by Fung King-yuen Debera. / On t.p., "2+" is superscript. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 102-112). / Acknowledgement --- p.i / Abstract --- p.ii / Table of Content --- p.iv / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- Problems of heavy metals in the environment --- p.1 / Chapter 1.1.2 --- Methods of removal of heavy metal from industrial effluent --- p.5 / Chapter 1.1.3 --- The properties of magnetites --- p.10 / Chapter 1.1.4 --- Role of magnetites in water treatment --- p.12 / Chapter 1.1.5 --- The advantages of using magnetites and further application of magnetites --- p.16 / Chapter 1.2 --- Objectives of the study --- p.21 / Chapter 2. --- Materials and methods --- p.23 / Chapter 2.1 --- Selection of the organisms --- p.23 / Chapter 2.2 --- Culture media and chemicals --- p.23 / Chapter 2.3 --- Growth of the bacterial cells --- p.25 / Chapter 2.4 --- Immobilization of the bacterial cells on magnetites --- p.27 / Chapter 2.4.1 --- Effects of chemical and physical factors on the immobilization of the bacterial cells on magnetites --- p.27 / Chapter 2.4.2 --- Effect of pH on the desorption of cells from magnetites --- p.28 / Chapter 2.5 --- Nickel ion uptake experiments --- p.28 / Chapter 2.6 --- Effects of operational conditions on the nickel removal capacity of the magnetite-immobilized bacterial cells --- p.29 / Chapter 2 .6.1 --- Effect of physical factors --- p.29 / Chapter 2.6.2 --- Effect of chemical factors --- p.30 / Chapter 2.7 --- Optimization of the nickel removal efficiency --- p.30 / Chapter 2.8 --- Nickel adsorption isotherm of the magnetite- immobilized cells of Enterobacter sp4-2 --- p.30 / Chapter 2.9 --- Recovery of adsorbed Ni2+ from the magnetite- immobilized cells of Enterobacter sp4-2 --- p.31 / Chapter 2.9.1 --- Multiple adsorption-desorption cycles of Ni2+ by using citrate buffer --- p.32 / Chapter 2.9.2 --- Multiple adsorption-desorption cycles of Ni2+ by using ethylenediaminetetraacetic acid (EDTA) --- p.33 / Chapter 2.10 --- Effect of acidic treatment --- p.33 / Chapter 2.10.1 --- Effect of acidic treatment on the nickel removal capacity of the magnetites and the magnetite- immobilized cells of Enterobacter sp4-2 --- p.33 / Chapter 2.10.2 --- Effect of acidic treatment on the recovery of the adsorbed Ni2+ from magnetites and the magnetite- immobilized cells Enterobacter sp4-2 --- p.34 / Chapter 2.11 --- Removal and recovery of Ni2+ from the electroplating effluent --- p.34 / Chapter 3. --- Results --- p.36 / Chapter 3.1 --- Effects of chemical and physical factors on the immobilization of the bacterial cells on magnetites --- p.36 / Chapter 3.1.1 --- Effect of pH --- p.36 / Chapter 3.1.2 --- Effect of cells to magnetites ratio --- p.36 / Chapter 3.1.3 --- Effect of temperature --- p.39 / Chapter 3.2 --- Effect of pH on the desorption of cells from magnetites --- p.39 / Chapter 3.3 --- Nickel ion uptake experiments --- p.44 / Chapter 3.4 --- Effects of operational conditions on the nickel removal capacity of the magnetite-immobilized bacterial cells --- p.44 / Chapter 3.4.1 --- Effect of reaction temperature --- p.44 / Chapter 3.4.2 --- Effect of retention time --- p.44 / Chapter 3.4.3 --- Effect of pH --- p.47 / Chapter 3.4.4 --- Effect of the presence of cations --- p.50 / Chapter 3.4.5 --- Effect of the presence of anions --- p.50 / Chapter 3.5 --- Optimization of the nickel removal efficiency --- p.55 / Chapter 3.6 --- Nickel adsorption isotherm of the magnetite- immobilized cells of Enterobacter sp4-2 --- p.55 / Chapter 3.7 --- Recovery of adsorbed Ni2+ from the magnetite- immobilized cells of Enterobacter sp4-2 --- p.59 / Chapter 3.7.1 --- Multiple adsorption-desorption cycles of Ni2+ by using citrate buffer --- p.59 / Chapter 3.7.2 --- Multiple adsorption-desorption cycles of Ni2+ by using ethylenediaminetetraacetic acid (EDTA) --- p.63 / Chapter 3.8 --- Effect of acidic treatment --- p.63 / Chapter 3.8.1 --- Effect of acidic treatment on the nickel removal capacity of the magnetites and the magnetite-immobilized