Pesquisa de colicinas em Escherichia coli produtora de toxina shiga
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2017-12-21
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Dissertação de mestrado
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Objetivo: Pesquisar a produção de colicinas por cepas de Escherichia coli produtora de toxina Shiga (STEC) de diferentes sorotipos, isoladas de infecções em humanos, do reservatório animal e do ambiente, buscando analisar sua importância como fator de virulência. Métodos: Um total de 251 cepas STEC, isoladas de diferentes origens, como humana (n=46), animal (n=195) e água (n=10) foram estudadas. Foram utilizados ensaios de expressão de colicinas e indução da expressão nas cepas negativas com Mitomicina C (MMC). A pesquisa das sequências genéticas para colicinas foi realizada utilizando primers para col Ia/Ib, col Ia, col Ib, col E2 e col V. As cepas que tiveram seus genes col identificados, foram testadas em ensaios de disco-difusão, para avaliação da sensibilidade a antimicrobianos. Resultados: A expressão de colicinas foi identificada em 62% das cepas STEC. Alta frequência de expressão de colicinas foi identificada nas cepas STEC de origem humana (91%), isoladas de água (100%) e variou de 48% a 100% de acordo com a espécie animal. A indução da expressão de colicinas foi observada em apenas cinco cepas do sorotipo O157:H7. A produção de colicinas foi identificada em uma grande diversidade de sorotipos, porém dentre as cepas deorigem humana os mais freqüentes foram O111:H8 (29%), O26:H11 (19%) O111:H- (19%); enquanto entre os bovinos prevaleceram os sorotipos O178:H19 (13%), O111:H8 (11%) e O116:H21 (9%) e dentre os caprinos, O5:H- e O174:H8. Dentre as cepas STEC colicinogênicas 49% carreavam alguma das sequências genéticas pesquisadas, sendo o gene col Ib o mais prevalente (59%) seguido de col Ia/Ib (19%) e col E2 (16%). A ocorrência de mais de um gene para colicina foi observada em cinco cepas STEC de origem humana e animal. A maioria (91%) das cepas STEC que apresentaram um dos genes col foi sensível aos antimicrobianos pesquisados. A análise da correlação entre o genótipo stx e a presença do gene col demonstrou que os genótipos stx2 e stx1stx2 foram os mais frequentes sendo observados em 45% e 41% das cepas, respectivamente. Conclusão: Uma alta freqüência na expressão de colicinas foi, pela primeira vez, identificada em cepas STEC de origem humana, de espécies animais e do ambiente no Brasil. Apesar da diversidade de sorotipos identificados entre as cepas colicinogênicas, a frequência de colicinas em sorotipos STEC responsáveis por causar severas infecções humanas poderia indicar a sua participação como fator de virulência. Além disso, a alta ocorrência de colicinas em cepas STEC isoladas de diferentes espécies animais e do ambiente sugere o seu envolvimento como mecanismo de persistência nestes reservatórios.
Aim: To investigate the production of colicins by Shiga toxin-producing Escherichia coli (STEC) strains of different serotypes isolated from human infections, from the animal reservoir and the environment, aiming to analyze its importance as a virulence factor. Methods: A total of 251 strains isolated from different sources, such as human (n = 46), animal (n = 195) and water (n = 10) were studied. Assays for expression of colicins and induction of expression in negative strains using Mitomycin C (MMC) were used. The search for the genetic sequences for colicins was carried out using primers for col Ia / Ib, col Ia, col Ib, col E2 and col V. The strains that had their col genes identified were tested in disc-diffusion assays, for evaluation of the susceptibility to antimicrobials. Results: Expression of colicins was identified in 62% of STEC strains. High frequency of colicin expression was identified in STEC strains of human origin (91%), isolated from water (100%) and ranged from 48% to 100% according to the animal species. Induction of colicin expression was observed in only five strains of the O157: H7 serotype. The production of colicins was identified in a wide variety of serotypes but the most frequent ones among strains of human origin were O111: H8 (29%), O26: H11 (19%) and O111: H- (19%); while the serotypes O178: H19 (13%), O111: H8 (11%) and O116: H21 (9%) prevailed among cattle isoaltes and O5: H- and O174: H8 among strains isolated from goat. Among the colicinogenic STEC strains, 49% carried some of the genetic sequences studied, being the most prevalent colIb gene (59%) followed by colIa / Ib (19%) and colE2 (16%). The occurrence of more than one gene for colicin was observed in five STEC strains of human and animal origin. The majority (91%) of the STEC strains that carried one of the col genes were sensitive to the antimicrobials tested. The analysis of the correlation between the stx genotype and presence of the col gene demonstrated that the stx2 and stx1stx2 genotypes were the most frequent, being observed in 45% and 41% of the strains, respectively. Conclusion: A high frequency in the expression of colicins was identified for the first time in STEC strains of human origin, animal species and the environment in Brazil. Despite the diversity of serotypes identified among colicinogenic strains, the frequency of colicins in STEC serotypes responsible for causing severe human infections could indicate its participation as a virulence factor. In addition, the high occurrence of colicins in STEC strains isolated from different animal species and the environment suggests its involvement as a mechanism of persistence in these reservoirs.
Aim: To investigate the production of colicins by Shiga toxin-producing Escherichia coli (STEC) strains of different serotypes isolated from human infections, from the animal reservoir and the environment, aiming to analyze its importance as a virulence factor. Methods: A total of 251 strains isolated from different sources, such as human (n = 46), animal (n = 195) and water (n = 10) were studied. Assays for expression of colicins and induction of expression in negative strains using Mitomycin C (MMC) were used. The search for the genetic sequences for colicins was carried out using primers for col Ia / Ib, col Ia, col Ib, col E2 and col V. The strains that had their col genes identified were tested in disc-diffusion assays, for evaluation of the susceptibility to antimicrobials. Results: Expression of colicins was identified in 62% of STEC strains. High frequency of colicin expression was identified in STEC strains of human origin (91%), isolated from water (100%) and ranged from 48% to 100% according to the animal species. Induction of colicin expression was observed in only five strains of the O157: H7 serotype. The production of colicins was identified in a wide variety of serotypes but the most frequent ones among strains of human origin were O111: H8 (29%), O26: H11 (19%) and O111: H- (19%); while the serotypes O178: H19 (13%), O111: H8 (11%) and O116: H21 (9%) prevailed among cattle isoaltes and O5: H- and O174: H8 among strains isolated from goat. Among the colicinogenic STEC strains, 49% carried some of the genetic sequences studied, being the most prevalent colIb gene (59%) followed by colIa / Ib (19%) and colE2 (16%). The occurrence of more than one gene for colicin was observed in five STEC strains of human and animal origin. The majority (91%) of the STEC strains that carried one of the col genes were sensitive to the antimicrobials tested. The analysis of the correlation between the stx genotype and presence of the col gene demonstrated that the stx2 and stx1stx2 genotypes were the most frequent, being observed in 45% and 41% of the strains, respectively. Conclusion: A high frequency in the expression of colicins was identified for the first time in STEC strains of human origin, animal species and the environment in Brazil. Despite the diversity of serotypes identified among colicinogenic strains, the frequency of colicins in STEC serotypes responsible for causing severe human infections could indicate its participation as a virulence factor. In addition, the high occurrence of colicins in STEC strains isolated from different animal species and the environment suggests its involvement as a mechanism of persistence in these reservoirs.
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MARCONDES, Caio Antonio Righetti. Pesquisa de colicinas em Escherichia coli produtora de toxina shiga. São Paulo, 2017. [101] p. Dissertação (Mestrado em Microbiologia e imunologia) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2017.