Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--University of Rhode Island, 2004. / Typescript. Includes bibliographical references (leaves 104-116).

Thesis (Ph. D., Microbiology, Molecular Biology and Biochemistry)--University of Idaho, August 2009. / Major professor: Carolyn H. Bohach. Includes bibliographical references. Also available online (PDF file) by subscription or by purchasing the individual file.

Thesis (Ph. D.)--University of Hong Kong, 1991.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Made available in DSpace on 2014-08-13T14:50:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-26Bitstream added on 2014-08-13T18:01:12Z : No. of bitstreams: 1 000735330_20140826.pdf: 26004 bytes, checksum: 80491d16dd561bc066d6dacbdc900451 (MD5) / A piometra canina é uma enfermidade caracterizada pela inflamação do útero com acúmulo de exsudatos, acometendo principalmente fêmeas adultas. Ocorre na fase lútea do ciclo estral em decorrência de alterações hormonais e infecção bacteriana. É reconhecida como uma das principais causas de morte em cadelas e a Escherichia coli é o principal patógeno associado a esta doença. O objetivo deste estudo foi isolar e identificar cepas de E. coli provenientes de conteúdo intra uterino, boca e fezes de cadelas diagnosticadas com piometra, estudar a prevalência de genes codificadores de fatores de virulência uropatogênicos das cepas obtidas testando ainda a semelhança genética entre as cepas isoladas de conteúdo intra uterino e boca e avaliar a susceptibilidade “in vitro” das bactérias isoladas frente a 12 agentes antimicrobianos. Setenta cepas de E. coli, 25 provenientes do conteúdo intra uterino, 26 provenientes da boca e 19 provenientes das fezes, isoladas de 6 cadelas foram examinadas por PCR. Entre as cepas examinadas, 67 (95,7%) foram positivas para o gene fim, 19 (27,1%) foram positivas para iss, 18 (25,7%) foram positivas para hly , 13 (18,5%) foram positivas para iuc e 12 (17,1%) foram positivas para usp. Três animais apresentaram grande similaridade entre as cepas de E. coli isoladas do conteúdo intra uterino e da boca, através da análise da diversidade genética realizada mediante emprego da técnica REP-PCR, ERIC-PCR e BOX-PCR. Resistência antimicrobiana predominante foi detectada para cefalotina (67,1%), ampicilina (65,7%), tetraciclina (61,4%) e nitrofurantoina (58,5%) entre as cepas isoladas. Resistência a múltiplos antimicrobianos foi detectada em 12 (48,0%) dos isolados do conteúdo intra uterino, em 22 (84,6%) dos isolados da boca e em 14 (73,6%) dos isolados das fezes... / Canine pyometra is a disease characterized by the inflammation of the uterus with accumulation of purulent discharge, affecting mainly adult animals. Occurs in the luteus phase of the estrus cycle in result of hormone alterations and generally is associated with bacterial infections. It is recognized as one of the main causes of disease and death in the bitch and Escherichia coli is the major pathogen associated with this disease. The aim of this study was to research, isolation and identification of Escherichia coli strains from intrauterine contents, mouth and feces of bitches diagnosed with pyometra, studying the prevalence of uropathogenic virulence factors genes in strains isolated, still testing the genetic similarity among strains isolated from intrauterine contents and mouth and evaluate the susceptibility in vitro of the isolated bacteria to 12 antimicrobial drugs. Seventy E. coli strains, 25 from intrauterine contents, 26 from mouth and 19 from feces isolated from six bitches were examined by PCR. Among the strains 67 (95.7%) were positive for fim, 19 (27.1%) were positive for iss, 18 (25.7%) were positive for hly, 13 (18.5%) were positive for iuc and 12 (17.1%) were positive for usp. Multiple antimicrobial resistance was detected for cephalothin (68.0%), nalidixic acid (56.0%) and ampicillin (56.0%) among the pyomera E. coli isolates. Multidrug resistance was found in 12 (48.0%) of the isolates from pyometra, in 22 (84.6%) of the isolates from mouth and in 14 (73.6%) of the isolates from feces...

