Magister Public Health - MPH / Avian Influenza (AI) is a contagious viral zoonotic disease with great public health implications and negative socioeconomic impact (WHO, 2006a). The highly pathogenic avian influenza (HPAI) infection is transmitted from birds to man mostly through contact with contaminated poultry and objects (INFOSAN, 2005), hence people who come in contact with birds such as live bird sellers (LBS) are the more vulnerable population (WHO, 2006a). Inadequate knowledge of AI health risks and poor practice of AI preventive measures amongst LBS increases the risk of spread of the infection in both humans and animals.The aim of this study was to describe and quantify the knowledge and practice of LBS with regards to avian influenza health risks and preventive activities in Agege, an urban area in Lagos State, Nigeria. / South Africa

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

Dentre as espécies do gênero Yersinia, Yersinia enterocolitica é a espécie mais prevalente como causa de doença em humanos e animais. Y. enterocolitica é dividida em seis biotipos. Os biotipos 1B, 2, 3, 4 e 5 compreendem linhagens associadas à doença em humanos e animais, enquanto o biotipo 1A consiste de linhagens consideradas não patogênicas. Apesar de Y. enterocolitica biotipo 2 ser de importância clínica, há uma escassez de estudos no país, o que dificulta avaliar o envolvimento dessa bactéria como causa de doença em humanos e em animais, bem como, determinar o impacto de sua presença no meio-ambiente. O objetivo deste trabalho foi investigar o potencial patogênico, determinar o perfil de suscetibilidade a antimicrobianos e verificar a diversidade genotípica de linhagens de Y. enterocolitica biotipo 2 isoladas no Brasil. Foram estudadas 40 linhagens de Y. enterocolitica biotipo 2, isoladas de humanos (5), ambiente (34) e animal (1), entre os anos de 1979 e 1998. Ademais, nas análises filogenéticas, foram acrescidas 26 linhagens de Y. enterocolitica pertencentes aos outros biotipos, com o intuito de comparar as linhagens de Y. enterocolitica biotipo 2 aos biotipos 1A, 1B, 3, 4 e 5. As linhagens de humanos e animal foram sensíveis a todos os 14 antimicrobianos testados. Dentre as 34 linhagens de ambiente, sete (20,6%) foram resistentes a um ou dois antimicrobianos, sendo esses, amicacina, cefoxitina, gentamicina, e sulfametoxazol - trimetoprima. Todas as linhagens apresentaram os genes inv, ail, ystA, hreP, tccC e myfA. Os genes fepD e fes foram detectados em 39 (97,5%) linhagens, o gene virF foi encontrado em três (7,5%) linhagens, os genes ystB e fepA não foram detectados em nenhuma linhagem. Todas as linhagens apresentaram comportamento relacionado à virulência frente aos testes fenotípicos de atividade da pirazinamidase, hidrólise da esculina e fermentação da salicina. O dendrograma de similaridade genética de Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) agrupou as linhagens de Y. enterocolitica biotipo 2 em cinco grupos denominados A, B, C, D e E. Todas as linhagens, com exceção de duas, apresentaram similaridade genética superior a 88,3%. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as linhagens de Y. enterocolitica biotipo 2 em três grupos denominados I, J e K. A maioria das linhagens (72,5%) apresentou similaridade ii genética superior a 78,3%. O dendrograma de similaridade genética de Multilocus variable number tandem repeat analysis (MLVA) agrupou as linhagens de Y. enterocolitica biotipo 2 em dois grupos denominados O e P com similaridade genética superior a 37,7%. Pode-se concluir que o potencial patogênico das linhagens de Y. enterocolitica biotipo 2 foi evidenciado pela prevalência da maioria dos marcadores de virulência, bem como, pelo comportamento relacionado à virulência frente aos testes fenotípicos pesquisados. Algumas linhagens apresentaram-se resistentes a antimicrobianos de primeira escolha no tratamento de yersiniose, o que pode acarretar em falha terapêutica. Os resultados de