monitor the adherence of bacteria cells to the roots was performed by scanning electron microscopy of roots collected along the experiment (i.e. 24, 48 and 72 hours after inoculation). Bacterial cells obtained in each treatment were firstly submitted to RNA extraction, followed by mRNA enrichment and RNA-Seq using 454-pyrosequencing technology. The broad gene expression profile obtained was mapped into the drafted genome, resulting in a total of 1.930 gene clusters. After that, these clusters were filtered according to their abundance and differential occurrence in each treatment resulting in 280 differential expressed genes. Functions related to methanol/etanol metabolism, cell division, oxidative stress response, siderophore production, peptidoglycan and hopanoid biosynthesis were induced in bacterial cells adhered to plant roots, while genes related to essential cell metabolism were observed mostly in control and planktonic treatment. Also, these data provide insights into the mechanisms modulating plant microbe-interaction, significantly distinguishing biofilm and planktonic treatment, showing that the physical contact is a crucial step on plant-microbe interactions. In conclusion, results allowed a strongly supported analysis of gene expression, based on the genome draft of an endophytic bacterium interacting with the host plant. Further studies should focus on the adaptive mechanisms present in this bacterium, like the methylotrophic lifestyle and other specific metabolisms which might support its behavior as an endophytic bacterium.

A bacteria Pectobacterium carotovora causa danos a diferentes hospedeiros por meio da produção de enzimas pectinolíticas que degradam o pectato de cálcio da lamela media próximo a parede celular, causando extravasamento do conteúdo celular, sintomas da podridão mole. Bactérias do gênero Burkholderia tem se mostrado capazes em controlar a podridão mole em orquídeas, no entanto, os aspectos moleculares envolvidos neste controle ainda não foram estudados. Neste trabalho, foram avaliados 602 transformantes quanto a sua habilidade em inibir os sintomas da podridão mole, onde foram observados 16 mutantes defectivos no controle da doença. Destes, foram identificados sete diferentes genes inativados pelo transposon Tn5, e estes genes podem estar envolvidos em processos de síntese de aleloquímicos, competição por nutrientes, adaptação a condições ambientais, e na interação com o hospedeiro e/ou entre microrganismos. No entanto, o envolvimento destes genes na perda da capacidade em controlar a podridão mole deve ser melhor estudado. / The bacterium Pectobacterium carotovora cause damage to different hosts and by production of pectic enzymes that degrade calcium pectate of the middle lamella near of the cell wall, causing overflow of cell content and consequently the soft rot. Burkholderia genus has proven able to control the soft rot in Orchids, however, the molecular aspects involved in the control have not been studied. In this work, 602 transformants were characterized for their ability to inhibit soft rot caused by P. carotovora. We identified 16 mutants showing shifts in inhibition pattern or lost of the ablitity to inhibit soft rot symptoms. Among these mutants, we identified 7 genes related to disease inhibition,and this genes may be involved in process of allelochemicals synthesis, competition for nutrients, adapting to environmental conditions, and interaction between the host and microorganisms. However, the involvement of these genes in loss of ability to control the soft rot disease is being further studied in details.

INTRODUÇÃO: As infecções osteoarticulares permanecem como um grande desafio para os profissionais de saúde envolvidos no seu manejo, a despeito do sucesso obtido com a introdução dos antimicrobianos para o tratamento das doenças infectocontagiosas no final da década de 1930. O Staphylococcus aureus (S. aureus) é o agente mais frequentemente encontrado nestas infecções e também é o agente mais estudado, porém possuímos poucas informações disponíveis na literatura médica a respeito das osteomielites por bacilos Gram-negativos (BGN). OBJETIVOS: A caracterização clínica e microbiológica dos episódios de osteomielite causadas por bacilos Gram-negativos. A determinação das diferenças evolutivas e dos fatores de risco para a ocorrência de osteomielite por bacilos Gram-negativos, quando comparadas à osteomielite causada por S. aureus. MÉTODOS: Análise retrospectiva dos casos de osteomielite causadas por bacilos Gram-negativos atendidos no Instituto de Ortopedia e Traumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de janeiro de 2007 a janeiro de 2009. Apenas amostras de osso ou aspirado de canal medular foram consideradas válidas. RESULTADOS: Foram incluídos 89 pacientes no grupo S. aureus e 101 pacientes no grupo BGN. Os pacientes do grupo BGN eram predominantemente do sexo masculino (63%), com mediana de 42 anos de idade. Apresentaram-se com osteomielite crônica (43%) e osteomielite aguda associada à fratura exposta (32%), nos membros inferiores (71%), cuja principal sintomatologia inicial foi a fistulização (69%). Quando comparado ao grupo S. aureus, o grupo BGN estava estatisticamente associado com o antecedente de fratura exposta (35% vs. 18%; p=0,0064), apresentando ainda um maior tempo de internação hospitalar (mediana 41 vs. 24 dias; p=0,0114), maior