by Rabi Biswas. / Publication date from spine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 122-143). / PREFACE --- p.2 / ACKNOWLEDGEMENTS --- p.3 / CONTENTS --- p.4 / GLOSSARY --- p.9 / ABSTRACT --- p.11 / INTRODUCTION --- p.12 / Chapter 1.1. --- OVERVIEW --- p.12 / Chapter 1.2. --- OBJECTIVES OF THE STUDY --- p.14 / LITERATURE REVIEW --- p.15 / Chapter 2.1. --- BACKGROUND OF THE STUDY --- p.15 / Chapter 2.2. --- ESCHERICHIA COLI : OVERVIEW --- p.17 / Chapter 2.2.1. --- Morphology --- p.18 / Chapter 2.2.2. --- Cultural characteristics --- p.18 / Chapter 2.2.3. --- Biochemical reactions --- p.19 / Chapter 2.2.4. --- Antigenic Structure --- p.19 / Chapter 2.2.5. --- Identification --- p.20 / Chapter 2.2.6. --- Classification of coli --- p.20 / Chapter 2.3. --- HISTORY OF EPEC --- p.22 / Chapter 2.3.1. --- E. coli as a cause of diarrhoea --- p.22 / Chapter 2.3.2. --- The first use of the term EPEC --- p.23 / Chapter 2.3.3. --- EPEC as a separate category of E. coli --- p.24 / Chapter 2.4. --- PATHOGENESIS OF EPEC --- p.25 / Chapter 2.4.1. --- Plasmid encoded virulence properties --- p.25 / Chapter 2.4.2. --- Characteristic interaction with intestinal mucosa --- p.26 / Chapter 2.4.3. --- Production of toxins --- p.28 / Chapter 2.5. --- EPIDEMIOLOGY OF EPEC --- p.29 / Chapter 2.6. --- EPIDEMIOLOGY OF EPEC IN CHINA AND HONG KONG --- p.32 / Chapter 2.7. --- CLINICAL INFECTION BY EPEC AND MANAGEMENT --- p.33 / Chapter 2.7.1. --- Epidemiological syndromes --- p.33 / Chapter 2.7.2. --- Infective dose --- p.33 / Chapter 2.7.3. --- Incubation period --- p.33 / Chapter 2.7.4. --- Host factors --- p.33 / Chapter 2.7.5. --- Reservoirs of infection --- p.33 / Chapter 2.7.6. --- Routes of transmission --- p.34 / Chapter 2.7.7. --- Seasonal variation --- p.34 / Chapter 2.7.8. --- Mechanism of diarrhoea --- p.34 / Chapter 2.7.9. --- Histology --- p.35 / Chapter 2.7.10. --- Clinical features --- p.35 / Chapter 2.7.11. --- Treatment --- p.35 / Chapter 2.7.12. --- Prevention --- p.36 / Chapter 2.8. --- DETECTION OF EPEC: LABORATORY METHODS --- p.36 / Chapter 2.8.1. --- O/H Serotyping --- p.36 / Chapter 2.8.2. --- Adhesion assay --- p.37 / Chapter 2.8.3. --- EAF probe --- p.37 / Chapter 2.8.4. --- FAS (Fluorescein Actin Staining) test --- p.37 / Chapter 2.8.5. --- ELISA (Enzyme Linked Immunosorbent Assay) --- p.38 / Chapter 2.8.6. --- eaeA gene probe --- p.38 / Chapter 2.8.7. --- bfpA probe --- p.39 / Chapter 2.8.8. --- PCR (Polymerase Chain Reaction) --- p.39 / MATERIALS AND METHODS --- p.41 / Chapter 3.1. --- PATIENT RECRUITMENT AND DATA COLLECTION --- p.41 / Chapter 3.1.1. --- Study site --- p.41 / Chapter 3.1.2. --- Study design --- p.41 / Chapter 3.1.3. --- Study period --- p.42 / Chapter 3.1.4. --- Study population --- p.42 / Chapter 3.1.5. --- Selection of patients --- p.42 / Chapter 3.1.6. --- Inclusion criteria for cases --- p.43 / Chapter 3.1.7. --- Exclusion criteria --- p.43 / Chapter 3.1.8. --- Selection of control group --- p.43 / Chapter 3.1.9. --- Collection of stool specimens --- p.44 / Chapter 3.1.10. --- Treatment of the study-patients --- p.45 / Chapter 3.1.11. --- Collection of date --- p.45 / Chapter 3.1.12. --- Ethical approval --- p.46 / Chapter 3.2. --- LABORATORY METHODS --- p.47 / Chapter 3.2.1. --- IN PWHLABORATORY --- p.47 / Chapter 3.2.2. --- IN AFRIMS LABORATORY --- p.48 / Chapter 3.3. --- DATA MANAGEMENT AND STATISTICAL METHODS --- p.63 / RESULT --- p.64 / Chapter 