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This study evaluated the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) analysis of fliC for typing flagella antigen (H) of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains isolated from different animals. The molecular typing of the H type was efficient in the determination of 93 (85%) strains. Two nonmotile (H-) E. coli strains showed a PCR-RFLP electrophoretic profile that did not match known H type patterns. The fliC nucleotide sequence of strains B2N and 4a revealed a nucleotide substitution at the restriction site and a nucleotide insertion that generated a stop codon, respectively. The results of this study showed that PCR-RFLP analysis of fliC is faster, less laborious and as efficient for the determination of H type E. coli isolated from animals, compared to serotyping and that it is useful in determining H type in nonmotile strains and strains expressing non-reactive H antigens. Moreover, the fliC sequence of strain B2N suggests that we could have found a new flagellin antigen type.

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.

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk. A presença de seqüências de DNA associadas à capacidade de captação de ferro (irp-2, fyuA, sitA, fepC, iucA), adesão (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) e de invasão (inv, ial) e a classificação dentro dos quatro grupos filogenéticos principais de Escherichia coli (Grupos A, B1, B2 e D) foram determinadas, através de PCR, em 30 amostras comensais de E. coli isoladas de frangos e de 49 linhagens APEC (24 isoladas de frangos com septicemia, 14 isoladas de frangos com síndrome da cabeça inchada e 11 isoladas de embriões de galinhas com onfalite). Nenhuma das linhagens apresentou os genes inv, ial, efa, e toxB. Os genes lpfA O157/O154, iucA, fepC e irp-2 foram encontrados em freqüências significativas entre as amostras patogênicas. O gene iucA foi associado com amostras causadoras de septicemia e de síndrome da cabeça inchada. Os genes fepC e irp-2 foram associados a amostras causadoras de síndrome da cabeça inchada. A análise filogenética demonstrou que linhagens comensais e causadoras de onfalite pertenceram principalmente ao Grupo filogenético A, não patogênico. Amostras causadoras de síndrome da cabeça inchada pertenceram, em sua maioria, ao Grupo patogênico D. Linhagens causadoras de septicemia pertenceram aos Grupos A e D. Estes dados sugerem que linhagens APEC causadoras de septicemia provavelmente têm uma origem ancestral múltipla: uma derivada de uma linhagem patogênica e outra de uma linhagem não patogênica que possivelmente evoluiu através da aquisição horizontal de genes de virulência. Amostras causadoras de síndrome da cabeça inchada possivelmente constituem um grupo clonal patogênico. Por outro lado, amostras causadoras de onfalite possivelmente constituem um grupo clonal não patogênico, que, possivelmente causam onfalite devido a uma infecção oportunista. A presença de genes de virulência também encontrados em E. coli de origem humana pode indicar a possível ocorrência de zoonoses causadas por APEC.

Streptococcus ssp are important components of the dental biofilm and Streptococcus crista is considered to be an interesting model of bacterial interactions taking place in this biofilm. In the present work, S. crista strains were isolated from the dental biofilm of Brazilian individuals and studied with respect to their biological characteristics and their molecular profile by means of AP-PCR techniques, using the RR2, 434, OPR2, OPR8, and OPR13 primers. Results allowed us to build a similarity dendrogram. Analysis of the similarity dendrogram allowed the separation of the studied strains into similarity groups. All isolates presented fibril tufts by Transmission Electron Microscopy (TEM). These isolates were able to bind to salivary amylase and to adhere to mouth epithelial cells. Some strains displaying fibril tufts and positive adherence were not able to co-aggregate with Fusobacterium nucleatum, suggesting that different adhesin groups are present in these strains.Streptococcus spp são importantes componentes do biofilme dental sendo Streptococus crista considerado um interessante modelo de interações bacterianas que nele ocorrem. No presente trabalho linhagens de S. crista, foram isoladas do biofilme dental de indivíduos brasileiros, e estudadas em relação a suas características biológicas e ao seu perfil molecular através da técnica do AP-PCR, usando-se os iniciadores RR2, 434, OPR2, OPR8 e OPR13. Os resultados nos permitiram construir um dendrograma de similaridade. A análise do dendrograma de similaridade permitiu a separação das linhagens estudadas em grupos de similaridade. Todos os isolados apresentaram tufo de fibrilas, quando estudados por Microscopia Eletrônica de Transmissão (MET). Estes isolados foram capazes de se ligar à amilase salivar e de se aderir a células epiteliais bucais. Algumas linhagens, que apresentam tufo de fibrilas e aderência positiva, não foram capazes de coagregar com a Fusobacterium nucleatum, sugerindo que diferentes grupos de adesinas estão presentes nestas amostras.

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.

Streptococcus ssp are important components of the dental biofilm and Streptococcus crista is considered to be an interesting model of bacterial interactions taking place in this biofilm. In the present work, S. crista strains were isolated from the dental biofilm of Brazilian individuals and studied with respect to their biological characteristics and their molecular profile by means of AP-PCR techniques, using the RR2, 434, OPR2, OPR8, and OPR13 primers. Results allowed us to build a similarity dendrogram. Analysis of the similarity dendrogram allowed the separation of the studied strains into similarity groups. All isolates presented fibril tufts by Transmission Electron Microscopy (TEM). These isolates were able to bind to salivary amylase and to adhere to mouth epithelial cells. Some strains displaying fibril tufts and positive adherence were not able to co-aggregate with Fusobacterium nucleatum, suggesting that different adhesin groups are present in these strains. Streptococcus spp são importantes componentes do biofilme dental sendo Streptococus crista considerado um interessante modelo de interações bacterianas que nele ocorrem. No presente trabalho linhagens de S. crista, foram isoladas do biofilme dental de indivíduos brasileiros, e estudadas em relação a suas características biológicas e ao seu perfil molecular através da técnica do AP-PCR, usando-se os iniciadores RR2, 434, OPR2, OPR8 e OPR13. Os resultados nos permitiram construir um dendrograma de similaridade. A análise do dendrograma de similaridade permitiu a separação das linhagens estudadas em grupos de similaridade. Todos os isolados apresentaram tufo de fibrilas, quando estudados por Microscopia Eletrônica de Transmissão (MET). Estes isolados foram capazes de se ligar à amilase salivar e de se aderir a células epiteliais bucais. Algumas linhagens, que apresentam tufo de fibrilas e aderência positiva, não foram capazes de coagregar com a Fusobacterium nucleatum, sugerindo que diferentes grupos de adesinas estão presentes nestas amostras.