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-31Bitstream added on 2014-06-13T19:22:44Z : No. of bitstreams: 1 maluta_rp_dr_jabo.pdf: 440520 bytes, checksum: 3c6ccf7fa3a09af508156250e6a377c0 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Canadian Bureau For International Education (Cbie) / Os ruminantes são reservatórios de cepas de Escherichia coli envolvidas na etiologia de doenças graves em humanos. Nesse estudo, a freqüência de E. coli Shigatoxigênica (STEC), E. coli enteropatogênica (EPEC) e E. coli enterotoxigênica (ETEC) foi determinada em fezes e carcaças de ovinos em três fazendas e um matadouro-frigorífico localizados no Estado de São Paulo e as cepas encontradas foram caracterizadas. A freqüência de STEC nas três fazendas foi similar, enquanto a freqüência de EPEC foi variável. Não foram encontradas amostras contendo ETEC. As cepas de STEC stx1- stx2+ revelaram-se geneticamente heterogêneos, possuindo freqüentemente as variantes Stx2a ou Stx2dact, as quais são relacionadas à doenças mais severas em humanos e ademais eles foram freqüentemente originados de amostras colhidas do matadouro-frigorífico. Adicionalmente, algumas cepas desse grupo possuíram novas variantes de Stx2 ou o subtipo Stx2e, que é relacionado à doença do edema em suínos. As cepas de STEC stx1+ stx2+ e stx1+ stx2- mostraram-se geneticamente mais homogêneas, a maioria possuindo os genes lpfAO113, iha e ehxA. As cepas de STEC stx1+ stx2- apresentaram comumente o gene relacionado à ExPEC tsh. As estirpes de EPEC foram heterogêneas, muitas possuíram os genes efa1, ehxA, lpfAO113 ou paa, que são associados à diarréia em humanos. As cepas de STEC e EPEC demonstraram-se geneticamente diversas quando analisadas por PFGE. Esses resultados demonstram que cepas de E. coli potencialmente patogênica para humanos estão presentes na microbiota intestinal de ovinos, com potencial de contaminar carcaças em abatedouro e conseqüentemente serem transmitidas por via alimentar / Ruminants are a reservoir of Escherichia coli which may cause severe disease in humans. Pathotypes related to intestinal disease include Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In this study, the prevalence of these pathotypes was examined in sheep feces and carcasses on three farms and at an abattoir. The strains were then characterized. The prevalence of STEC on the three farms was similar, whereas that of EPEC varied between farms. No ETEC were detected. STEC stx1- stx2+ strains were genetically heterogeneous, more frequently possessing Stx2 variant Stx2a or Stx2dact related to more severe disease in humans, and often originated from the abattoir rather than the farms. In addition, some strains of this group possessed new Stx2 variants or Stx2e, the subtype related to porcine edema disease. STEC stx1+ stx2+ and stx1+ stx2- strains were genetically more homogeneous, mostly possessed the genes lpfAO113, iha and ehxA. The STEC strains stx1+ stx2- commonly harbored ExPEC-related gene tsh. The EPEC strains were heterogeneous, several possessing efa1, ehxA, lpfAO113 or paa, genes associated with diarrhea in humans. STEC and EPEC strains were genotypically diverse by PFGE. These results demonstrate that E. coli potentially pathogenic for humans are present in the sheep intestinal microflora, particularly at the abattoir, underlining the potential for foodborne transmission

Orientador: Luiz Augusto do Amaral / Banca: Max Ternero Gangani / Banca: Juliana Bega Junqueira / Banca: Karina Paes Bürger / Banca: Angela Cleusa de Fátima Banzatto de Carvalho / Resumo: Os objetivos do estudo foram avaliar a qualidade do efluente quanto a dinâmica da população de Escherichia coli, presença de E. coli shigatoxigênicas e eliminação de ovos de helmintos. Além de avaliar a produção e os potenciais de produção de biogás pelo processo de codigestão anaeróbia dos dejetos de bovinos leiteiros com e sem adição de 7% de caldo de cana-de-açúcar e biorremediador. Para o ensaio 1, foram utilizados dez biodigestores bateladas divididos em dois tratamentos, dejeto sem caldo de cana-de-açúcar (DSC) e dejeto com caldo (DCC), com tempo de retenção hidráulica (TRH) foi de 90 dias. O ensaio 2, foi realizado concomitante ao primeiro, com a mesma distribuição de tratamentos, para o monitoramento periódico da dinâmica da população E. coli e presença E. coli shigatoxigênicas, do pH e da acidez volátil. Para tanto, foram abastecidos mais 36 biodigestores bateladas, construídos de garrafas de material plástico de um litro, sendo analisadas duas repetições por tratamento a cada dez dias. As reduções das populações de E. coli foram significativas no DSC (60 dias) e no DCC (20 dias). Para E. coli shigatoxigênicas ocorreu em um período mais curto, 40 dias no DSC e menos de 10 dias no DCC. Posteriormente, foram realizados mais dois ensaios para avaliar a produção e potencial de produção de biogás e contagem de ovos de helmintos. Foram utilizados 20 biodigestores bateladas, com tempo de retenção hidráulica (TRH) de 90 dias, para cada ensaio. Os tratamentos foram distribuíd... