Clonal relationship among atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans.

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Enteroaggregative Escherichia coli from humans and animals differ in major phenotypical traits and virulence genes.

Enteroaggregative Escherichia coli (EAEC) is characterized by the expression of the aggregative adherence pattern to cultured epithelial cells. In this study, we determined the phenotypic and genotypic relationships among 86 EAEC strains of human and animal (calves, piglets and horses) feces. Serotypes and the presence of EAEC virulence markers were determined, and these results were associated with ribotyping. Strains harboring aggR (typical EAEC) of human origin were found carrying several of the searched markers, while atypical EAEC harbored none or a few markers. The strains of animal origin were classified as atypical EAEC (strains lacking aggR) and harbored only irp2 or shf. Strains from humans and animals belonged to several different serotypes, although none of them prevailed. Sixteen ribotypes were determined, and there was no association with virulence genes profiles or serotypes. Relationship was not found among the strains of this study, and the assessed animals may not represent a reservoir of human pathogenic typical EAEC.

Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR.

Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR technique and their serotypes were determined. The ERIC-PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A-D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC-PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)-PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates.

Characterization of extraintestinal pathogenic Escherichia coli isolated from captive wild felids with bacteremia.

Diseases caused by extraintestinal pathogenic Escherichia coli (ExPEC) in wild felids are rarely reported. Although urinary tract infections are infrequently reported in domestic cats, such infections when present are commonly caused by ExPEC. The present work characterized ExPEC strains isolated from 2 adult felines, a snow leopard (Panthera uncia) and a black leopard (Panthera pardus melas), that died from secondary bacteremia associated with urinary tract infections. Isolates from both animals were classified into the B2 phylogenetic group and expressed virulence genotypes that allowed them to cause severe disease. In addition, strains from the black leopard showed multidrug resistance.

Atypical enteropathogenic Escherichia coli genomic background allows the acquisition of non-EPEC virulence factors.

Atypical enteropathogenic Escherichia coli (aEPEC) has been associated with infantile diarrhea in many countries. The clonal structure of aEPEC is the object of active investigation but few works have dealt with its genetic relationship with other diarrheagenic E. coli (DEC). This study aimed to evaluate the genetic relationship of aEPEC with other DEC pathotypes. The phylogenetic relationships of DEC strains were evaluated by multilocus sequence typing. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The phylogram showed that aEPEC strains were distributed in four major phylogenetic groups (A, B1, B2 and D). Cluster I (group B1) contains the majority of the strains and other pathotypes [enteroaggregative, enterotoxigenic and enterohemorrhagic E. coli (EHEC)]; cluster II (group A) also contains enteroaggregative and diffusely adherent E. coli; cluster III (group B2) has atypical and typical EPEC possessing H6 or H34 antigen; and cluster IV (group D) contains aEPEC O55:H7 strains and EHEC O157:H7 strains. PFGE analysis confirmed that these strains encompass a great genetic diversity. These results indicate that aEPEC clonal groups have a particular genomic background--especially the strains of phylogenetic group B1--that probably made possible the acquisition and expression of virulence factors derived from non-EPEC pathotypes.

Development and implementation of a secure, integrated management system for medical images and electronic clinical records for small hospitals.

The field of Medical Informatics is currently experiencing increasing demands for new models of the Picture Archiving and Communication Systems (PACS) and Digital Imaging and Communications in Medicine (DICOM) protocols. Despite of the considerable advantages of current systems, implementation in hospitals is remarkably slow, due primarily to difficulties in integration and relatively high costs. Even though the success of DICOM standards has greatly contributed to the development of PACS, many hospitals remain unable to support it or to make full use of its potential because various imaging modalities in use at these sites generate images that cannot be stored in the PACS and cannot be managed in a centralized manner without DICOM standardization modules. Furthermore, the imaging modalities being used in such smaller centers are expensive and unlikely to be replaced, making DICOM compliance untenable. With this in mind, this paper describes the design, development, and implementation of a management system for medical diagnostic imaging, based on the DICOM standard and adapted to the needs of a small hospital. The system is currently being implemented in the San Rafael Hospital at A Coruna in Spain, and integrated with the existing hospital information system (HIS). We have studied the networking infrastructure of the hospital and its available image generation devices, and have subsequently carried out a series of measurements including transmission times, image file size, compression ratios, and many others that allow us to analyze the behavior of the system. Results obtained from these investigations demonstrate both the flexibility of using such a "small-hospital" DICOM-based framework as well as the relative cost-effectiveness of the system. In this regard, the approach, described herein, might serve as a model for other small, and possibly mid-sized, medical centers.