4.1. --- DEMOGRAPHY OF THE PATIENTS --- p.64 / Chapter 4.1.1. --- Age distribution of the patients --- p.64 / Chapter 4.1.2. --- Sex distribution of the patients --- p.64 / Chapter 4.1.3. --- Ethnic origin of the patients --- p.65 / Chapter 4.1.4. --- Distribution of area of abode in Hong Kong --- p.66 / Chapter 4.1.5. --- School attendance of the patients --- p.66 / Chapter 4.2. --- PREDISPOSING FACTORS FOR DIARRHOEA --- p.67 / Chapter 4.2.1. --- History of breast feeding of the patients --- p.67 / Chapter 4.2.2. --- History of contact with diarrhoea --- p.68 / Chapter 4.2.3. --- Travel history within last two weeks preceding onset of diarrhoea --- p.68 / Chapter 4.2.4. --- Source of drinking water --- p.69 / Chapter 4.3. --- CLINICAL FEATURES --- p.70 / Chapter 4.3.1. --- Duration of diarrhoea at the time of admission --- p.70 / Chapter 4.3.2. --- Frequency of stool --- p.71 / Chapter 4.3.3. --- Nature and contents of stool --- p.72 / Chapter 4.3.4. --- Condition of the perineum --- p.72 / Chapter 4.3.5. --- Duration of vomiting at the time of admission --- p.73 / Chapter 4.3.6. --- Frequency of vomiting --- p.73 / Chapter 4.3.7. --- Level of dehydration in cases --- p.74 / Chapter 4.3.8. --- Urine output during illness --- p.74 / Chapter 4.3.9. --- Fever associated with illness --- p.75 / Chapter 4.4. --- HISTORY OF HOME- MANAGEMENT --- p.77 / Chapter 4.4.1. --- Main food taken at home during illness --- p.77 / Chapter 4.4.2. --- Supplementary fluid taken at home during illness --- p.77 / Chapter 4.4.3. --- Duration of hospital stay --- p.78 / Chapter 4.4.4. --- Recruitment of patients in different months --- p.79 / Chapter 4.5. --- RESULTS OF GENE PROBING FOR E. COLI --- p.80 / Chapter 4.6. --- DETAILS OF THE EAF+ EPEC CASES --- p.82 / Chapter 4.6.1. --- Associated infections in EAF+ cases --- p.83 / Chapter 4.7. --- AETIOLOGY OF DIARRHOEA --- p.84 / Chapter 4.7.1. --- Age distribution --- p.85 / Chapter 4.7.2. --- Seasonal distribution --- p.86 / Chapter 4.7.3. --- Clinical features --- p.87 / Chapter 4.7.4. --- Different groups of Salmonella --- p.88 / Chapter 4.7.5. --- Dual infection among enteropathogens isolated --- p.89 / DISCUSSION --- p.90 / Chapter 5.1. --- RISK FACTORS ASSOCIATED WITH DIARRHOEA --- p.91 / Chapter 5.1.1. --- Age and sex of the patients --- p.91 / Chapter 5.1.2. --- Nutritional status --- p.91 / Chapter 5.1.3. --- Breast feeding --- p.92 / Chapter 5.1.4. --- Travelling --- p.93 / Chapter 5.2. --- SEVERITY OF DIARRHOEA IN HONG KONG --- p.93 / Chapter 5.3. --- MOLECULAR EPIDEMIOLOGY OF EPEC IN HONG KONG --- p.94 / Chapter 5.3.1. --- EAF probe --- p.94 / Chapter 5.3.2. --- EAF and EPEC virulence --- p.95 / Chapter 5.3.3. --- eaeA probe --- p.96 / Chapter 5.3.4. --- FAS test --- p.97 / Chapter 5.3.5. --- bfpA probe --- p.97 / Chapter 5.3.6. --- Comparison and contrast among the probes --- p.97 / Chapter 5.3.7. --- Probes in the present study --- p.99 / Chapter 5.3.8. --- Role of serogrouping at present --- p.100 / Chapter 5.3.9. --- EPEC in Hong Kong --- p.100 / Chapter 5.4. --- PREVALENCE OF OTHER CATEGORIES OF E. COLI --- p.101 / Chapter 5.5. --- COMMON AETIOLOGY OF DIARRHOEA IN HONG KONG --- p.102 / Chapter 5.5.1. --- Rotavirus as the most common cause --- p.102 / Chapter 5.5.2. --- Non-typhoid Salmonella as the major bacterial pathogen --- p.102 / Chapter 5.5.3. --- Campylobacter and Shigella as cause of diarrhoea --- p.103 / Chapter 5.5.4. --- Role of parasites