ERIC-PCR e PFGE mostraram a alta similaridade entre as linhagens de Y. enterocolitica biotipo 2, sugerindo que as mesmas pouco se diferenciaram ao longo dos 19 anos e que possivelmente o meio ambiente tem sido uma fonte de contaminação para humanos e animais no Brasil. A técnica de MLVA agrupou as linhagens de Y. enterocolitica biotipo 2 quanto à sua origem e a técnica de ERIC-PCR agrupou as linhagens de Y. enterocolitica biotipos 1A, 1B, 2, 3, 4, e 5 quanto às diferentes patogenicidades características de cada biotipo. / Among the species of the genus Yersinia, Yersinia enterocolitica is the most prevalent species that cause illness in humans and animals. Y. enterocolitica is divided into six biotypes. Biotypes 1B, 2, 3, 4 e 5 comprise strains associated to illness in humans and animals, while biotype 1A comprise strains considered nonpathogenic. Despite of the fact that Y. enterocolitica biotype 2 is of clinical importance, there is a paucity of studies in this country, which makes difficult to assess the involvement of this bacteria as a cause of illness in humans and animals, as well as to determine the impact of its presence in the environment. The aim of this work was to investigate the pathogenic potential, to determine the antimicrobial resistance profile and to verify the genetic diversity of Y. enterocolitica biotype 2 strains isolated in Brazil. Forty strains of Y. enterocolitica biotype 2 isolated from humans (5), environment (34) and animal (1), between 1979 and 1998 were studied. Besides, in the phylogenetic analyzes it was added 26 Y. enterocolitica strains belonging to the other biotypes, in order to compare the Y. enterocolitica biotype 2 strains to biotypes 1A, 1B, 3, 4 e 5. Humans and animals strains showed susceptibility to all 14 antibiotics tested. Among the 34 environment strains, seven (20.6%) were resistant to one or two antibiotics used such as amikacin, cefoxitin, gentamicin and sulfamethoxazole-trimethoprim. All the strains presented the genes inv, ail, ystA, hreP, tccC and myfA. Genes fepD and fes were detected in 39 (97.5%) strains, virF was found in three (7.5%) strains, and ystB and fepA were not detected in any strains. All the strains exhibited behavior related to virulence against the phenotypic tests of pyrazinamidase activity, esculin hydrolysis and salicin fermentation. The dendrogram of genetic similarity of Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) grouped the Y. enterocolitica biotype 2 strains in five groups, designated A, B, C, D and E. All the strains, except two, showed a genetic similarity of more than 88.3%. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the Y. enterocolitica biotype 2 strains in three groups, designated I, J and K. The majority of the strains (72.5%) showed a genetic similarity of more than 78.3%. The dendrogram of genetic similarity of Multilocus variable number tandem repeat analysis (MLVA) grouped the Y. enterocolitica iv biotype 2 strains in two groups, designated O and P with a genetic similarity of more than 37.7%. It is possible to conclude that the pathogenic potential of the Y. enterocolitica biotype 2 strains was highlighted by the prevalence of the majority of the virulence markers searched, as well as by the behavior related to virulence against the phenotypic tests. Some strains were resistant to antimicrobials that are the first choice for yersiniosis treatment, which can result in therapeutic failure. The results of ERIC-PCR and PFGE showed a high genetic similarity between the Y. enterocolitica biotype 2 strains, suggesting that the strains differed little over 19 years, and that the environment has been possibly a source of humans and animals infections in Brazil. The MLVA technique grouped the Y. enterocolitica biotype 2 strains according their origins, and the ERIC-PCR technique grouped the Y. enterocolitica biotypes 1A, 1B, 2, 3, 4 and 5 strains according to the different pathogenicity characteristics of each biotype.