Bundle-forming pili and EspA are involved in biofilm formation by enteropathogenic Escherichia coli.

Microcolony formation is one of the initial steps in biofilm development, and in enteropathogenic Escherichia coli (EPEC) it is mediated by several adhesins, including the bundle-forming pilus (BFP) and the EspA filament. Here we report that EPEC forms biofilms on plastic under static conditions and a flowthrough continuous culture system. The abilities of several EPEC isogenic mutants to form biofilms were assessed. Adhesins such as BFP and EspA, important in microcolony formation on epithelial cells, are also involved in bacterial aggregation during biofilm formation on abiotic surfaces. Mutants that do not express BFP or EspA form more-diffuse biofilms than does the wild type. We also determined, using gfp transcriptional fusions, that, consistent with the role of these adhesins in biofilms, the genes encoding BFP and EspA are expressed during biofilm formation. Finally, expression of espA is controlled by a quorum-sensing (QS) regulatory mechanism, and the EPEC qseA QS mutant also forms altered biofilms, suggesting that this signaling mechanism plays an important role in EPEC biofilm development. Taken together, these studies allowed us to propose a model of EPEC biofilm formation.

Characterization of monkey enteropathogenic Escherichia coli (EPEC) and human typical and atypical EPEC serotype isolates from neotropical nonhuman primates.

Enteropathogenic Escherichia coli (EPEC) has been associated with infantile diarrhea and mortality in humans in developing countries. While diarrhea is also a major problem among primates kept in captivity, the role of E. coli is unclear. This study was designed to characterize diarrheagenic E. coli recovered from the feces of 56 New World nonhuman primates, primarily marmosets (Callithrix spp.). Seventeen of the 56 primates had signs of diarrhea and/or enteritis. E. coli recovered from feces from these animals was tested by PCR for genes encoding virulence factors of diarrheagenic E. coli and for patterns of adherence to HeLa cells. In addition, isolates were characterized by the fluorescence actin staining test and by their ability to induce attaching and effacing lesions. PCR for the eae gene was positive in 10 of the 39 (27%) apparently healthy animals and in 8 of the 17 (47%) animals with diarrhea and/or enteritis. Colonies of eae(+) E. coli were serotyped and examined by PCR for genes encoding EPEC virulence markers. The eae(+) E. coli isolates recovered from both healthy and sick nonhuman primates demonstrated virulence-associated attributes similar to those of EPEC strains implicated in human disease and are designated monkey EPEC. The results presented here indicate that EPEC may be a significant pathogen for nonhuman primates, deserving further investigation. The similarities between the affected animals investigated in this study and human EPEC infections suggest that marmosets may represent an important model for EPEC in humans.

Typing of avian pathogenic Escherichia coli strains by REP-PCR

A técnica de REP (Repetitive extragenic palindrome)-PCR foi utilizada para avaliar a variabilidade genética de 49 amostras de Escherichia coli patogênicas para aves (APEC), isoladas de aves de corte (frangos) em diferentes surtos de septicemia (n=24), síndrome da cabeça inchada (n=14) e onfalite (n=11). Trinta amostras comensais, isoladas de frangos sem sinais de doença, foram utilizadas como controle. A análise do perfil eletroforético obtido por reação de REP-PCR utilizando DNA purificado das amostras evidenciou a amplificação de 0 a 15 bandas de DNA com pesos moleculares variando entre 100 pb e 6.1 Kb. A análise deste padrão permitiu a construção de um dendrograma demonstrando o agrupamento das 79 amostras em 49 perfis distintos. Embora a técnica de REP-PCR tenha apresentado grande poder discriminatório, as amostras patogênicas e não patogênicas não foram discriminadas entre si assim como não foi observado o agrupamento de amostras causadoras do mesmo tipo de doença. Por outro lado, demonstramos recentemente que outras técnicas tais como ERIC-PCR e a análise de isoenzimas foram eficientes quando utilizadas para esta mesma finalidade. Concluindo, REP-PCR parece não ser uma técnica eficiente e universal para discriminar entre amostras APEC. Porém, a estrutura clonal populacional obtida com o uso de REP-PCR não deve ser desprezada, particularmente se considerarmos que os mecanismos de patogenicidade de APEC ainda não são completamente conhecidos.