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objectives of the study were to evaluate the effluent quality in a dynamic situation of the Escherichia coli population, the presence of E. coli shigatoxigenic and the elimination of helminth eggs. In addition, evaluating the production and potential of biogas production by the process of anaerobic codigestion of the waste of dairy cattle with and without addition of 7% of sugarcane broth and bioremediator. For test 1, ten batch biodigesters were divided in two treatments, with no sugarcane broth (W) and waste with sugarcane broth (WWSC), with hydraulic retention time (HRT) of 90 days. Experiment 2 was carried out concomitantly with the first one, it had the same distribution of treatments, to realize the periodic monitoring of dynamic population of E. coli and presence of E. coli shigatoxigenic, pH and volatile acidity. To this end, 36 batch biodigesters were used, consisting of bottles of one liter plastic material, and two replicates per treatment were analyzed every ten days. Reductions of E. coli populations were significant in W (60 days) and in WWSC (20 days). For E. coli shigatoxigenic occurred in a shorter period, 40 days in W and less than 10 days in WWSC. Subsequently, two more tests were carried out to evaluate the production and potential of biogas production and helminths egg counting. 20 batch biodigesters with 90 days hydraulic retention time (HRT) were used for each test. The treatments were distributed in a completely randomized design, in a 2x2 factoria... (Complete abstract click electronic access below) / Doutor

Non-canonical base pairs that escape the proof-reading activity of the DNA polymerase emerge from DNA replication as DNA mismatches. To promote genomic integrity, these DNA mismatches are corrected by a secondary protection system, called DNA mismatch repair (MMR). Understanding the details of MMR is important for human health as defects in mismatch repair can result in cancer (e.g. hereditary nonpolyposis colorectal cancer, also known as Lynch syndrome). Being normally stochastic in nature, mismatches can emerge at random locations in a chromosome. Therefore, using a molecular tool to generate substrates for the MMR system at a defined locus has been particularly useful in my study of DNA mismatch repair in vivo. In this study, I have used a CTG•CAG repeat array, also called the “TNR array”, to generate frequent substrates for the MMR system in Escherichia coli. In E. coli, the MMR system searches for hemimethylated GATC motifs around a mismatch to initiate removal of the faulty nascent (un-methylated) strand. Analysing the usage of GATC motifs around the TNR array, I have found that the MMR system preferentially utilizes the GATC motifs on the origin distal side of the TNR array demonstrating that the bidirectionality of MMR in vitro is constrained in live cells. My results suggest that in vivo MMR operates by searching for the nearest hemimethylated GATC site located between the mismatch and the replication fork and excision of the nascent strand occurs directionally away from the fork towards the mismatch. Previous in vitro studies have established that the excision reaction during MMR terminates at a discrete point about 100 bp beyond a mismatch. However, in vivo recombination at a 275 bp tandem repeat, which has been proposed to be mediated by single stranded DNA generated during the excision reaction, has suggested that the end point of the excision reaction in live cells may extend much further from the mismatch than this. I have used this assay for extended excision to determine the influence of GATC sites on excision tracts. In this study, modification of the GATC motifs on the origin proximal side of the TNR has shown that the excision reaction does not stop at a GATC motif on the origin proximal side of the mismatch. In addition, sequential modifications of GATC motifs on the origin distal side of the TNR array, thereby shifting the start point of the excision reaction to a greater distance, have suggested that the length of an excision tract is a function of the distance it covers from the start point rather than from a mismatch. My observation of directionality with respect to DNA replication in the recognition of GATC sites suggested that MMR and DNA replication might be coupled in some way and that perhaps active (or blocked) MMR might impede the progress of the replication fork. However, no replication intermediates were detected using two-dimensional agarose gel electrophoresis of genomic DNA fragment containing the TNR array upon restriction digestion. I was therefore unable to support the hypothesis that active or blocked MMR led to a slowing down of DNA replication. Given my observation of a decrease in MMR by separating the mismatch from the closest origin distal GATC site, I set out to test whether MMR caused any selection pressure for the genomic distribution of GATC motifs. To do this, I generated artificial model genomes using a Markovian algorithm based on the nucleotide composition and codon usage in E. coli. Strikingly, the comparison of the distribution of GATC motifs in the E. coli genome with those from artificial sequences has shown that GATC motifs are distributed randomly in E. coli genome, except for a small clustering effect which has been detected for short spaced (0-40 basepairs) GATC motifs. The observed distribution of slightly over-represented GATC motifs in the E. coli genome appears to be a function of the total number of GATC motifs and it seems that the DNA mismatch repair system has evolved to utilize the natural distribution of GATC motifs to maintain genomic integrity.

