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

Abstract

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.

Specific Properties of Enteropathogenic Escherichia coli Isolates from Diarrheal Patients and Comparison to Strains from Foods and Fecal Specimens from Cattle, Swine, and Healthy Carriers in Osaka City, Japan

“…Group I strains were further divided into subgroups Ia and Ib depending on whether they contained the gene with the strongest association with diarrhea, efa1 (lifA). The 14 pairs of primers (including three variants of lpfA) and the PCR conditions used in this study are listed in Table 1 (15)(16)(17)(20)(21)(22)(23)(24).…”

Molecular and Phenotypic Characterization of Escherichia coli O26:H8 among Diarrheagenic E. coli O26 Strains Isolated in Brazil

“…Moreover, EPEC strains may carry a large plasmid known as the EPEC adherence factor plasmid (pEAF) (5,6), which encodes the bundle-forming pilus (BFP) and plasmid-encoded regulator, a complex regulator of virulence genes (4,7). Thus, the EPEC pathotype has been subdivided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), with the basic difference being the presence and absence of pEAF and bfp, and BFP expression, respectively (4,(8)(9)(10).…”

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

“…Bacterial strains -Seventy-two aEPEC isolates were used in this study, all of which have been previously characterized by polymerase chain reaction (PCR) analysis as containing the eae gene and lacking the EAF plasmid, the bfpA gene and BFP expression (Bueris et al 2007, Abe et al 2009, Nara et al 2010. These isolates belong to a wide range of serotypes and exhibit distinct patterns of adhesion to HEp-2 cells (Abe et al 2009).…”