A resistência bacteriana a antibióticos é um grave e crescente problema de saúde pública de âmbito mundial. O principal, e mais eficiente, mecanismo de resistência aos &#946-lactâmicos em bacilos Gram-negativos é a produção de &#946-lactamases, que possuem a capacidade de hidrolisar o anel &#946-lactâmicos e consequentemente inativar essa classe de antibióticos. Vale ressaltar, que atualmente os antibióticos &#946-lactâmicos são os mais utilizados clinicamente, particularmente em infecções graves. Dentre as &#946-lactamases existentes destacam-se as carbapenemases, enzimas capazes de inativar a maioria dos antibióticos &#946-lactâmicos. Uma grande preocupação é o fato dessas enzimas, em sua maioria, serem codificadas por plasmídeos, o que propicia a disseminação desses genes de resistência; portanto, é de extrema importância a realização de um rápido e efetivo monitoramento da presença de patógenos portadores desses genes de resistência, para que assim se possa prevenir a disseminação desses determinantes. Foram incluídos neste estudo 230 amostras únicas de Acinetobacter e Pseudomonas aeruginosa resistentes a imipenem detectados em pacientes internados em hospitais privados da cidade de São Paulo durante o período de fevereiro a outubro de 2013. As amostras foram avaliadas quanto à hidrólise de imipenem por espectrofotometria, quanto à presença de genes de carbapenemases por PCR e sequenciamento, e quanto à clonalidade por eletroforese em campos pulsados (PFGE) ou ERIC-PCR. Foram realizados ensaios de conjugação, transformação e sequenciamento completo de plasmídeos. Dentre as amostras de Acinetobacter spp. 80% (88) foram capazes de hidrolisar o imipenem. Dentre esses 76,1% (67) foram positivos para blaOXA-51-like, 19,3% (17) foram positivos para blaOXA-72. blaOXA-23, blaOXA-482 e blaIMP-1 foram detectados isoladamente em isolados distintos. O gene blaIMP-1 foi detectado em A. ursingii inserido em integron de classe 1 e representa a primeira descrição no Brasil. Uma nova carbapenemase OXA-482-like foi detectada em A. baumanii. Utilizando-se ERIC-PCR, observou-se uma grande diversidade de grupos clonais, com o máximo de quatro isolados por grupo. Dentre as amostras de P. aeruginosa, apenas 35,3% foram capazes de hidrolisar o imipenem. Dessas amostras, 14 possuíam o gene blaSPM-1, e isolados únicos possuíam, individualmente, os genes blaIMP, blaVIM, blaKPC-2 ou blaGES-23. O gene blaKPC-2 foi detectado inserido em contexto genético diferente dos descritos anteriormente, em plasmídeo IncU de 32 Kb, mobilizável, mas não conjugativo. Esta é a primeira descrição da sequencia completa de plasmídeo albergando o gene blaKPC-2 em P. aeruginosa no Brasil. Nas demais amostras (20) com atividade hidrolítica, não foram detectados genes de carbapenemase conhecidos, o que sugere a presença de genes de carbapenemase ainda não descritos. Em três amostras foi possível obter transformantes com plasmídeos, resistentes a carbapenêmicos. As amostras com blaSPM-1 apresentaram perfis de PFGE estreitamente relacionados. Em contraste, os perfis de PFGE das amostras com potenciais novas carbapenemases apresentaram índice de similaridade de Dice inferior ix a 80%, evidenciando grande diversidade clonal. Nossos achados evidenciam que a carbapenemase não intrínseca predominante em Acinetobacterem hospitais privados da cidade de São Paulo é OXA-72, e em hospitais privados há uma grande diversidade clonal. Em P. aeruginosa, a carbapenemase predominante é SPM-1, cuja disseminação é mediada por um único clone. Há