Os mexilhões são alimentos marinhos freqüentemente ingeridos crus, ou parcialmente cozidos, e o hábito de aferventar estes bivalves somente até que abram as valvas, é insuficiente para eliminar os microrganismos patogênicos eventualmente presentes neste molusco. Após levantamento inicial, e visando melhorar a qualidade do mexilhão Perna perna cultivado e comercializado no município de Ubatuba, SP, esta pesquisa estudou o crescimento de Staphylococcus aureus e Bacillus cereus em mexilhões in natura e pré-cozidos, e a eliminação dos mesmos por meio de tratamentos térmicos, avaliando, posteriormente, as características físico-químicas e sensoriais dos produtos. Em ambos os casos, lotes de 1 kg de mexilhão foram inoculados, individualmente, com cepas de S. aureus e B. cereus e mantidos, por 10 horas, a temperatura ambiente (25ºC±1ºC) e sob refrigeração (7ºC±1ºC). Posteriormente, foram estabelecidos seis tipos de tratamentos térmicos, sendo três sob vapor (5, 10 e 15 min) e três por imersão em água (5, 10 e 15 min), buscando estabelecer o binômio que proporcionasse a eliminação dos mesmos, e avaliando o rendimento, os aspectos físico-químicos e sensoriais. Para ambos microrganismos, ocorreu crescimento durante as 10 horas de estudo, sendo este mais evidente, nos tratamentos mantidos a temperatura ambiente. No mexilhão pré-cozido ocorreram as maiores contagens microbianas, se comparado ao mexilhão in natura. Com relação aos tratamentos térmicos, todos foram eficientes, eliminando os microrganismos da ordem de, pelo menos 2 ciclos logarítmicos, no entanto, os tratamentos térmicos por imersão em água, permitiram melhores resultados do que os tratamentos sob vapor. As análises fisico-químicas e sensoriais, não apresentaram diferença estatística entre os tratamentos térmicos estudados. Com o emprego de altas temperaturas por um determinado período, obteve-se perda de alguns minerais, como Potássio e Boro, tendo outros, não apresentado alteração com relação ao tempo de exposição ao calor. Já, quanto ao rendimento, houve diferença, em nível de 5%, sendo os melhores rendimentos alcançados nos menores tempos de exposição ao calor e, os tratamentos por imersão, apresentaram resultados melhores que os tratamentos sob vapor. Concluiu-se que o tratamento térmico, binômio tempo-temperatura, de 10 min em água à ebulição, é suficiente para reduzir os microrganismos, permitindo a retenção dos nutrientes e um rendimento de 54,36%, podendo, portanto, ser recomendado para os produtores, visando melhorar a qualidade do mexilhão, via adequação do manejo atualmente empregado. / Mussels are seafood frequently ingested raw or partially cooked and the habit of boiling bivalves only to open the valves, is insufficient to eliminate several species of pathogenic bacteria. Seeking to improve the quality of the cultivated and marketed Perna perna mussel in the district of Ubatuba, SP, this research studied the microbiological growth of Staphylococcus aureus and Bacillus cereus in fresh and pre-cooked mussels, and the elimination by thermal treatments, being evaluated its physicochemical and sensorial characteristics. For such, lots of 1 kg of mussel was inoculated individually with strains of S. aureus and B. cereus, and maintained by 10 hours at environmental temperature (25ºC±1ºC) and under refrigeration (7ºC±1ºC). Six thermal treatments were established, 3 in steam (5, 10 and 15 min) and 3 in boiling water (5, 10 and 15 min), being looked in the elimination of B. cereus and S. aureus, and also evaluating the performance, and physical-chemical and sensorial aspects. Microbiological growth was verified after 10 hours for both microorganisms, and this being more evident in the treatments maintained at environmental temperature. Pre-cooked mussel obtained the largest microbial developments, if compared to fresh mussels. About the thermal treatments, everyone was efficient, eliminating at least 2 logarithmic cycles, however, thermal treatments in boiling water obtained better results than the steam treatments. The physical-chemical and sensorial analyses, didn't present statistical difference among the thermal treatments studied. Use of high temperatures for a determinate period, obtained lost in some minerals, like potassium and boron, and others minerals not presented alteration in relation to the heat time exposure. Already in the performance, it was obtained statistical difference, being the best performances reached in the smallest times of heat exposition, and the treatments in boiling water presented better results than the steam treatments. The thermal treatment, binomial time-temperature, of 10 min in boiling water, is enough to reduce the microorganisms, allowing the retention of the nutrients and performance of 54.36%, could be recommended for the producers seeking to improve the traditional handling.