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Sorotipagem de amostras de Streptococcus suis isoladas de suínos em granjas dos Estados de Säo Paulo, Minas Gerais e Paraná / Serotyping of Streptococcus suis strains isolated from pigs in the States of Säo Paulo, Minas Gerais e Paraná, Brazil

Infecçöes causadas por Streptococcus suis säo muito comuns em países onde a indústria de carne suína é desenvolvida. Estas infecçöes estäo relacionadas a casos clínicos de broncopneumonia, meningite, artrite, pericardite, miocardite, endocardite, poliserosite fibrinosa, septicemia, rinite e aborto. Esta bactéria também foi descrita como patógeno de ruminantes e humanos. No Brasil há evidências clínicas da existência de processos infecciosos causados por S. suis afetando mais de 50 por cento das granjas em Estados como Säo Paulo, Minas Gerais e Paraná. No presente estudo foram isoladas 51 amostras de S. suis de granjas do Estados acima referidos, coletadas de diferentes casos clínicos como septicemia, meningite, artrite e pneumonia, tendo sido obtidas ou em cultura pura ou como patógeno de maior predominância nos tecidos de suínos. Este material foi semeado em Columbia ágar sangue adicionado de 5 por cento de sangue bovino e incubado a 37°C por 24 horas. Para a identificaçäo bioquímica as colônias que apresentavam a-hemólise, bem como as amostras padräo, foram submetidas a testes convencionais para a confirmaçäo da espécie S. suis, tais como: hidrólise de arginina, teste de Voges-Proskauer, e produçäo de ácido a partir de vários carboidratos (inulina, salicina, trealose, lactose, sacarose, sorbitol, manitol e glicerol). As amostras também foram testadas para habilidade de crescimento em meio de TSA com 6,5 por cento de NaCl e para a produçäo de amilase. Todas as amostras que fizeram parte desta pesquisa foram testadas pelo sistema Api 20 Strep para confirmaçäo dos resultados obtidos nos testes convencionais. Para a sorotipagem foram produzidos antissoros de 1 a 8. Outras amostras näo pertencentes a estes sorotipos também foram sorotipadas. O antissoro produzido em coelhos foi titulado pelo teste de aglutinaçäo em tubo com 2-mercaptoetanol e pelo teste de reaçäo capsular e, quando adequados, foram usados no teste de co-aglutinaçäo, para a sorotipagem das amostras de S. suis. A sorotipagem das 51 amostras isoladas mostraram os seguintes resultados: 30 (58,8 por cento) foram classificadas como sorotipo 2, 11 (21,6 por cento) das amostras como sorotipo 3, sete (13,72 por cento) como sorotipo 7, duas (3,92 por cento) como sorotipo 1 e uma amostra como pertencente ao sorotipo14 (1,96 por cento). Este é o primeiro relato do isolamento de um grande número de amostras de S. suis no Brasil, de casos típicos de processos infecciosos causados por esta bactéria

Incidência de Escherichia coli enterotoxigênica (ETEC), rotavírus e Clostridium perfringens de casos de diarréia em crianças, na regiäo de Campinas, SP, Brasil / Incidence of enterotoxigenic Escherichia coli (ETEC) rotaviruses and clostridium perfringens from cases of infantile diarrhea, in the region of Campinas, SP, Brazil

