Adhesion properties, fimbrial expression and PCR detection of adhesin-related genes of avian Escherichia coli 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. (c) 2005 Elsevier B.V. All rights reserved.1064173227528

Clonal distribution of an atypical MRP+, EF*, and suilysin(+) phenotype of virulent Streptococcus suis serotype 2 strains in Brazil

Streptococcus suis is considered one of the most important bacterial swine pathogens worldwide. The distribution of the 35 described serotypes in diseased animals may vary in different regions. Data regarding S. suis isolation from pigs in South America is not available. In the present study, 51 isolates of S. suis recovered in pure culture or as the predominant species from diseased animals in Brazil, were analyzed. These isolates were classified as serotypes 2 (58.8%), 3 (21.5%), 7 (13.7%), 1 (3.9%), and 14 (2%). Serotype 2 isolates were further studied for their production of virulence-related proteins muramidase-released protein (MRP), extracellular factor (EF), and suilysin. In addition, the genetic diversity was studied by randomly amplified polymorphic DNA. All but 1 of the serotype 2 isolates showed a clonal distribution of an atypical phenotype (MRP+, EF*, suilysin(+)), different from the known European (MRP+, EF+, suilysin(+)), and North American (MRPv, EF-, suilysin(-)), phenotypes.671525

Determination of the clonal structure of avian Escherichia coli strains by isoenzyme and ribotyping analysis

Forty-nine avian Escherichia coli strains isolated from different outbreak cases of septicemia (24), swollen head syndrome (14) and omphalitis (11), and 20 strains isolated from poultry with no signs of the mentioned illnesses, for a total of 69 strains, were typed by isoenzyme profile and ribotyping analysis by restriction fragment length polymorphism (RFLP). Isoenzyme analysis discriminated better among strains (0-0.07 degree of genetic dissimilarity) than ribotyping analysis (0- 0.02 degree of genetic dissimilarity). The enzyme profiles of the E. coli isolates allowed the identification of 33 clones that were organized into six main clusters (A-F). Cluster A comprised 87% of the pathogenic strains and had no commensal strains, while commensal strains were assigned to clusters B-F. The ribotyping analysis resulted in a more heterogenous distribution of strains but most of those that cause the same type of infection were kept close together. Taken as a whole, these results demonstrate that pathogenic clones are more similar to one another when compared with commensal strains and suggest a correlation between the genetic background and the pathogenic characteristics of avian pathogenic E. coli strains.502636

Frequency of Shiga toxin-producing Escherichia coli (STEC) isolates among diarrheic and non-diarrheic calves in Brazil

The occurrence of Shiga toxin (Stx) gene sequences was examined in 344 fecal samples from diarrheic (n = 139) and non-diarrheic (n = 205) calves from 12 beef farms in Sao Paulo State, Brazil to study the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains. Forty-four (12.7%) animals were found to be positive for stx. The frequency of carriage of stx was higher in diarrheic calves (28/139, 20%) than in non-diarrheic animals (16/205, 7.8%) (P < 0.001). Among the 24 STEC strains recovered from the animals, 12 isolates carried stx1, four stx2, and 8 carried both stx1 and stx2 genes. The eae and the enterohaemolysin (Ehly) gene sequences occurred at high frequencies in these STEC strains (41.6 and 50.0%, respectively). A total of 16 serotypes were identified. The serotypes O111:NM (four isolates), O111:H8 (two) and O118:H16 (one), currently described as enterohaemorrhagic E. coli (EHEC), were isolated from cattle in Brazil for the first time. These findings reinforce the importance of cattle as a reservoir of EHEC strains in Brazil. (C) 2003 Elsevier B.V. All rights reserved.974167110310

Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (lily) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1 h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin. (C) 2002 Elsevier Science B.V. All rights reserved.891293

eae-negative attaching and effacing Escherichia coli from piglets with diarrhea

One hundred and ninety strains of Escherichia toll 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 ene 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. (C) 2001 Editions scientifiques et medicales Elsevier SAS.1521758

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E-coli (EPEC)

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of Sao Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for epsilon, one for alpha, one for K, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations. (C) 2004 Elsevier B.V. All rights reserved.101426927