Os prejuízos causados por Escherichia coli patogênica para aves (APEC) na produção de aves é justificativa de pesquisas realizadas no mundo todo há décadas. Recentemente, o potencial zoonótico e a multirresistência das cepas desse patotipo têm sido alvo frequente dos trabalhos realizados com APEC. O objetivo desse trabalho foi caracterizar 225 cepas APEC isoladas de perus condenados por aerossaculite em abatedouros em relação à resistência a 14 antimicrobianos, perfil de virulência e grupos filogenéticos. 92% das amostras apresentaram perfil de multirresistência (MDR) e os índices mais altos de resistência foram a sulfonamidas (94%), tetraciclina (83%), eritromicina (82%), estreptomicina (60%), amoxicilina (53%) e ácido nalidíxico (48%). Metade das cepas foram classificadas no grupo filogenético B2 (50%), seguido por B1 (28,6%), grupo A (17,1%) e grupo D (4,8%). Os genes de virulência pesquisados tiveram prevalência de iroN (95%), iss (93%), cvi/cva (67%), iucD (67%), tsh (56%), irp2 (51%), ibeA (31%), vat (24%), neuS (19%), astA (17%) e papC (15%). Considerando o potencial zoonótico e a associação a um maior número de genes de virulência quando comparadas aos outros grupos filogenéticos, as cepas B2 foram selecionadas para pesquisa de integrons de classe 1 e clonalidade. As 112 amostras B2 eram pertencentes a 83 diferentes perfis ERIC, sendo classificadas como multiclonais. Os integrons de classe 1 estiveram presentes em 107 isolados (95,5%). Esses resultados demonstram que a maioria das cepas pesquisadas pertencia ao grupo mais virulento (B2) e relacionado a cepas ExPEC humanas. Aliado ao alto índice de multirresistência encontrado, esses dados sugerem que as aves podem servir como reservatório de cepas patogênicas e multirresistentes, tanto para humanos como para animais, reforçando a ideia de que as aves representem importante papel na cadeia epidemiológica das ExPEC. / Avian Pathogenic Escherichia coli (APEC) has been studied for decades because of its economic impact on the poultry industry. Recently, the zoonotic potential of APEC and multidrug-resistant strains have emerged. The aim of this study was to investigate the virulence profile, phylogenetic background and antimicrobial resistance in 225 strains of Escherichia coli isolated from turkeys presenting airsacculitis. The results showed that 92% of strains presented a multidrug-resistance (MDR) profile, and the highest levels of resistance were to sulfamethazine (94%), tetracycline (83%). Half of these strains (112/225) were classified in phylogenetic group B2, followed by groups B1 (28.6%), A (17.1%) and D (4.8%). The prevalence of virulence genes was as follows: iroN (95%), iss (93%), cvi/cva (67%), iucD (67%), tsh (56%), irp2 (51%), ibeA (31%), vat (24%) neuS (19%), astA (17%) and papC (15%). Considering the zoonotic potential and association to a greater number of virulence genes when compared to other phylogenetic groups, B2 strains were selected for screening of class 1 integrons and clonality. The 112 samples belonging to 83 ERIC profiles and classified as multiclonal. Class 1 integrons were present in 107 isolates (95.5%).These results demonstrate that the majority of the investigated strains belonged to group B2, which is more virulent and is related to human ExPEC strains. Coupled with the high rate of multidrug-resistance found, these data suggest that turkeys may serve as a reservoir for pathogenic and multidrug-resistance strains, for humans and animals, reinforcing the idea that poultry plays an important role in the epidemiological chain of ExPEC.