Twenty-three Escherichia coli O26 strains from humans, cattle, sheep, pigs and chicken were investigated for virulence markers and for genetic similarity by pulsed field gel electrophoresis and multi locus sequence typing. Two groups of genetically closely related O26 strains were defined. One group is formed by enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli strains, which do not ferment rhamnose and dulcitol and most of these carry a plasmid encoding enterohemolysin. The other group consists of rhamnose and dulcitol fermenting EPEC strains, which carry plasmids encoding alpha-hemolysin. Multiple species of domestic animals were shown to serve as a reservoir for human pathogenic O26 EPEC and EHEC strains.

Forty-nine avian pathogenic Escherichia coli (APEC) strains obtained from chickens suffering from septicemia (24), swollen head syndrome (14) and omphalitis (11), isolated from individuals in different regions of Brazil and from different outbreaks, were studied for their adhesion to trachea epithelial cells, fimbrial expression and hemagglutination capacity to different erythrocyte types. These results were compared with their content of fimbriae-related genes as detected by polymerase chain reaction (PCR) using specific pair of primers. The aim of these assays was to determine the importance of expression of adhesins in the pathogenic strains and to evaluate the presence of adhesin genes either previously described or not yet recognized for APEC strain. Thirty commensal strains isolated from poultry showing no signs of any of the above diseases were used to compare the results with the pathogenic isolates. The PCR assay demonstrated that septicaemic and swollen head syndrome strains had the highest number of adhesion-related genes of recognized importance in pathogenicity. Using different media for growth conditions, 40 different D-mannose resistant haemagglutination patterns were observed in this study, what indicates the expression of a great variability of surface agglutinins in these bacterial strains. Our results also showed that adhesion, whether D-mannose resistant (MRA) or D-mannose sensitive (MSA), is a characteristic observed in both pathogenic and commensal strains. Several strains with positive adherence had no genetic sequences related to the studied adhesin genes what indicates that our APEC strains probably possess a genome with adhesins genes besides those describe elsewhere and that have not yet been described.

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.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.

Infecções causadas por Streptococcus suis são muito comuns em países onde a indústria de carne suína é desenvolvida. Estas infecções estão 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% das granjas em Estados como São 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% de sangue bovino e incubado a 37°C por 24 horas. Para a identificação bioquímica as colônias que apresentavam a-hemólise, bem como as amostras padrão, foram submetidas a testes convencionais para a confirmação da espécie S. suis, tais como: hidrólise de arginina, teste de Voges-Proskauer, e produção 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% de NaCl e para a produção de amilase. Todas as amostras que fizeram parte desta pesquisa foram testadas pelo sistema Api 20 Strep para confirmação dos resultados obtidos nos testes convencionais. Para a sorotipagem foram produzidos antissoros de 1 a 8. Outras amostras não pertencentes a estes sorotipos também foram sorotipadas. O antissoro produzido em coelhos foi titulado pelo teste de aglutinação em tubo com 2-mercaptoetanol e pelo teste de reação capsular e, quando adequados, foram usados no teste de co-aglutinação, para a sorotipagem das amostras de S. suis. A sorotipagem das 51 amostras isoladas mostraram os seguintes resultados: 30 (58,8%) foram classificadas como sorotipo 2, 11 (21,6%) das amostras como sorotipo 3, sete (13,72%) como sorotipo 7, duas (3,92%) como sorotipo 1 e uma amostra como pertencente ao sorotipo14 (1,96%). 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. Também foi realizada a sorotipagem dos isolados, mostrando uma alta prevalência do sorotipo 2, quando comparada com a dos demais sorotipos encontrados.

Department of Microbiology, Institute of Biomedical Sciences, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Department of Clinical Analysis, School of Pharmacy, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Institute of Biological Sciences, Universidade Federal do Para, Belem, PA, Brazil; School of Public Health, Universidade de Sao Paulo, Sao Paulo, SP, Brazil; Environmental Company of Sao Paulo State (CETESB), Sao Paulo, SP, Brazil

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0–11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.

One strain (S32) of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI) of 25.15 ± 1.26 (mean ± 1 standard error of the mean). Treatment of the bacterial cells with trypsin (0.25mg/ml) decreased in 70%-80% the AI and metaperiodate (10mg/ml) abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100ºC/5 min) before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1: 640, at least. The piglet ileal loop assay, carried out with strains S32 and Jab-1 (negative control) demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microcopy (TEM) demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhuteniun red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies.Uma amostra (S32) de Clostridium perfringens tipo A foi isolada de um caso de enterite catarral em leitões. Esta amostra foi capaz de aderir a células HeLa mostrando um índice de adesão (AI) de 25,15 ± 1,26 (media ± 1 erro padrão da media). Tratamento das células bacterianas com tripsina (0,25mg/ml) diminuiu 70%-80% e metaperiodato (10mg/ml) aboliu significantemente a adesão, sugerindo que a estrutura responsável por esta adesão era provavelmente uma glicoproteína. O tratamento pelo calor das suspensões bacterianas (100ºC/5min) diminuiu o AI ao nível dos controles negativos. Soro de coelho anti-S32 inibiu a aderência a células HeLa até a diluição de 1:640, pelo menos. O teste da alça ligada de leitão recem nascido demonstrou que a amostra S32 era capaz de aderir às células epiteliais intestinais, conforme demonstrado pela coloração de Gram de secções histológicas do intestino dos animais inoculados. O estudo em Microscópio Eletrônico de Transmissão demonstrou que a amostra S32 de Cl. perfringens mostrava um material de natureza fibrilar frouxa, ao contrário da amostra Jab-1 (controle negativo) que demonstrava uma aparência "nua ou lisa". A estabilização das células bacterianas com antissoro homólogo (S32), seguida de coloração com vermelho de rutenio, revelou de maneira mais nítida que longos materiais fibrilares, de aparência frouxa, se estendendo para longe da célula bacteriana, ligando por vezes estas células entre si. A possibilidade desta estrutura ser uma adesina para esta amostra de Cl. perfringens tipo A, talvez desempenhando um papel na patogenia da enterite catarral de leitões, depende de mais estudos.

Six hundred and ninety three male inmates from three penitentiaries, two (A and B) maximum-security systems and one (C) minimum-security facility, located in Campinas, State of São Paulo, Brazil were studied for the presence of human immunodeficiency virus (HIV) antibodies, using a cross-sectional design. The search for anti-HIV antibodies in 693 samples of sera collected was carried out by two serological tests: (a) the Microparticle enzyme immunoassay-HIV-1 and HIV-2 (MEIA) (Abbott Laboratories) and (b) the Western Blot-HIV-1 (WB) (Cambridge Biotech Corporation) to confirm positive results with MEIA. Sera reactivity for HIV antibodies was 14.4%. The highest frequency of anti-HIV antibodies was found in the A and B maximum-security prisons: 17% and 21.5%, respectively. In prison C, the frequency of reagents was 10.9%. Seventy three inmates, initially negative in the MEIA test, were checked again five and seven months later. Three of them, all from the maximum-security facilities, became reactive in the MEIA test, with confirmation in the WB, suggesting that serological conversion had occurred after imprisonment.

