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

• Published in: Journal of applied microbiology 2010-07, Vol.109 (1), p.35-43
• Main Authors: Nara, J.M., Cianciarullo, A.M., Culler, H.F., Bueris, V., Horton, D.S.P.Q., Menezes, M.A., Franzolin, M.R., Elias, W.P., Piazza, R.M.F.
• Format: Article
• Language: English
• Subjects: Animals; Assaying; Bacterial Typing Techniques - methods; Biological and medical sciences; bundle‐forming pilus; Calcium chloride; Cellulose esters; Cellulose nitrate; Colonies; Differentiation; E coli; Enterobacteriaceae; enteropathogenic E. coli; Enteropathogenic Escherichia coli - classification; Enteropathogenic Escherichia coli - isolation & purification; Escherichia coli; Ethylenediaminetetraacetic acids; expression; Fimbriae, Bacterial - chemistry; Fundamental and applied biological sciences. Psychology; Immunoblotting; Immunoblotting - methods; immunodetection; Immunoelectron microscopy; Microbiology; Pili; rabbit polyclonal antiserum; Rabbits; Salts; Sodium chloride
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2009.04625.x

Aims:  The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle‐forming pilus (BFP) expression. Methods and Results:  Anti‐BFP antiserum was raised in rabbits and its reactivity 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. Conclusion:  The assay enables reliable identification of BFP‐expressing isolates and contributes to the differentiation of typical and atypical EPEC. Significance and Impact of the Study:  The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.