Detection and identification of E. coli producing heat-labile enterotoxin type I by enzymatic amplification of a specific DNA fragment

Abstract

The polymerase chain reaction (PCR) was used to identify strains of Escherichia coli which produce heat-labile toxin type I (LTI). Amplification primers were designed to detect E. coli strains of human as well as porcine origin. This assay was used to test the ATCC 37218 strain, which carries a recombinant plasmid with the genetic information for production of porcine LTI (pLTI). In addition, three clinical E. coli isolates of human and one of porcine origin were tested. All clinical isolates were reported to produce heat-labile enterotoxin (hLTI and pLTI, respectively) when tested by the Y1 adrenal cell method and/or by the CHO cell method. All strains yielded the expected 275 bp DNA fragment after enzymatic amplification. This fragment was further identified by allele specific oligonucleotide hybridization. Alternatively, the fragment was identified by a SmaI restriction enzyme site which is present in the genes of both the E. coli isolated from humans and pigs. The detection limit determined in water with the ATCC 37218 strain was 20 bacteria. The amplified sequence included a CfoI polymorphism which allowed to distinguish between the genes coding for pLTI and hLTI. All of the strains tested showed this polymorphism as expected. Depending on the identification method chosen, SmaI digestion or oligonucleotide hybridization, pure water can be analysed within 8 h or 12 h, respectively. This method may be adapted to environmental and food samples.