Haemagglutination of purified F42 fimbriae was found to be inhibited by N-acetyl-galactosamine. Purified F42 fimbrial adhesin reacted with distinct membrane components from chicken erythrocytes (35, 37 and 40 kDa) in immunoblot analysis, suggesting that the binding occurred to proteins or glycoproteins.

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.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:59:20Z No. of bitstreams: 1 S0103-84782002000200017.pdf: 42603 bytes, checksum: a7bcabcb2df7232a1af99c4afde7749b (MD5) Made available in DSpace on 2013-08-22T18:59:20Z (GMT). No. of bitstreams: 1 S0103-84782002000200017.pdf: 42603 bytes, checksum: a7bcabcb2df7232a1af99c4afde7749b (MD5) Previous issue date: 2002-04-01 Made available in DSpace on 2013-09-30T19:56:04Z (GMT). No. of bitstreams: 2 S0103-84782002000200017.pdf: 42603 bytes, checksum: a7bcabcb2df7232a1af99c4afde7749b (MD5) S0103-84782002000200017.pdf.txt: 12155 bytes, checksum: 3b43b2b7a8caed3f070d86139ef17fdd (MD5) Previous issue date: 2002-04-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:14:36Z No. of bitstreams: 2 S0103-84782002000200017.pdf: 42603 bytes, checksum: a7bcabcb2df7232a1af99c4afde7749b (MD5) S0103-84782002000200017.pdf.txt: 12155 bytes, checksum: 3b43b2b7a8caed3f070d86139ef17fdd (MD5) Made available in DSpace on 2014-05-20T15:14:36Z (GMT). No. of bitstreams: 2 S0103-84782002000200017.pdf: 42603 bytes, checksum: a7bcabcb2df7232a1af99c4afde7749b (MD5) S0103-84782002000200017.pdf.txt: 12155 bytes, checksum: 3b43b2b7a8caed3f070d86139ef17fdd (MD5) Previous issue date: 2002-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Examinando 52 espécimes fecais de bezerros com diarréia de fazendas da região de Jaboticabal, SP, Brasil, uma das amostras de Escherichia coli isoladas, quando analisada pela Reação em Cadeia da Polimerase (PCR), demonstrou a presença dos genes que codificam para a enterotoxina termolábil do tipo LT-II. Este é o primeiro relato de amostra de E. coli enterotoxigênica, isolada de bezerros com diarréia no Brasil, contendo os genes para codificação da enterotoxina LT-II. Encontram-se apenas citações de isolamento no Brasil de amostras de E. coli LT-II+ de alimentos de origem animal. Examining 52 samples of stools from calves with diarrhea from the region of Jaboticabal, SP, Brazil , one of the strains of Escherichia coli isolated, as examined by the Polymerase Chain Reaction (PCR), harbored the genes encoding for the LT-II thermolabile enterotoxin. This is the first report on the isolation of enterotoxigenic E. coli from calves with diarrhea in Brazil harboring the genes for LT-II enterotoxin. In our country, strains of LT-II + E. coli have been reported to occur only in foods of animal origin. Universidade de Campinas Instituto de Biologia Curso de Biologia Molecular e Genética de Microorganismos Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Microbiologia e Imunologia USP Instituto de Ciências Biomédicas Departamento de Microbiologia Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Microbiologia e Imunologia

The 157-kb conjugative plasmid pEO5 encoding alpha-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire alpha-hlyCABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited alpha-hly determinants. The alpha-hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli alpha-hly plasmid pHly152, in particular, the structural alpha-hlyCABD genes (99.2% identity) and the regulatory hlyR regions (98.8% identity). pEO5 and alpha-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural alpha-hlyCABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS2 element upstream of the hlyC gene in pHly152. The presence of transposon-like structures at both ends of the alpha-hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.

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. 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.

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.

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.

One hundred and ninety strains of Escherichia coli that were isolated from pigs with diarrhea in the state of Sao Paulo, Brazil, and that were negative for enterotoxins and cytotoxins were investigated. Strains which adhered to HeLa cells were examined for fluorescence actin staining (FAS), the ability to induce attaching and effacing (A/E) lesions on HEp-2 cells detectable by transmission electron microscopy and the presence of eae gene sequences detected by PCR. Intimin production was detected by western blot and serogrouping was performed. Forty-seven isolates adhered to HeLa cells in several patterns, but none adhered in a localized adherence pattern. However, seven of the 47 adherent strains were positive for the FAS reaction, although the reactions were usually weak or atypical. One FAS-negative and three FAS-positive strains, which were examined for their ability to induce A/E lesions, were all positive. Subsequently, testing of these strains for the eae gene showed that they all lacked this gene. These findings, along with earlier reports of eae-negative A/E E. coli, suggest that higher quantities of E. coli in this category might be detected if more reliance were placed on phenotypic tests rather than on gene detection tests alone.

One strain (S32) of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI) of 25.15 ± 1.26 (mean ± 1 standard error of the mean). Treatment of the bacterial cells with trypsin (0.25mg/ml) decreased in 70%-80% the AI and metaperiodate (10mg/ml) abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100ºC/5 min) before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1: 640, at least. The piglet ileal loop assay, carried out with strains S32 and Jab-1 (negative control) demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microcopy (TEM) demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhuteniun red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies. Uma amostra (S32) de Clostridium perfringens tipo A foi isolada de um caso de enterite catarral em leitões. Esta amostra foi capaz de aderir a células HeLa mostrando um índice de adesão (AI) de 25,15 ± 1,26 (media ± 1 erro padrão da media). Tratamento das células bacterianas com tripsina (0,25mg/ml) diminuiu 70%-80% e metaperiodato (10mg/ml) aboliu significantemente a adesão, sugerindo que a estrutura responsável por esta adesão era provavelmente uma glicoproteína. O tratamento pelo calor das suspensões bacterianas (100ºC/5min) diminuiu o AI ao nível dos controles negativos. Soro de coelho anti-S32 inibiu a aderência a células HeLa até a diluição de 1:640, pelo menos. O teste da alça ligada de leitão recem nascido demonstrou que a amostra S32 era capaz de aderir às células epiteliais intestinais, conforme demonstrado pela coloração de Gram de secções histológicas do intestino dos animais inoculados. O estudo em Microscópio Eletrônico de Transmissão demonstrou que a amostra S32 de Cl. perfringens mostrava um material de natureza fibrilar frouxa, ao contrário da amostra Jab-1 (controle negativo) que demonstrava uma aparência "nua ou lisa". A estabilização das células bacterianas com antissoro homólogo (S32), seguida de coloração com vermelho de rutenio, revelou de maneira mais nítida que longos materiais fibrilares, de aparência frouxa, se estendendo para longe da célula bacteriana, ligando por vezes estas células entre si. A possibilidade desta estrutura ser uma adesina para esta amostra de Cl. perfringens tipo A, talvez desempenhando um papel na patogenia da enterite catarral de leitões, depende de mais estudos.