cells of Enterobacter sp4-2 --- p.63 / Chapter 3.8.2 --- Effect of acidic treatment on the recovery of the adsorbed Ni2+ from the magnetites and the magnetite-immobilized cells of Enterobacter sp4-2 --- p.66 / Chapter 3.9 --- Removal and recovery of Ni2+ from the electroplating effluent --- p.69 / Chapter 4. --- Discussion --- p.72 / Chapter 4.1 --- Selection of the organisms --- p.72 / Chapter 4.2 --- Effects of chemical and physical factors on the immobilization of the bacterial cells on magnetites --- p.72 / Chapter 4.2.1 --- Effect of pH --- p.72 / Chapter 4.2.2 --- Effect of cells to magnetites ratio --- p.74 / Chapter 4.2.3 --- Effect of temperature --- p.75 / Chapter 4.2.4 --- Effect of pH on the desorption of cells from magnetites --- p.76 / Chapter 4.3 --- Nickel ion uptake experiments --- p.78 / Chapter 4.4 --- Effects of operational conditions on the nickel removal capacity of the magnetite-immobilized bacterial cells --- p.80 / Chapter 4.4.1 --- Effect of reaction temperature --- p.80 / Chapter 4.4.2 --- Effect of retention time --- p.81 / Chapter 4.4.3 --- Effect of pH --- p.82 / Chapter 4.4.4 --- Effect of the presence of cations --- p.83 / Chapter 4.4.5 --- Effect of the presence of anions --- p.84 / Chapter 4.5 --- Optimization of the nickel removal efficiency --- p.85 / Chapter 4.6 --- Nickel adsorption isotherm of the magnetite- immobilized cells of Enterobacter sp4-2 --- p.86 / Chapter 4.7 --- Recovery of adsorbed Ni2+ from the magnetite- immobilized cells of Enterobacter sp4-2 --- p.87 / Chapter 4.7.1 --- Multiple adsorption-desorption of Ni2+ --- p.89 / Chapter 4.7.2 --- Effect of acidic treatment on the nickel removal capacity and recovery --- p.91 / Chapter 4.8 --- Removal and recovery of Ni2+ from the electroplating effluent --- p.93 / Chapter 5. --- Conclusion --- p.96 / Chapter 6. --- Summary --- p.99 / Chapter 7. --- References --- p.102

Emerging pathogens are organisms whose incidence has increased within the past two decades. In the last 40 years, several pathogens have emerged to cause infectious waterborne and foodborne diseases, thus causing a significant public health concern. Enterobacter sakazakii and Naegleria fowleri are emerging pathogens that have been documented to cause fatal infections. E. sakazakii is an emerging foodborne pathogen that represents a significant health risk by causing infections resulting in septicemia, meningitis and necrotizing enterocolitis in neonates, premature infants and also elderly immunocompromised individuals. Naegleria fowleri is a water-based protozoan flagellate that is the cause of primary amoebic meningoencephalitis; a fatal disease that mostly infects children and young adults through water-related recreational activities. The focus of this dissertation is to identify environmental reservoirs of Enterobacter sakazakii and to determine inactivation strategies to control Naegleria fowleri by chlorine and ultraviolet disinfection. In Appendix A, samples from various household kitchens were collected to determine the presence of E.sakazakii. The highest percentage of E.sakazakii was isolated from kitchen sponges (8%; n=50) and dishrags (10%; n=50). This study provided information on the presence of E.sakazakii on environmental surfaces in the kitchen. In Appendix B, our recent research has determined that N. fowleri is present in 8% (n=143) of municipal drinking water wells in central and southern Arizona. Therefore, guidelines need to be established for treatment of water with various disinfectants to control the growth and proliferation of N.fowleri. In Appendix C, the Ct values (concentration (mg/l) × exposure time) for chlorine inactivation of N. fowleri trophozoites and cysts were determined using the Efficiency Hom Kinetic Model (EHM). The Ct values for 99% inactivation of trophozoites and cysts were estimated to be 9 and 31, respectively. The ultraviolet light dose required for the 99% inactivation of N.fowleri trophozoites and cysts was determined to be 63 mW.sec/cm² and 13 mW.sec/cm², respectively.

