IDENTIFICAÇÃO DOS GENES QUE CODIFICAM PARA A ENTEROTOXINA TERMOLÁBIL LT-II em AMOSTRAS DE ESCHERICHIA COLI ISOLADAS DE BEZERROS COM DIARRÉIA NA REGIÃO DE JABOTICABAL, SP, BRASIL

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

O objetivo do trabalho foi determinar a ocorrência de fatores de virulência, tais como, a expressão de fímbrias, produção de hemolisina, colicina e aerobactina em 100 cepas de Escherichia coli isoladas de pacientes ambulatoriais e hospitalizados de um hospital universitário de nível de atendimento terciário, entre os meses de julho e agosto de 2000, que apresentavam sinais clínicos e laboratoriais de infecção do trato urinário (ITU). Foram pesquisados os genes pap, afa e sfa responsáveis pela expressão de fímbrias através da técnica de PCR. A freqüência dos fatores de virulência entre as cepas estudadas foi de 96,0%, 76,0% e 24,0% para hemolisina, aerobactina e colicina respectivamente, e a prevalência dos genes para os sistemas de adesinas fimbriais foi de 32,0%, 19,0% e 11,0% para os genes pap, sfa e afa respectivamente. As cepas isoladas dos pacientes ambulatoriais exibiram um número maior de fatores de virulência quando comparadas com aquelas provenientes de indivíduos hospitalizados.

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

Aims: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil.Methods and Results: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%).Conclusion: STEC isolates were found in bovine mastitic milk in Brazil.Significance and Impact of the Study: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.

FUNCTIONAL ALPHA-TROPOMYOSIN PRODUCED IN ESCHERICHIA-COLI - A DIPEPTIDE EXTENSION CAN SUBSTITUTE THE AMINO-TERMINAL ACETYL GROUP

Unlike the muscle protein, alpha-tropomyosin expressed in Escherichia coli does not bind actin, does not exhibit head-to-tail polymerization, and does not inhibit actomyosin ATPase activity in the absence of troponin. The only chemical difference between recombinant and muscle tropomyosins is that the first methionine is not acetylated in the recombinant protein (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). We expressed three fusion tropomyosins in E. coli with 2, 3, and 17 amino acids fused to its amino terminus. Ah three fusions restored actin binding, head-to-tail polymerization, and the capacity to inhibit the actomyosin ATPase to these unacetylated tropomyosins. Unlike larger fusions, the small fusions of 2 and 3 amino acids do not interfere with regulatory function. Therefore the presence of a fused dipeptide at the amino terminus of unacetylated tropomyosin is sufficient to replace the function of the N-acetyl group present in muscle tropomyosin. A structural interpretation for the function of the acetyl group, based on our results and the coiled coil structure of tropomyosin, is presented.

Use of vaccine and probiotic in the control of swine diarrhea caused by enterotoxigenic Escherichia coli

A total of 42 pregnant sows were divided into eight groups and submitted to the following treatments: group I with seven unvaccinated sows whose piglets did not receive probiotic, was used as control, group II with five vaccinated sows whose piglets did not receive probiotic, groups III, IV and V with five vaccinated sows each whose piglets received probiotic for 5, 15 and 28 days, respectively, and groups VI, VII and VIII with five unvaccinated sows each whose piglets received probiotic for 5, 15 and 28 days, respectively. Each animal in the vaccinated groups received subcutaneously Two doses of 5.0ml of vaccine containing pill K88, K99, 987P and F42 of Escherichia coli. The probiotic contained Lactobacillus acidophilus at the dose of 2.0x10(8) live cells in 20ml of milk and was administered orally. All animals were observed clinically and bacteriologically and the titers of anti-K88, anti-K99, anti-987P and anti-F42 antibodies were determined in serum and colostrum. The results showed that the vaccine associated to the probiotic administered for 28 days was the most effective treatment for the control of diarrhea caused by enterotoxigenic Escherichia coli.

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the sigma(s) subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44-50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iciR, and fadR) achieved only medium cell densities (15-21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth, on a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the sigma(s) regulon to grow to high cell densities, at least not under the conditions tested.

LACK OF VIRULENCE FACTORS IN ESCHERICHIA-COLI STRAINS OF ENTEROPATHOGENIC SEROGROUPS ISOLATED FROM WATER

Thirty-eight Escherichia coli strains belonging to 14 human enteropathogenic serogroups were isolated from 33 of 208 water samples studied. No virulence factor or virulence-related gene sequences were found in any of the 38 strains analyzed. The results point out the importance of detecting specific virulence factors before incriminating water as a source of human diarrhea.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli ( EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non- human primates belong to the serogroups and/ or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non- human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/ serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non- human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non- human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Type 2 heat-labile enterotoxin (LT-II)-producing Escherichia coli isolated from ostriches with diarrhea

The culture supernatant of Escherichia coli, isolated from ostriches with diarrhea in Brazil, caused elongation in Vero cell, rounding in Chinese hamster ovary (CHO) cells and a cytoplasmic vacuolation in ostrich embryo fibroblasts (OEF), but it was not cytotoxic for chicken embryo fibroblasts (CEF). These effects were not neutralized by antiserum to cholera toxin. Polymerase chain reaction assays showed that the ostrich E.coli contained the gene encoding (eltII-A), but not those for type 1 heat-labile enterotoxin (eltA), heat-stable enterotoxins (estA, estB), verocytotoxins (stx-I, stx-II), or cytotoxic necrotizing factors (cnf 1, cnf 2). All isolates belonged to serotype O15:H8. The enteropathogenic relevance of LT-II in ostrich diarrhea remains undetermined. (C) 2004 Elsevier B.V. All rights reserved.