O presente trabalho objetivou pesquisar e caracterizar Escherichia coli patogênica (E. coli produtora de toxina Shiga STEC; E. coli aviária patogênica - APEC) em fragatas (Fregata magnificens) da costa do Estado de São Paulo e, desta forma, contribuir para a compreensão de aspectos epidemiológicos deste importante grupo de enfermidades zoonóticas bacterianas, as colibaciloses. Para tanto, foram realizadas três expedições científicas aos dois sítios de nidificação de fragatas no estado de São Paulo, Ilha Principal do Arquipélago de Alcatrazes (24°06´S 45°41´W) e Ilha de Castilho (25°16´S 47°57´W), nas quais foi colhido um total de 42 amostras de \"swabs\", sendo 21 cloacais e 21 de coanas, oriundos de 18 filhotes de sexo indeterminado, 2 fêmeas adultas e 1 macho adulto de fragatas. Das 42 amostras clinicas estudadas, foram identificadas 18 com crescimento de E.coli. Destas, foram isoladas 67 cepas, que foram então submetidas à pesquisa de genes de virulência e caracterização de grupos filogenéticos através da técnica de PCR. Em seguida, as cepas que exibiram fatores de virulência foram analisadas através de sorotipagem. Para investigar os padrões de resistência e sensibilidade a antimicrobianos, foram realizados antibiogramas para 18 tipos distintos de antimicrobianos para uma cepa de cada amostra selecionada de E. coli. Os resultados alcançados mostraram que foi possível identificar 15 diferentes perfis de virulência, com positividade para os seguintes genes: fimH (98,3%), malX (52,4%), traT (31,1%), cvaC (22,9%), fyuA (19,6%), ibeA (19,6%), aer (13,1%) e papC (13,1%). A maioria dos isolados de fragatas foi caracterizado entre os grupos filogenéticos D e B2, e os principais sorotipos encontrados foram O1:H6, O2:H7, O25:H4, e O102:H10. Em relação à resistência a antimicrobianos, 60,1%% dos isolados foram sensível aos 18 diferentes antimicrobianos testados; por outro lado, o antimicrobiano que apresentou maior resistência foi a Tetraciclina, que se revelou inefetiva em 20,1% das amostras pesquisados. Até o momento não foram identificadas cepas de STEC nos indivíduos pesquisados; por outro lado, os isolados albergam genes que caracterizam as ExPEC. Tais resultados mostram que a maioria das cepas isoladas é potencialmente patogênica para as aves, podendo representar significativo risco para sanidade das aves marinhas, além de se configurar como agentes zoonóticos relevantes, enfatizando a importância de se investigar a eventual participação das aves selvagens, em especial as marinhas, na cadeia epidemiológica de doenças ocasionadas por E. coli. Tais informações poderão nortear as pesquisas de doenças selecionadas nas populações das fragatas, assim como embasar a adoção de eventuais ações em relação a aspectos de saúde animal e humana, que tenham as fragatas como um dos elos. / The present study is to investigate and characterize pathogenic Escherichia coli (E. coli Shiga toxin-producing - STEC avian pathogenic E. coli - APEC) on frigates (Fregata magnificens) from the coast of São Paulo and contribute to the understanding of epidemiological aspects in this important group of zoonotic bacterial diseases, the colibacillosis. To this, there were three scientific expeditions to two nesting sites of frigates in the state of Sao Paulo, the main island of Alcatraz (24 ° 06\'S - 45 ° 41\'W) and Castilho´s island (25 ° 16\'S - 47 ° 57\'W), in which it was collected a total of 42 samples of swabs, and 21 cloacal and choanal 21, coming from 18 offspring of indeterminate sex, 2 adult females and 1 adult male frigate. Of the 42 clinical samples studied, 18 were identified with growth of E.coli. Of these, 67 strains were isolated, which were then tested for virulence genes and characterization of phylogenetic groups by PCR technique. Then the strains that exhibited virulence factors were analyzed by serotyping. To investigate the patterns of resistance and sensitivity to antimicrobial susceptibility tests were performed for 18 different types of antimicrobials for one strain of each selected sample of E. coli. The results showed that it was possible to identify 15 different profiles of virulence, tested positive for the following genes: fimH (98.3%), malX (52.4%) traT (31.1%), cvaC (22.9 %), fyuA (19.6%), ibeA (19.6%), aer (13.1%) and papC (13.1%). Most isolates of frigates was characterized between phylogenetic groups D and B2, and the main serotypes were O1:H6, O2:H7, O25:H4, and O102:H10. For resistance to antibiotics, 60.1%% of the isolates were sensitive to 18 different antimicrobial agents tested on the other hand, the antimicrobial with the highest resistance was to tetracycline, which has proved ineffective in 20.1% of the samples studied. So far not been identified in STEC strains studied subjects on the other hand, isolates harbor genes that characterize ExPEC. These results show that most of the strains are potentially pathogenic to birds and may represent significant risk to health of sea birds, including how to configure agents relevant, emphasizing the importance of investigating the possible involvement of wild birds, especially navies in the epidemiological chain of diseases caused by E.coli. Such information could guide the research of selected diseases in populations of the frigates, as well as basing the adoption of any action in relation to aspects of animal and human health, which have the frigates as a link.