To the Editor: We report the first isolation of a multiple antimicrobial drug–resistant strain of Shiga toxin–producing Escherichia coli (STEC) O118:H16 from cattle in Latin America. The strain was isolated during a study of fecal STEC in 205 healthy and 139 diarrheic cattle on 12 beef farms in the state of Sao Paulo, Brazil, in February 2000; one case of STEC was found in a 1-month-old calf with diarrhea. This bovine STEC O118:H16 strain showed resistance to eight antimicrobial substances; the following resistance (R)-genes were detected: ampicillin (blaTEM1-like), kanamycin and neomycin (aphA1), streptomycin (strA/B), sulphametoxazol (sul2), tetracyclin (tet[A]), trimethoprim (no dfrA1, A5, A7, A12, A14, or A17), and trimethoprim/sulphamethoxazol. The STEC O118:H16 strain from Brazil was found to be similar for virulence genes (Shiga toxin 1 [stx1], intimin beta 1 [eae β1], and EHEC-hemolysin [E-hlyA]) and for antimicrobial drug resistance to STEC O118:H16 strains, which were isolated in different countries of Europe (1). Beginning in 1986, STEC O118:H16 was identified as an emerging pathogen for calves and humans in Belgium and Germany (2–4). Cattle and human STEC O118:H16 isolates were similar in virulence attributes and antimicrobial drug resistance and belonged to a distinct genetic clone (1). Transmission of these pathogens from cattle to humans on farms was observed (5). Beginning in 1996, STEC O118:H16 has become important as an emerging pathogen in humans and has been associated with bloody diarrhea and hemolytic uremic syndrome (2). Analysis of the antimicrobial resistance profiles showed that >96% of the European STEC O118:H16 strains showed resistance to one or more antimicrobial drugs in contrast to the 10% to 15% drug-resistant strains that were detected among STEC belonging to other serotypes (1,6,7). STEC O118:H16 showing multiresistance in up to eight different antimicrobial drugs predominated among younger isolates, indicating that drug resistance genes have accumulated over time in STEC O118:H16 strains. The frequency of antimicrobial drug resistance in STEC and Stx-negative E. coli in humans and animals was compared in a study by Schroeder et al. (8). Among human clinical E. coli isolates, antimicrobial resistance was less frequently observed in STEC than in Stx-negative strains, whereas in cattle, antibiotic-resistant strains were found at similar frequencies in both groups of E. coli. The relatively higher frequency of antimicrobial-resistant STEC in cattle was explained by the use of antimicrobial drugs in cattle production, whereas human infections with STEC are generally not treated with antibiotics (8). Cattle could thus be an important source of new emerging antibiotic-resistant STEC strains such as O118:H16. The genetic basis of antimicrobial resistance in STEC O118:H16 is broad, including R-plasmids, integrons, transposons, and chromosomally inherited drug-resistance genes. Fluoroquinolone resistance has also been acquired by some STEC O118:H16 strains (1). The heterogenicity of antimicrobial drug–resistance patterns, the increase of multidrug-resistant strains over time of isolation, and the evidence for multiple aquisition and genetic location of R-determinants indicate that strains belonging to the STEC O118:H16 clone have a propensity to acquire and accumulate R-genes. The finding that multidrug-resistant STEC O118:H16 is isolated from cattle in South America indicates the global spread of this new emerging EHEC type.

Seventeen strains of E. coli, isolated from chickens with colisepticaemia, were studied with respect to their pathogenic characteristics including: serum resistance, toxin production, pathogenicity for one-day-old chicks, colicin production, adherence to and invasiveness of HeLa cells, plasmid DNA profile and SDS-PAGE electrophoresis of membrane proteins, as well as electron microscope studies and hemagglutination tests for fimbriae. We concluded that the adherence to and the invasiveness of HeLa cells were not related to the pathogenicity of these strains for chickens. Plasmid profiles were not related to the bactericidal activity of the serum. Toxin production was correlated to the highest levels of pathogenicity. Some of the strains had mannose-resistant fimbriae. SDS-PAGE of membrane proteins of all the strains which were either not pathogenic or which had a very high LD50 lacked two major protein subunits of 40.7 kDa and 28.8 kDa found only in pathogenic strains.

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 test), results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the IIM one. No unspecific reaction was obtained in the IH with 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 specific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data.

Erratum to ` Pathogenic characteristics of Escherichia coli strain isolated from newborn piglets with diarrhoea in Brazil'' [Vet. Microbiol. 76 (2000) 51±59] Maria de FaÂtima Martins, Nilce M. Martinez-Rossi, Alessandra Ferreira, Marcelo Brocchi, Tomamasa Yano, Antonio Fernando P. Castro, Wanderley D. Silveira Depto. de NutrcEaAo e ProducEaAo Animal, Faculdade de Medicina VeterinaÂria e Zootecnia, USP, 13630-970 Pirassununga, SP, Brazil Depto. de GeneÂtica, Faculdade de Medicina de RebeiraAo Preto, USP, 14049-900 RebeiraAo Preto, SaAo Paulo, SP, Brazil Depto. de Parasitologia, Microbiologia e Immunologia, Faculdade de Medicina de RebeiraAo Preto, USP, 14049-900 RebeiraAo Preto, SaAo Paulo, SP, Brazil Depto. de Microbiologia e Immunologia, IB, UNICAMP, CP.6109, Campinas SP, Brazil Instituto de CieÂncias BiomeÂdicas, Universidade de SaAo Paulo, SaAo Paulo, SP, Brazil

