Contribution of AIDA-I to the pathogenicity of a porcine diarrheagenic Escherichia coli and to intestinal colonization through biofilm formation in pigs Madhu Ravi a , Musangu Ngeleka b, * , Sang-Hyun Kim c , Carlton Gyles c , Fre ´de ´ric Berthiaume d , Michael Mourez d , Dorothy Middleton a , Elemir Simko a a Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada b Prairie Diagnostic Services, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada c Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada d GREMIP, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec J2S 7C6, Canada Received 1 February 2006; received in revised form 25 October 2006; accepted 31 October 2006 Abstract In order to evaluate the role of the AIDA-I of porcine diarrheagenic Escherichia coli strain PD20 serogroup O143 (AIDA-I + , STb + ), a mutant strain PD20M (AIDA-I  , STb + ) was generated from strain PD20 by an allelic exchange procedure. In addition, the full-length aidA gene was reintroduced into strain PD20M to generate the complemented strain PD20C (pT aidA, AIDA-I + , STb + ). A non-pathogenic E. coli strain PD71 was used as negative control. Each strain was inoculated to newborn pigs via stomach tube. Severity of diarrhea was evaluated clinically and intestinal colonization was assessed by histology, immuno- histochemistry (IHC), and transmission electron microscopy (TEM) including immunogold electron microscopy (IGEM). The adhesion pattern to HeLa cells, bacterial auto-aggregation and biofilm formation were evaluated in vitro. Pigs infected with strains PD20 or PD20C developed diarrhea 16 and 28 h after inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71. Histology, IHC, TEM and IGEM examinations showed heavy bacterial colonization with biofilm formation in the large intestine, and marked in vivo expression of AIDA-I protein in pigs infected with strains PD20 or PD20C in contrast to pigs infected with strains PD20M or PD71. The in vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial auto-aggregation and significant biofilm formation ( p < 0.05) by the AIDA-I + strains, when compared to AIDA-I  strains. These results demonstrate that expression of AIDA-I is essential for intestinal colonization and in vitro bacterial autoaggregation and biofilm formation. Thus, AIDA-I may be considered a significant virulence determinant in development of diarrhea caused by porcine diarrheagenic AIDA-I + E. coli PD20 in piglets. # 2006 Elsevier B.V. All rights reserved. Keywords: Adhesin involved in diffuse adherence; AIDA-I Escherichia coli; AIDA-I E. coli pathogenesis; AIDA-I E. coli biofilm www.elsevier.com/locate/vetmic Veterinary Microbiology 120 (2007) 308–319 * Corresponding author. Tel.: +1 306 966 7250; fax: +1 306 966 2488. E-mail address: musangu.ngeleka@usask.ca (M. Ngeleka). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.10.035