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QUANTITATIVE BIOFILM ASSAY USING A MICROTITER PLATE TO SCREEN FOR ENTEROAGGREGATIVE ESCHERICHIA COLI

The gold standard for identification of Enteroaggregative Escherichia coli (EAEC) remains the HEp-2 cell adherence test, which is time-consuming and requires specialized facilities. We evaluated the usefulness of a quantitative biofilm assay to screen for EAEC from a total of 1,042 E. coli strains from children with diarrhea. Bacteria were incubated overnight in high-glucose Dulbecco’s modified Eagle’s medium using a polystyrene microtiter plate. The plate was stained with crystal violet after washing, and the biofilm was quantified using an enzyme-linked immunosorbent assay plate reader. The aggR gene was evaluated by a polymerase chain reaction. Forty-eight (77.4%) of 62 strains with an optical density at 570 nm (OD₅₇₀) > 0.2 were identified as EAEC by the HEp-2 adherence test, while no EAEC was found in strains with an OD₅₇₀ 0.2. Twenty-one aggR+ and 27 aggR - EAEC strains could be screened by an OD₅₇₀ > 0.2 using this assay. Although confirmation by a HEp-2 cell adherence test is needed, this biofilm assay is convenient and useful in screening for EAEC.

Received February 17, 2004. Accepted for publication April 14, 2004.

Acknowledgments: We thank Dr. Yasumitsu Baba, Dr. Takuya Ikeda, and all of the staff at the Clinical Laboratory of Kagoshima City Medical Association for collecting the E. coli strains.

Financial support: This study was supported by the Research Fund of the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 12470168).

Authors’ addresses: Naoko Wakimoto, Junichiro Nishi, Jav Sarantuya, Mayumi Iwashita, Kunihiro Manago, Koichi Tokuda, Masao Yoshinaga, and Yoshifumi Kawano, Department of Pediatrics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan, Telephone: 81-99-275-5354, Fax: 81-99-265-7196, E-mail: nishi1{at}m2.kufm.kagoshima-u.ac.jp. Jalaluddin Sheikh and James P. Nataro, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201.

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