potencialmente um número significativo de novas carbapenemases em Acinetobacter e P. aeruginosa, algumas delas mediadas por plasmídeos. / Bacterial resistance to antibiotics is a serious and growing public health problem worldwide. The main and most efficient mechanism of resistance to &#946-lactams in Gram-negative bacilli is the production of &#946-lactamases, which have the ability to hydrolyze the &#946-lactam ring and consequently inactivate this class of antibiotics. It is worth mentioning that currently &#946-lactam antibiotics are the most used clinically, particularly in severe infections. Among the existing &#946-lactamases, carbapenemases are capable of inactivating most &#946-lactam antibiotics. A major concern is that these enzymes are mostly encoded by plasmids, which facilitates the spread of these resistance genes; therefore, it is of extreme importance to carry out a rapid and effective monitoring of the presence of pathogens bearing these resistance genes, in order to prevent the dissemination of these determinants. This study included 230 unique samples of imipenem-resistant Acinetobacterand Pseudomonas aeruginosa detected in patients hospitalized in private hospitals in the city of São Paulo during the period from February to October 2013. The samples were evaluated for the imipenem hydrolysis by spectrophotometry, the presence of carbapenemase genes by PCR and sequencing, and concerning clonality by pulsed field electrophoresis (PFGE) or ERIC-PCR. Conjugation, transformation and complete sequencing of plasmids were performed. Among Acinetobacter spp. samples, 80% (88) were able to hydrolyze imipenem. Among these, 76.1% (67) were positive for blaOXA-51-like genes and 19.3% (17) were positive for blaOXA-72. The blaOXA-23, blaOXA-482 and blaIMP-1 genes were detected alone in distinct isolates. The blaIMP-1 gene was detected in A. ursingii inserted in class 1 integron and represents the first description in Brazil. A novel OXA-482-like carbapenemase was detected in A. baumanii. Using ERIC-PCR, a great diversity of clonal groups was observed, with a maximum of four isolates per group. Among P. aeruginosa samples, only 35.3% were able to hydrolyze imipenem. Of these samples, 14 had the blaSPM-1 gene, and single isolates individually possessed the blaIMP, blaVIM, blaKPC-2 or blaGES-23 genes. The blaKPC-2 gene was found inserted in a genetic context different from those described previously, in a mobilizable, but not conjugative, 32 Kb IncU plasmid. This is the first description of the complete nucleotide sequence of a plasmid harboring the blaKPC-2 gene in P. aeruginosa in Brazil. In the remaining samples (20) with hydrolytic activity, no known carbapenemase genes were detected, suggesting the presence of carbapenemase genes not yet described. In three samples it was possible to obtain transformants with plasmids, resistant to carbapenems. Samples with blaSPM-1 showed closely related PFGE profiles. In contrast, the PFGE profiles of the samples with potential new carbapenemases showed Dice similarity index lower than 80%, evidencing a great clonal diversity. Our findings show that the predominant non-intrinsic carbapenemase in Acinetobacter in the city of São Paulo is OXA-72, and in private hospitals there is great clonal diversity. In P. aeruginosa, the predominant carbapenemase is SPM-1, the spread of this enzyme is mediated by a single clone. There are potentially a significant number of new carbapenemases in Acinetobacter and P. aeruginosa, some of them plasmid mediated.