in childhood diarrhoea in Hong Kong --- p.104 / Chapter 5.6. --- CONTROL MEASURES FOR DIARRHOEAL DISEASES --- p.105 / Chapter 5.6.1. --- Prevention of diarrhoea through improved nutrition --- p.105 / Chapter 5.6.2. --- Fluid supplementation in diarrhoea --- p.106 / Chapter 5.6.3. --- Strategies to control diarrhoea --- p.106 / Chapter 5.6.4. --- Health education --- p.107 / CONCLUSION --- p.108 / APPENDIX --- p.109 / Chapter 7.1. --- QUESTIONNAIRE --- p.109 / Chapter 7.2. --- LABORATORY METHODS --- p.111 / Chapter 7.2.1. --- Routine culture of stool specimens for bacteria --- p.111 / Chapter 7.2.2. --- "Serotyping of Salmonella, Shigella and Vibrio cholerae" --- p.114 / Chapter 7.2.3. --- Microscopic examination of ova and cysts --- p.117 / Chapter 7.2.4. --- Laboratory diagnosis of rotavirus --- p.118 / Chapter 7.3. --- INVESTIGATION REQUISITION FORM --- p.121 / REFERENCES --- p.122 / GRADUATE SEMINARS & PUBLICATIONS --- p.144 / Chapter a. --- Graduate seminars --- p.144 / Chapter b. --- Publications --- p.144

Orientadores: Wanderley Dias da Silveira, Eliana Guedes Stehling / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T23:32:04Z (GMT). No. of bitstreams: 1 Pace_Fernandade_D.pdf: 4667016 bytes, checksum: c841e6b899f0efc5569c9c6c6ce46935 (MD5) Previous issue date: 2011 / Resumo: Linhagens de Escherichia coli patogênica para aves (APEC) causam infecções extraintestinais e são responsáveis por significativas perdas econômicas na indústria avícola mundial. Recentemente, foram descritos isolados de APEC geneticamente relacionados a diversas outras E.coli extraintestinais (ExPEC) de origem humana, indicando a possibilidade das mesmas constituírem risco zoonótico para humanos. Alguns dos conhecidos fatores de virulência de APEC incluem adesinas, sistema de aquisição de ferro, citotoxinas, entre outros. Nesse trabalho, demonstramos que a linhagem de APEC SEPT 362, isolada do fígado de uma ave apresentando sinais clínicos de septicemia, expressa o Sistema de Secreção Tipo VI (SST6), causa rearranjo do citoesqueleto de células epiteliais cultivadas in vitro, é capaz de aderir e invadir células HeLa e é viável dentro de macrófagos. Para estudar o envolvimento do SST6 na patogênese da linhagem SEPT362, foram deletados três genes desse sistema: hcp, que codifica para uma proteína estrutural e secretada, clpV, que codifica para uma ATPase e icmF (intracellular multiplication factor), gerando três mutantes, respectivamente. Todos os mutantes demonstraram uma diminuição nos processos de adesão e invasão a células HeLa, formação de biofilme e virulência in vivo. Estudos de transcriptoma mostraram que a expressão da fímbria tipo 1 encontra-se diminuída nesses mutantes, o que poderia ser responsável pela diminuição do processo de adesão e invasão às células epiteliais. Nesse trabalho, demonstramos que o SST6 é importante para o processo de patogenicidade, visto que todos os mutantes tiveram sua virulência atenuada em experimentos realizados in vivo com uma significativa diminuição de características relacionadas à patogenicidade in vitro. Esses resultados demonstram que os genes estudados do SST6 influenciam a expressão da fímbria tipo 1 e contribuem para a patogênese desta linhagem APEC / Abstract: Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins, among others. Here we demonstrated that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS), causes cytoskeleton rearrangements, invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated three mutants, ?hcp (which encodes a protein suggessed to be both secreted and a structural component of the T6SS), ?clpV (encoding the T6SS ATPase) and ?icmF (intracellular multiplication factor). All mutants showed decreased adherence and invasion to HeLa cells and decrease in several other pathogenicity related characteristics. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants (?hcp and ?clpV) were attenuated in an infection model in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to the pathogenesis of this APEC strain pathogenesis / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Orientadores: Wanderley Dias da Silveira, Marcelo Brocchi / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T05:13:36Z (GMT). No. of bitstreams: 1 Paiva_JacquelineBoldrinde_D.pdf: 5619127 bytes, checksum: 79177e4233fdbb891e6530eb0f3438bd (MD5) Previous issue date: 2014 / Resumo: Escherichia coli patogênica para aves (APEC) é responsável por inúmeras perdas no setor avícola mundial, por causar uma série de doenças nas aves que se apresentam de forma sistêmica ou localizada as quais são, coletivamente, denominadas colibacilose. Os mecanismos de virulência destas linhagens patogênicas para aves e, possivelmente, patogênicas para seres humanos ainda não foram totalmente elucidados. Este trabalho foi desenvolvido com o intuito de estudar genes possivelmente envolvidos com a patogenicidade de uma linhagem APEC causadora de Síndrome da Cabeça Inchada (SCI-07) ONT:H31, a partir de resultados obtidos em um microarranjo realizado in vitro, o qual comparou a linhagem em estudo à linhagem padrão E. coli EHEC 8624 (linhagem enterohemorrágica). Nove genes, detectados como sendo super-expressos no microarranjo, nas condições estudadas, foram selecionados para construção de mutantes nulos e de seus complementos [feoA (transporte de ferro), nirC (transportador de nitrito), flgE ( gancho flagelar), tyrR (regulador de transcrição da síntese de aminoácidos aromáticos), potF (subunidade periplasmática do transportador putrescina), yehD (possível fimbria), bfr (bacterioferrina), csgA ( subunidade principal da curlina) e entD (enteroquelina)]. Os mutantes construídos foram avaliados quanto as suas capacidades de adesão e invasão em cultivos celulares, e quanto ao seu potencial patogênico em aves de um dia de idade em comparação à cepa selvagem. As linhagens ?bfr, ?csgA e ?nirC apresentaram diminuição da capacidade de adesão em fibroblastos de aves (linhagem FEG) em comparação à linhagem selvagem em ambos os modelos adotados: na presença e ausência de alpha-D-mannopyranoside; a linhagem ?potF apresentou diminuição da adesão apenas na ausência de alpha-D-mannopyranoside. Os mutantes ?csgA e ?tyrR apresentaram redução na capacidade de invadir linhagem de células de trato respiratório humano (linhagem Hep-2). Nenhum mutante avaliado mostrou alteração na capacidade de invadir fibroblastos de aves (linhagem CEC-32). Os mutantes ?flgE e ?tyrR mostraram diminuição na capacidade de invadir e sobreviver em macrófagos de aves (linhagem HD11). A motilidade das linhagens mutantes ?csgA, ?bfr, ?yehD, ?potF, ?entD, ?nirC e ?feoA foi aumentada enquanto o mutante ?tyrR mostrou redução de motilidade e o mutante ?flgE tornou-se imóvel. Nenhuma linhagem mutante obtida mostrou a mesma capacidade da linhagem selvagem em causar mortalidade em aves de um dia; ?feoA tornou-se hipervirulenta