Os antibióticos têm sido utilizados como aditivos na alimentação animal para aumentar o desempenho e manter a saúde dos animais, como suínos e frangos, destinadas à produção de alimentos de origem animal. No entanto, desde 2006, a Comunidade Europeia proibiu o uso de antibióticos para esse proposito devido ao desenvolvimento de resistência bacteriana aos antibióticos. Como resultado, várias alternativas foram estudadas e propostas para substituir os antibióticos utilizados na alimentação animal. Os óleos essenciais têm recebido considerável atenção devido às suas propriedades antimicrobianas. Portanto, o objetivo do presente trabalho foi avaliar in vitro a atividade antibacteriana dos óleos essenciais contra a microbiota patogênica e probiótica de ocorrência no trato gastrointestinal de suínos e aves, destinadas à produção de alimentos de origem animal. A atividade antibacteriana seletiva, a qual, significou uma alta atividade antibacteriana contra bactérias patogênicas e reduzida ou nenhuma atividade sobre bactérias probióticas, foi avaliada como característica fundamental dos óleos essenciais com alto desempenho. Esta característica foi avaliada nos óleos essenciais usados individualmente e em combinações binarias. Inicialmente, no Capítulo 2, uma triagem de vinte e oito óleos essenciais (OEs) através do método de difusão em disco mostrou que Eucalyptusglobulus, E. exserta, Pimenta pseudocaryophylllus, Orange Oil Phase Essence, e CitrusTerpens (Os dois últimos OEs foram subprodutos do processamento da laranja para obtenção de suco) tiveram uma atividade antibacteriana seletiva sobre a bactéria patogênica Salmonella Enteritidis e a bactéria probiótica Lactobacillus plantarum. Numa fase posterior, esses cinco óleos foram avaliados individualmente e em misturas binárias, contra cinco bactérias patogênicas e três bactérias probióticas. Os melhores resultados foram observados quando os OEs foram avaliados isoladamente e não em misturas. Assim, Orange Oil Phase Essence e Citrus Terpens destacaram-se por ter a melhor atividade antibacteriana seletiva contra essas bactérias. No Capítulo 3, uma análise mais detalhada da atividade antibacteriana dos óleos essenciais foi realizada utilizando Orange Oil Phase Essence e a mistura composta pelos óleos de E. globulus e P. pseudocaryophyllus. Estes dois óleos foram selecionados com base nos resultados do Capitulo 2 e da disponibilidade de OEs em nosso estoque. Ambos, óleo e a mistura foram avaliados sobre a bactéria patogênica mais resistente, E. faecalis, e a bactéria probiótica menos resistente L. rhamnosus, como observado no Capitulo 2. A avaliação da Concentração Inibitória Mínima e Concentração Bactericida Mínima do Orange Oil Phase Essence e da mistura mostrou que não tiveram um efeito antibacteriano seletivo sobre E. faecalis e L. rhamnosus. Finalmente, no Capitulo 4, foi avaliada a atividade antibacteriana individual e combinada dos óleos de E. globulus e P. pseudocaryophyllus sobre E. faecalis e L. rhmanosus. Os resultados mostraram que a combinação destes dois OEs, avaliadas pelo método checkerboard, não potencializou a atividade antibacteriana seletiva dos dois OEs. Portanto, observou-se que o óleo de E. globulus isoladamente apresentou a melhor atividade antibacteriana seletiva contra E. faecalis e L. rhamnosus. Em conclusão, este trabalho permitiu identificar óleos essenciais com perfil antibacteriano