O presente trabalho objetivou pesquisar e caracterizar Escherichia coli patogênica (E. coli produtora de toxina Shiga STEC; E. coli aviária patogênica - APEC) em fragatas (Fregata magnificens) da costa do Estado de São Paulo e, desta forma, contribuir para a compreensão de aspectos epidemiológicos deste importante grupo de enfermidades zoonóticas bacterianas, as colibaciloses. Para tanto, foram realizadas três expedições científicas aos dois sítios de nidificação de fragatas no estado de São Paulo, Ilha Principal do Arquipélago de Alcatrazes (24°06´S 45°41´W) e Ilha de Castilho (25°16´S 47°57´W), nas quais foi colhido um total de 42 amostras de \"swabs\", sendo 21 cloacais e 21 de coanas, oriundos de 18 filhotes de sexo indeterminado, 2 fêmeas adultas e 1 macho adulto de fragatas. Das 42 amostras clinicas estudadas, foram identificadas 18 com crescimento de E.coli. Destas, foram isoladas 67 cepas, que foram então submetidas à pesquisa de genes de virulência e caracterização de grupos filogenéticos através da técnica de PCR. Em seguida, as cepas que exibiram fatores de virulência foram analisadas através de sorotipagem. Para investigar os padrões de resistência e sensibilidade a antimicrobianos, foram realizados antibiogramas para 18 tipos distintos de antimicrobianos para uma cepa de cada amostra selecionada de E. coli. Os resultados alcançados mostraram que foi possível identificar 15 diferentes perfis de virulência, com positividade para os seguintes genes: fimH (98,3%), malX (52,4%), traT (31,1%), cvaC (22,9%), fyuA (19,6%), ibeA (19,6%), aer (13,1%) e papC (13,1%). A maioria dos isolados de fragatas foi caracterizado entre os grupos filogenéticos D e B2, e os principais sorotipos encontrados foram O1:H6, O2:H7, O25:H4, e O102:H10. Em relação à resistência a antimicrobianos, 60,1%% dos isolados foram sensível aos 18 diferentes antimicrobianos testados; por outro lado, o antimicrobiano que apresentou maior resistência foi a Tetraciclina, que se revelou inefetiva em 20,1% das amostras pesquisados. Até o momento não foram identificadas cepas de STEC nos indivíduos pesquisados; por outro lado, os isolados albergam genes que caracterizam as ExPEC. Tais resultados mostram que a maioria das cepas isoladas é potencialmente patogênica para as aves, podendo representar significativo risco para sanidade das aves marinhas, além de se configurar como agentes zoonóticos relevantes, enfatizando a importância de se investigar a eventual participação das aves selvagens, em especial as marinhas, na cadeia epidemiológica de doenças ocasionadas por E. coli. Tais informações poderão nortear as pesquisas de doenças selecionadas nas populações das fragatas, assim como embasar a adoção de eventuais ações em relação a aspectos de saúde animal e humana, que tenham as fragatas como um dos elos. / The present study is to investigate and characterize pathogenic Escherichia coli (E. coli Shiga toxin-producing - STEC avian pathogenic E. coli - APEC) on frigates (Fregata magnificens) from the coast of São Paulo and contribute to the understanding of epidemiological aspects in this important group of zoonotic bacterial diseases, the colibacillosis. To this, there were three scientific expeditions to two nesting sites of frigates in the state of Sao Paulo, the main island of Alcatraz (24 ° 06\'S - 45 ° 41\'W) and Castilho´s island (25 ° 16\'S - 47 ° 57\'W), in which it was collected a total of 42 samples of swabs, and 21 cloacal and choanal 21, coming from 18 offspring of indeterminate sex, 2 adult females and 1 adult male frigate. Of the 42 clinical samples studied, 18 were identified with growth of E.coli. Of these, 67 strains were isolated, which were then tested for virulence genes and characterization of phylogenetic groups by PCR technique. Then the strains that exhibited virulence factors were analyzed by serotyping. To investigate the patterns of resistance and sensitivity to antimicrobial susceptibility tests were performed for 18 different types of antimicrobials for one strain of each selected sample of E. coli. The results showed that it was possible to identify 15 different profiles of virulence, tested positive for the following genes: fimH (98.3%), malX (52.4%) traT (31.1%), cvaC (22.9 %), fyuA (19.6%), ibeA (19.6%), aer (13.1%) and papC (13.1%). Most isolates of frigates was characterized between phylogenetic groups D and B2, and the main serotypes were O1:H6, O2:H7, O25:H4, and O102:H10. For resistance to antibiotics, 60.1%% of the isolates were sensitive to 18 different antimicrobial agents tested on the other hand, the antimicrobial with the highest resistance was to tetracycline, which has proved ineffective in 20.1% of the samples studied. So far not been identified in STEC strains studied subjects on the other hand, isolates harbor genes that characterize ExPEC. These results show that most of the strains are potentially pathogenic to birds and may represent significant risk to health of sea birds, including how to configure agents relevant, emphasizing the importance of investigating the possible involvement of wild birds, especially navies in the epidemiological chain of diseases caused by E.coli. Such information could guide the research of selected diseases in populations of the frigates, as well as basing the adoption of any action in relation to aspects of animal and human health, which have the frigates as a link.