e todos os demais mutantes apresentaram atenuação em diferentes graus, sendo a linhagem ?entD totalmente atenuada e, portanto, promissora quanto ao seu uso como linhagem vacinal.para o combate à colibacilose em aves, ou como linhagem carreadoras de epítopos presentes em outras linhagens APEC / Abstract: Avian Pathogenic Escherichia coli (APEC) is responsible for significant economic loses in the poultry industry worldwide, by cause a range of systemic or localized diseases in poultry collectively termed colibacillosis. The virulence mechanisms of these pathogenic strains for poultry and possibly pathogenic for humans have not been fully elucidated. This work was developed in order to study genes potentially involved in the pathogenicity of an APEC strain isolated from a Swollen Head Syndrome case (SCI- 07) ONT:H31; since the results obtained in a microarray performed in vitro, which compared the SCI-07 strain to the standard strain E. coli 8624 EHEC (enterohemorrhagic strain). Nine overexpressed genes in microarray under the conditions studied were selected for construction of null mutants and their complements [feoA (iron transport), nirC (nitrite transporter), flgE (flagellar hook), tyrR (transcriptional regulator of the aromatic amino acids biosynthesis), potF (periplasmic putrescine transporter subunit), yehD (putative adhesin), bfr (bacterioferritin), csgA (major curling subunit) and entD (enterochelin)]. The mutants constructed were evaluated for their capacity for adhesion and invasion in cell cultures, and for its pathogenic potential in one-day-old chickens in comparison to the wild type strain (WT). The ?bfr, ?csgA and ?nirC strains showed decreased adhesion capacity on avian fibroblasts (CEF cells) compared to the WT in both models adopted: in the presence and absence of alpha-D-mannopyranoside, the ?potF strain showed decrease on adhesion only in the absence of alpha-D-mannopyranoside. The ?csgA and ?tyrR mutants had reduced ability to invade human larynx cell line (Hep-2 cells). No mutant showed changes in the capacity of invade avian fibroblasts birds (CEC-32cells). The ?flgE and ?tyrR mutants showed decreased ability to invade and survive into avian macrophages (HD11 cells). The motility of mutant strains ?csgA, ?bfr, ?yehD, ?potF, ?entD, ?nirC and ?feoA was increased while the ?tyrR mutant showed reduced motility and the mutant ?flgE became nonmotile. No mutant strain showed the same capacity of the WT in cause mortality in one-day-old chickes; ?feoA became hipervirulenta and all other mutants showed attenuation in different degrees, including the ?entD that was completely attenuated and a promising vaccine candidate strain to combat colibacillosis in poultry, or as a carrier strain of epitopes present in other APEC strains / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Escherichia coli enteropatogênica (EPEC) é uma das principais causas de diarreia em crianças. Na carência de fosfato (Pi), um conjunto de genes conhecido como regulon PHO é induzido. Esse regulon é controlado pelo sistema Pst, que além de ser um transportador de Pi, reprime a expressão de PHO quando Pi é abundante, e pelo sistema de dois componentes PhoB/PhoR. A deleção de pst reduziu a adesão de EPEC à células epiteliais in vitro, pois diminuiu da expressão dos reguladores PerA/PerC, que por sua vez controlam a expressão de genes envolvidos na adesão. Este efeito foi exclusivo de pst e não