tempo para a obtenção da primeira cultura positiva (mediana 10 vs. 6,5 dias; p=0,0042), antibioticoterapia mais prolongada (mediana 40 vs. 24 dias; p=0,0329), maior número de procedimentos cirúrgicos (média 3,41 vs. 2,47; p=0,0173) e maior uso de reparo do revestimento cutâneo (31% vs. 9%; p=0,0005). O grupo S. aureus estava estatisticamente associado com as osteomielites da coluna vertebral (23,6% vs. 6,9%; p=0,0008). Foram isolados 121 agentes Gram-negativos de 101 amostras clínicas e os agentes mais frequentes foram Enterobacter spp. (24,7%), Acinetobacter baumannii (21,4%), Pseudomonas aeruginosa (19,8%) e Klebsiella pneumoniae (8,2%). CONCLUSÕES: Os 101 pacientes portadores de osteomielite por BGN eram na sua maioria jovens, do sexo masculino, vítimas de traumas nos membros inferiores e que desenvolveram osteomielite aguda e crônica associadas a fraturas expostas. Os pacientes do grupo BGN necessitaram de um número maior de procedimentos cirúrgicos, maior uso de reparo do revestimento cutâneo, permaneceram internados por mais tempo, necessitaram de um número de dias maior para o isolamento do agente infeccioso e utilizaram antibioticoterapia mais prolongada, quando comparados aos pacientes do grupo S. aureus. O antecedente de fratura exposta foi o principal fator de risco para o desenvolvimento de osteomielite por um BGN, quando comparado ao grupo S. aureus / INTRODUCTION: Bone and joint infection remains a serious therapeutic challenge, despite the high success rate observed with antibiotic therapy in most bacterial disease since the end of 1930 decade. Staphylococcus aureus (S. aureus) is the most studied and the most frequently isolated pathogen, but there is insufficient information in medical literature regarding Gram- negative bacilli (GNB) osteomyelitis. OBJECTIVES: Describe clinical and microbiological characteristics of Gram-negative bacilli osteomyelitis. Establish evolving differences and risk factors for the occurrence of GNB osteomyelitis, compared to S. aureus osteomyelitis. METHODS: Retrospective analysis of all patients with GNB osteomyelitis treated at Institute of Orthopedics and Traumatology, Hospital das Clínicas - School of Medicine, Universidade de São Paulo from january 2007 to january 2009. Only bone or bone marrow aspirate samples were included. RESULTS: 89 patients were included in S. aureus group and 101 patients were included in GNB group. Patients in GNB group were mostly male (63%), with median age of 42 years. At presentation, they had chronic osteomyelitis (43%) and acute open-fracture associated osteomyelitis (32%), in the lower limbs (71%), with a discharging sinus as the main clinical sign (69%). When compared to S. aureus group, GNB group was statistically associated with a previous history of open-fracture (35% vs. 18%; p=0.0064), showed a longer length of hospital stay (median 41 vs. 24 days; p=0.0114), a higher number of days to isolate the infective bacteria (median 10 vs. 6,5 days; p=0.0042), a longer use of antibiotics (median 40 vs. 24 days; p=0.0329), a higher number of surgical procedures (mean 3,41 vs. 2,47; p=0.0173) and a higher rate of soft- tissue reconstruction (31% vs. 9%; p=0.0005). S. aureus group was statistically associated with spine osteomyelitis (23,6% vs. 6.9%; p=0.0008). 121 Gram-negative pathogens were isolated from 101 clinical samples and the most frequent agents were Enterobacter spp. (24.7%), Acinetobacter baumannii (21.4%), Pseudomonas aeruginosa (19.8%) and Klebsiella pneumoniae (8.2%). CONCLUSIONS: Patients with GNB osteomyelitis were mainly young, male, with lower limb trauma and developed chronic and open- fracture associated osteomyelitis. Patients in GNB group had a higher number of surgical procedures, a higher rate of soft-tissue reconstruction, a longer length of hospitalization, a longer time to isolate the infective bacteria and a prolonged use of antibiotics, when compared to patients in S. aureus group. A previous history of open-fracture was the main risk factor to development of GNB osteomyelitis, compared to S. aureus group

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Several compound present in fruits as polyphenols are able to kill or inhibit the growth of microorganisms. These proprieties are relevant mainly in tropical areas, as Amazonian region where infectious are highly prevalent. Therefore, this study investigated the tucumã Amazonian fruit antimicrobial activity against 37 microorganisms. The potential role of oxidative metabolism imbalance was also studied as causal mechanism of antimicrobial activity. The results showed antibacterial effect of pulp and peel tucumã hydro-alcoholic extracts on three gram-positive bacteria (Enteroccocus faecalis, Bacillus cereus, Listeria monocytogenes) and antifungical effect against Candida albicans. The antimicrobial contribution of main chemical compounds (quercetin, rutin, β-carotene and gallic, caffeic and chlorogenic acids) found in tucumã extracts was also investigated showing an inhibitory effect depending of the organism mainly by quercetin in bacteria and rutin in C.albicans. Analysis of kinetic of DNA releasing in extracellular medium by fluorescence using DNA Pico Green assay® and reactive oxygen species production (ROS) showed potential oxidative imbalance contribution on tucumã inhibitory effect. In B.cereus and C.albicans this effect was