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Made available in DSpace on 2015-12-06T23:05:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2005 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O Enterobacter spp. é um patógeno clinicamente importante em infecções de corrente sangüínea, e está freqüentemente associado à resistência às cefalosporinas de amplo espectro. Neste microrganismo, a resistência geralmente ocorre devido à desrepressão do gene ampC, mas outros mecanismos de resistência também podem existir, como a produção de beta-lactamases de espectro ampliado mediada por plasmídios (ESBL), o que limita as opções da terapêutica antimicrobiana. Objetivos: A análise do perfil de sensibilidade, da resistência à cefalosporina de quarta geração, da similaridade genética, e da adequação da terapia antimicrobiana entre amostras de Enterobacter spp. isoladas em infecções de corrente sangüínea no complexo Hospital São Paulo/ UNIFESP, em 2003 e 2004. Métodos: Um total de 93 amostras de bacteremia, consecutivamente coletadas entre janeiro de 2003 e março de 2004, foram testadas quanto ao perfil de sensibilidade para 7 agentes antimicrobianos, utilizando a metodologia do Etest e, seguindo o procedimento para os testes de ágar difusão, descritos pelo NCCLS. Foram utilizadas fitas de Etest ESBL screen para a detecção de beta-lactamases de espectro ampliado nos isolados de Enterobacter spp.. As amostras de Enterobacter spp. resistentes à cefepima foram selecionadas para a avaliação da similaridade genética através da ribotipagem automatizada, e os dados clínicos e demográficos dos pacientes com hemocultura positiva para esses isolados foram avaliados quanto a adequação da terapia antimicrobiana. Resultados: As taxas de resistência em Enterobacter spp. variaram de 28% para ceftazidima, 18,3% para cefotaxima e 12,9% para cefepima. O imipenem foi o antimicrobiano mais ativo (100% de sensibilidade), mesmo contra os isolados AmpC estavelmente desreprimidos. A ordem decrescente de atividade (% sensibilidade) contra os 93 isolados testados foi: imipenem (100%) >amicacina (86,0%) >cefepima (84,9%) = gatifloxacina (84,9%) >piperacilina/tazobactam (81,7%) >ceftazidima (72,0%) >cefotaxima (68,8%). Apenas 46,2% dos isolados de Enterobacter spp. resistentes à ceftazidima apresentaram sensibilidade à cefepima. A resistência cruzada com outros antimicrobianos foi comum entre as amostras resistentes à ceftazidima e àquelas resistentes à cefepima. A produção de ESBL foi encontrada em 5 isolados de Enterobacter spp.. Foram observados 7 ribotipos distintos entre as 14 amostras de Enterobacter spp. resistentes à cefepima. Os pacientes com bacteremia por Enterobacter spp. resistente à cefepima, em sua maioria, possuíam doença de base potencialmente ou rapidamente fatal e xvi encontravam-se internados em unidades de terapia intensiva ou unidades cirúrgicas. Cinco entre 16 pacientes com bacteremia por Enterobacter spp. com sensibilidade reduzida à cefepima receberam terapia antimicrobiana inicial apropriada. A taxa de mortalidade foi maior no grupo com terapia antimicrobiana empírica inadequada comparado aquele com terapia antimicrobiana adequada. Conclusões: Este estudo mostrou altas taxas de resistência à cefepima em Enterobacter spp. isolados de bacteremia, devido a presença de mecanismos adicionais de resistência além da produção de ESBL, e sugere que o uso prévio das cefalosporinas de amplo espectro pode estar relacionado com a emergência de isolados resistentes, e que a terapia antimicrobiana empírica inadequada pode estar associada a maior mortalidade entre esses pacientes. A grande variabilidade genética encontrada entre as amostras de Enterobacter spp. resistentes à cefepima alerta à importância da política do uso apropriado dos agentes antimicrobianos, particularmente das cefalosporinas de amplo espectro, para restringir a seleção de isolados resistentes. / Background: Enterobacter spp. is an important clinical pathogen in nosocomial bloodstream infections that frequently exhibits resistance to high spectrum cephalosporins. In this microrganism, resistance is usually due to derepression of AmpC locus, but other resistance mechanisms can also occur, like plasmid-encoded extended-spectrum beta-lactamases (ESBLs), which makes antimicrobial therapy options much more limited. Purpose: The main objectives of this study were to evaluate susceptibility profile, fourth-generation cephalosporin resistance, genetic similarity, and antimicrobial therapy adequacy among Enterobacter