One of the greatest threats to human health is increasing antimicrobial resistance among pathogens, and finding alternatives for treatment of bacterial infections is of highest importance together with a more controlled use of antibiotics. Porphyrins and heme have both been shown to be a promising class of compounds for inactivation of bacteria; porphyrins by their excellent properties to act as a photosensitizer, and heme by its importance as an iron source during a bacterial infection in vertebrates. This thesis describes the development of analytical methods for the identification and determination of porphyrins and heme using liquid chromatography coupled to tandem mass spectrometry. Subsequently, these developed methods were applied to bacterial samples to investigate different culture conditions and additives effect to the intracellular porphyrin and heme composition. Singlet oxygen production of three naturally occurring porphyrins have been determined together with the photosensitivity for blue light and the porphyrin content in E. coli. Toothbrushes equipped with a LED, emitting light with a wavelength of 450 nm, were used in an eight week randomized clinical trial to investigate any positive periodontal effect of blue light. Porphyrin and heme content in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were highly affected by the different cultivation conditions. The culture age of A. actinomycetemcomitans affected the porphyrin profile, while only small changes were observed for P. gingivalis during growth. A large change of the porphyrin profile could be observed when the bacteria were passaged onto a new growth medium. Additional porphyrins were detected and the total porphyrin content increased up to 28 times. These findings highlight the need for more standardized cultivation procedures when performing in vitro experiments. Heme content in Escherichia coli was affected when different additives related to biosynthesis of heme were added to the growth medium. The uptake of heme could be reduced with 52% when a compound that chemically looks similar to heme was added to the growth medium. Since heme acquisition is important for many pathogens, this could be a promising target for antimicrobial drugs. E. coli showed no sensitivity for 405 nm light using light doses up to 172.8 J/cm2 and only low concentrations of porphyrins could be quantified. By adding a porphyrin precursor to E. coli the intracellular concentration of porphyrins increased remarkably and a light dose of 57.6 J/cm2 reduced the bacterial number with &gt; 5 log10 steps. This shows that E. coli can be killed due to their endogenous porphyrins. In the clinical study we could see a weak trend that the 450 nm LED toothbrush possessed a phototherapeutic effect for three clinical indices. All indices were decreased in the intervention group, but there were no statistically significant difference compared to the control group. However, four inflammation markers were significantly decreased in the intervention group while only one decreased significantly in the control group. In conclusion, this thesis has shown that porphyrins and heme are produced endogenously in microorganisms and that the porphyrin profiles vary depending on culture conditions and different additives. Furthermore, porphyrins may be used as endogenous photosensitizers to inactivate bacteria, but more research is necessary to determine if there is a specific porphyrin that contributes more to the photosensitivity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

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.

A Escherichia coli enteropatogênica atípica (aEPEC) é um dos principais agentes causadores de diarreia infantil e às vezes é necessário o tratamento com antimicrobianos. O objetivo do presente trabalho foi verificar o perfil de resistência aos antimicrobianos de 72 amostras de aEPEC, correlacionando com o perfil genético de resistência e a produção de &#946;-lactamases de espectro extendido (ESBL). As aEPEC apresentaram resistência aos &#946;-lactamâmicos, sulfonamidas, tetraciclinas, aminoglicosídeos e ansamicina, verificadas através de disco difusão. Multirresistência foi identificada em 27,8%. Treze amostras (18,1%) produziram ESBL. Os genes de resistência encontrados através de PCR foram: sul1 - 50%, sul2 - 86,4%, tetA - 42,1%, tetB - 68,4%, tetC- 5,3%, blaCTX-M - 26,1%, blaTEM -78,3%, blaSHV - 4,4% e intl - 35,7%. O conhecimento sobre o perfil de resistência e produção de ESBLs é muito importante na orientação do tratamento adequado quando se fizer necessário, além de conhecer o potencial de resistência que poderiam vir a ser transferidos para outras bactérias. / The atypical Enteropathogenic Escherichia coli (aEPEC) is one of the most common childhood diarrhea`s agent, and sometimes the antimicrobials treatment may be necessary. The aim of this study was verify the antimicrobial resistance profile of 72 aEPEC strains, correlating with the resistance genetic profile, and the extended-spectrum &#946;-lactamases (ESBL) production. The aEPEC strains presented resistance to &#946;-lactams, Sulfonamides, Tetracycline, Aminoglycoside, and Ansamycin, verified through disc diffusion. Multiresistance was identified in 27.8%. Thirteen strains (18.1%) produced ESBL. The resistance genes found through PCR were: sul1 - 50%, sul2 86.4%, tetA 42.1%, tetB 68.4%, tetC- 5.3%, blaCTX-M 26.1%, blaTEM -78.3%, blaSHV 4.4%, and intl 35.7%. The knowledge about resistance profile´s and ESBL production is very important in guiding the most appropriate treatment when it is necessary, in addition to knowing the resistance potential that could be transferred to other bacteria.

