Cultivation-Independent Analysis of Bacteria in IDEXX Quanti-Tray/2000 Fecal Indicator Assays

Abstract

Monitoring microbiological water quality is important for protecting water resources and the health of swimmers. Routine monitoring relies on cultivating fecal indicator bacteria (FIB), frequently using defined substrate technology. Defined substrate technology is designed to specifically enrich for FIB, but a complete understanding of the assay microbiology requires culture-independent analysis of the enrichments. This study aimed to identify bacteria in positive wells of Colilert and Enterolert Quanti-Tray/2000 (IDEXX Laboratories) FIB assays in environmental water samples and to quantify the degree of false-positive results for samples from an urban creek by molecular methods. Pooled Escherichia coli-and Enterococcus-positive Quanti-Tray/2000 enrichments, either from urban creek dry weather flow or municipal sewage, harbored diverse bacterial populations based on 16S rRNA gene sequences and terminal restriction fragment length polymorphism analyses. Target taxa (coliforms or enterococci) and nontarget taxa (Vibrio spp., Shewanella spp., Bacteroidetes, and Clostridium spp.) were identified in pooled and individual positive Colilert and Enterolert wells based on terminal restriction fragments that were in common with those generated in silico from clone sequences. False-positive rates of between 4 and 23% occurred for the urban creek samples, based on the absence of target terminal restriction fragments in individual positive wells. This study suggests that increased selective inhibition of nontarget bacteria could improve the accuracy of the Colilert and Enterolert assays.Quantifying fecal pollution in recreational waters is important for protecting the health of swimmers. Current standards for microbiological water quality in the United States and elsewhere are based on culturable fecal indicator bacteria (FIB), i.e., total coliforms, fecal coliforms, or Escherichia coli, and enterococci (21,53,57). Various methods are used to quantify FIB, including multiple-tube fermentation, membrane filtration, and defined substrate technologies (9,16,45). All rely on temperature, substrate, and selective growth inhibitors to select for FIB (23,29). The commercially available defined substrate technologies Colilert and Enterolert (IDEXX Laboratories, Westbrook, ME) are accepted by the U.S. Environmental Protection Agency as alternatives to the multiple-tube fermentation and membrane filtration methods for fresh, marine, and estuarine surface waters (54). Specific enzyme-substrate relationships are the basis of both assays (45). In the Colilert assay, total coliforms and E. coli are indicated by a yellow or a yellow and a fluorescent metabolite, respectively. Positive Enterolert assays are indicated by a fluorescent metabolite. The manufacturer-supplied tray format is more convenient than multiple-tube fermentation and membrane filter techniques (20, 45). However, there is evidence that the Colilert and Enterolert assays are not specific (2,6,17,43,51).False-positive Colilert or Enterolert readings could occur when there i...

Holding-time and method comparisons for the analysis of fecal-indicator bacteria in groundwater

“…The bacterial communities in groundwater are likely much different than those found in surface water. In a more recent surface water study, Sercu et al (2011) found noncoliform bacteria, such as Lactobacillales and Clostridiales, which exhibit enzyme activity causing a positive reaction in the Enterolert method. Disagreement between the results of the two methods may be due to the presence of bacteria producing false-positive results by one method.…”

Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

“…While the use of multiple volumes can be mathematically equivalent to using serial dilutions, most work in this area has treated each dilution separately. 23–29,33 Combining results from wells of multiple volumes increases accuracy and can be achieved using the maximum likelihood estimate (MLE), or Most Probable Number (MPN) theory 42–49 as it is known in culture-based bacterial quantification systems. Three replicates (i.e.…”

“…wells) at three dilutions has long been the standard for this method, though it has been expanded to include increased numbers of wells to improve quantification using multivolume approaches. 47–49 Such a “three replicates at three dilutions” approach has been used to perform “MPN-PCR” based quantification, 13–15 although current digital PCR systems 25–32 have used only elements of MPN for analysis 31,32 of serial dilution experiments. Microfluidic devices for multivolume digital PCR, and appropriate statistical methods for proper analysis, have not been previously developed.…”

Comparison ofEnterococcusSpecies Diversity in Marine Water and Wastewater Using Enterolert and EPA Method 1600

“…[10]. False positive rates have been found to vary between 4% and 26% for Enterolert [11] and between 11% and 26% for mEI agar [10]. …”