spp. isolated from bloodstream infections at Hospital São Paulo complex, in 2003 and 2004. Methods: A total of 93 bloodstream isolates consecutively collected between January 2003 and March 2004 were susceptibility tested for 7 broad-spectrum agents by using Etest and following the NCCLS procedures for agar difusion tests. ESBL Etest strips for the detection of extended-spectrum beta-lactamases were used to determine ESBL phenotypes in Enterobacter spp. strains. The bloodstream cefepime-resistant Enterobacter spp. were further selected for molecular typing by automated ribotyping, and the medical records of all patients with positive blood culture for these cefepime resistant pathogens were examined to evaluate antimicrobial therapy adequacy and the effect of inappropriate empirical antibiotic therapy on patients outcome. Results:. Resistance rates among Enterobacter spp. ranged from 28% for ceftazidime, 18,3% for cefotaxime and 12,9% for cefepime. Imipenem showed the highest susceptibility rate (100.0% susceptible) and was active even against the stably derepressed AmpC-producers. The overall rank order of spectrum (% susceptible) was: imipenem (100%) amikacin (86,0%) >cefepime (84,9%) = gatifloxacin (84,9%) >piperacillin/tazobactam (81,7%) >ceftazidime (72,0%) >cefotaxime (68,8%). Only 46,2% of ceftazidime-resistant Enterobacter spp. were susceptible to cefepime. Co-resistance to other antimicrobial agents was common amog ceftazidime-resistant and cefepime-resistant isolates. Five strains of Enterobacter spp. were phenotypically detected as ESBL producers. Seven distinct ribotyping patterns were observed among the 14 cefepime-resistant Enterobacter spp.. Most of cefepimeresistant Enterobacter spp. bacteremias were from patients with severe comorbidities and admitted in intensive care units or surgical units. Of the 16 patients infected with cefepime-susceptibility-reduced Enterobacter spp., 5 received appropriate initial antimicrobial therapy. The mortality rate was higher in the inadequately treated group. Conclusions: This study shows high rates of cefepime resistance in bloodstream Enterobacter spp. isolates due to the presence of additional mechanisms of antimicrobial resistance, other than ESBL production; and suggests that previous broadspectrum cephalosporin administration could be related to resistance emergence, and empirical inappropriate antimicrobial therapy could adversely affect patients outcome. The great genomic variability found among cefepime-resistant Enterobacter spp. highlights the importance of appropriate use of antimicrobial agents, particularly broadspectrum cephalosporins, to restrict selection of resistant isolates. / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2016-03-15T14:15:03Z (GMT). No. of bitstreams: 2 222.pdf: 7280517 bytes, checksum: 5c1c665cf2ba85668951a0c1daaafd4e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / O Parque Nacional Serra da Capivara (PNSC) é um ambiente propício para o desenvolvimento e conservação de grande número de espécies endêmicas da caatinga, além de ser uma região de importância ímpar, pois é o maior sítio arqueológico do Brasil. A água nesta região é escassa e o contato das pessoas e seus animais domésticos e de criação com os animais silvestres faz com que aumente a possibilidade de circulação de possíveis patógenos, possibilitando a emergência de doenças tanto no homem quanto em animais. Este trabalho teve como objetivo analisar a presença e circulação de bactérias potencialmente enteropatogênicas e zoonóticas na região do Parque Nacional Serra da Capivara, localizado no sudeste do Estado do Piauí Brasil. Coletas de amostras de água, fezes humanas e de animais silvestres e de criação foram realizadas em diferentes regiões no interior e no seu entorno,durante as idas ao campo, computando um montante de 170 amostras coletadas. (...) Não foi possível o isolamento de amostras do gênero Campylobacter, importante enteropatógeno de impacto para a Saúde Pública. O percentual de resistência das amostras a pelo menos uma das drogas testadas foi de 71 por cento sendo mais representativos para os fármacos: penicilina, cefoxitina e tetraciclina. A vigilância dos sorovares de Salmonella em ambientes aquáticos constitui um importante elemento para o monitoramento de infecções humanas e animais. / Neste estudo foram identificados 11sorovares de Salmonella: Worthington, Panama, Brooklyn, Glasgow, Braenderup, Isangi, Grumpensis, Rubislaw, Miami, Cerro, Thompson e duas amostras identificadas como Salmonella enterica