Depuis quelques années, des Toxi-Infections Alimentaires Collectives causées par la contamination de produits frais, comme les laitues, par des bactéries pathogènes entériques (Salmonella, Escherichia coli productrice de shigatoxines -ou STEC-) apparaissent de plus en plus nombreuses. La présence de ces bactéries dans cet environnement inhabituel est un risque sanitaire émergent majeur, d'autant plus que les bactéries entériques, pathogènes ou non, présentent fréquemment des résistances aux antibiotiques. Afin d’étudier la persistance des bactéries antibiorésistantes ou pathogènes sur des laitues, la caractérisation de plasmides de résistance portés par des souches de E. coli issues d’environnements aquatiques contaminés a été réalisé pour, par la suite, étudier leur implication potentielle dans l’adhésion des souches-hôtes sur différentes variétés de laitues. L’étude de la survie et de l’adhésion de souches de E. coli environnementales et de laboratoire, transformées avec les plasmides d’intérêt, sur de jeunes plants de laitues a permis de mettre en évidence trois points : 1) plus le temps de contact entre bactéries et feuilles augmente et moins la survie bactérienne est importante ; 2) il existe une différence de survie et d’adhésion selon les variétés de laitues étudiées ; 3) il existe une différence de survie et d’adhésion entre les souches de laboratoire et les souches environnementales, ces dernières étant en meilleur état métabolique et montrant une adhésion plus importante durant les 11-12 jours d’expérimentation. Après ces constatations de persistance des E. coli antibiorésistantes en conditions contrôlées, des études en champs sur 4 exploitations maraîchères normandes, possédant des itinéraires techniques différents, ont été réalisées. La recherche de pathogènes entériques, Salmonella et STEC, a été effectuée sur les laitues et une recherche de E. coli, témoin de contamination fécale, a été réalisée sur les laitues ainsi que dans l’eau d’irrigation d’un des sites. Les résultats révèlent une qualité microbiologique satisfaisante des parcelles étudiées (selon l’arrêté européen N°2073/2005) bien que des E. coli aient été régulièrement retrouvées au niveau des laitues, dont certaines antibiorésistantes. L’analyse de l’eau d’irrigation a montré la présence continue de E. coli, dont des souches présentant des profils d’antibiorésistance communs à ceux retrouvés sur les laitues, montrant que l’eau d’irrigation est l’une des sources critiques de contamination des végétaux en champs. / In recent years, foodborne diseases caused by fresh products contaminated, such as lettuce, with enteric pathogenic bacteria (Salmonella, Shigatoxin-producing Escherichia coli-or STEC-) increasingly. The presence of these bacteria in this unusual environment is a major emerging health risk, especially since enteric bacteria, whether pathogenic or not, are frequently resistant to antibiotics. To study the persistence of antibiotic-resistant or pathogenic bacteria on lettuce, the characterization of resistance plasmids carried by E. coli strains from contaminated aquatic environments was carried out in order to study their potential involvement in adhesion of host strains on different varieties of lettuce. The study of the survival and adhesion of environmental and laboratory E. coli strains, transformed with the plasmids of interest, on young lettuce plants allowed to highlight three points: 1) more time contact between bacteria and leaves increases and less bacterial survival is important; 2) there is a difference in survival and adhesion depending on the varieties of lettuce studied; 3) there is a difference in survival and adhesion between laboratory strains and environmental strains, the latter being in better metabolic state and showing greater adhesion during the 11-12 days of experimentation. After the persistence of antibiotic-resistant E. coli strains under controlled conditions, field studies on 4 Normandy vegetable farms, with different technical itineraries, were carried out. The search for enteric pathogens, Salmonella and STEC, was carried out on lettuce and a search for E. coli, a control of fecal contamination, was realized on the lettuce as well as in the irrigation water of one of the sites. The results reveal a satisfactory microbiological quality of the agricultural plots studied (according to the European decree N ° 2073/2005) although E. coli strains were regularly found at the lettuce level, including some antibiotic resistant. Analysis of the irrigation water showed the continued presence of E. coli strains, including strains with common antimicrobial resistance profiles to those found on lettuce, showing that irrigation water is one of the critical sources of plant contamination in the field.