Os prejuízos causados por Escherichia coli patogênica para aves (APEC) na produção de aves é justificativa de pesquisas realizadas no mundo todo há décadas. Recentemente, o potencial zoonótico e a multirresistência das cepas desse patotipo têm sido alvo frequente dos trabalhos realizados com APEC. O objetivo desse trabalho foi caracterizar 225 cepas APEC isoladas de perus condenados por aerossaculite em abatedouros em relação à resistência a 14 antimicrobianos, perfil de virulência e grupos filogenéticos. 92% das amostras apresentaram perfil de multirresistência (MDR) e os índices mais altos de resistência foram a sulfonamidas (94%), tetraciclina (83%), eritromicina (82%), estreptomicina (60%), amoxicilina (53%) e ácido nalidíxico (48%). Metade das cepas foram classificadas no grupo filogenético B2 (50%), seguido por B1 (28,6%), grupo A (17,1%) e grupo D (4,8%). Os genes de virulência pesquisados tiveram prevalência de iroN (95%), iss (93%), cvi/cva (67%), iucD (67%), tsh (56%), irp2 (51%), ibeA (31%), vat (24%), neuS (19%), astA (17%) e papC (15%). Considerando o potencial zoonótico e a associação a um maior número de genes de virulência quando comparadas aos outros grupos filogenéticos, as cepas B2 foram selecionadas para pesquisa de integrons de classe 1 e clonalidade. As 112 amostras B2 eram pertencentes a 83 diferentes perfis ERIC, sendo classificadas como multiclonais. Os integrons de classe 1 estiveram presentes em 107 isolados (95,5%). Esses resultados demonstram que a maioria das cepas pesquisadas pertencia ao grupo mais virulento (B2) e relacionado a cepas ExPEC humanas. Aliado ao alto índice de multirresistência encontrado, esses dados sugerem que as aves podem servir como reservatório de cepas patogênicas e multirresistentes, tanto para humanos como para animais, reforçando a ideia de que as aves representem importante papel na cadeia epidemiológica das ExPEC. / Avian Pathogenic Escherichia coli (APEC) has been studied for decades because of its economic impact on the poultry industry. Recently, the zoonotic potential of APEC and multidrug-resistant strains have emerged. The aim of this study was to investigate the virulence profile, phylogenetic background and antimicrobial resistance in 225 strains of Escherichia coli isolated from turkeys presenting airsacculitis. The results showed that 92% of strains presented a multidrug-resistance (MDR) profile, and the highest levels of resistance were to sulfamethazine (94%), tetracycline (83%). Half of these strains (112/225) were classified in phylogenetic group B2, followed by groups B1 (28.6%), A (17.1%) and D (4.8%). The prevalence of virulence genes was as follows: iroN (95%), iss (93%), cvi/cva (67%), iucD (67%), tsh (56%), irp2 (51%), ibeA (31%), vat (24%) neuS (19%), astA (17%) and papC (15%). Considering the zoonotic potential and association to a greater number of virulence genes when compared to other phylogenetic groups, B2 strains were selected for screening of class 1 integrons and clonality. The 112 samples belonging to 83 ERIC profiles and classified as multiclonal. Class 1 integrons were present in 107 isolates (95.5%).These results demonstrate that the majority of the investigated strains belonged to group B2, which is more virulent and is related to human ExPEC strains. Coupled with the high rate of multidrug-resistance found, these data suggest that turkeys may serve as a reservoir for pathogenic and multidrug-resistance strains, for humans and animals, reinforcing the idea that poultry plays an important role in the epidemiological chain of ExPEC.

