Characterisation of Enterococci in the Christchurch Oxidation Ponds

Sewage entering the Christchurch wastewater treatment plant at Bromley is treated sequentially through screening, primary sedimentation, trickling filters, contact tanks, and clarifiers, then through a series of six shallow oxidation ponds. The final effluent is discharged into the Heathcote estuary after a three-week flow period through the ponds. As the estuary is used for recreational purposes, the discharge is monitored twice weekly for levels of the indicator bacteria (faecal coliforms and enterococci) to ensure it complies with the discharge limits set by Environment Canterbury.

Between December 2005 and May 2006, unusually high levels of enterococci were detected in the final stage oxidation pond immediately prior to discharge into the estuary. Interestingly, these peaks occurred at a time when faecal coliform levels showed no increase, remaining well under the consent levels of less than 2000 colony forming units (cfu) / 100ml. The following questions were raised;

• Had these enterococci broken through from the treatment process (indicating human sewage contamination and a significant health risk)?
• Were the enterococci from the 20-30,000 wildfowl that occupy the ponds? or,
• Were the enterococci from some other source?

To investigate this further we undertook antibiotic resistance analysis (ARA) of the enterococci. ARA involves isolating individual enterococci from suspected sources then determining the ability of individual enterococci to grow on media containing 12 different antibiotics at up to four different concentrations. Statistical comparison of the antibiotic resistance profiles of populations of enterococci from different sources can allow the sources of these enterococci to be distinguished. This is an example of a library-based method.

MAR method

Antibiotic resistance profiles were generated on 520 enterococci from raw sewage entering the plant, 530 enterococci from wildfowl in the vicinity of the plant (Canadian geese, ducks), 50 enterococci from ponds during "normal" levels of enterococci, and 440 enterococci from final discharge pond during "high" levels of enterococci.

Over 400 different antibiotic resistance profiles were generated from enterococci from raw sewage and wildfowl, enabling these two populations to be distinguished from one another with average correct classification of 84%. In contrast, the enterococci from periods of high levels in the ponds had only 11 different antibiotic resistance profiles, with over half of the isolates having the same resistance profile. This very surprising observation suggested that the enterococci were not from either the human sewage or the wildfowl, but that they had potentially grown in the ponds! Genetic analysis of isolates using pulsed field gel electrophoresis supported the clonal nature of these isolates, and the conclusion that these enterococci may have grown in the ponds, rather than being faecally associated. Enterococci have now been removed from the consent conditions and replaced by E. coli, although levels are still monitored.

We published a paper describing this work which can be accessed following the link below.

Elaine Moriarty, Fariba Nourozi, Beth Robson, David Wood, and Brent Gilpin. (external link)
Evidence for Growth of Enterococci in Municipal Oxidation Ponds, Obtained Using Antibiotic Resistance Analysis.
Applied & Environmental Microbiology. 2008. 74, 7204-7210.