Análise dos processos de formação de biofilme e interação de Escherichia coli produtora de toxina Shiga com superfícies bióticas e abióticas.
Data
2013
Autores
Guimaraes, Cecilia Matheus 
Orientadores
Guth, Beatriz Ernestina Cabilio
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A capacidade de formacao de biofilme a superficies abioticas (poliestireno e vidro) e interacao a celulas eucarioticas de origem humana (HeLa, Caco-2 e T84) foi avaliada em um conjunto de amostras de Escherichia coli produtora de toxina Shiga (STEC) O157 (n=11) e nao-O157 (n=8) assim como em tres amostras O157, desprovidas desta toxina, isoladas do couro e carcacas de bovinos. Adicionalmente, a presenca de sequencias geneticas associadas a fimbrias curli (csgA e crl ), fimbria do tipo 1 (F1) (fimH ), celulose (bscA), proteinas autotransportadoras EhaA (ehaAα e ehaβ), Sab (sab), Cah (cah) e Antigeno 43 (Ag43) (flu) foi pesquisada em todas as amostras, assim como a expressao destes genes foi avaliada nas amostras STEC formadoras de biofilme. A caracterizacao genotipica demonstrou que todas as amostras carreavam os genes pesquisados, com excecao de sab e flu que foram identificados, respectivamente, em uma e oito das amostras nao-O157. Quatro amostras STEC O157 e duas nao-O157 foram capazes de formar biofilme em vidro porem, apenas uma das amostras STEC O157 tambem formou biofilme na superficie de poliestireno. Ensaios de RT-PCR demonstraram que a expressao dos genes avaliados foi comum dentre as amostras formadoras de biofilme com excecao dos genes ehaAα, cah e flu. De modo geral, a presenca de curli, F1 e celulose nao foi identificada dentre as amostras O157, atraves de ensaios fenotipicos, exceto por uma amostra que expressou celulose. Por outro lado, no conjunto de amostras nao-O157, a frequencia detectada foi de 50%, 87% e 87%, respectivamente. Vinte amostras apresentaram capacidade de aderencia a celulas HeLa, sendo que o padrao de aderencia localizada-like ocorreu em todas as amostras O157, enquanto dentre as nao-O157, prevaleceu o padrao de aderencia difusa (75 %). Dentre as amostras aderentes, padroes mistos com ocorrencia de aderencia agregativa foram observados em uma amostra O157 e em tres amostras nao-O157. Nos ensaios de invasao realizados em celulas Caco-2 nao-diferenciadas, apenas cinco amostras, todas STEC O157, apresentaram potencial invasor. Quando estas amostras foram submetidas a ensaios em celulas Caco-2 e T84 diferenciadas, observou-se que apenas tres (60%) e quatro (80%) amostras, respectivamente, apresentaram potencial de invasao. Duas amostras STEC (O157:H7 e O105:H18) que apresentaram elevada capacidade de formacao de biofilme em superficies abioticas foram selecionadas para avaliacao da habilidade de interagir com folhas de ruculas (Eruca sativa). Adicionalmente, ensaios de recombinacao homologa, utilizando o sistema Lambda Red, foram empregados para avaliar a participacao do flagelo e das proteinas Cah, Sab e Ag43 nas interacoes destas amostras com diferentes superficies. A delecao do flagelo reduziu a capacidade de interacao da amostra O157 com todas as superficies avaliadas, com excecao de folhas de rucula. Assim como a delecao de Cah na amostra O157 reduziu sua habilidade de aderir e invadir celulas humanas. Entretanto, a ausencia desta proteina provocou aumento na interacao com superficies abioticas e vegetal. A delecao de Sab na amostra O105 reduziu a capacidade de interacao com a maioria das superficies, com excecao da aderencia a celulas HeLa e ao vidro. Alem disso, a delecao de sab provocou um aumento no potencial invasor a celulas Caco-2. A ausencia do gene flu na amostra O105 reduziu drasticamente a habilidade da cepa de interagir com as superficies avaliadas, em especial, a capacidade de interacao com a superficie vegetal. Foi possivel observar que, dependendo da superficie analisada, algumas das alteracoes no genoma das cepas STEC O157 e O105 levaram a ocorrencia de mecanismos compensatorios os quais promoveram maior capacidade de interacao. Em conjunto, os dados obtidos demonstram que a expressao de estruturas associadas a aderencia e formacao de biofilme por amostras STEC varia de acordo com a superficie com a qual estas amostras estejam interagindo. Tambem foi possivel determinar que para este grupo de amostras, nao existiu correlacao entre interacao com superficies bioticas e a capacidade de formar biofilme em superficies abioticas. A ocorrencia de amostras STEC isoladas de fezes ou da carcaca de bovinos capazes de formar biofilme em superficies abioticas e vegetal, de aderir e invadir celulas humanas ressalta uma importante habilidade deste patogeno persistir no ambiente e interagir com o hospedeiro.
