Differentiation of F18ab+ from F18ac+ Escherichia coli by Single-Strand Conformational Polymorphism Analysis of the Major Fimbrial Subunit Gene (fedA)

Toxin-producing Escherichia coli expressing F18 fimbriae colonizes the small intestines of weaned pigs and causes diarrhea, edema disease, or both. The F18 family is composed of two antigenic variants, F18ab and F18ac. Because many strains do not express F18 fimbriae in vitro, identification and differentiation of these two variants are difficult. Single-strand conformational polymorphism (SSCP) analysis is a rapid method for identifying genetic mutations and polymorphisms. The F18 major fimbrial subunit genes (fedA) of 138 strains were amplified by PCR, and genetic differences were detected by SSCP analysis. The SSCP analysis of the fedA gene differentiated F18ab⁺ strains from F18ac⁺ strains. Most strains classified as F18ab⁺ by SSCP analysis contained Shiga toxin 2e and enterotoxin genes. Most strains classified as F18ac⁺ by SSCP analysis contained only enterotoxin genes. The SSCP analysis was a useful method for predicting the antigenicity of F18⁺ E. coli and could also be used for analysis of other virulence genes in E. coli and other pathogenic bacteria.Enterotoxigenic Escherichia coli (ETEC) and E. coli organisms that produce Shiga toxin 2e (STEC) colonize the porcine small intestine and cause diarrhea and edema disease, respectively. The fimbrial adhesins of K99, F41, K88, and 987P fimbriae mediate adherence and promote ETEC colonization of the neonatal pig’s small intestine. Of these four fimbriae, only K88 is frequently detected in ETEC isolated from both weaned and neonatal pigs (24). The F18 fimbria mediates colonization of both ETEC and STEC in weaned, but not neonatal, pigs. The F18 fimbrial family is composed of two antigenic variants, F18ab and F18ac, and has been previously referred to as F107, 2134P, Av24, and 8813 (1–4, 11, 14, 15, 19, 20, 25).Differentiation of strains expressing F18ab from those expressing F18ac may be important in development and selection of effective vaccines for ETEC and STEC infections in weaned pigs. Differentiation of strains producing F18ab and F18ac is also important because of a correlation between the type of toxin produced and clinical sequelae in infected swine. F18ab⁺ strains are generally STEC and are associated with edema disease, while F18ac⁺ strains are generally ETEC and are associated with diarrhea (4, 15, 26). Monospecific polyclonal antisera and the monoclonal antibody 6C7/C1, which is specific for F18ac⁺ strains, can differentiate between these two antigenic variants (3, 4, 15, 19). However, serologic differentiation is not always possible because many strains do not express F18 when cultured in vitro under standard culture conditions (1, 8, 25, 26). The gene encoding the major fimbrial subunit of F18 (fedA) in both F18ab⁺ and F18ac⁺ strains has been sequenced, and differences have been found (9). A PCR-restriction fragment length polymorphism (RFLP) test consisting of amplification of the fedA gene followed by digestion with the restriction enzyme NgoMI has been used to differentiate F18ab⁺ from F18ac⁺ strains (9, 15, 19). This PCR-RFLP test avoided the problems of serologic differentiation, which requires in vitro pilus expression, and was based upon the DNA sequences of seven different fedA genes, fedA and fedA.1 to fedA.6 (9). These seven different sequences were determined by sequencing of the fedA genes of only 10 unique strains (9), demonstrating that the fedA gene is highly polymorphic.Single-strand conformational polymorphism (SSCP) analysis can rapidly identify polymorphisms in a gene and is useful when a large number of samples are being analyzed. Single-strand DNA migrates according to size and shape in a nondenaturing gel. The shape is dependent upon folding due to intermolecular interactions which are DNA sequence dependent (5). The major objective of this study was to determine if SSCP analysis of the fedA gene could differentiate F18ab⁺ from F18ac⁺ strains.