Orientador: Rodrigo Tavanelli Hernandes / Resumo: A Escherichia coli enteroagregativa (EAEC) é um importante agente causador de diarreia aguda e persistente em crianças e adultos em todo o mundo. EAEC é definida como isolados de E. coli que produzem o padrão de aderência agregativa (AA) em células epiteliais (HeLa e/ou HEp-2) cultivadas in vitro. O patotipo EAEC pode ser dividido em típico e atípico com base na presença do gene aggR, que codifica um ativador transcricional, presente apenas no primeiro grupo. O objetivo deste estudo foi caracterizar uma coleção de isolados de EAEC obtidos de pacientes com diarreia durante um período de 7 anos de vigilância epidemiológica (2010-2016). Um total de 220 isolados de EAEC (194 típicas e 26 atípicas) foi classificado nos distintos grupos filogenéticos de E. coli, e caracterizados quanto aos sorotipos (O:H), padrão de aderência produzidos em células HeLa, sensibilidade aos antimicrobianos, e a presença de 25 genes responsáveis por codificarem fatores de virulência no patotipo EAEC. A maioria dos isolados de EAEC foi classificada nos grupos filogenéticos A (44,1%; 97/220) e B1 (21,4%; 47/220). Em relação ao padrão de aderência, observamos que 92,7% (204/220) produziram o padrão AA. Além disso, foram identificados nove isolados (4,1%; 9/220) que produziram a aderência em cadeia (CLA), dos quais seis produziram concomitantemente o padrão AA, além de isolados de EAEC que produzem um padrão de aderência indefinido (1,4%; 3/220) e isolados não aderentes (3,6%; 8/220). Foi identificado some... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Enteroaggregative Escherichia coli (EAEC) is an important agent that causes acute and persistent diarrhea in children and adults worldwide. EAEC is defined as E. coli isolates that produce the aggregative adherence pattern (AA) on epithelial cells (HeLa and/or HEp-2) cultured in vitro. The EAEC pathotype can be divided in typical and atypical based on the presence of the aggR gene, which encodes a transcriptional activator, in the former group. The aim of this study was to characterize a collection of EAEC isolates obtained from diarrheal patients over a 7-year period of surveillance (2010-2016). A total of 220 EAEC isolates (194 typical and 26 atypical) were evaluated regarding the phylogenetic classification, serotypes, adherence pattern produced on HeLa cells, susceptibility to antimicrobial drugs and the presence of 25 virulence factor-encoding genes. The majority of the EAEC isolates were assigned to phylogroups A (44.1%; 97/220) and B1 (21.4%; 47/220). Regarding the adherence pattern, was observed that 92.7% (204/220) produced AA. Moreover, we identified nine isolates (4.1%; 9/220) that produced the chain-like adherence (CLA), with six of them producing concomitantly the AA pattern, besides EAEC isolates producing an undefined adherence (1.4%; 3/220) and isolates non-adherent (3.6%; 8/220). Only 0.9% (2/220) of the EAEC isolates studied presented the multidrug resistance phenotype. The genes encoding for the major pilin subunit of all five previously described aggregati... (Complete abstract click electronic access below) / Doutor

Orientador: Rodrigo Tavanelli Hernandes / Resumo: Escherichia coli uropatogênica (UPEC) causa a maioria das infecções do trato urinário (ITU), incluindo cistite e pielonefrite, no hospedeiro humano. A UPEC utiliza numerosos fatores de virulência para entrar, aderir, colonizar, adquirir nutrientes essenciais, multiplicar e causar danos ao ambiente do trato urinário. Estudos recentes demonstraram que alguns isolados de UPEC carregam fatores de virulência associados à patótipos diarreiogênicos de E. coli (DEC), como EAEC (E. coli enteroagregativa) e EPEC (E. coli enteropatogênica). Uma grande preocupação nas infecções por UPEC é o aumento da