Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP...

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Published in:	Applied and Environmental Microbiology Vol. 70; no. 8; pp. 4478 - 4485
Main Authors:	Johnson, L.K, Brown, M.B, Carruthers, E.A, Ferguson, J.A, Dombek, P.E, Sadowsky, M.J
Format:	Journal Article
Language:	English
Published:	Washington, DC American Society for Microbiology 01-08-2004
Subjects:	Animals Biological and medical sciences Cats Cattle Comparative analysis DNA fingerprinting DNA Fingerprinting - methods DNA primers Dogs Escherichia coli Escherichia coli - classification Escherichia coli - genetics Escherichia coli - isolation & purification Escherichia coli Infections - microbiology Feces Feces - microbiology Fundamental and applied biological sciences. Psychology Gene Library Genetic diversity Genetic Variation Genotype Genotype & phenotype horizontal fluorophore enhanced repetitve extragenic palindromic polymerase chain reaction Humans Image Processing, Computer-Assisted livestock Microbiology polymerase chain reaction Polymerase Chain Reaction - methods Public Health Microbiology Repetitive Sequences, Nucleic Acid - genetics Sample Size strain differences strains Water Pollution wild animals Human Natural population Sample Escherichia coli Genotype Genetic diversity Coliforms Animal Pollution source Bacteria Isolate Feces Enterobacteriaceae
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Summary:	A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 Present address: Minnesota Department of Agriculture, St. Paul, MN 55107. Corresponding author. Mailing address: Department of Soil, Water, and Climate, University of Minnesota, 439 Borlaug Hall, 1991 Upper Buford Cir., St. Paul, MN 55108. Phone: (612) 624-2706. Fax: (612) 625-2208. E-mail: Sadowsky@soils.umn.edu.
ISSN:	0099-2240 1098-5336
DOI:	10.1128/AEM.70.8.4478-4485.2004