Escherichia (E.) coli ist als kommensales Bakterium ein wichtiger Bestandteil des Mikrobioms von Säugern, jedoch zudem der häufigste Infektionserreger des Menschen. Entsprechend des Infektionsortes werden intestinal (InPEC) und extraintestinal pathogene E. coli (ExPEC) unterschieden. Die Pathogenese von E. coli-Infektionen ist durch Virulenzfaktoren determiniert, welche von jeweils spezifischen virulenzassoziierten Genen (inVAGs und exVAGs) kodiert werden. Häufig werden exVAGs auch in E. coli-Isolaten aus dem Darm gesunder Wirte nachgewiesen. Dies führte zu der Vermutung, dass exVAGs die intestinale Kolonisierung des Wirtes durch E. coli unterstützen. Das Hauptziel dieser Arbeit bestand darin, das Wissen über den Einfluss von exVAGs auf die Besiedlung und damit die Adhäsion von E. coli an Epithelzellen des Darmtraktes zu erweitern. Die Durchführung einer solch umfassenden E. coli-Populationsstudie erforderte die Etablierung neuer Screeningmethoden. Für die genotypische Charakterisierung wurden mikropartikelbasierte Multiplex-PCR-Assays zum Nachweis von 44 VAGs und der Phylogenie etabliert. Für die phänotypische Charakterisierung wurden Adhäsions- und Zytotoxizitätsassays etabliert. Die Screeningmethoden basieren auf der VideoScan-Technologie, einem automatisierten bildbasierten Multifluoreszenzdetektionssystem. Es wurden 398 E. coli-Isolate aus 13 Wildsäugerarten und 5 Wildvogelarten sowie aus gesunden und harnwegserkrankten Menschen und Hausschweinen charakterisiert. Die Adhäsionsassays hatten zum Ziel, sowohl die Adhäsionsraten als auch die Adhäsionsmuster der 317 nicht hämolytischen Isolate auf 5 Epithelzelllinien zu bestimmen. Die Zytotoxizität der 81 hämolytischen Isolate wurde in Abhängigkeit der Inkubationszeit auf 4 Epithelzelllinien geprüft. In den E. coli-Isolaten wurde eine Reihe von VAGs nachgewiesen. Potentielle InPEC, insbesondere shigatoxinproduzierende und enteropathogene E. coli wurden aus Menschen, Hausschweinen und Wildtieren, vor allem aus Rehen und Feldhasen isoliert. exVAGs wurden mit stark variierender Prävalenz in Isolaten aus allen Arten detektiert. Die größte Anzahl und das breiteste Spektrum an exVAGs wurde in Isolaten aus Urin harnwegserkrankter Menschen, gefolgt von Isolaten aus Dachsen und Rehen nachgewiesen. In Isolaten der phylogenetischen Gruppe B2 wurden mehr exVAGs detektiert als in den Isolaten der phylogenetischen Gruppen A, B1 und D. Die Ergebnisse der Adhäsionsassays zeigten, dass die meisten Isolate zelllinien-, gewebe- oder wirtsspezifisch adhärierten. Ein Drittel der Isolate adhärierte an keiner Zelllinie und nur zwei Isolate adhärierten stark an allen Zelllinien. Grundsätzlich adhärierten mehr Isolate an humanen sowie an intestinalen Zelllinien. Besonders Isolate aus Eichhörnchen und Amseln sowie aus Urin harnwegserkrankter Menschen und Hausschweine waren in der Lage, stark zu adhärieren. Hierbei bildeten die Isolate als Adhäsionsmuster diffuse Adhäsion, Mikrokolonien, Ketten und Agglomerationen. Mittels statistischer Analysen wurden Assoziationen zwischen exVAGs und einer hohen Adhäsionsrate ersichtlich. So war beispielsweise das Vorkommen von afa/dra mit einer höheren Adhäsionsrate auf Caco-2- und 5637-Zellen und von sfa/foc auf IPEC-J2-Zellen assoziiert. Die Ergebnisse der Zytotoxizitätsassays zeigten eine sehr starke und zeitabhängige Zerstörung der Monolayer aller Epithelzelllinien durch die α-Hämolysin-positiven Isolate. Auffallend war die hohe Toxizität hämolytischer Isolate aus Wildtieren gegenüber den humanen Zelllinien. Mit den innerhalb dieser Arbeit entwickelten Screeningmethoden war es möglich, große Mengen an Bakterien zu charakterisieren. Es konnte ein Überblick über die Verbreitung von VAGs in E. coli aus unterschiedlichen Wirten gewonnen werden. Besonders Wildtiere wurden sowohl durch den Nachweis von VAGs in den entsprechenden Isolaten, verbunden mit deren Adhäsionsfähigkeit und ausgeprägter Zytotoxizität als Reservoire pathogener E. coli identifiziert. Ebenso wurde eine zelllinienspezifische Adhäsion von Isolaten mit bestimmten exVAGs deutlich. Damit konnte der mögliche Einfluss von exVAGs auf die intestinale Kolonisierung bestätigt werden. In weiterführenden Arbeiten sind jedoch Expressions- und Funktionsanalysen der entsprechenden Proteine unerlässlich. Es wird anhand der Mikrokoloniebildung durch kommensale E. coli vermutet, dass Adhäsionsmuster und demzufolge Kolonisierungsstrategien, die bisher pathogenen E. coli zugeschrieben wurden, eher als generelle Kolonisierungsstrategien zu betrachten sind. Das E. coli-α-Hämolysin wirkt im Allgemeinen zytotoxisch auf Epithelzellen. Ein in der Fachliteratur diskutierter adhäsionsunterstützender Mechanismus dieses Toxins ist demnach fragwürdig. Innerhalb dieser Arbeit konnte gezeigt