resistência antimicrobiana, levando à falha do tratamento em algumas ITUs causadas por esse patógeno. Nesse estudo, um total de 118 isolados de UPEC de amostras ambulatoriais de urina de pacientes atendidos no Hospital das Clinicas da Faculdade de medicina de Botucatu entre março e maio de 2018. Reação em cadeia da polimerase (PCR) foi usada para detectar 29 genes que codificam fatores de virulência, bem como marcadores de DEC (escN, stx1/2, aatA e aggR); além de genes que codificam adesinas e toxinas associadas ao patótipo EAEC. Os isolados de UPEC foram designados nos diferentes filogrupos de E. coli, utilizando um PCR quadruplex; e a determinação do perfil de susceptibilidade antimicrobiana foi realizada pelo método de disco difusão. Entre os isolados estudados, 39,8% foram atribuídos ao filogrupo B2, enquanto UPEC dos filogrupos B1 (14,4%), A (14,4%), D (12,7%), F (8,5%), G (3,4%), E ( ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Uropathogenic Escherichia coli (UPEC) cause the majority of urinary tract infections (UTIs), including cystitis and pyelonephritis, in the human host. UPEC utilizes numerous virulence factors to entry, adhere, colonize, acquire essential nutrients, multiply and cause damage in the urinary tract environment. Recent studies have shown that some UPEC isolates carry virulence factors associated with the diarrheagenic E. coli (DEC) pathotypes, such as EAEC (enteroaggregative E. coli) and EPEC (enteropathogenic E. coli). A major concern in UPEC infections is the constant increasing of antimicrobial resistance, thus leading to treatment failure in some UTIs caused by this pathogen. In this study a total of 118 UPEC isolates were obtained from outpatient urine samples, attended at University Hospital of Botucatu Medical School between March and May of 2018. Polymerase chain reaction (PCR) was used to detect 29 virulence factor-encoding genes, diarhoeagenic E. coli markers, (escN, stx1/2, aatA and aggR), as well as genes encoding adhesins and toxins associated with the EAEC pathotype. The UPEC isolates were assigned in the distinct E. coli phylogroups, using a quadruplex PCR; and the determination of the antimicrobial resistance profile was performed using the diskdiffusion method. Among the isolates studied, 39.8% were assigned to phylogroup B2, while UPEC isolates from other phylogroups were detected as follows: B1 (14,4%), A (14,4%), D (12,7%), F (8,5%), G (3,4%), E ( 2,5%), E. cla... (Complete abstract click electronic access below) / Mestre

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

As part of recognizing the interconnected nature of different sectors in relation to health, AMR (antimicrobial resistance) has emerged as an issue of high global importance. E. coli isolates were taken from pig farms in Thailand, which serves as a point of interest in the study of ARGs (antimicrobial resistance genes) in emerging economies. The fecal samples were collected from pigs, humans who came in contact with the pigs, and humans who did not have contact with pigs to be analyzed for ARGS, virulence genes, and plasmids. Data was analyzed with an automated pipeline in the form of ARG-MATEE, the Antimicrobial Resistance Gene Multi-Analysis Tool for Enteric E. coli, a tool designed in this study to be used here and in future investigations. ARG-MATEE regulates and records internal software versions in a produced report which also includes data tables for all non phylogeny results in Boyce–Codd normal form and data visualizations for plasmids, ARGs, virulence genes, and phylogeny. Through the use of ARG-MATEE, the iss virulence gene was seen to be significantly different between testing groups as it is present in only human testing groups, suggesting the loss of function of the iss gene in pigs, showing host specialization.