subsp. houtenae. Quando analisadas quanto ao seu perfil filogenético, observou-se 14 clones distintos. Isolados com mesma origem clonal circulam em diferentes pontos de coletas no interior e entorno do Parque Nacional Serra da Capivara. Os dados obtidos neste estudo são relevantes para a Saúde Pública, pois contribuem para o monitoramento ambiental a partir do conhecimento da epidemiologia dos enteropatógenos circulantes no ambiente, impactando animais e a população que habita na região do PNSC. / The National Park Serra da Capivara (PNSC) is a proper environment for the developmentand conservation of a great number of species of the caatinga and endemic species, it is also aregion of singular importance for being the largest archaeological site in Brazil. The water inthis region is scant and the contact of people and of their animal whether they are pets or forbreeding, with the wild animals incresases the probability of circulation of pathogens, makingit possible for the appearance of deseases in men and in other animals. This study had theobjective of analisyng the presence and circulation of bacterias potencially enteropathenogenic and zoonotic in the region of the National Park Serra da Capivara ,located in the southeast of the state of Piaui Brazil. Water samples, feces of humans and ofbreeding and wild animals were collected in different regions inside and in the surroundings of the PNSC, during trips to the field mounting up to 170 samples collected. Five Hundredand twenty two isolated were obtained to the following genus/species of the family Enterobacteriaceae: Escherichia coli (32.6 percent); Enterobacter spp. (22.8 percent); Klebsiella spp (11.8 percent); Salmonella spp (10 percent), Citrobacter spp. (9.2 percent) amongst others. The isolation of thesample of the genus Campylobacter, important enteropathogen of public health impact, wasnot possible. The percentage of resistence of the samples to, at least, one of the drugs testedwas from 71 percent and the percentages of resistence were more representative for the drugs: penicilin, cefoxitin and tetracyclin. The surveillance of Salmonella serovars in aquatic environment constitutes an important element for the monitoring of animal and humaninfections. (AU)^ien / In this study 11 different Salmonella serovars were identified: Worthington, Panama, Brooklyn, Glasgow, Braenderup, Isangi, Grumpensis, Rubislaw, Miami, Cerro, Thompson and 2 samples identified as S. enterica subsp. houtenae. When analysed accordingto their filogenetic profile, they produced 14 different clones. Isolate samples with sameclonal origen circulate in distinct points of collect inside and in the surroundings of the PNSC. The data obtained in this study are relevant for public health, because they contribute to the environmental monitoring based on epidemiology knowledge of the enteropathogens circuling in the environment, causing impact on the animals and in the population living in the area of the PNSC. (AU)^ien

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-29Bitstream added on 2014-06-13T19:35:22Z : No. of bitstreams: 1 sato_vs_me_jabo.pdf: 522350 bytes, checksum: 34c42a9a6f174fa4f2002e3d48b07c50 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A capacidade de bactérias endofíticas em solubilizar fosfato inorgânico é alvo de grande interesse por parte dos microbiologistas, uma vez que as fosfatases são responsáveis por hidrolisar compostos orgânicos produzindo fósforo solúvel. Dessa forma, a fosfatase ácida ligada à membrana (MBAP) foi obtida a partir de Enterobacter sp. isolada de raízes de Cattleya walkeriana (Orchidaceae) e identificada pelo seqüenciamento do gene 16S rRNA. A expressão da enzima mostrou-se estritamente regulada pelo fósforo (expressão ideal em 7 mm). O pH ótimo aparente (3,5) não foi afetado pela concentração de p-nitrofenilfosfato. Em pH 3,5, a enzima é uma fosfomonidrolase inespecífica capaz de hidrolisar os substratos PNPP (61,2 U/mg), ATP (19,7 U/mg), e o pirofosfato (29,7 U/mg), com K0.5 de 0,06 mM, 0,11 mM e 0,08 mM, respectivamente. A enzima exibi cinética Michaelina para o pNPP (n=1,2). Para o ATP e o pirofosfato interações sítio-sítio foram observadas com n=1,6 e 2,3, respectivamente. Os íons de magnésio foram potentes estimuladores (K0.5=2,2 mM), enquanto o arsenato e o fosfato foram potentes inibidores competitivos. A atividade PNPPase foi inibida pelo EDTA, mas não pelo cálcio, levamisol, zinco, cobalto e phidroximercuribenzoato. A entalpia de inativação térmica foi da ordem de 77,5 kcal.mol- 