Orientadora: Profa. Dra. Jane Martha Graton Mikcha

Data da Defesa: 23/04/2013



 INTRODUCTION. Although milk is a highly consumed food, its contamination and the development of pathogenic microorganisms due to its intrinsic features are rife. Escherichia coli not only indicates contamination from fecal sources but may also be pathogenic. Among the several pathotypes of E. coli that may cause diarrhea in humans, E. coli as a producer of Shiga toxic (STEC) causes food-transmitted diseases worldwide. Serious implications to health may occur in humans and may range from mild diarrhea to hemorrhagic colitis to severe extra-intestine complication, such as Uremic Hemolytic Syndrome (UHS) and Thrombotic Thrombocytopenic Purpura. Virulence factors, such as Shiga toxins, intimine and enterohemolisin, are involved in STEC pathogenesis and several types of food from animal or vegetal origin cause STEC episodes. Although most food contamination episodes caused by STEC are linked to non-pasteurized milk consumption and its derivates, episodes related to the consumption of pasteurized milk have been reported. Genotype methods such as Polymerase Chain Reaction (PCR) have been employed to study E. coli epidemiology and to research on virulent, fast and sensitive genes.
AIMS. Current research evaluates the genetic similarity and research genes stx1, stx2, eae, ehxA in E. coli isolated from pasteurized cattle milk.
MATERIALS AND METHODS. Eighty-seven isolates of E. coli from pasteurized bovine milk from 22 dairies in the northwestern region of the state of Paraná, Brazil, were analyzed. Isolates were stored in Tryptic Soy Broth (TSB, Difco) with glycerol at -20 ° C, the samples were identified biochemically before extraction of DNA, which was in agreement with Swanenburg et al. (1998), with modifications. Technical enterobacterial repetitive sequence Intergenic Consensus (ERIC-PCR) and sequence repetitive extragenic palindromic (REP-PCR) were used for the genetic similarity. Values equal to or greater than 98% similarity were considered related. The discriminatory power (D) of ERIC-PCR and REP-PCR was calculated using Simpson's diversity index. For the detection of virulence genes was used PCR, primers and amplification conditions were specific to each gene stx1, stx2, eae, ehxA.
RESULTS AND DISCUSSION. ERIC-PCR and REP-PCR differentiated the isolates of E. coli genomic profiles 76 and 81, respectively. Analysis of ERIC-PCR and REP-PCR showed high genetic diversity among the isolates of E. coli could be explained by different sources of contamination by E. coli in bovine milk. REP-PCR mustered only bacterial isolates from the same samples of milk, ERIC PCR showed that the isolates from different samples of milk were similar. Both techniques not grouped bacterial isolates from processed milk in dairy different. Were not detected virulence genes stx1, stx2, eae, ehxA in isolates of E. coli analyzed, possibly by the low incidence of these pathogens in pasteurized dairy products.
CONCLUSION. ERIC and REP-PCR revealed a high genetic diversity of E. coli isolated from bovine milk pasteurized and non-detection of genes stx1, stx2, eae, ehxA normally present in STEC in bacterial isolates. Knowledge of the epidemiology of. diarrheagenic E. coli in pasteurized milk is highly relevant to understanding the transmission and provides information for the development and implementation of control measures throughout the production chain. The research on virulence factors in other pathotypes of E. coli is important to evaluate the potential of pasteurized commercially available as vector bacterium analyzed.
Key Words: Escherichia coli Shiga toxin-producing (STEC), PCR (Polymerase Chain Reaction), virulence genes, Enterobacterial Repetitive Intergenic Consensus Sequence Polymerase Chain Reaction (ERIC-PCR), Repetitive Extragenic Palindromic Sequence Polymerase Chain Reaction (REP-PCR).

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