Abstract The ability to form biofilm to abiotic surfaces (polystyrene and glass) and the interaction with human eukaryotic cells (HeLa, Caco2 and T84) were evaluated among O157 (n=11) and nonO157 (n=8) Shiga toxinproducing Escherichia coli (STEC) strains, as well as on three O157 strains devoid of this toxin, isolated from hide and carcass of cattle. All strains were analyzed for the presence of genetic sequences associated to curli fimbriae (csgA and crl), type 1 fimbriae (F1) (fimH), cellulose (bscA), autotransporter proteins EhaA (ehaAα and ehaβ), Sab (sab), Cah (cah) and Antigen 43 (Ag43) (flu), and the expression of these genes was evaluated in biofilmforming STEC strains. Genotypic characterization demonstrated that all strains carried the genes studied, except for flu and sab that were identified, respectively, in one and eight nonO157 STEC strains. Four O157 and two nonO157 STEC strains were able to form biofilm on glass surface, and only one O157 STEC strain also formed biofilm on polystyrene surface. RTPCR assays demonstrated that the expression of evaluated genes was common among biofilmforming strains, except for ehaAα, cah and flu genes. Generally, presence of curli, F1 and cellulose was not detected by phenotypical assays on O157 strains, except for one strain that expressed cellulose. On the other hand, among nonO157 strains, the detected frequency was 50%, 87% and 87%, respectively. Twenty strains were able to adhere to HeLa cells, and all O157 strains presented the localized adherencelike pattern, while among nonO157 strains the diffuse pattern prevailed (75%). Mixed patterns with aggregative adherence were observed on one O157 and three nonO157 adherent STEC strains. On invasion assays using nondifferentiated Caco2 cells, only five strains, all belonging to O157 STEC serogroup, presented ability to invade. When these strains were submitted to assays using differentiated Caco2 and T84 cells, only three (60%) and four (80%) strains, respectively, presented invasive potential. Two STEC strains (O157:H7 and O105:H18) presenting strong ability to form biofilm on abiotic surfaces were further selected to evaluate their ability to interact with rocket leaves (Eruca sativa). Additionally, homologe recombination assays, using Lambda Red System, were employed to evaluate the participation of flagella and Cah, Sab and Ag43 proteins on the interaction of these strains with different surfaces. Deletion of flagella reduced the ability of O157 strain to interact with all studied surfaces, except for interaction with rocket leaves. Similarly, deletion of Cah reduced the ability of O157 strain to adhere and invade human cells. On the other hand, absence of this protein increased interactions with abiotic and vegetal surfaces. The absence of Sab on O105 strain reduced its interaction with most surfaces, except for the adherence to HeLa cells and on glass. Furthermore, deletion of sab increased the invasive potential of the strain to Caco2 cells. Lacking of flu reduced the ability of O105 strain to interact with the surfaces studied, specially with the vegetal surface. It has been observed that depending on the evaluated surface 11 some changes on the genome of O157 and O105 STEC strains lead to a compensatory mechanism which promoted higher rates of interaction. Taken together, these results demonstrated that the expression of structures associated to adherence and biofilm formation by STEC strains varies according to the interacted surface. It was also possible to observed that, for this group of strains, no correlation between the interaction with biotic surfaces and the ability to form biofilm on abiotic surfaces was found. The occurrence of STEC strains isolated from cattle feces or carcass able to form biofilm on abiotic and vegetal surfaces, to adhere and invade human cells, highlights an important ability of this pathogen to persist in the environment and to interact with the host.