1. Os resultados sugerem que a produção da fosfatase ácida ligada à membrana representa um mecanismo de solubilização do fosfato mineral aumentando a disponibilidade de nutrientes para as plantas / The ability of endophytic bacteria in solubilizing inorganic phosphate is of great interest by microbiologists since phosphatases are responsible for catalyzing the hydrolysis of organic compounds producing soluble phosphorus. Thus, the membranebound acid phosphatase (MBAP) was obtained from Enterobacter sp. isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis. The enzyme expression was demonstrated to be strictly regulated by phosphorus (optimal expression at 7 mM). The enzyme was obtained by centrifugation at 100.000g for 1 h at 4ºC. The apparent optimal pH (3.5) was not affect by p-Nitrophenyl phosphate concentration. At pH 3.5, the enzyme showed a broad substrate specificity hydrolyzing different substrates such as PNPP (61.2 U/mg), ATP (19.7 U/mg), and pyrophosphate (29.7 U/mg), with K0.5 values of 0.06 mM, 0.11 mM and 0.08 mM, respectively. The hydrolysis of PNPP by the enzyme exhibited Michaelian kinetics with n= 1.2. For ATP and pyrophosphate site-site interactions were observed with n= 1.6 and 2.3, respectively. Although magnesium ions were stimulatory (K0.5= 2.2 mM), arsenate and phosphate were a powerful competitive inhibitor. The PNPPase activity was inhibited EDTA but not by calcium, levamisole, zinc, cobalt and phydroxymercurybenzoate. The ΔH for thermal inactivation was 77.5 kcal.mol-1. Our results suggest that the production of a membrane-bound acid phosphatase might be one mechanism of mineral phosphate solubilization turn it´s nutrients availability to plants

Orientador: Tomomasa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T07:05:53Z (GMT). No. of bitstreams: 1 Kota_DanielJiro_M.pdf: 555074 bytes, checksum: 910a5d4735f12d01edac8766de74e9c5 (MD5) Previous issue date: 2008 / Resumo: A bactéria Enterobacter cloacae tem se destacado como importante agente de infecção hospitalar, muitas vezes levando ao óbito de pacientes imunodeprimidos. Estudando as propriedades de virulência desta bactéria em nosso laboratório, foi detectado que 18% das amostras estudadas demonstraram atividade citotóxica sobre células Vero. A citotoxina, de peso molecular de 100 KDa, foi purificada da amostra com maior atividade citotóxica (118.9) por cromatografia de troca iônica e exclusão molecular, não mostrou atividade letal em camundongos e não causou acúmulo de fluido em intestino de camundongos recém-nascidos. Além disso, não apresentou atividade leucotóxica. Quanto à sua propriedade hemolítica, constatou-se que a toxina purificada apresentou atividade hemolítica sobre eritrócitos de coelho, carneiro e boi, sendo não hemolítica sobre eritrócitos humanos, com presença ou ausência de ~mercaptoetanol. O mecanismo de ação desta toxina parece estar relacionado com o comprometimento da mitocôndria, o que ativaria a via apoptótica e a lise da membrana das células Vero. Quand~ aquecida por 100°C por 30 minutos, esta perdeu toda sua atividade citotóxica, demonstrando ser termolábil. Apesar da similaridade em sua citotoxicidade sobre as células Vero com os efeitos causados por verotoxinas, os genes STX (Shiga-Toxin) 1 e 2 e VT (Verotoxin)1 e 2, característicos de verotoxinas, não foram amplificados pela PCR / Abstract: The bacterium Enterobacter cloacae has been recognized as an important infectious agent in hospitaIs, sometimes leading patients to obit. Studying its virulence properties in our laboratory, 18% of the strains were characterized as cytotoxic against Vero cells. The cytotoxin was purified from the most cytotoxic strain (118.9) by means ionic exchange and molecular exclusion chromatography techniques respectively, increasing its relative activity in 62.5%. The purified toxin did not display either lethal activity nor did it cause fluid accumulation in neonatal mice intestine. Furthermore, no leukotoxic activity was detected and regarding its hemolytic activity, the purified toxin showed hemolytic activity against rabbit, bovine and sheep erythrocytes, but not against human erythrocytes, with or without beta-mercaptoethanol. The toxicity mechanism seems to be associated with a failure in the mitochondria's function, which would activate the apoptotic pathway and membrane lyses. When heated for 100°C for 30 minutes, the toxin lost completely its cytotoxicity. Despite the fact that the toxin displayed similar effects in Vero cells compared to verotoxins, PCR did not