clear since after 24 hours the ROS levels were higher when compared to negative control group. In conclusion, tucumã extracts present antimicrobial activity to four microorganisms that have large problems of drug resistance, and the possible mechanism of action of this Amazon fruit is related to REDOX imbalance. / Aguns compostos, como por exemplo, os polifenóis presentes em várias frutas, são capazes de matar ou inibir o crescimento de microrganismos. Estas propriedades estão presentes principalmente em frutos de áreas tropicais, como a região amazônica. Portanto, este estudo investigou a atividade antimicrobiana contra 37 microrganismos de um fruto amazônico conhecido como tucumã. O potencial papel de desequilíbrio do metabolismo oxidativo também foi estudado como mecanismo causal da atividade antimicrobiana. Os resultados mostraram efeito antibacteriano para os extratos hidroalcóolicos da polpa e da casca do tucumã em três bactérias gram- positivas (Enterococcus faecalis, Bacillus cereus, Listeria monocytogenes) e efeito antifúngico contra Candida albicans. A contribuição antimicrobiana dos principais compostos químicos (quercetina, rutina, β- caroteno, ácidos clorogênicos, cafeico e gálico) encontrados em extratos do tucumã também foi investigada, apresentando um efeito inibidor dependendo principalmente do microrganismo, em bactérias pela quercetina e no fungo pela rutina. Análise da cinética da liberação de DNA no meio extracelular por fluorescência utilizando o ensaio de DNA Pico Green® e produção de espécies reativas de oxigênio (ROS) mostrou efeito inibitorio do tucumã no potencial desequilíbrio oxidativo. Em B. cereus e C. albicans este efeito foi claro após as 24 horas onde os níveis de ROS foram maiores quando comparados ao grupo controle negativo. Em conclusão, os extratos de tucumã apresentam atividade inibitória de quatro microrganismos que possuem grandes problemas de resistência aos fármacos, e o possível mecanismo de ação deste fruto amazônico está relacionada com o desequilíbrio REDOX.

La surexpression des pompes d’efflux (PE) appartenant à la famille Resistance-Nodulation-Division (RND) est l’un des contributeurs majeurs de la multirésistance (MDR) et la pathogénicité des bactéries Gram-négatives. Ces transporteurs sont capables d'expulser à l’extérieur de la cellule bactérienne différentes classes d'antibiotiques, ce qui contribue de manière significative à l'échec thérapeutique du traitement des maladies infectieuses. Dans ce contexte, les PEs sont des cibles intéressantes pour la découverte de nouveaux antimicrobiens. Afin de combattre ce mécanisme de résistance, des inhibiteurs des pompes d’efflux (EPIs) sont développés comme adjuvants d'antibiotiques dans le but de restaurer ou d'améliorer leur activité. L'archétype AcrAB-TolC est particulièrement répandu chez les espèces d’Enterobacter pertinentes en clinique (pathogènes « ESKAPE »). Cette étude décrit une stratégie basée sur des analogues des fluoroquinolones pour le drug design des EPIs, contre la pompe AcrB chez E. aerogenes. Ainsi, la synthèse et l'évaluation microbiologique des dérivés de quinazoline-4(3H)-one ont été effectuées. Les propriétés structurales et moléculaires des composés testés (i.e. rigidité et flexibilité) ont également été étudiées. Pour cela, de nouveaux scaffolds ont été évaluées. Plusieurs molécules ont montré une augmentation de la sensibilité des bactéries à la norfloxacine et au chloramphénicol. Les résultats obtenus, appuyés par la modélisation moléculaire, suggèrent que la flexibilité moléculaire et la nature des fonctions chimiques des EPIs jouent un rôle essentiel dans l'amélioration de l'activité et la sélectivité vis-à-vis des fluoroquinolones. / Overexpression of Resistance-Nodulation-Division (RND) efflux pumps (EP) is a major contributor in multidrug resistance (MDR) and pathogenicity in Gram-negative bacteria. These transporters are able to expel out of the bacterial cell clinically important antibiotic classes, contributing in a significant manner to the treatment failure of infectious diseases. With the worrying levels of bacterial resistance reported worldwide and the continuous spreading of MDR pathogens, EPs are interesting targets for the discovery of new antimicrobial drugs. Therefore, to overcome this mechanism, efflux pump inhibitors (EPIs) are being developed as adjuvants in order to restore or improve the activity of usual antibiotics. The AcrAB-TolC archetype is particularly widespread in Enterobacter spp. presenting clinical relevance (ESKAPE pathogens). In this study, we described the drug design strategy based on fluoroquinolone antibiotic analogs, against the AcrB pump of E. aerogenes. Thus, synthesis and microbiological evaluation of quinazolin-4(3H)-one derivatives were performed. The structural and molecular properties of the tested compounds (i. e. rigidity and flexibility) were also investigated. In this purpose, a scaffold hopping of the quinazolinone core to homologous benzoquinazolinones and precursors benzamides were carried out. Several molecules increased the bacterial susceptibility towards norfloxacin and chloramphenicol. The obtained results, supported by molecular modeling, suggest that molecular flexibility and the nature of chemical functions play a critical role to improve activity and selectivity on fluoroquinolone potentiation targeting AcrB efflux pump.

Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative motile curved rod that inhabits the gastric mucosa of the human stomach. The organism chronically infects billions of people worldwide and is one of the most genetically diverse of bacterial species. Infection with the organism potentially induces chronic gastritis and peptic ulcer disease. In addition, H. pylori plays a role in the etiology of gastric cancer and gastric MALT lymphoma. The risk of infection is increased in those living in the developing world, which has been ascribed to precarious hygiene standards, crowded households, and deficient sanitation common in this part of the world. Thus, the aim of this study was to identify the risk factors in the transmission of H. pylori in our environment, i.e. in Nkonkobe Municipality in the Eastern Cape Province, South Africa. Faecal samples were collected from 356 apparently healthy subjects, consisting of 168 males and 188 females aged from 3 months to 60 years (Mean = 31 years). A standardized questionnaire was applied, it described demographic characteristics including age, sex, household hygiene, socioeconomic status, area of residence, duration of stay in the area, sharing bath water, sharing tooth brush, habit of sucking thumb, medication currently being taken or medication taken within the past three months, source of water, type of toilet used, education and occupation. A sandwich-type enzyme immunoassay amplification technology (Amplified IDEIA TM Hp StAR TM , Oxoid, UK) was used to analyze the faecal samples for the detection of H. pylori antigens using monoclonal antibodies specific for H. pylori antigens. To assess the possibility of faecal oral route with tap water as an intermediary link, PCR targeting the ureC (glmM), a highly conserved gene in H. pylori ii was carried out to detect H. pylori DNA in faecal samples of already positive samples by HpSA test as well as in direct tap water used by the H. pylori positive subjects. QIAamp DNA stool mini kit was used to extract DNA from faecal samples. Tap water samples were then obtained using sterile bottles from areas inhabited by H. pylori positive subjects as determined by HpSA test and PCR. DNA extraction from water samples was done using UltraCleanTM Water DNA Isolation Kit (0.22μm) according to the manufacturer’s instructions. PCR with primers specific for H. pylori glmM gene was carried out with both positive and negative controls incorporated. Fisher’s exact test was used to assess the univariate association between H. pylori infection and the possible risk factors. Odds ratio (OR) and the corresponding 95 percent confidence interval (CI) were calculated to measure the strength of association using EPI INFO 3.41 package. P values of < .05 were required for significance. The precision rate of the diagnostic tests used was also determined. H. pylori antigen was detected in 316 of the 356 subjects giving an overall prevalence of 88.8 percent. Prevalence increased with age from 75.9 percent in children < 12 years age to 100 percent in the age group from 13 years to 24 years, also 100 percent prevalence of H. pylori was recorded in young adults aged 25-47 years and subjects aged 60 years (P < .05). H. pylori prevalence was higher in females than in males. Of 188 females who participated in the study, H. pylori antigen was detected in 172 (91.5 percent) versus 144 (85.7 percent) of 168 males (P > .05). Interestingly, H pylori antigen was detected more often (100 percent) in the high socioeconomic group than in those of low socioeconomic group (85.9 percent). Sixteen (66.7 percent) of twenty four faecal samples that had previously tested positive for the organism by HpSA test were confirmed positive by PCR. However none of the treated tap water samples tested positive for the organism by PCR. The present iii study revealed a high prevalence of H. pylori in faecal samples of asymptomatic individuals in the Nkonkobe Municipality, an indication of active infection. The obtained results also revealed that direct treated tap water might not be playing a crucial role in the oral transmission of H. pylori in the studied population.