werden, dass die entwickelten Screeningmethoden umfassende Analysen einer großen Anzahl an E. coli-Isolaten ermöglichen. / Escherichia (E.) coli is as commensal bacterium an important component of the microbiome of humans and animals, but also the most common infectious agent of human. According to the site of infection intestinal pathogenic (InPEC) and extraintestinal pathogenic E. coli (ExPEC) are differentiated. The pathogenesis of E. coli infections is determined by virulence factors encoded by specific virulence-associated genes (inVAGs and exVAGs). Frequently, exVAGs also be detected in E. coli isolates from the intestine of clinically healthy hosts. This led to the assumption that exVAGs support the intestinal colonization of the host by E. coli. The main objective of this work was to extend the knowledge about the influence of exVAGs on the settlement and adhesion of E. coli to epithelial cells of the intestinal tract. The implementation of such a comprehensive E. coli population study required the establishment of new screening methods. For the genotypic characterization novel microbead-based multiplex PCR assays were established to detect 44 VAGs and phylogeny. For the phenotypic characterization novel in vitro adhesion and cytotoxicity assays were established. These screening methods based on the VideoScan technology, which is an automated image-based multi-fluorescence detection system. There have been characterized 398 E. coli isolates from 13 wild mammal species and 5 species of wild birds as well as from healthy and urinary diseased humans and domestic pigs. The adhesion assays were aimed at both the adhesion rates and the adhesion patterns of the 317 non-hemolytic isolates on intestinal human Caco-2 and porcine IPEC-J2 cells and on human urinary bladder 5637, porcine kidney PK-15 epithelial and HEp-2 cells. The cytotoxicity of 81 hemolytic isolates was compared on the human intestinal epithelium LoVo, and on 5637, IPEC-J2 and PK-15 according to the incubation period. The E. coli isolates showed a series of VAGs. Potential InPEC, especially shigatoxin-producing and enteropathogenic E. coli were isolated from humans, domestic pigs and wild animals, especially from deers (Capreolus capreolus) and hares (Lepus europaeus). exVAGs were detected with widely varying prevalence in E. coli isolates from all species studied. The largest number and the widest range of exVAGs were shown in isolates from urine of urinary diseased patients, followed by isolates from badgers (Meles meles) and deer. Within the isolates of the phylogenetic group B2 more exVAGs were detected as within the isolates of the phylogenetic groups A, B1, and D. Adhesion of the E. coli isolates was specific to cells, host, and tissue, though it was also unspecific. A third of the isolates adhered to any cell line and only two isolates adhered strongly to all cell lines. Basically, more bacteria adhered to human as well as to intestinal cell lines. Especially isolates from squirrels (Sciurus vulgaris) and blackbirds (Turdus merula) and from the urine of urinary diseased humans and domestic pigs were able to strongly adhere. Commensal and pathogenic isolates can adhere in various forms, including diffuse distribution, microcolonies, chains and clumps. Using statistical analyzes associations between the occurrence of some VAGs and a high adhesion rates were seen. Several known adhesins were associated with host cell specific adhesion. Other new potential adhesion genes were described. The results of the cytotoxicity assays showed a very strong and time-dependent degradation of the epithelial cell monolayer of all the α-hemolysin-positive E. coli isolates. The high toxicity of hemolytic isolates from wild animals against the human cell lines was striking. The screening methods enabled both, the genotypic and phenotypic characterisation of large amounts of bacterial isolates. An overview of the distribution of VAGs in E. coli from different hosts was obtained. Especially wild animals were either by the detection of VAGs in the corresponding E. coli isolates, combined with the adhesion and marked cytotoxicity identified as reservoirs of pathogenic E. coli. As well, a cell-line specific adhesion of E. coli isolates with certain exVAGs became clear. Thus, the possible influence on the intestinal colonization of exVAGs could be confirmed. In further work, however, expression and functional analysis of the corresponding proteins are essential. It is suspected on the basis of microcolony formation by commensal E. coli that adhesion patterns and consequently colonization strategies that were previously attributed to pathogenic E. coli, are to be regarded rather as a general colonization strategy. The E. coli α-hemolysin acts generally cytotoxic to epithelial cells. An in the literature discussed adhesion supporting mechanism of this toxin is therefore questionable. Within this work it was shown that the developed screening methods enable comprehensive analyzes of a large number of E. coli isolates.