The K99 and F41 fimbrial antigens were extracted from Escherichia coli B41 strain by heating, and semi-purified by precipitation with ammonium sulfate and treatment with sodium desoxicolate (DOC). The semi-purified antigens K99 and F4I were used to prepare an oil emulsified vaccine against bovine colibacillosis. The vaccine was made up containing in each dose 1,500, 750 or 380 HU (Haemagglutinating Units). The test of the vaccine efficiency was measured by gel immunodiffusion and ELISA test. Challenge of calves from vaccinated and control cows was carried out with the virulent Escherichia coli B4I. Our findings suggested that 750 HU vaccine, induces better production of anti-K99-F4l antibodies in vaccinated cows, and the transference of these antibodies to calves through the colostrum, gave full protection to challenge. Furthermore no adverse effects were observed in any of the vaccinated cows. Os antígenos K99-F41 foram extraídos da amostra de Escherichia coli B41 por aquecimento e semi-purificados pela precipitação com sulfato de amônio e tratamento com desoxicolato de sódio (DOC). Os antígenos semi-purificados foram utilizados na produção de uma vacina oleosa contra a colibacilose bovina. Foram preparadas vacinas contendo em cada dose 1.500 HU (Unidades Hemaglutinantes), 750 HU e 380 HU. A eficiência da vacina foi avaliada através do ensaio de imunodifusão dupla, ensaio imunoenzimático (ELISA) e por um desafio, em que a amostra de Escherichia coli virulenta foi inoculada nos bezerros nascidos de vacas vacinadas e não vacinadas. Observamos que a vacina contendo 750 HU foi a que melhor induziu a produção de anticorpos nas vacas vacinadas, e que estes mostraram-se protetores, uma vez que os bezerros nascidos de vacas vacinadas e que mamaram o colostro, nada sofreram no desafio. Não se verificou nenhum efeito colateral nas vacas vacinadas.

Foi realizada uma pesquisa na região de Campinas, SP, Brasil, sobre a presença de Escherichia coli enterotoxigênica (ETEC), rotavírus e Clostridium perfringens enterotoxigênico em fezes diarréicas de crianças com até 2 anos de idade. Dos 132 espécimens fecais examinados quanto à 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 modifi cado. Entre as 183 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, Illb, IVa, de acordo com a classificação por nós adotada. Apenas 113 amostras de fezes foram examinadas para a presença de C. perfringens enterotoxigênico. Para a detecção da enterotoxina nos sobrenadantes das culturas foram utilizadas as técnicas de hemaglutinação passiva reversa e inoculação intravenosa em camundongos, sendo encontradas 12 (10,61%) amostras entero-toxigênicas. Diante destes resultados é chamada a atenção sobre o valor apenas relativo de uma coprocultura convencional para fins de diagnóstico, ressaltando-se a importância da criação de métodos simplificados que favoreçam a detecção e identificação dos grupos de agentes enteropatogênicos estudados na presente pesquisa.

Three enterotoxigenic Escherichia coli (ETEC) strains (coded 62, 104, and 567/7) isolated from piglets with neonatal diarrhea produced only a thermostable enterotoxin. Although these strains showed mannose-resistant microhemagglutination (MRMH), the responsible factor was serologically different from the known hemagglutinating colonization factors from porcine strains (K88, K99, and F41). Bacterial cells from these strains adhered to HeLa cells and pig brush borders. Electron microscope studies revealed the presence of fimbria-like structures on bacterial cells grown at 37 C but not on cells grown at 18 C. The antiserum prepared from partially purified fimbrial antigen (provisionally called F42) inhibited chicken erythrocyte MRMH caused by these strains as well as adherence of strain 567/7 to HeLa cells and to pig brush borders. These data taken together suggest the existence of a new hemagglutinating adhesin that is different from those so far described for porcine ETEC.

Neste estudo, 26 amostras de ostras (Crassostrea gigas) comercializadas na cidade de São Paulo e em alguns pontos do litoral de São Paulo, e 36 amostras de mexilhões (Perna perna) colhidas mensalmente em 3 pontos do litoral de Ubatuba - SP, foram submetidas à pesquisa de vibrios potencialmente patogênicos. As amostras desses moluscos eram submetidas a enriquecimento em água peptonada alcalina sem cloreto de sódio e com 1 por cento de cloreto de sódio, e GSTB. O isolamento foi realizado em ágar TCBS. Colônias sacarose positivas e negativas, sugestivas de espécies de Vibrio foram identificadas presuntivamente em meio de ágar ferro de Kligler, sendo confirmadas através de provas bioqufmicas complementares. Uma parte das amostras de vibrios potencialmente patogênicos isoladas foi submetida ao teste de Dean e teste de alça ligada em íleo de coelhos. Os vibrios potencialmente patogênicos encontrados em amostras de ostras foram V. alginolyticus (81 por cento ), V.parahaemolyticus (77 por cento ), V. cholerae não 0:1 (31 por cento ), V. fluvialis (27 por cento ), V. furnissii (19 por cento ), V. mimicus (12 por cento ) e V. vulnificus (12 por cento ) e em amostras de mexilhões foram V. alginolyticus(97 por cento ), V. parahaemolyticus(75 por cento ), V. fluvialis (47 por cento ), V. vulnificus (11 por cento ), V. cholerae não 0:1 (6 por cento ), V. furnissii (6 por cento ) e V. mimicus (6 por cento ). Observou-se acúmulo de fluido em alça ligada de íleo de coelho entre 0,25 e 0,49 ml/cm em 6,9 por cento das amostras, entre 0,5 e 0,99 ml/cm em 15,6 por cento e maior ou igual a 1 ml/cm em 15,1 por cento , e/ou intestino de camundongos lactentes (Teste de Dean) em 26,6 por cento das amostras testadas, confirmando o elevado potencial desses microrganismos em causar gastrenterite. Verificou-se ausência de variação sazonal e também, de correlação entre os vibrios potencialmente patogênicos isolados e os indicadores de contaminação fecal, confirmando que a presença desses microrganismos ocorre de forma autóctone e que, as condições climáticas foram favoráveis à sobrevivência dessas espécies em todas as épocas do ano. Considerando-se os resultados obtidos no presente estudo e o fato de que ostras e mexilhões são habitualmente ingeridos crus ou insuficientemente cozidos, pode-se concluir que sua ingestão constitui-se em um determinado grau de risco para a saúde do consumidor. In this work, 26 oysters samples (Crassostrea gigas), found in the market of São Paulo city and some coastal areas of São Paulo State, and 36 mussels samples (Perna perna), that were collected monthly in 3 coastal areas of Ubatuba city - SP., were analyzed for the potential patogenic vibrios occurrence. Samples were enriched in alcalin peptone water with (1 per cent ) and without sodium cloride and GSTB. Isolation was performed on TCBS agar. suspect sacharosis positive and negative colonies, resembling vibrio species, were presumptively identified on Kligler iron agar, and confirmed by complementary biochemical tests. Some of this potential patogenic vibrios were submitted to suckling mouse assay and rabbit ileal loop assay. Potential patogenic vibrios isolated from oyster samples were: V. alginolyticus (81 per cent ), V. parahaemolyticus (77 per cent ), V. cholerae non 0:1 (31 per cent ), V. fluvialis (27 per cent ) I V. furnissii (19 per cent ), V. mimicus (12 per cent ) and V. vulnificus (12 per cent ) and from mussels samples were: V. a.1ginolyticus (97 per cent ), V. parabaemolyticus (75 per cent ), V. fluvialis (47 per cent ), V. vulnificus (11 per cent ), V. cholerae non 0:1 (6 per cent ), V. furnissii (6 per cent ) and V. mimicus (6 per cent ). It was found 6,9 per cent of samples between 0,25 and 0,49 ml/cm of fluid accumulation in ileal loop assay, 15,6 per cent between 0,5 and 0,99 ml/cm and 15,1 per cent was equal or higher than 1 ml/cm. Among the samples assayed for suckling mouse 26,6 per cent were positive. These results confirm the high potential of these microrganisms to induce gastroenteritis. Seasonal variation as well as correlation between the potential patogenic vibrios isolated and the fecal contamination indicators were not found, confirming that the presence of such microrganisms occurs autochthonously and that the climate conditions were favourable to these species survival during the whole year. with the results of this work and considering that oyster and mussels are usually ingested raw or insufficiently cooked, the conclusion is that the ingestion of such mollusks presents a certain degree of risk for the consumer\'s health.