devido a expressão constitutiva dos genes de PHO causada pela deleção de pst. A expressão da fímbria BFP, PerA e PerC também dependem da síntese de ppGpp, uma molécula de alarme envolvida na regulação de genes relacionados à carência nutricional. ppGpp regula positivamente a expressão de PerA e PerC. Entretanto, RpoS, o fator relacionado à resposta ao estresse, afetou negativamente o nível de adesão de EPEC e a expressão de BFP. / Enteropathogenic E. coli (EPEC) is one of the causes of diarrhea in children. Phosphate (Pi) shortage induces transcription of the genes known as the PHO regulon. These genes are controlled by the Pst system, that is also a high-affinity Pi transporter, and represses PHO expression under Pi-replete conditions. PHO is also controlled by the two-component system PhoB/PhoR. Deletion of the pst operon reduced the adhesion of EPEC to epithelial cells in vitro due to a decrease in the expression of the regulators PerA and PerC that in turn control the expression of genes related to adhesion. The constitutive expression of the PHO genes in the pst mutant was not the cause of adhesion inhibition. Expression of bfp and the regulators PerA and PerC was also dependent on ppGpp, an alarmone involved in the regulation of genes related to nutrient limitation. On the other hand, RpoS, the factor that controls the general stress response, negatively affected EPEC adhesion and bfpA expression.

Escherichia coli enteropatogênica (EPEC) é uma das principais causas de diarreia em crianças. Na carência de fosfato (Pi), um conjunto de genes conhecido como regulon PHO é induzido. Esse regulon é controlado pelo sistema Pst, que além de ser um transportador de Pi, reprime a expressão de PHO quando Pi é abundante, e pelo sistema de dois componentes PhoB/PhoR. A deleção de pst reduziu a adesão de EPEC à células epiteliais in vitro, pois diminuiu da expressão dos reguladores PerA/PerC, que por sua vez controlam a expressão de genes envolvidos na adesão. Este efeito foi exclusivo de pst e não devido a expressão constitutiva dos genes de PHO causada pela deleção de pst. A expressão da fímbria BFP, PerA e PerC também dependem da síntese de ppGpp, uma molécula de alarme envolvida na regulação de genes relacionados à carência nutricional. ppGpp regula positivamente a expressão de PerA e PerC. Entretanto, RpoS, o fator relacionado à resposta ao estresse, afetou negativamente o nível de adesão de EPEC e a expressão de BFP. / Enteropathogenic E. coli (EPEC) is one of the causes of diarrhea in children. Phosphate (Pi) shortage induces transcription of the genes known as the PHO regulon. These genes are controlled by the Pst system, that is also a high-affinity Pi transporter, and represses PHO expression under Pi-replete conditions. PHO is also controlled by the two-component system PhoB/PhoR. Deletion of the pst operon reduced the adhesion of EPEC to epithelial cells in vitro due to a decrease in the expression of the regulators PerA and PerC that in turn control the expression of genes related to adhesion. The constitutive expression of the PHO genes in the pst mutant was not the cause of adhesion inhibition. Expression of bfp and the regulators PerA and PerC was also dependent on ppGpp, an alarmone involved in the regulation of genes related to nutrient limitation. On the other hand, RpoS, the factor that controls the general stress response, negatively affected EPEC adhesion and bfpA expression.