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Orientador: Márcio Garcia Ribeiro / Resumo: Escherichia coli é o principal agente de mastite clínica bovina de origem ambiental, caracterizado pela complexidade de fatores de virulência (FV). O patógeno causa sinais clínicos que variam desde alterações exclusivamente no leite (grau 1 ou leve), no quarto afetado (grau 2 ou moderado), até manifestações sistêmicas (grau 3 ou grave). No entanto, até o momento, não está estabelecido o perfil de genes deste patógeno relacionados à virulência em infecções mamárias em vacas, tampouco com a gravidade clínica dos casos. Neste cenário, o presente estudo investigou 18 genes associados com E. coli extraentérica (ExPEC), o perfil “in vitro” de motilidade swimming e swarming, e a sensibilidade/resistência aos antimicrobianos em 114 isolados de E. coli obtidos de vacas com mastite clínica com escores de gravidade 1 (45/114=39,5%), 2 (62/114=54,4%) e 3 (7/114=6,1%). Os principais genes codificadores de FV detectados foram de adesinas (fimH, 114/114=100%; ecpA, 73/114=64,0%; fimA, 36/114=31,6%), resistência ao soro (traT, 93/114=81,6%; ompT, 40/114=35,1%), sideróforos (irp2, 11/114=9,6%) e hemolisina (hlyA, 8/114=7%). Os isolados apresentaram 99,1% (113/114) de resistência in vitro a bacitracina e cloxacilina, 98,2% (112/114) a lincosamina e 54,4% (62/114) a eritromicina. Do total de isolados, 98,2% (n=112/114) foram multirresistentes pelo cálculo do índice de resistência múltipla aos antimicrobianos (IRMA). Não houve diferença estatística significante entre as medianas para motilidade ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Escherichia coli is the major pathogen involved in the etiology of bovine mastitis from the environment origin. This pathogen is characterized by a complexity of virulence factors (VF). Mammary infections by E. coli has shown a wide range of clinical signs causing changes in milk (score 1 or mild), quarters (score 2 or moderate), and systemic signs (score 3 or severe). Nevertheless, to date, the profile of the genes related to the virulence of this pathogen in mammary infections and the severity scores of the cases are not fully understood. In this scenario, a panel of 18 genes associated with extra-intestinal E. coli (ExPEC) were investigated, in addition to in vitro swimming and swarming motility profile, and antimicrobial susceptibility/resistance pattern among 114 E. coli strains isolated from cows with clinical mastitis showing severity scores 1 (45/114=39.5%), 2 (62/114=54.4%) and 3 (n=7/114=6.1%). The main genes related to VF harbored by isolates were adhesins (fimH, 114/114=100%; ecpA, 73/114=64.0%; fimA, 36/114=31.6%), serum resistance (traT, 93/114=81.6%; ompT, 40/114=35.1%), siderophores (irp2, 11/114=9.6%) and hemolysin (hlyA, 8/114=7%). Among studied isolates, 99.1% (113/114) showed in vitro resistance to bacitracin and cloxacillin, 98.2% (112/114) to lincosamin, and 54.4% (62/114) to erytromycin. Out of the total isolates, 98.2% (112/114) were considered multidrug resistant based on multiple antimicrobial resistance (MAR) index. No statistical difference was obs... (Complete abstract click electronic access below) / Doutor

A DNA microarray capable of detecting 445 virulence factors (VFs) and antibiotic resistance genes was used to assess human and animal fecal E. coli isolates for pathogenic potential and host specificity. The only enteric pathotype detected was atypical EPEC, which was found in 3.7% of all isolates. 17% of human isolates were extraintestinal pathotypes, with the majority of these being uropathogenic. Isolates from humans and chickens were the most likely to have resistance to at least one class of antibiotic. VFs that were found almost exclusively in human isolates, when compared to one other group, included sat (10% of human isolates and no animal isolates), iucD and iut (24% of human, <1% mammal) and iha (16% human, <1% wild avian). Decision trees utilizing multiple probes to identify the source of an E. coli isolate were able to correctly identify the source of 79% of validation isolates in a human vs. animal comparison. / Environment Canada

