DIRECT-DETECTION OF EAE-POSITIVE BACTERIA IN HUMAN AND VETERINARY COLORECTAL SPECIMENS BY PCR

A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous s equences in enteropathogenic Escherichia call and Citrobacter spp, (am plification of eae open reading frame, 178 bp) in sections of the inte stines of humans and animals with colonic lesions, Positive PCR result s were observed with eae-positive reference strains off. coli and Citr obacter rodentium (Citrobacter freundii biotype 4280). Known eae-negat ive reference strains off. call and other laboratory strains of enteri c bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli and C. rodentium was be tween 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were mo dified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extran eous bacterial DNA from the PCP amplification cocktail without added t emplate, Sections of colon from three pigs experimentally affected wit h colon lesions due to enteropathogenic (attaching and effacing) E. ca ll were PCR positive for bacterial eae genome. Sections from control a nimals were negative. Sections of colon from one of 18 biopsies from c onfirmed AIDS patients and from 22 of 35 colorectal cancer patients we re PCR positive for bacterial eae genome, The PCR test was a simple an d quick method of detecting bacterial eae genome in human and veterina ry clinical specimens. This method may remove the need for initial cul ture and detection of the gene by DNA probing from potential associate d lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar rel ationship is possible for human patients having similar lesions.