Identification of enteroaggregative Escherichia coli in infants with acute diarrhea based on biofilm production in Manipal, south India

Background: Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes persistent diarrhea among infants, both in developing and industrialized countries. The EAEC strains adhere to epithelial cell surface, to the glass substratum and to each other in a distinctive stacked brick-formation. Thus, gold standard for identification of EAEC remains the HEp-2 cell adherence test, which is time consuming and requires specialized facilities. Aim: To evaluate the usefulness of quantitative biofilm assay to screen for EAEC from children with acute diarrhea. Materials and Methods: A total of 100 E. coli strains were collected from acute diarrheal cases from December 2005 to November 2006. The strains were screened for biofilm production using microtiter plate method. The biofilm in the microtiter plate was visualized after staining with crystal violet and was quantified using enzyme immunosorbent assay plate reader. The Aggregative plasmid and Heat stable toxin genes were evaluated by a multiplex polymerase chain reaction. The strains were identified as EAEC with an optical density at 570 nm (OD 570 ) > 0.2. Results: Of the total 100 Escherichia coli strains, 28 were positive by Polymerase Chain Reaction for two genes, AggR and EAST. Of the 28 PCR-positive strains screened for biofilm, 25 (89.2%) showed positive results by microtiter plate method. Conclusion: The quantitative biofilm assay using microtiter plate is convenient and economical and can be used as a screening method to screen E. coli isolates from acute diarrheal cases. The best use of this test is to screen large number of isolates quickly, and if positive this can be confirmed by multiplex PCR for AggR and EAST genes. This assay may contribute to demonstrating the true incidence of EAEC with and without AggR among clinically isolated E. coli strains, which can cause acute diarrhea