L’opéron métabolique frz code les sous-unités d’un transporteur PTS de la sous-famille du fructose, un activateur transcriptionnel des systèmes PTS de la famille MgA (FrzR), deux cétoses-1,6-bisphosphate aldolases de type II, une kinase spécifique des sucres (famille ROK) et une protéine de la superfamille des cupines. Il est fortement associé aux souches d’Escherichia coli à virulence extra-intestinale. Nous avons montré qu’il procure un avantage aux bactéries lors de conditions de stress (peu d’oxygène, phase stationnaire de croissance, croissance dans le sérum et l’intestin) et qu’il est impliqué dans l’adhérence et l’internalisation de la bactérie dans diverses cellules eucaryotes, en régulant l’expression des fimbriae de type 1. L’activateur (FrzR) est impliqué dans ces phénotypes. A l’aide de microarrays, une série de gènes sous la dépendance du système Frz ont été identifiés. Nos données suggèrent que frz code un senseur de l’environnement permettant à E. coli de s’adapter à un environnement fluctuant en régulant notamment certains gènes de virulence et d’adaptation à l’hôte. Un modèle de régulation est présenté. / The metabolic frz operon codes for three subunits of a PTS transporter of the fructose sub-family, for a transcriptional activator of PTS systems of the MgA family (FrzR), for two type II ketose-1,6-bisphosphate aldolases, for a sugar specific kinase (ROK family) and for a protein of the cupin superfamily. It is highly associated with Extra-intestinal Pathogenic Escherichia coli strains. We proved that frz promotes bacterial fitness under stressful conditions, (such as oxygen restriction, late stationary phase of growth or growth in serum or in the intestinal tract). Furthermore, we showed that frz is involved in adherence to and internalization of E. coli in several eukaryotic cells by regulating the expression of type 1 fimbriae. The FrzR activator is involved in these phenotypes. Microarrays, experiments allowed the identification of several genes under the dependence of the frz system. Our data suggest that frz codes for a sensor of the environment allowing E. coli to adapt to a fluctuating environment by regulating some virulence and host adaptation genes. A regulation model is presented.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-09-21Bitstream added on 2014-06-13T19:35:22Z : No. of bitstreams: 1 paula_cjs_me_jabo.pdf: 334942 bytes, checksum: 17d2a17239376692690b930753f05b71 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Cachorros têm sido propostos como possíveis reservatórios de cepas de Escherichia coli patogênicas que causam infecções em cachorros e em humanos. Entre Janeiro e Dezembro de 2006, 92 cepas de E. coli foram analisadas para a detecção da presença de genes produtores de toxina Shigalike (stx 1 e stx 2) e o gene da intimina (eae). Doze cepas de E. coli Shigatoxigênica (STEC) foram detectadas por PCR como portadoras dos genes de Shiga toxina, 7 cepas do gene stx 1 (7,6%), 5 do stx 2 (5,4%) e nenhuma delas apresentando ambos os genes. Nove (9,8%) das cepas de E. coli apresentaram o gene eae e não eram produtoras de Shiga toxina. Os isolados de E. coli foram também examinados para a detecção dos genes codificadores de adesinas (pap, sfa e afa), hemolisina e aerobactina. A freqüência dos genes de virulência detectados nestes isolados foi de 12,0% pap, 1,0% sfa, 10,0% hemolisina e 6,5% aerobactina. Seis destes isolados foram caracterizados como cepas de E.coli patogênica extraintestinal (ExPEC). Entre as cepas de STEC e ExPEC foi encontrado um alto nível de resistência a agentes antimicrobianos (estreptomicina com 81% e 100% e cefalotina com 85,7% e 50% respectivamente) e algumas delas foram caracterizadas como E. coli resistentes a múltiplas drogas (MDREC), o que representa um motivo de preocupação devido ao risco de disseminação dos genes de resistência para a microbiota dos seres humanos. / Dogs have been proposed as a possible reservoir of the pathogenic Escherichia coli that causes infection in dogs and human beings. From January to December 2006, 92 E. coli isolates from 25 diarrheic dogs were analyzed by screening for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twelve Shiga toxin-producing E. coli (STEC) isolates were detected by PCR to harbor the Shiga toxin genes, 7 isolates the stx 1 (7.6%); 5 the stx 2(5.4%) and none both of them. Nine (9.8%) of the E. coli isolates studied were eae positive non Shiga toxin-producing. The E. coli isolates also were screened for the presence of adhesion-encoding genes (pap, sfa, afa), hemolysin and aerobactin genes. Virulence gene frequencies detected in those isolates were: 12.0% pap, 1.0% sfa, 10.0% hemolysin and 6.5% aerobactin. Ten isolates were characterized as extraintestinal pathogenic E. coli (ExPEC) strains. Among STEC and ExPEC isolates was found high level of resistance to antimicrobial agents and some of them as characterized as multidrug resistant E. coli (MDREC), what represent a reason for concern due the risk of dissemination of antimicrobial resistant genes to the microbiota of human beings.