Comparison of Enterococcus Species Diversity in Marine Water and Wastewater Using Enterolert and EPA Method 1600

EPA Method 1600 and Enterolert are used interchangeably to measure Enterococcus for fecal contamination of public beaches, but the methods occasionally produce different results. Here we assess whether these differences are attributable to the selectivity for certain species within the Enterococcus group. Both methods were used to obtain 1279 isolates from 17 environmental samples, including influent and effluent of four wastewater treatment plants, ambient marine water from seven different beaches, and freshwater urban runoff from two stream systems. The isolates were identified to species level. Detection of non-Enterococcus species was slightly higher using Enterolert (8.4%) than for EPA Method 1600 (5.1%). E. faecalis and E. faecium, commonly associated with human fecal waste, were predominant in wastewater; however, Enterolert had greater selectivity for E. faecalis, which was also shown using a laboratory-created sample. The same species selectivity was not observed for most beach water and urban runoff samples. These samples had relatively higher proportions of plant associated species, E. casseliflavus (18.5%) and E. mundtii (5.7%), compared to wastewater, suggesting environmental inputs to beaches and runoff. The potential for species selectivity among water testing methods should be considered when assessing the sanitary quality of beaches so that public health warnings are based on indicators representative of fecal sources. 1. Introduction EPA Method 1600 and Enterolert (IDEXX, Westbrook, ME, USA) are two EPA-approved methods that are often used to measure Enterococcus for recreational bathing water quality assessments [1]. The methods have been used interchangeably for (1) regulatory monitoring to detect possible fecal contamination of water; (2) epidemiology studies to correlate swimmer’s illness rates with densities of Enterococci in water, and (3) microbial source tracking studies to reduce fecal inputs to protect public health. A number of studies have found that these two methods generally produce comparable results [2–5]. However, several authors have found that the results may be markedly different [6–9]. There are several reasons, other than sample variability, that may explain the inconsistency between methods. EPA Method 1600 is a membrane filtration approach, where water is passed through a membrane that is subsequently placed atop Enterococcus Indoxyl-β-D-glucoside (mEI) agar and, following incubation, examined for colonies with blue halos. Enterolert is a defined substrate methodology that measures a fluorescent endpoint based