Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression

ABSTRACT

The aim of study was to develop a colony immunoblot assay to differentiatetypical from atypical enteropathogenic Escherichia coli (EPEC) by detectionof bundle-forming pilus (BFP) expression. Anti-BFP antiserum was raised in rabbits and itsreactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. The assay enables reliable identification of BFP-expressing isolatesand contributes to the differentiation of typical and atypical EPEC.The colony immunoblot for BFP detectiondeveloped in this study combines the simplicity of an immunoserologicalassay with the high efficiency of testing a large number of EPECcolonies.