25º Congresso Brasileiro de Microbiologia

25º Congresso Brasileiro de Microbiologia

ResumoID:1104-1

Área: Patogenicidade Microbiana ( Divisão D )

INTRACELLULAR SURVIVAL AND PERSISTENCE OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) WITHIN CULTURED HUMAN ENTEROCYTES

Fabiana Cordeiro (UERJ); Thais Locha Zangali Vargas Stampe (UERJ); Aloysio de Mello Figueiredo Cerqueira (UFF); Alice Gonçalves Martins Gonzalez (UFF); Beatriz Ernestina Cabilio Guth (UNIFESP); Joao Ramos Costa Andrade (UERJ)

Resumo

STEC colonizes the intestinal tract of healthy cattle and is mostly transmitted to humans by raw meat and milk contaminated by bovine feces. STEC serotypes usually involved in outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome are known as EHEC (Enterohaemorrhagic E. coli). Most EHEC strains induce "attaching and effacing" (A/E) lesions in the intestinal mucosa due the expression of several genes located in a pathogenicity island called LEE (locus of enterocyte effacement). However, some LEE-negative STEC serotypes also produce disease and their virulence abilities are not fully understood. It was observed in our laboratory that both LEE-negative and LEE-positive STEC strains can invade human enterocytic cell lineages, such as Caco-2. We studied the invasive abilities of seven strains carrying the LEE island (serotypes O26:H11, O103:H2, O111a,c:H-, O153:H25, O157:H7) and five LEE-negative strains (serotypes O8:H19, O22:H8, O113:H21). Aminoglycoside exclusion tests showed that bacterial invasion was at least 10 times higher in the enterocytic cell lines Caco-2 and T-84 than in the non-enterocytic HEp-2 and HeLa human cells. Invasion of all STEC strains tested was significantly higher (P<0.05) in non-polarized, non-differentiated Caco-2 cells than in fully polarized cell monolayers. STEC strains of serotypes O8:H19, O26:H11, O113:H21 and O157:H7 were used to study the invasion process. The kinetics of the invasion showed that intracellular bacteria could be detected as early as 5 minutes of infection (0.2%-0.5% of the inoculated bacteria) and steadily increased until 180 min. (1% of initial inocula). Double-immunofluorescence staining showed individual intracellular bacteria in 5 min. and intracellular bacterial groups from then on, until reach 180 min. of infection. STEC persisted in the intracellular compartment of Caco-2 cells until 96 hours without evidence of cell death, while the virulent Salmonella Typhimurium strain used as an invasive control, fully destroyed the cell monolayer in 48 hours. The study of the STEC intracellular behaviour was carried out with the BacLight bacterial viability test. It was shown that the live sub-population of intracellular bacteria slowly decreases until 48 h. of infection and then increases, suggesting adaptation to the intracellular environment and active multiplication. The relatively harmless intracellular localization of STEC can be an efficient strategy to prevent its elimination from the bovine intestinal tract. Bacterial escape for the extracellular space and/or the invasion of the neighbouring enterocytes could assure the continuous re-infection of the intestinal mucosa.

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Palavras-chave: STEC, enterocytes, persistence, intracellular survival

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