Use of repetitive DNA sequences and the PCR To differentiate Escherichia coli isolates from human and animal sources

Abstract

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.

FIG. 1

rep-PCR DNA fingerprint patterns of E. coli strains obtained from beef and dairy cows. (A) PCR DNA fingerprint patterns generated with primer BOX A1R. Lanes A and L contained an external standard, a 1-kb molecular weight ladder. (B) PCR DNA fingerprint patterns generated with primers REP 1R and REP 2I. Lanes A and L contained an external standard, a 1-kb molecular weight ladder. The E. coli strains used for the fingerprint analysis shown in panel B are identical to the strains used for the analysis shown in panel A, except that the strains in lanes O and T are reversed.

FIG. 2

Dendrogram showing the relatedness of E. coli strains isolated from humans, geese, ducks, sheep, pigs, chickens, and cows as determined by a PCR DNA fingerprint analysis performed with primer BOX A1R. Relationships were determined by using Jaccard similarity coefficients and the neighbor-joining clustering method.

FIG. 3

MANOVA of BOX-derived PCR DNA fingerprints from E. coli strains obtained from animal and human sources. Binary band-matching character tables were analyzed by MANOVA, accounting for the covariance structure. The E. coli isolates were obtained from humans (●), geese (□), ducks (○), sheep (⧫), pigs (■), chickens (▴), and cows (▵). The first discriminant is represented by the distance along the x axis, and the second discriminant is represented by the distance along the y axis.