seletivo para eles serem possíveis alternativas botânicas aos antibióticos utilizados na alimentação animal. / Antibiotics have been used in animal feed to maintain health and increase performance, as in the case of pigs and poultry intended for food production of animal origin. However, since 2006 the European Community has banned the use of antibiotics for this purpose due to the emergence and increase of antibiotic-resistant bacteria. As a result, several alternatives have been studied and proposed to substitute antibiotics used in animal feed. Essential oils have received considerable attention due to their antimicrobial properties. Therefore, the objective of this work was to evaluate in vitro the antibacterial activity of essential oils against pathogenic and probiotic bacteria that occur in the gastrointestinal tract of swine and poultry, intended for food production of animal origin. The selective antibacterial activity, which means high antibacterial activity on pathogenic bacteria and reduced or no activity on probiotic bacteria, was evaluated as a fundamental feature of the highest-performance essential oils. This feature was evaluated in essential oils used individually and in binary combinations. Initially, in Chapter 2, the screening of twenty-eight essential oils (EOs) by disk diffusion method showed that Eucalyptus globulus, E. exserta, Pimenta pseudocaryophylllus, Orange Oil Phase Essence, and Citrus Terpens (the last two EOs were by-products of orange juice production) had a selective antibacterial activity against the pathogenic Salmonella Enteritidis and probiotic Lactobacillus plantarum. At a later stage those five oils were evaluated, individually and in binary blends, against five pathogenic bacteria and three probiotic bacteria. Better results were observed when the EOs were checked alone and not in blends. Orange Oil Phase Essence and Citrus Terpens stood out for having the two best selective antibacterial activities against those bacteria. In Chapter 3, a more detailed analysis of essential oil antibacterial activities was perfomed using Orange Oil Phase Essence and the blend composed of E. globulus and P. pseudocaryophyllus. These two oils were selected based on the results of Chapter 2 and from the availability of our EO stock. Both oil and blend were checked on the most resistant pathogenic bacterium, E. faecalis, and on the less resistant probiotic bacterium of the Lactobacillus genus, L. rhamnosus, as observed in Chapter 2. The evaluation of Minimal Inhibitory Concentration and Minimal Bactericidal Concentration for Orange Oil Phase Essence and the blend showed that there was not a selective antibacterial effect against E. faecalis and L. rhamnosus. Finally, in Chapter 4, the individual and combined antibacterial activities of E. globulus and P. pseudocaryophyllus essential oils on E. faecalis and L. rhmanosus were evaluated. The results showed that the combination of two EOs evaluated by checkerboard method did not potentiate the selective antibacterial activity of the two EOs. Therefore, it was observed that the E. globulus essential oil alone had the best selective antibacterial activity against E. faecalis and L. rhamnosus. In conclusion, this work enabled the identification of essential oils with selective antibacterial profile that can become possible botanical alternatives to antibiotics used in animal feed.