Neste estudo, mostramos a possibilidade de identificação dos AgH de ETEC pela caracterização molecular do gene fliC pela análise de seu polimorfismo de restrição. Um único alelo de fliC de ETEC foi encontrado para cada antígeno flagelar, utilizando-se a endonuclease RsaI, com exceção do H21. Além de cepas móveis, isolados imóveis também puderam ser caracterizados por essa técnica molecular. A alta tipabilidade da PCR-RFLP foi comprovada por meio de sua aplicação não só a amostras de ETEC com AgH previamente desconhecidos, mas também a outras linhagens de E. coli. Observamos que após identificação do antígeno flagelar das amostras de ETEC pela PCR-RFLP, a determinação do antígeno somático pôde ser direcionada, diminuindo assim, o número de anti-soros utilizados para a pesquisa do AgO. A técnica de PCR-RFLP, em nosso estudo, mostrou uma sensibilidade de 83% e 100% de especificidade. Esta técnica foi mais rápida na identificação do AgH de E. coli (2 dias) em comparação à sorologia clássica (7 ou mais dias, dependendo da motilidade da cepa). Acreditamos que o método de soroaglutinação para determinação do AgH será substituído rapidamente pela PCR-RFLP. Contudo, a soro aglutinação não poderá ser totalmente dispensada em curto prazo. No futuro, com o perfil molecular obtido dos alelos de cepas procedentes de estudos epidemiológicos, novos padrões serão definidos para as cepas diarreiogênicas de E. coli, permitindo o abandono da sorologia para AgH. In this study, we showed that the H antigens of ETEC can be characterised by restriction analysis of the polymorphism of the fliC gene. Only one allele of the fliC gene of ETEC was found for each flagellar antigen when restriction endonuclease RsaI was used, with the exception of H21 . Additionally, non-motile strains could also be characterised using this molecular technique. The high typeability of this technique was demonstrated by the fact that it can not only be applied to ETEC samples with previously unknown H antigens but also to all other lineages belonging to the E. coli species. Moreover, the determination of the somatic antigen was guided by the identification of the flagellar antigen of ETEC strains by PCR-RFLP, thus reducing the number of anti-AgO sera used. In this study, the PCR-RFLP technique showed a sensitivity of 83% and a specificity of 100%. This technique proved to be quicker for the identification of the E. coli AgH, taking 2 days to complete, in comparison to the 7 or more days necessary when using classic serotyping. We believe that the determination of the AgH by seroagglutination will soon be substituted by the PCR-RFLP technique. However, serotyping will still have to be used in the short run, for further studies involving PCR-RFLP must be carried out. In the future, with the determination of the molecular profiles of alleles of strains obtained in epidemiological studies, new patterns will have been described for the diarrhoeagenic strains of E. coli, thus permitting the abandonment of AgH serotyping for good.

Pseudomonas aeruginosa é um dos principais agentes de infecção hospitalar que tem adquirido um caráter endêmico decorrente a sua resistência intrínseca e/ou adquirida aos antibacterianos comercialmente disponíveis. O objetivo do estudo foi avaliar, in vitro, opções terapêuticas baseadas na atividade sinérgica. Dez cepas de P. aeruginosa clonalmente não relacionadas, previamente caracterizadas como produtoras de metalo-beta-lactamase (MbL) do tipo SPM-1, VIM-1 e PA GIM-1, foram avaliadas. O efeito sinérgico foi investigado por Checkerboard e Time-Kill. As combinações [Piperacilina/Tazobactam x Aztreonam] e [Tigeciclina x nanofragmentos de bicamada de brometo de dioctadecildimetilamônio (DDA)] mostraram atividade sinérgica para 90 e 100% das cepas, respectivamente. Os resultados respaldam o uso terapêutico combinado de [Piperacilina/Tazobactam x Aztreonam], contra infecções produzidas por cepas de P. aeruginosa multirresistentes produtoras de MbLs, além disso, [DDA/Tigeciclina] pode constituir a base para consolidar uma nova forma farmacêutica de uso clínico. Pseudomonas aeruginosa is a leading cause of nosocomial infections, which it has acquired an endemic status due to their intrinsic or acquired resistance to antibacterial agents commercially available. The aim of the study was to evaluate in vitro therapeutic options based on the synergistic activity. Ten clonally unrelated strains of P. aeruginosa, previously characterized as metallo-beta-lactamase (MbL) producers (i.e., SPM-1, VIM-1 and PA GIM-1) were evaluated. The synergistic effect was investigated by checkerboard and Time-Kill assays. The combinations [Piperacillin/Tazobactam x Aztreonam] and [Tigecycline x bilayer fragments of dioctadecyldimethylammonium bromide (DDA)] showed synergistic activity for 90 and 100% of the strains, respectively. The results support the use of combined therapy by using [Piperacillin/Tazobactam x Aztreonam] against infections produced by strains of P. aeruginosa producing MbLs, moreover, [DDA / Tigecycline] may be the basis for build a new pharmaceutical form for clinical use.