amplified the STXl and -2 and VTl and 2 genes, found in verotoxin expressing strains / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Enterobacter sakazakii é um bacilo Gram-negativo, pertencente à família Enterobacterieceae. Este microrganismo vem ganhando a atenção das autoridades de saúde pública ao redor do mundo, não tanto pela morbidade, que é baixa, mas pela elevada taxa de mortalidade que varia de 40-80%. O patógeno afeta principalmente recém-nascidos de baixo peso e bebês com até seis meses de idade. Em comum, estas crianças têm o fato de serem alimentadas com fórmula infantil desidratada, a base de leite. Em nosso país ainda não existem muitos estudos sobre a ocorrência deste patógeno em fórmulas infantis, nem no ambiente de preparo das mesmas. O objetivo deste trabalho foi avaliar as condições de produção de mamadeiras para recém-nascidos em maternidades da Grande São Paulo, além de determinar a população de E. sakazakii em fórmulas infantis desidratadas e reidratadas. A população de Enterobacteriaceae e a presença de E. sakazakii também foram avaliadas em amostras ambientais, de utensílios e mão de manipuladores. Avaliou se ainda o comportamento do patógeno em fórmula infantil reidratada simulando as condições de oferecimento aos bebês. Coletou-se amostras de três hospitais maternidades diferentes (A-escola/B-público/C-particular) e analisou-se a presença de E. sakazakii usando método ISO. Para fórmulas desidratadas e reidratadas usou-se a mesma metodologia e a técnica de número mais provável (NMP). A população de Enterobacteriaceae foi determinada usando-se PetrifilmTM 3M. E. sakazakii foi detectada em duas amostras do Hospital A (na sobra da mamadeira que voltou do berçário e de uma amostra da lata lacrada da fórmula infantil desidratada. A população nesta amostra foi de 0,03 NMP/100g.) No Hospital B, foi detectada em apenas uma amostra (na esponja de lavagem das mamadeiras contaminadas). No Hospital C, E. sakazakii não foi detectada nas amostras analisadas. Quanto à população de Enterobacteriaceae nos lactários, observou-se uma variação, sendo que as amostras colhidas no hospital C foram as que apresentaram populações mais elevadas. As cepas de E. sakazakii isoladas apresentaram comportamento similar àquele da cepa padrão, ocorrendo um aumento de 2 log na população do patógeno quando simulou-se as condições de serviço das fórmulas, via naso-gástrica, aos bebês nos berçários. / Enterobacter sakazakii is a bacillus belonging to the Enterobacteriaceae family. It is considered an opportunistic pathogen that has been gaining attention from health authorities all over the world. While morbidity associated with this bacterium is low, mortality rates can range from 40-80%. The pathogen affects mainly low-birth-weight neonates (first 28 days), but babies less than 6 month old are also at risk. Powdered infant formula has been incriminated as the possible source of the microorganism to the infected babies. ln Brazil, as in several other countries, there is scarce information regarding the incidence of E. sakazakii in powdered infant formula, in reconstituted formula, and in milk kitchens areas in hospitals. The objective of this study was to evaluated the presence of E. sakazakii in the environment, utensils, handlers, powdered and rehydrated infant formula from milk kitchens from different maternity wards in Sao Paulo, Brazil. Moreover, it was evaluated the behavior of the pathogen in rehydrated infant formula. Samples were collected from 3 hospitals maternities (A-school/B-public/C-particular) and analyzed for E. sakazakii using the ISO method. For formula (powdered or rehydrated) the MPN technique was used. Enterobacteriaceae population was determined using PetrifilmTM 3M. E. sakazakii was found in one unopened formula can collected from Hospital A (0,03 MPN/100g), although the pathogen could not be detected in other cans from the same lot. E. sakazakii was also found in leftovers from one nursing bottle from the same hospital and from one cleaning sponge from Hospital B. E. sakazakii was not detected in none of the samples from Hospital C. A variation in Enterobacteriaceae population in milk kitchens was observed. Samples collected in Hospital C presented the highest population. Isolated strains of E. sakazakii presented similar behavior to standard strains, When spiked in rehidrated infant formula. A 2 log increase in the population of the pathogen was observed when simulating the conditions of formula administration to the babies by naso-gastric tubing.