Escherichia coli enteropatogênica atípica (aEPEC) faz parte de um grupo de patógenos capazes de formar um tipo de lesão característica em cultura de tecidos epiteliais, denominada attaching and effacing (A/E). Os genes que são necessários para produção da lesão A/E estão localizados em uma ilha de patogenicidade denominada região LEE (locus of enterocyte effacement). A transcrição de genes da região LEE está sujeita a regulação por vários fatores, entre eles quorum sensing, termo utilizado para designar um mecanismo de regulação gênica dependente da concentração celular. Esse mecanismo é usado por bactérias Gram-positivas e Gram-negativas e em ambos os casos envolve a produção e detecção de moléculas sinalizadoras extracelulares, denominadas autoindutores. Até o momento, pelo menos quatro sistemas de quorum sensing foram descritos, entre eles o sistema de autoindutor AI-3 encontrado em bactérias Gram-positivas e Gram-negativas. Diversos mecanismos celulares, entre eles a expressão de fatores de virulência em amostras de EPEC e EHEC, são regulados por esse fenômeno. O principal objetivo deste estudo foi verificar se existe uma possível regulação por quorum sensing na interação in vitro de uma amostra de E. coli da microbiota intestinal com amostras de aEPEC. Após a confirmação da produção de AI-3 por amostras de E.coli da microbiota intestinal foram realizados ensaios de adesão e quantificação utilizando meio pré-condicionado com esta amostra, epinefrina e bloqueadores que confirmaram que os padrões de adesão de aEPEC obtidos em menor tempo são devidos a presença de AI-3 no meio pré-condicionado, indicando a participação de quorum sensing nessa interação. Além disso, foi observado um fenômeno citotóxico nas células que não é produzido pelo AI-3. / Atypical Enteropathogenic Escherichia coli (aEPEC) are part of a group of pathogens capable of forming a type of lesion characteristic of epithelial tissues in culture, called attaching and effacing (A/E). The genes that are required for production of A/E lesion are located in a pathogenicity island called LEE region (locus of enterocyte effacement). The transcription of LEE genes in the region is subject to regulation by various factors, including quorum sensing, a term used to describe a mechanism of gene regulation dependent on cell concentration. This mechanism is used by Gram-positive and Gram-negative and in both cases involves the production and detection of extracellular signaling molecules, called autoinducers. So far, four systems of quorum sensing have been described, including the system of autoinducers AI-3 found in Gram-positive and Gram-negative bacteria. Several cellular mechanisms, including expression of virulence factors in EPEC and EHEC are regulated by this phenomenon. The main objective of this study was to determine whether there is a possible regulation by quorum sensing in the in vitro interaction of a strains of E. coli of the intestinal microbiota with strains aEPEC. After confirming the production of AI-3 in E. coli of the intestinal microbiota were performed adhesion assays and quantification using means preconditioned with this strains, epinephrine, and blockers who confirmed that patterns of adherence of aEPEC obtained in less time are due to the presence of AI-3 in the preconditioned means, indicating the involvement of quorum sensing in this interaction. Furthermore, we observed a phenomenon that cytotoxic cells is not produced by AI-3.

Chlamydia is the most common bacterial sexually transmitted infection in the United States and worldwide. It often goes unnoticed due to lack of symptoms and left untreated it can ascend the female genital tract to cause sequelae like pelvic inflammatory disease and irreversible tubal infertility. In reproductive-aged women, female sex hormones estrogen (E2) and progesterone (P4) concentrations fluctuate during the menstrual cycle and are influenced by hormonal contraceptives and hormone replacement therapy. E2 and P4 influence genital Chlamydia infection in women and mice, but these multifactorial interactions are not entirely mapped out. The complex interplay of E2 and P4 with Chlamydia and the host response demand further study to determine the effect of hormonal environment and host susceptibility to Chlamydia. E2 primarily signals through estrogen receptors (ER) ERα and ERβ. We used ERα or ERβ knockout (KO) mice to study the role of E2 and ERs in chlamydial progression and examined the host immune response at day 9 post-infection, when we expected the immune response to be the most robust. ERαKO, but not ERβKO mice had significant differences in the progression of Chlamydia and the host immune response. Future studies should test the immune response at additional timepoints, and a model should be utilized wherein ERα and ERβ are simultaneously silenced by chemical knockdown of ERβ in ERα knockout mice using ER agonist ICI 182, 680. 3 Mice are widely used in Chlamydia research, but due to its short estrus cycle, infection cannot be established naturally before infected cells are shed. To overcome this, mice are pretreated with depot medroxyprogesterone acetate (DMPA), an exogenous progesterone that halts the estrus cycle. However, a mouse model not reliant on DMPA pretreatment is needed because 1.) DMPA can affect the immune response and 2.) the hormonal environment in women is not static. Our model uses mice that are ovariectomized to stop the production of endogenous E2 and P4, then treated with physiologically relevant levels of E2 and P4 via implantation of a hormone-filled capsule. We observed that E2 protected mice from Chlamydia, making our model a good alternative for in vivo Chlamydia studies.

