PCR for detection of cdt-III and the relative frequencies of cytolethal distending toxin variant-producing Escherichia coli isolates from humans and cattle

Abstract

A PCR assay that uses primers whose sequences were obtained from the published sequence of the cdt-III gene was developed to determine the frequencies of the cdt-I, cdt-II, and cdt-III genes in Escherichia coli isolates from humans and animals. E. coli isolates producing cytolethal distending toxin (CDT) were infrequently detected. The cdt-I gene was preferentially detected in strains with the cnf1 gene, while the cdt-III gene was found in strains carrying the cnf2 gene. The cdt-III genotype was more prevalent in animal isolates, while the cdt-I and cdt-II genotypes were more evident in human isolates. The presence of further cdt gene variants was indicated by the presence of toxin activity in cell culture in the absence of PCR amplification of the cdt-I, cdt-II, or cdt-III gene.

FIG. 1.

Sensitivities of cdt-specific primers. (A) Different concentrations of boiled cell preparations. The isolates in lanes 1 to 6 were amplified with cdt-I-specific primers, the isolates in lanes 7 to 12 were amplified with cdt-II-specific primers, and the isolates in lanes 14 to 19 were amplified with cdt-III-specific primers. The numbers of boiled bacterial cells serving as templates for each lane were as follows: lanes 1, 7, and 14, 1.25 × 10⁵; lanes 2, 8, and 15, 1.25 × 10⁴; lanes 3, 9, and 16, 1.25 × 10³; lanes 4, 10, and 17, 1.25 × 10²; lanes 5, 11, and 18, 12.5; lanes 6, 12, and 19, 1.25. Lane 13, 100-bp ladder. (B) Different concentrations of extracted DNA preparations. The isolates in lanes 2 to 7 were amplified with cdt-I-specific primers, the isolates in lanes 8 to 13 were amplified with cdt-II-specific primers, and the isolates in lanes 14 to 19 were amplified with cdt-III-specific primers. The amounts of DNA in each lane were as follows: lanes 2, 8, and 14, 500 ng; lanes 3, 9, and 15, 50 ng; lanes 4, 10, and 16, 5 ng; lanes 5, 11, and 17, 0.5 ng; lanes 6, 12, and 18, 0.05 ng. Lanes 7, 13, and 19, negative controls containing water instead of the DNA template used for each PCR; lanes 1 and 20, 100-bp ladders.