Made available in DSpace on 2015-12-06T22:55:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2010 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Programa de Apoio a Núcleos de Excelência (PRONEX) / O perfil de resistência a antimicrobianos e análise da diversidade genética foi analisado em 32 amostras de Escherichia coli produtoras de toxina Shiga (STEC) isoladas de infecção humana (n = 21) e das fezes de bovinos saudáveis (n = 11) e em 12 amostras de E. coli de origem humana isoladas de sítios extraintestinais, exceto uma delas isolada de diarréia. As amostras foram isoladas no Estado de São Paulo. Multiresistência (resistência a ≥3 grupos de antimicrobianos) foi observada tanto nas amostras de STEC (21/32 - 65,6%) como nas demais E. coli estudadas (8/12 - 66.7%). As amostras de STEC foram resistentes à tetraciclina (100%), seguida à estreptomicina (78,1%) e trimetoprim-sulfametoxazol (56,2%). Onze amostras de STEC resistentes a ampicilina carreavam a enzima β-lactamase TEM (blaTEM) e foram sensíveis a todas as cefalosporinas e quinolonas analisadas. As amostras de E. coli associadas a infecções intestinais e extraintestinais foram resistentes a um maior número de grupos de antimicrobianos. Foi observado resistência à estreptomicina (91,7%), tetraciclina (75%) e trimetoprim-sulfametoxazol (66,7%). Resistência à ampicilina (58,3%) foi também identificada. Três amostras de E. coli mostraram resistência a cefalosporinas de terceira geração, uma delas isolada de infecção intestinal carreava blaCTX-M-14 e blaTEM-1 e outra amostra isolada de secreção traqueal carreava blaCTX-M-15 e blaOXA-1. Cinco das 12 amostras de E. coli mostraram resistência à quinolonas. O gene da integrase associada ao integron classe 1 (intI1) foi encontrado em 6 (28,6%) das 21 amostras de STEC isoladas de humanos pertencentes ao sorogrupo O111 e em uma amostra isolada de bovino (9,1%) pertencente ao sorogrupo O118. Oito das 12 amostras de E. coli associadas a infecções intestinais e extraintestinais apresentaram intI1 e pertenciam a vários sorotipos. As amostras intI1 positivas carreavam plasmídeos com uma diversidade de tamanho, mas perfil plasmidial similar foi observado em algumas delas. O ensaio de hibridização indicou que intl1 estava localizado em plasmídeos em 5 das 7 amostras de STEC e em 6 das 8 demais amostras de E. coli. Análise dos integrons por PCR-RFLP revelou perfil idêntico em 4 amostras de STEC e em uma E. coli extraintestinal. Esses integrons tinham tamanho uniforme e continham o cassete gênico aadA1. O agrupamento filogenético mostrou que a maioria das amostras de STEC pertencia ao grupo B1, enquanto os grupos A, B2 e D foram observados entre as demais amostras de E. coli em 33,3%, respectivamente. Quatro amostras de E. coli isoladas de infecções extraintestinais foram denominadas como típicas E. coli patogênicas extraintestinais (ExPEC). Entre as 44 amostras de E. coli estudadas 54,5% apresentaram serino-proteases autotransportadoras (SPATE). A toxina vacuolizante Vat foi identificada somente em 3 das 12 E. coli associadas a infecções intestinais e extraintestinais, e essas amostras apresentaram fímbria tipo 1, fator necrosante citotóxico, Alfa hemolisina e a adesina de membrana externa, Iha (100%, 8,3%, 8,3% e 25%, respectivamente). A tipagem por PFGE das amostras de STEC O111 e O118 mostrou uma diversidade de perfis, mas a maioria das amostras foi agrupada em um mesmo grupo (80% a 97% de similaridade). Por outro lado, a tipagem molecular confirmou que a maioria das amostras de E. coli associadas a infecções intestinais e extraintestinais era epidemiologicamente não relacionada (similaridade genética ≥80%), exceto 2 amostras de E. coli isoladas do mesmo paciente, no mesmo dia, e que apresentaram padrões de PFGE com similaridade de 93,3%. Os plasmídeos das amostras de E. coli associadas a infecções intestinais e extraintestinais carreando genes de resistência e/ou virulência, acarretaram um custo biológico de pequeno a neutro por geração, quando inseridos às amostras laboratoriais de E. coli. Em conclusão, apesar da aquisição de genes ter interferido pouco na capacidade das amostras de E. coli laboratoriais em se multiplicarem e consequentemente se disseminarem, a presença de integrons e de outros elementos genéticos móveis tanto em amostras de STEC como entre as amostras de E. coli extraintestinais é preocupante, principalmente em relação as