MSc (Microbiology) / Department of Microbiology / Background: Escherichia coli is a facultative anaerobic bacterium that forms part of the gut microbiota. It is used as an indicator that confirms recent faecal contamination. E. coli have been identified amongst the pathogens that are mostly responsible for moderate to severe diarrheal outbreaks in the low and middle-income countries. With South Africa facing an issue in water scarcity, issues concern poor sanitation and hygiene practices results in serious public health problems and allows E. coli to be transmitted from infected human or animal faeces to a new susceptible host using environmental reservoirs such as soil, water, hands as the transmission pathway. Objective: The primary objective of the study was to characterize E. coli strains from rural communities of Vhembe district, Limpopo, South Africa. Methodology: Households of 7 villages in the Vhembe district were randomly selected. A total of 81 households (HHs) were part of the study. In each household, a structured questionnaire was used to background information on WASH practices. Samples taken from each HH included toilet seat swabs, floor swabs, child and mother handwash samples, stored water samples and running tap water samples. A total of 399 samples were analysed using Colilert® Quanti-trays®/2000 method to detect the presence of Escherichia coli. Positive E. coli samples were further identified using multiplex polymerase chain reaction (m-PCR) to determine the pathogenic strains of E. coli. Transmission pathways were established using identified strains. Results: Data from the structured questionnaires showed common problems of availability of running tap water; lack of provision of sanitation; open practice on defaecation and very little hand hygiene practices. A total of 91 (22.81%) samples tested positive for E. coli with the Colilert® Quanti-trays®/2000 method. The mothers’ handwash samples had the most E. coli prevalence followed by stored water samples. The most prevalent E. coli pathotype was EPEC with the virulence gene eae. Atypical EPEC (60.44%) outnumbered the typical EPEC (5.49%). The pathotype ETEC was detected in 41.76% samples and EHEC in 9.89% samples. Transmission pathway was observed from the different households; with eae gene (aEPEC) being the most detected from samples looking at the LT gene (ETEC). v | P a g e Discussion: All 7 villages are facing common issues such as lacking running water, poor sanitation and improper hand hygiene practices. The mothers were the most contaminated and it was observed that its due to the daily activities that they perform around the house. It is of importance for them to practice proper hand hygiene to prevent transmission of pathogenic E. coli to the children via direct or indirect transmission route. The pathogenic E. coli was detected from all different samples collected from the households including the floor and toilet seat samples. EPEC was detected the most, and studies have shown that this strain is known to cause diarrheal infections in young children from developing countries. Conclusion: The members of the study village households were aware of the WASH services and its importance. However, proper implementation into their day-to-day life is lacking due to the high number of TC and E. coli detected from handwash samples and stored water samples from the households. Recommendation: Appropriate WASH strategies should be established to ensure good health at the village households. Further studies should be done to check possible transmission pathways from more village households. / NRF

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.

