Carcaterização denotípica e genotípica de Escerichia coli produtora de toxina shiga (STEC) isoladas de bovinos de corte no Estado do Paraná
Data
2008-01-29
Autores
Pigatto, Caroline Peters [UNESP]
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ISSN da Revista
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Escherichia coli produtoras de toxina Shiga (STEC) são reconhecidas como agentes causadores de infecções em humanos em todo o mundo. O principal reservatório é o bovino. Neste trabalho, cepas de STEC previamente isoladas de fezes bovinas foram caracterizadas usando PCR multiplex para determinar os genes de virulência (stx1, stx2, ehxA, eaeA e saa), soroaglutinação passiva reversa em látex (RPLA-VTEC screen) para avaliar a expressão da toxina Shiga, PCR-RFLP e sequenciamento para obter os subtipos e a variabilidade dos genes stx2, respectivamente. Foram determinados também os sorotipos, o perfil de sensibilidade e a viabilidade das cepas de STEC em queijo minas frescal. A freqüência de STEC nas amostras de fezes bovinas foi de 37%. Foram encontrados trinta e quatro sorotipos de STEC sendo os mais freqüentes o ONT:H7 (10%), O22:H8, O22:H16 e ONT:H21 (7% cada). Onze sorotipos encontrados não tinham sido associados com STEC até o momento. A maioria das STEC (96%) foi susceptível a todos os antimicrobianos testados. A produção de toxina Shiga determinada pelo ensaio RPLA foi de 89%. Os marcadores de virulência foram encontrados em 11 diferentes combinações, a mais freqüente foi stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% cada). Foram detectados 8 subtipos de stx2: stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc e stx2O48. Os genes que apresentaram maior freqüência foram: stx2 e stx2c. As seqüências parciais obtidas sugerem a presença de elevada variabilidade nos genes do tipo stx2 nas STEC analisadas. A viabilidade de STEC não-O157 em queijo minas revelou que diferentes cepas de STEC podem ser detectadas nos queijos após 10 dias de armazenamento sob refrigeração. Os dados encontrados neste trabalho sugerem isolados com alto potencial de patogenicidade oferecendo risco de desencadear graves infecções à população.
Shiga toxin-producing Escherichia coli (STEC) is recognize worldwide as an organism capable to cause human diseases. Cattle are the main source of STEC. In this research, STEC strains previously isolated were analyzed using multiplex-PCR for virulence genes, the RPLA assay to detect the Shiga toxin production and serotyping. PCR-RFLP and nucleotide sequence were analyzed to detect stx2 genes subtypes and their variability. Moreover tests for antimicrobial susceptibility and the vialbility of STEC in Minas Frescal cheese were done. The frequency of cattle shedding STEC was 37%. Thirty-four serotypes of STEC were found, the most frequent being ONT:H7 (10%), O22:H8, O22:H16 and ONT:H21 (7% each). Eleven serotypes had not been associate with STEC until the moment. Most of the strains (96%) were susceptible to all antimicrobial agents tested. Production of Shiga toxin by the RPLA assay was detected in most (89%) of the STEC strains. The frequency of virulence markers were found in 11 diferent combinations: stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% each). Eigth stx2 subtypes were detect (stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc; stx2O48) and the most frequent were: stx2; stx2c. The partial sequences of stx2 genes suggested a high variability of stx2 types in the STEC analyzed. The STEC viability in cheese could be detected after 10 days of storage under refrigeration. The results found in this work suggest strains with high potential of pathogenicity offering risk to lead serious infections to the population.
Shiga toxin-producing Escherichia coli (STEC) is recognize worldwide as an organism capable to cause human diseases. Cattle are the main source of STEC. In this research, STEC strains previously isolated were analyzed using multiplex-PCR for virulence genes, the RPLA assay to detect the Shiga toxin production and serotyping. PCR-RFLP and nucleotide sequence were analyzed to detect stx2 genes subtypes and their variability. Moreover tests for antimicrobial susceptibility and the vialbility of STEC in Minas Frescal cheese were done. The frequency of cattle shedding STEC was 37%. Thirty-four serotypes of STEC were found, the most frequent being ONT:H7 (10%), O22:H8, O22:H16 and ONT:H21 (7% each). Eleven serotypes had not been associate with STEC until the moment. Most of the strains (96%) were susceptible to all antimicrobial agents tested. Production of Shiga toxin by the RPLA assay was detected in most (89%) of the STEC strains. The frequency of virulence markers were found in 11 diferent combinations: stx2 (27%), stx1 stx2 e stx1 stx2 ehxA saa (16% each). Eigth stx2 subtypes were detect (stx2OX3a/O111; stx2; stx2c; stx2(vha); stx2(vhb); stx2OX3b; stx2vnb/vhc; stx2O48) and the most frequent were: stx2; stx2c. The partial sequences of stx2 genes suggested a high variability of stx2 types in the STEC analyzed. The STEC viability in cheese could be detected after 10 days of storage under refrigeration. The results found in this work suggest strains with high potential of pathogenicity offering risk to lead serious infections to the population.
Descrição
Palavras-chave
Escherichia coli, Toxina Shiga, Shiga toxin
Como citar
PIGATTO, Caroline Peters. Carcaterização denotípica e genotípica de Escerichia coli produtora de toxina shiga (STEC) isoladas de bovinos de corte no Estado do Paraná. 2008. xiv, 84 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2008.