Seasonal variation in accurate identification of Escherichia coli within a constructed wetland receiving tertiary-treated municipal effluent

Authors: McLain, Jean; Williams, Clinton

Submitted to: Water Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2008
Publication Date: 5/20/2008
Citation: Mclain, J.E., Williams, C.F. 2008. Seasonal variation in accurate identification of Escherichia coli within a constructed wetland receiving tertiary-treated municipal effluent. Water Research. doi:10.1016/j.watres.2008.06003. 42:4041-4048

Interpretive Summary: There is an increasing trend worldwide to require more efficient use of water resources and thus, a large number of communities are now utilizing reclaimed water, an increasingly safe and reliable water source. In the United States alone, an estimated 1.7 billion gallons (6.4 million m3) per day of wastewater is reused. With the growth in use of recycled water, accurate monitoring of water quality, including the presence of E. coli, increases in importance. Commercially available enzyme-based chromogenic media are often used for detection of E. coli, both because they are easy for water utility workers to use and interpret, and because they provide rapid results. The unique chemistry of reclaimed waters, however, could potentially hinder accurate detection of E. coli using this enzyme-based technology. Over seven months, we monitored biological and chemical water quality in a constructed wetland filled with reclaimed municipal wastewater. E. coli levels were quantified using m-Coli Blue 24® broth, a commercially available assay. Confirmatory testing of E. coli isolates revealed a 41% rate of false positive identification using the chromogenic broth over the sampling period. Seasonal differences were evident, as false positive rates averaged 32% during warm summer months, but rose sharply to 75% in the late fall and winter. Water temperature was among the variables significantly correlated to the false-positive identification of E. coli, confirming the observed seasonal trend. This is the first study to report that false positive rates using chromogenic media may be strongly affected by seasonal changes in water variables. This work also highlights the extreme importance of validation of microbiological results from chromogenic media for accurate analysis of reclaimed water quality.

Technical Abstract: The reuse of municipal wastewater has been escalating worldwide as a means to extend increasingly limited water supplies. With the growth in use of recycled water, accurate monitoring of water quality parameters, including Escherichia coli (E. coli), increases in importance. Chromogenic media, because they are easy to use and provide rapid sample analysis, are often used for detection of E. coli in environmental samples. The presence of unique levels of organic and inorganic compounds, however, alters the chemistry of reclaimed waters, potentially hindering accurate detection of E. coli using enzyme-based chromogenic technology. Over seven months, we monitored biological and chemical water quality in a constructed wetland filled with tertiary-treated municipal effluent. E. coli levels were quantified using m-Coli Blue 24® broth, a commercially available assay. No E. coli were isolated in the wetland source waters, but E. coli, total coliforms, and heterotrophic bacteria each increased dramatically within the wetland on all sampling dates, most probably due to fecal inputs from resident populations of waterfowl and mammals. Confirmatory testing of isolates presumptive for E. coli by culturing and polymerase chain reaction (PCR) revealed a 41% rate of false positive identification using m-Coli Blue 24® broth over the entire sampling period. Seasonal differences were evident, as false positive rates averaged 35% (35 of 100 colonies) during warm summer months, but rose sharply to 75% (75 of 100 colonies) in the late fall and winter. During the cooler months, the heterotrophic plate count was most closely correlated to the identification of false positives, indicating that competition from non-target bacteria contributed to the failure of the broth to identify E. coli. This is the first study to report that false positive rates using chromogenic media may show strong seasonal differences. This work also highlights the extreme importance of validation of microbiological results from chromogenic media for accurate analysis of reclaimed water quality.