O Objetivo deste trabalho foi desenvolver uma estratégia vacinal de utilizando linhagens de salmonellas capazes de expressar uma forma atoxica da principal toxina associada a linhagens de EHEC que causa a síndrome hemolítica urémica. Estas linhagens de salmonellas diferem na expressão de flagelina, a principal unidade estrutural do flagelo. As linhagens foram capazes de expressar uma forma atóxica da proteína \"Shiga-like\" no interior da bactéria e no espaço extracelular, apresentaram alta estabilidade plasmidial in vitro e in vivo e foram capazes de aumentar o grau de colonização intestinal. Além disso, avaliamos o potencial imunogênico de estas linhagens e encontramos que a imunização com três doses de salmonelas foram capazes de gerar anticorpos sistêmicos (IgG) e locais (IgA) com propriedades neutralizantes in vitro e proteção parcial em condições in vivo frente a ensaios de desafios com a toxina nativa. Não fomos capazes de encontrar diferencias significativas em lãs propriedades imunogênicas entre as linhagens que diferem na expressão de flagelina. Nossos resultados indicam que formas atóxicas da proteína Stx2 expressadas em vetores biológicos podem ser uma alternativa de estratégia vacinal para controlar a SHU. The goal of this study was to develop a vaccine strategy of using Salmonella strains capable of expressing a nontoxic form of the main toxin associated with EHEC strains that cause hemolytic uremic syndrome. These strains of Salmonella differ in flagellin expression, the main structural unit of the flagellum. The strains were capable of expressing a nontoxic form of the protein \"Shiga-like\" within the bacteria and the extracellular space, showed high plasmid stability in vitro and in vivo and were able to increase the degree of intestinal colonization. Furthermore, we evaluated the immunogenic potential of these strains and found that immunization with three doses of salmonella were able to generate (IgA) humoral and (IgG) systemic antibodies with neutralizing properties in vitro and partial protection in vivo conditions facing trials challenges with the native toxin. We were unable to find significant differences in the immunogenic properties between these strains that differ in flagellin expression. Our results indicate that nontoxic forms of Stx2 expressed in biological vectors can be an alternative vaccine strategy to control the SHU.

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

As ITU são as mais freqüentes infecções ocasionadas por ExPEC. Entre os fatores de virulência (FV) encontram-se nestas cepas adesinas, invasinas, toxinas, sideróforos, e evasinas, localizados em plasmídios ou ilhas de patogenecidade. O objetivo deste estudo foi caracterizar 45 cepas de E. coli isoladas de 33 cães e 7 gatos com ITU, quanto aos sorotipos, FV e grupos filogenéticos. Dos sorogrupos relacionados às ITU foram encontrados O6 (20%), O2 (16%), O25 (4%), O4 e O11 (4% cada um). Entre os genes pesquisados, foram encontrados fimH (100%), pap (47%), sfa (33%) e iha (4%); ibeA (29%); cnf1 (31%), hlyA (27%); fyuA (80%), iucD (22%); traT (51%); cvaC (20%) e malX (67%). Os isolados felinos foram agrupados em B2 (89%) e D (11%), enquanto os caninos em A (5,5%), B1 (19,5%), B2 (55,5%) e D (19,5%). Estes resultados sugerem que as ExPEC isoladas de cães e gatos apresentam potencial patogênico para ocasionar doenças mais graves que as ITU, assim como ocorre em humanos. Além disso, a similitude com as amostras humanas reforça a hipótese acerca de seu potencial zoonótico. The ability of ExPEC to cause extraintestinal infections in humans, dogs, and cats is associated with the expression of a variety of virulence factors (VF). The aim of this study was to evaluate the frequency of VF related to ExPEC, serotypes, and phylogenetic groups in 45 strains isolated from 33 dogs and 7 cats with UTI. These strains presented serogroups related with extraintestinal infections, e.g. O6 (20%), O2 (16%), O25 (4%), O4 e O11 (each one) and the following genes: fimH (100%), pap (47%), sfa (33%) e iha (4%); ibeA (29%); cnf1 (31%), hlyA (27%); fyuA (80%), iucD (22%); traT (51%); cvaC (20%) e malX (67%), cvaC (20%), and malX (67%). All feline strains were concentrated in B2 (89%) and D (11%) phylogenetic groups, whereas the canine ones were distributed in the four groups, A (5,5%), B1 (19,5%), B2 (55,5%) and D (19,5%). These findings suggesting that ExPEC isolated from dog and cat contain virulence markers to cause diseases, more severe than UTI, likewise in humans. Besides, these the close similarity between human and animal ExPEC supports the hypotesis of zoonotic potencial of them.

Ovinos são considerados reservatórios de Escherichia coli produtora da toxina Shiga (STEC). Este estudo pesquisou amostras de 100 carneiros no Estado de São Paulo. PCR foi empregada para detecção de stx1, stx2, eae, ehx, e intiminas. RFLP-PCR foi realizado para identificação das variantes de stx1/stx2 e fliC. Cinco isolados eae+stx- de sorotipos O128:H2, O145:H2, O153:H7 e O178:H7 apresentaram intiminas b, g e e. Cinqüenta e seis STEC foram obtidos e os genótipos foram stx1cstx2d-O118 (56,1%), stx1c (33,3%), stx2d-O118 (7,6%), e stx1cstx2dOX3a (3%). Os sorotipos mais freqüentes foram ONT:H8, ONT:H-, O75:H-, O174:H8 e O5:H-. Gene fliC codificando para H2, H8, H14, H16, H19, H21 ou H28 foi identificado em 25/33 isolados. ehx foi detectado em três/cinco EPEC Atípicas e em 38 (57,6%) STEC. Sensibilidade aos antimicrobianos foi observada em 77,2% dos isolados STEC. A análise da similaridade genética por PFGE revelou 24 perfis entre 40 isolados STEC e EPEC atípicas. O estudo demonstrou que ovinos saudáveis podem se comportar como reservatórios de STEC e EPEC atípica. Lambs are reported as carriers of Shiga toxin-producing Escherichia coli (STEC) worldwide. This study surveyed 100 sheep in São Paulo, Brazil. PCR was used for detection of stx1, stx2, eae, ehx, and intimins. RFLP-PCR investigated the stx1/stx2 variants and flagellar antigen (fliC). Five isolates were eae+/stx-, belonging to serotypes O128:H2, O145:H2, O153:H7 and O178:H7, harboring b, g and e intimins. Fifty-six STEC isolates were recovered, and stx genotypes were stx1cstx2d-O118 (56.1%), stx1c (33.3%), stx2d-O118 (7.6%), and stx1cstx2dOX3a (3%). A diversity of STEC serotypes was observed, but most frequent were ONT:H8, ONT:H-, O75:H-, O174:H8 and O5:H-. fliC gene coding for H2, H8, H14, H16, H19, H21 or H28 was identified in 25/33 isolates. ehx gene was detected in three Atypical EPEC and in 38 (57,6%) STEC. Fifty-one (77.2%) STEC were susceptible to antimicrobials tested. Pulsed field gel electrophoresis (PFGE) revealed 24/40 profiles. The study showed that healthy ovine can be carrier of STEC and atypical EPEC in Brazil.

