The Effects of Indicator Organism Type on Phenotypic Characterization of Host-Specificity and the Implications for Microbial Source Tracking

The objective of this study was to investigate the effects of indicator organism type on the host specificity of fatty acid methyl ester (FAME) profiles. For that purpose, the FAME profiles of enterococcus, Escherichia coli, fecal coliform (FC), and total coliform (TC) isolates cultured from fecal samples of 5 hosts (cow, swine, poultry, deer, and water fowl) and sewage were developed. These four known-source libraries, one library for indicator organism, contained the FAME profiles of 1,733 isolates cultured from 250 samples collected over a period of 40 months. The known-source library of enterococcus included 511 isolates from 76 samples, while that of E. coli was comprised of 605 isolates from 78 samples. The fecal coliform known-source library size was 314 isolates from 48 samples and TC library included 303 isolates from 48 samples. The statistical significance of the host-specific differences were determined using discriminant analysis (DA), where percentages of isolates correctly classified into their respective host categories were calculated for each of the known-source libraries. Several different DA scenarios were tested for each known-source library including ones where the isolates from similar sources were pooled into larger groups. Overall, 17 different FAMEs were identified from 1,733 isolates. Eight of these FAMEs were common in four indicator organisms. The qualitative comparison of FAMEs showed that only TC and fecal coliform FC isolates carried signature FAMEs, FAMEs associated exclusively with only one host group. Most notably, three hydroxy FAMEs extracted from a significant fraction of FC isolates were associated exclusively with sewage samples. Similarly, two hydroxy FAMEs were found only in some of the TC isolates cultured from sewage. The host specific differences among E. coli and enterococcus isolates were solely quantitative differences; that is, no signature FAME was identified and variations in relative masses of FAMEs common among different host groups were the only host specific differences. The most accurate classification of known-source isolates into their respective hosts was achieved when FC and TC were used as indicator organisms. The lowest accuracy of classification was within E. coli isolates. Particularly, 95% accuracy of discriminating FC isolates of sewage origin against those of livestock and wildlife is significant. The accuracy increased to 100% when FC isolates were pooled into human versus non-human categories. These results suggests that E. coli may not be best choice of indicator organisms when FAME profiling is used for microbial source tracking and subsequent bacterial total maximum daily load (TMDL) allocations.