amostras de E. coli extraintestinais considerando a provável aquisição de resistência a outros antimicrobianos, como recentemente descrito aos carbapenens. / Phenotypic and genotypic characterization of resistance to antimicrobials and genetic diversity were analyzed in 32 Shiga Toxin-Producing Escherichia coli (STEC) strains isolated from human infections (n = 21) and cattle feces (n = 11), and 12 human extraintestinal E. coli strains, except one isolated from diarrhea. The E. coli strains were isolated in Sao Paulo State. Multiresistance (resistance to ≥3 antimicrobial groups) was observed in both STEC (21/32 – 65.6%) and ExPEC strains (8/12 - 66.7%). The STEC strains were resistant to tetracycline (100%) followed by streptomycin (78.1%) and trimethoprimsulfamethoxazole (56.2%). The eleven STEC strains resistant to ampicilin possessed β-lactamase TEM (blaTEM) enzyme and all strains were susceptible to third-generation cephalosporins, and also to quinolones. On the other hand, E. coli strains associated with intestinal and extraintestinal infections were resistant to a larger number of antimicrobial groups than STEC. Resistance was observed to streptomycin (91.7%), tetracycline (75%) and trimethoprimsulfamethoxazole (66.7%). Resistance to ampicillin (58.3%) was also identified. Three E. coli strains were non-susceptible to third-generation cephalosporins, one isolated from diarrhea carried blaCTX-M-14 and blaTEM-1 whereas other, isolated from tracheal secretion, carried blaCTX-M-15 and blaOXA-1. Five of the 12 E. coli strains showed resistance to quinolones. Integrase associated with class 1 integron (intI1) was detected in six (28.6%) of the 21 human STEC strains belonging to O111 serogroup and in one (9.1%) bovine strain belonging to O118 serogroup. Eight of the 12 E. coli strains associated with intestinal and extraintestinal infections presented intI1 and belonged to various serotypes. The intI1-positive isolates carried plasmids showing a diversity of sizes, but similar plasmid profiles were observed in some strains. Southern blot hybridization assay with intI1-specific probe indicated that intl1 was located on plasmids in five out of the seven STEC strains and in six out of the eight E. coli strains. Analysis of integrons by PCR-RFLP revealed identical profiles in 4 STEC strains and in one extraintestinal E. coli strain. These integrons had a uniform size and contained a single gene cassette aadA1. The phylogenetic grouping showed that most of the STEC strains belonged to B1 group, while groups A, B2 and D (33.3%), respectively were observed among the other E. coli isolates. Four E. coli strains isolated from extraintestinal infection were classified as typical extraintestinal pathogenic E. coli (ExPEC). Among 44 E. coli strains studied, 54.5% presented the Spate protease autotransporter. The Vat vacuolating toxin was identified only in 3 of the 12 E. coli strains associated with intestinal and extraintestinal infections, and these strains presented type-1 fimbriae, cytotoxic nectrotizing factor, alpha-hemolysin and the IrgA homologue adhesin, Iha (100%, 8.3%, 8.3% e 25%, respectively). PFGE analysis of O111 and O118 STEC strains showed a diversity of profiles, but most of the strains were grouped in the same cluster (80% to 97% of similarity). On the other hand, PFGE analysis revealed that most E. coli strains associated with intestinal and extraintestinal infections were epidemiologically unrelated, and were not grouped in any cluster with significant similarity (≥80%), except 2 E. coli strains, isolated at the same day from the same patient, and that presented PFGE patterns with similarity of 93.3%. The plasmids of the E. coli strains associated with intestinal and extraintestinal infections carrying resistance and/or virulence genes, resulted in a low or neutral biological cost per generation, when inserted into laboratory E. coli strains. In conclusion, despite the acquisition of genes has shown a low interference in the ability of laboratory E. coli strains to grow and consequently to spread, the presence of integrons and other mobile genetic elements in both STEC and E. coli associated with extraintestinal infections is worrisome, mainly in relation to extraintestinal E. coli strains considering the probable acquisition of resistance to other antimicrobials, as recently reported to carbapenems. / BV UNIFESP: Teses e dissertações