Escherichia coli enterohemorrágicas (EHEC) que possuem a Ilha de Patogenicidade LEE são importantes patógenos humanos. A habilidade de adesão, invasão e perfil genético têm sido estudados, já que sorotipos que não possuem LEE tem sido isolados de pacientes com doença severa e intracelularmente. Das nove amostras relacionadas com doença, quatro (44.5%) apresentaram stx2+. Todas foram positivas para o gene lpfA e iha, e negativas para toxB. Três (33,3%) apresentaram saa e cinco hly. A maioria apresentou padrão de adesão difusa e invasão negativa em células HEp-2. Em CaCO-2 apresentaram aderência com variados graus de intensidade, e a maioria das amostras testadas apresentou invasão maior que 3,3%. Frente ao inibidor de polimerização de actina citocalasina D, houve significativa redução nos níveis de invasão, sugerindo que estas amostras utilizam um mecanismo da célula hospedeira para internalização, e que provavelmente, fatores de virulência como adesinas, favorecem a adesão das mesmas, compensando a ausência do LEE, e facilitando a instalação da infecção. Enterohaemorrhagic Escherichia coli (EHEC) that possess the pathogenic island LEE are important human pathogens. The adhesion ability, invasion and genetic profile have been studied, since serotypes that do not possess LEE have been isolated from humans with severe disease and found intracellular. Nine strains related with SHU, four (44,5%) were stx2+. All strains were positive for IpfA and iha genes and negative for toxB. Three (33,3%) showed saa and five hly. The most strains showed a diffusely adhesion pattern and negative invasion in HEp-2 cells. It presented various degrees of adhesion, and the most tested strains showed invasion high than 3,3% in CaCO-2. That was a significant reduction of invasion in the presence of actin polymerization inhibitor Citochalasin D, suggesting that these strains use a host cell mechanism to invade, and probably virulence factors, like adhesins, favors this adhesion and compensate LEE absence, promoting the installation of infection.

As EPEC típicas apresentam padrão de adesão localizada (LA) em células epiteliais, formando de microcolônias compactas. As EPEC atípicas aderem no padrão de adesão localizada-like (LAL), no qual não se observa microcolônias e que ocorre tardiamente em relação à LA. O objetivo deste estudo foi caracterizar a interação de EPEC atípica que apresenta o padrão LAL com a célula epitelial. A cinética da formação da lesão A/E, característica da patogênese de EPEC, demonstrou um atraso na adesão e na formação da lesão em EPEC atípica, que correspondeu à expressão tardia de Intimina, Tir e EspA. A complementação de EPEC atípica com o regulador perABC antecipou a expressão destes fatores e a formação da lesão A/E. Não foi observada relação entre outras adesinas descritas em DEC com o padrão LAL, bem como a mobilização dos receptores eucarióticos nucleolina e b1-Integrina para o foco de adesão. Observou-se a possível interação da Intimina com um peptídeo da membrana da célula epitelial de 300 kDa, podendo tratar-se de um provável receptor para essa adesina. Typical EPEC exhibit a localized adherence (LA) pattern in epithelial cells, where tight bacterial clusters are produced. Atypical EPEC produce a localized-like (LAL) adhesion pattern, in which the compact clusters are not formed and that occurs in a later stage of the infection. The aim of this study was to characterize the interaction of LAL-adherent atypical EPEC with the epithelial cell. The kinetics of the A/E lesion, the main feature of EPEC pathology, showed a clear delay in its formation by the atypical strains, probably a consequence of the delay observed in the expression of Intimin, Tir and EspA. The complementation of atypical EPEC with the perABC regulator revealed to be effective in not only advancing the expression of these factors, as well the formation of A/E lesion. No correlation was found between the presence of different adhesins already described in DEC and the LAL pattern. The analysis of the possible interaction of Intimin with epithelial cell receptors showed the presence of a 300 kDa peptide that it seems to interact with this adhesin.

O sistema de captação de oligopeptideos (Opp), responsável pela captação de peptídeos com 3 ou mais resíduos de aminoácidos, representa um mecanismo importante de obtenção de nutrientes em bactérias. O operon Opp é constituído por 5 genes, sendo OppD e F responsáveis pela codificação dos componentes geradores, OppB e C codificantes para as proteínas que delimitam o poro da membrana e OppA que codifica o componente ligante. Neste trabalho identificamos uma alta identidade entre as proteínas OppA expressas por diferentes cepas de E. coli, 4 espécies do gênero Shigella ( 99 %) e diferentes sorovares de Salmonella enterica (85%) mas registramos a ocorrência de vários sítios polimórficos inter-específicos. A presença do gene OppA foi confirmada em 58 cepas diarreiogênicas de E. coli, Shigella e Salmonella. A partir da proteína OppA recombinante foi obtido soro policlonal específico que revelou a presença da proteína em todas as linhagens estudadas. Desta forma, concluímos que a proteína OppA está presente e conversada em espécies e linhagens dos três gêneros de Enterobacteriaceae estudados. The oligopeptide uptake system (Opp), involved with the uptake of peptides formed by 3 or more amino acid residues, represent important nutrient uptake mechanism. The Opp operon is usually represented by 5 structural genes, including OppD and OppF encoding proteins involved generation of energy , OppB and OppC, encoding membrane proteins delimiting a pore and OppA encoding the protein responsible both for specificity and affinity of the transport system toward different peptide substrates. In this study, we demonstrated that the OppA proteins expressed by different E. coli strains,4 Shigella species (99%) and different serovars of Salmonella enterica (85%) were quite conserved but the occurrence of inter-species polymorphism was demonstrated. The OppA gene was detected in 58 diarrheogenic E. coli, Shigella and Salmonella strains. Using a recombinant OppA protein produced in E. coli, specific polyclonal sera were generated and successfully applied in the immunological detection of the proteins expressed by the tested strains. Thus, we conclude that the OppA protein is present and conserved among species and strains of the three test Enterobacteriaceae genera.