Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of Escherichia coli Isolates To Identify Nonpoint Fecal Sources

Abstract

Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.

Sample Size, Library Composition, and Genotypic Diversity among Natural Populations of Escherichia coli from Different Animals Influence Accuracy of Determining Sources of Fecal Pollution

“…Thus, there was a 21.7% reduction in the average rate of correct classification by using the unique DNA fingerprint library, relative to that seen with the complete library and less than we and others have previously reported with smaller libraries of E. coli strains containing duplicate DNA fingerprints from the same individual animal (4, 6, 23). Our results indicate that the clonal nature of E. coli (11,20,33) originating from the same source animal artificially biases the average rate of correct classification, alters the fidelity of the database, and overestimates the ability of the database to assign isolates to their correct source group. Influence of library size on usefulness of DNA fingerprint libraries.…”

Temporal variations in patterns ofEscherichia colistrain diversity and antimicrobial resistance in the migrant Egyptian vulture

“…Also, ribotyping is suitable in epidemiological studies for determining relatedness and discrimination of strains [25,26]. …”

Genetic Diversity of Escherichia coli Isolated from Urban Rivers and Beach Water

“…Studies have employed DNA fingerprinting to discern characteristic patterns and associations among E. coli strains in order to classify the strains according to the host from which they were derived (7,11,17,23,27,31). Overall, studies that have assessed the relationships among strains by either cluster analysis (17) or discriminant analysis using banding patterns (7,27) have found that strains from a host source do not form an exclusive group of genetically similar strains but rather can display a wide range of diversity.…”