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Quantitative biofilm assay using microtiter plate to screen for enteroaggregative Escherichia coli

Quantitative biofilm assay using microtiter plate to screen for enteroaggregative Escherichia coli

Abstracts of the General Meeting of the American Society for Microbiology 103: C-367

Background: Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen defined by an aggregative adherence to HEp-2 cells. The gold standard for EAEC identification remains the HEp-2 adherence test. However, this test requires specialized facilities and can only be conducted in reference laboratories. Although a DNA probe CVD432 and an aggR-PCR method have been used to simplify the identification, some EAEC isolates are probe- or aggR-negative. EAEC is known to produce a bacterial film on the polystyrene surface. Recently, Sheikh and Nataro reported the quantitative analysis of EAEC biofilms. We applied this method to screening for EAEC. Objective: To evaluate the usefulness of the quantitative biofilm assay to screen for EAEC in clinical E. coli isolates. Methods: A total of 555 E. coli strains from sporadic diarrheal children were examined for biofilm and the aggR gene. For quantitative biofilm assay, bacteria were incubated overnight (18h) in DMEM with 0.45% glucose in flat-bottom polystyrene microtiter plate. The plate was stained with crystal violet after washing. Biofilm was quantified in duplicate using ELISA plate reader at 570nm. EAEC 042 was used as a positive control. All the strains showing OD570 >0.1 and 48 strains less than 0.1 were examined for HEp-2 adherence test. The aggR gene was evaluated by PCR. Results: The examined strains showed absorbance in the range from 0 to 2.02, while EAEC 042 showed 1.75. The strains were classified into three groups according to the absorbance: group A (0.2<), 31 strains; B (0.1apprx0.2), 32; C (<0.1), 492. The incidences of EAEC in the A and B group were 64.5% (20/31) and 3.1% (1/32), respectively. Forty-eight strains examined for HEp-2 assay in the group C were not EAEC. The incidences of aggR were as follows: A, 32.3% (10/31); B, 0% (0/32); C, 0.4% (2/492). All aggR-positive strains in the group A were EAEC, while two in the group C were not. Ten aggR-positive and eleven aggR-negative EAEC strains were screened by this assay. Conclusion: Although the confirmation by HEp-2 assay is needed to identify EAEC, the quantitative biofilm assay using microtiter plate is useful to screen for EAEC.

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