ORIGINAL ARTICLE Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC) J.R. Alves 1 , A.C.M. Pereira 1 , M.C. Souza 1 , S.B. Costa 1 , A.S. Pinto 1 , A.L. Mattos-Guaraldi 1 , R. Hirata-Ju ´ nior 1 , A.C.P. Rosa 1 and L.M.B.O. Asad 2 1 Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Cie ˆ ncias Me ´ dicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 2 Departamento de Biofı ´sica e Biometria, Instituto de Biologia Roberto Alca ˆ ntara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Introduction Enteroaggregative Escherichia coli (EAEC) is a diarrhoeal pathogen defined by its aggregative adherence (AA) to HEp-2 cells in culture (Nataro et al. 1987), characterized by a ‘stacked brick’ formation of bacteria attached to host cells. The AA pattern was first significantly associated with diarrhoea among children (Nataro et al. 1987). Cur- rently, EAEC has been implicated as a leading diarrhoeal pathogen in people of all ages in both developing and industrialized nations (Okeke and Nataro 2001; Huang et al. 2006). EAEC pathogenesis studies have suggested three stages: (i) abundant adherence to the intestinal mucosa by aggregative adherence fimbria (AAF), (ii) increased pro- duction of mucus and biofilm on the surface of the enterocytes and (iii) induction generation of mucosal inflammation (Huang et al. 2004). Although this three- stage model highlights key phenotypes associated with EAEC pathogenesis, precise virulence mechanisms remain to be determined. Laboratory studies on prototype EAEC strains revealed that the best-known EAEC putative virulence factors are encoded by genes on a partially conserved plasmid, pAA (Harrington et al. 2006). EAEC adheres to the intestinal mucosal surface and stimulates mucus secretion, forming a thick aggregating biofilm (Sheikh et al. 2001). Biofilm formation has been described as a common phenomenon among EAEC iso- lates derived from travellers with or without diarrhoea (Mohamed et al. 2007). Keywords 2,2-dipyridyl, biofilm, EAEC, HEp-2 cell, iron. Correspondence Ana Cla ´ udia de Paula Rosa, Departamento de Microbiologia, Imunologia e Parasitologia, Faculdade de Cie ˆ ncias Me ´ dicas, UERJ. Av. 28 de Setembro, 87 – Fundos, 3 o andar. Vila Isabel, Rio de Janeiro, RJ, Brazil. E-mail: anarosa@uerj.br 2009 ⁄ 0052: received 9 January 2009, revised 17 April 2009 and accepted 18 May 2009 doi:10.1111/j.1365-2672.2009.04417.x Abstract Aims: The aim of this study was to investigate the influence of low iron avail- ability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases. Methods and Results: The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea. Conclusions: Low iron availability may modulate biofilm formation and adhe- sive properties of EAEC strains to HEp-2 cells. Significance and Impact of the Study: The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains. Journal of Applied Microbiology ISSN 1364-5072 246 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 246–255 ª 2009 The Authors