Foi realizada uma pesquisa na regiäo de Campinas, S-, Brasil, sobre a presença de Escherichia coli enterotoxigência (ETEC), rotavírus e clostridium perfringens enterotoxigênico em fezes diarréicas de crianças com até 2 anos de idade. Dos 132 especimes fecais examinados quanto a presença de ETEC 27 (20,45%) foram positivos. Destes foram isoladas 41 amostras de ETEC, das quais 40 produziram apenas a enterotoxina termolábil (LT) detectada pelo teste de imuno-hemólise radial modificado. Entre as 1983 amostras de fezes examinadas para rotavírus, 29 (15,84%) foram positivas pelas técnicas de eletroforese em gel de poliacrilamida (PAGE) e ensaio imunoenzimático (EIE), sendo que destas, 15 (51,7%) foram provenientes de materiais coletados nos meses de inverno. Todas as amostras pertenciam ao grupo A e, através da técnica de PAGE, pode-se observar que o tipo eletroforético mais freqüente (9 amostras) foi designado Ib, IIc, IIIb, IVa, de acordo com a classificaçäo por nós adotada. Apenas 113 amostras de fezes foram examinadas para a presença de C. perfringens enterotoxigênico. Para a detecçäo da enterotoxina nos sobrenadantes das culturas foram utilizadas as técnicas de hemaglutinaçäo passiva reversa e inoculaçäo intravenosa em camundongos, sendo encontradas 12(10,62%) amostras enterotoxigências. Diante destes resultados é chamada a atençäo sobre o valor apenas relativo de uma coprocultura convencional para fins de diagnóstico, ressaltando-se a importância da criaçäo de métodos simplificados que favoreçam a detecçäo e identificaçäo dos grupos de agentes enteropatogênicos estudados na presente pesquisa

Use of an indirect haemagglutination test, for the detection of Clostridium perfringens type A enterotoxin

An indirect haemagglutination (IH) test is described for the detection of Clostridium perfringens type A enterotoxin, produced by strains isolated from human cases of food poisoning and from contaminated food. Though no strict relationship could be observed between titers in the IH test and the time it took mice to die from the intravenous inoculation of mice (IIM), results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the ILM one. No unspecific reaction was obtanined int he IH wirh a negative control and the inhibitions of the IH and IIM tests by specific antiserum against C. perfringens enterotoxin showed that the IH test is very spcific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data

Reproduçäo experimental da colibacilose suína em leitöes / Experimental reproduction of colibacillosis in piglets

Foi tentada a reproduçäo experimental da colibacilose suína neonatal, em leitöes recém-nascidos, usando-se para tal 4 grupos de amostras de Escherichia coli enterotoxigênicas (ETEC), a saber: 1) Duas amostras do sorogrupo 0149:K81, produtoras da enterotoxina termolábil (LT) e do fator de colonizaçäo K88; 2) Duas amostras do sorogrupo 0101:K30, produtoras da enterotoxina termoestável (STa) e do fator de colonizaçäo K99; 3) Uma amostra do sorogrupo 0157:K?, produtora da enterotoxina termoestável do tipo STb e do fator de colonizaçäo K88, e 4) Uma amostra do sorogrupo 08:K?, produtora da enterotoxina termoestável(STa) e de um novo fator de colonizaçäo, denominado F42. Todos os 14 leitöes inoculados por via oral com estas amostras de ETEC desenvolveram doença clínica com morte até 42 horas após a inoculaçäo, tendo sido possível detectar em todos eles a colonizaçäo do intestino delgado pelas amostras do ETEC inoculadas, através de técnica de imunofluorescência indireta. A produçäo de STa "in vivo", por amostras dos grupos 2 e 4 foi um fator importante na comprovaçäo de que a reproduçäo experimental da doença por estas amostras realmente ocorreu. De fato, a coprocultura, quer das fezes diarréicas, quer do conteúdo intestinal dos animais, revelou um alto índice de recuperaçäo de colônias LT+ -K88+ e Sta+ -K99+. Embora tenham ocorrido entre os diversos materiais examinados algumas diferenças quantitativas, a recuperaçäo de colônias STa+ -F42+ foi menor do que nos casos anteriores, porém os achados referentes a doença clínica, produçäo de STa "in vivo" e colonizaçäo do intestino delgado dos leitöes inoculados, comprovaram que o antígeno F42 é, sem dúvida, um novo fator de colonizaçäo em amostras de ETEC envolvidas na colibacilose suína