Abstract The ability to form biofilm to abiotic surfaces (polystyrene and glass) and the interaction with human eukaryotic cells (HeLa, Caco2 and T84) were evaluated among O157 (n=11) and nonO157 (n=8) Shiga toxinproducing Escherichia coli (STEC) strains, as well as on three O157 strains devoid of this toxin, isolated from hide and carcass of cattle. All strains were analyzed for the presence of genetic sequences associated to curli fimbriae (csgA and crl), type 1 fimbriae (F1) (fimH), cellulose (bscA), autotransporter proteins EhaA (ehaAα and ehaβ), Sab (sab), Cah (cah) and Antigen 43 (Ag43) (flu), and the expression of these genes was evaluated in biofilmforming STEC strains. Genotypic characterization demonstrated that all strains carried the genes studied, except for flu and sab that were identified, respectively, in one and eight nonO157 STEC strains. Four O157 and two nonO157 STEC strains were able to form biofilm on glass surface, and only one O157 STEC strain also formed biofilm on polystyrene surface. RTPCR assays demonstrated that the expression of evaluated genes was common among biofilmforming strains, except for ehaAα, cah and flu genes. Generally, presence of curli, F1 and cellulose was not detected by phenotypical assays on O157 strains, except for one strain that expressed cellulose. On the other hand, among nonO157 strains, the detected frequency was 50%, 87% and 87%, respectively. Twenty strains were able to adhere to HeLa cells, and all O157 strains presented the localized adherencelike pattern, while among nonO157 strains the diffuse pattern prevailed (75%). Mixed patterns with aggregative adherence were observed on one O157 and three nonO157 adherent STEC strains. On invasion assays using nondifferentiated Caco2 cells, only five strains, all belonging to O157 STEC serogroup, presented ability to invade. When these strains were submitted to assays using differentiated Caco2 and T84 cells, only three (60%) and four (80%) strains, respectively, presented invasive potential. Two STEC strains (O157:H7 and O105:H18) presenting strong ability to form biofilm on abiotic surfaces were further selected to evaluate their ability to interact with rocket leaves (Eruca sativa). Additionally, homologe recombination assays, using Lambda Red System, were employed to evaluate the participation of flagella and Cah, Sab and Ag43 proteins on the interaction of these strains with different surfaces. Deletion of flagella reduced the ability of O157 strain to interact with all studied surfaces, except for interaction with rocket leaves. Similarly, deletion of Cah reduced the ability of O157 strain to adhere and invade human cells. On the other hand, absence of this protein increased interactions with abiotic and vegetal surfaces. The absence of Sab on O105 strain reduced its interaction with most surfaces, except for the adherence to HeLa cells and on glass. Furthermore, deletion of sab increased the invasive potential of the strain to Caco2 cells. Lacking of flu reduced the ability of O105 strain to interact with the surfaces studied, specially with the vegetal surface. It has been observed that depending on the evaluated surface 11 some changes on the genome of O157 and O105 STEC strains lead to a compensatory mechanism which promoted higher rates of interaction. Taken together, these results demonstrated that the expression of structures associated to adherence and biofilm formation by STEC strains varies according to the interacted surface. It was also possible to observed that, for this group of strains, no correlation between the interaction with biotic surfaces and the ability to form biofilm on abiotic surfaces was found. The occurrence of STEC strains isolated from cattle feces or carcass able to form biofilm on abiotic and vegetal surfaces, to adhere and invade human cells, highlights an important ability of this pathogen to persist in the environment and to interact with the host.
Descrição
Citação
GUIMARÃES, Cecília Matheus. Análise dos processos de formação de biofilme e interação de Escherichia coli produtora de toxina Shiga com superfícies bióticas e abióticas. 2013. 126f. Tese (Doutorado em Microbiologia e Imunologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2013.