Des contaminations par Enterobacter gergoviae, dans les formulations cosmétiques diverses soulèvent le problème de la résistance bactérienne aux biocides. J'ai pu mettre en évidence, que certains épisodes de contaminations apparus au sein de l'entreprise partenaire, étaient dus à un même isolat persistant suite à un système de conservation/désinfection inefficace. Aux concentrations maximales autorisées par la commission européenne, les conservateurs ont des effets bactériostatiques mais ne présentent pas d'effets synergiques en association. Par conséquent, l'adaptation aux produits cosmétiques est favorisée. Divers mécanismes de résistance peuvent être mis en place par E. gergoviae : enzymatique (synthèse de peroxyredoxine), modifications de l'enveloppe (expression d'appendices externes : flagelles ou fimbriae/pili), modification de la mobilité, et formation de biofilms. L'apparition de ces mécanismes a aussi favorisé les résistances croisées conservateurs /désinfectants-détergents. / Contaminations by Enterobacter gergoviae in various cosmetic formulations raise the problem of bacterial resistance to biocides of the species, which represent the third source of cosmetics bacterial contaminations. The collaborative cosmetic company was concerned by unrelated contaminations but some contamination events were due to persistence of isolates in internal system process, despite disinfection protocols and use of preservatives. At the maximum levels allowed by the European Commission, preservatives tested have bacteriostatic effects and do not exhibit synergistic effects in combination. Therefore, adaptation in cosmetics is facilitated. Various resistance mechanisms can be implemented by E. gergoviae: Enzymatic (peroxyredoxin synthesis), changes in the envelope (expression of external appendages: flagella or fimbriae/pili), changes in mobility and biofilm formation. The appearance of such mechanisms has also promoted a cross-resistance conservative / disinfectant - detergent.

Abiotic stresses such as salt stress are the major limiting factors for agricultural productivity, and cause global food insecurity. It is well known that plant associated beneficial microorganisms can stimulate plant growth and enhance resistance to abiotic stresses. In this context, bacterial endophytes are a group of bacteria that colonize the host plant and play a fundamental role in plant growth enhancement under stress condition. Recently, our group reported that the beneficial bacteria Enterobacter sp.SA187 induces plant growth in Arabidopsis under salt stress conditions by manipulation of the plant ethylene signaling pathway. We therefore compared inoculation of plants by SA187 with virulent and non-virulent strains Pst DC3000. Although both strains inhibit plant growth at ambient conditions, Pst DC3000 hrcC-, but not Pst DC3000, induced salt stress tolerance, suggesting that Pst DC3000 hrcC- also contains plant growth promoting activity under stress conditions. Our results indicate that Pst DC3000 hrcC- shares features with beneficial bacteria by inducing salt tolerance through reduction of the shoot and root Na+/K+ ratio. To further elucidate the underlying mechanisms of this interaction with Arabidopsis, RNAseq, hormone and biochemical analyses were performed. Genetic studies also show that Pst DC3000 hrcC- induced salt stress tolerance involving several phytohormone pathways, including auxin, ethylene and salicylic acid. Transcriptome and genetic analyses indicate that glucosinolates play an important role in this beneficial interaction. We found that indolic and alkyl glucosinolates act as negative factors on Pst DC3000 hrcC-, alkyl glucosinolates are positive and indolic glucosinolates negative regulators in SA187 interaction with Arabidopsis. These results reveal that besides a repertoire of effectors, Pst DC3000 hrcC- also produces factors that can be beneficial for plant growth under certain stress conditions, as observed with Enterobacter sp. SA187.