Mälaren är Stockholms största dricksvattentäkt och den förser årligen 1,7 miljoner människor med rent dricks-, tvätt- och badvatten genom Stockholm Vattens vattenverk Norsborg och Lovö och Norrvattens vattenverk Görväln. Även om det finns reservvattentäkter som Bornsjön så räcker inte de till om något skulle hända med Mälaren som t.ex. oljeutsläpp från något fartyg som befinner sig i närheten av vattenintagen. Idag förekommer det ständigt föroreningar från alla möjliga utsläppskällor som enskilda avlopp, industrier, avloppsreningsverk, fritidsbåtar, jordbruk m.m. och alla dessa bidrar till en sämre vattenkvalitet. Syftet med den här rapporten är att identifiera och lokalisera möjliga utsläppspunkter för patogena mikroorganismer inom ett område som kan orsaka problem för Stockholm Vattens vattenverk Norsborg och Lovö. Rapporten ska senare användas för att undersöka vilka riskpunkter som utgör de största hoten mot vattenverken. Området innefattar till största del Ekerö kommun och Botkyrka kommun men även Salems kommun finns inom riskområdet. Ekerö släpper ständigt ut renat och orenat avloppsvatten från olika reningsverk och anläggningar som kan innehålla stora mängder patogena mikroorganismer. De enskilda avloppen står även för en del utsläpp eftersom det fortfarande finns hus och anläggningar med avloppslösningar som inte är godkända enligt dagens krav på utsläpp från enskilda avlopp. Kraven på avloppslösningar innefattar inte patogena mikroorganismer utan inriktas på eutrofierande ämnen som kväve och fosfor. Även jordbruket är en potentiell källa för utsläpp av patogena mikroorganismer då antingen gödsel eller slam kan användas som näringsämnen för åkrar. Även avföring från betesmarker kan spolas ut i vattentäkter med dagvattnet. En av fördelarna med Mälaren som vattentäkt är att dess vattenflöde är så pass stort att föroreningar som släpps ut späds ut över så stora vattenmängder. För att försäkra sig om att patogena mikroorganismer inte kan ta sig igenom vattenverken så finns det två modeller som är framtagna för att beräkna vilken motståndskraft verket har mot olika patogena mikroorganismer. De modellerna heter ODP (Optimal disinfektionspraxis) och MRA (Mikrobiologisk riskanalys). Informationen som uppsamlas i rapporten ska i ett senare skede användas i dels SeaTrack som SMHI skapat för att se hur mikroorganismer sprids i vatten och MRA för att utvärdera hur stora risker dessa utsläppspunkter medför till vattenverken. / Mälaren is Stockholms largest source of drinking water and is supplying 1,7 million people with drinking-, washing- and bathing water. The water is distributed through the companies Stockholm Vatten and Norrvatten from their water purification plants Norsborg, Lovö and Görväln. There is also a reserve fresh water source called Bornsjön but it’s not big enough to supply 1,7 million people which mean it can only replace Mälaren for a short time  if something would happen. Today it is always occurring pollutions from a set of different sources for example private sewage, factories, sewage treatment works, boats and agriculture. All those contribute with a lower water quality. The purpose with this report was to identify and find possible emission points for pathogenic micro-organisms inside a certain area that can cause problems for Stockholm Vattens water purification plants Norsborg and Lovö. The report will later be used to evaluate which risk points can be a threat to the water purification plants.  The risk area holds the biggest part of Ekerö, Botkyrka and also Salems. Ekerö is constantly emitting processed sewage water from all the different kind of sewage treatment works and that water can contain high amounts of pathogenic micro-organisms.  The private sewage is also contributing with emissions because some of them are not good enough to be approved by the standards today.  The demands for private sewage is not including pathogenic micro-organisms but is more directed to limit the emission of nutrients like phosphorus and nitrogen. Agriculture is also a possible source of emission of pathogenic micro-organisms when both manure and sludge can be used as nutrients for fields. Even the excrements from pasture lands can reach Mälaren with the surface water.A large advantage with Mälaren as a source of drinking water is the large water flow is diluting the pollutions that is emitted in to it. To ensure no pathogenic micro-organisms can pass through the water purification plants two different models exist to calculate a water purification plants resistance to different kinds of pathogenic micro-organisms. The models are called ODP (Optimal disinfection Praxis) and MRA (Microbiologic Risk Assessment). The information gathered in the report is later on going to be used in another computer model SeaTrack created by SMHI to predict particles movement in the eastern Mälaren and later on MRA to evaluate what kind of risks the different emission points have.