Rapid tests for detection and quantitation of Enterococcus contamination in recreational waters

Abstract

Presently, growth-based tests are used for the detection and quantitation of microbiological contaminants in the environment. These tests take a minimum of 24 h to generate a result, which compromises the ability to take the most appropriate action. This report describes a rapid test for Enterococcus in recreational water as an indicator of faecal contamination. This method involves (1) isolation and lysis of the target organism, (2) purification of ribosomal RNA (rRNA) from the lysate and (3) amplification and detection of the purified rRNA. rRNA is used as the target since, in contrast to DNA, there are hundreds to thousands of copies in the cell. The rRNA is purified from the lysate by target capture onto magnetic microspheres, which removes interfering substances present in the sample. The rRNA is then quantitated using transcription-mediated amplification (TMA) with real-time homogeneous detection of amplicon using a fluorescent oligonucleotide probe. Compared to polymerase chain reaction (PCR) amplification, TMA is isothermal, more rapid, and ideally suited to RNA detection. The test described here demonstrates sensitive detection and quantitation of enterococci over a wide dynamic range with a high level of analytical specificity. The latter is particularly important for accurate and relevant monitoring both for protecting public health and for source tracking. Many conventional microbiological tests are time-consuming, exhibit limited dynamic range and are known to lack specificity. This assay demonstrates the advantages achievable by the application of TMA of rRNA targets to current environmental testing challenges.

Choices in Recreational Water Quality Monitoring: New Opportunities and Health Risk Trade-Offs

“…The need for a more rapid estimate of water quality has been repeatedly emphasized by beach mangers, public health officials, and the scientific community. ,,, As a result, U.S. EPA’s recently released 2012 RWQC include the option of monitoring for QENT, a rapid, molecular test for enterococci that can be completed in 4–6 h . Several studies have explored the sample analytical inhibition and related problems associated with the qPCR analysis and have expressed some concern about its relationship to standard culturable indicators, its cost, and its practicality for monitoring programs. , Only a few studies, however, have explored the human health risk end point in relation to the monitoring options available. , The epidemiological studies conducted as part of establishing the 2012 RWQC found a link between QENT concentrations and gastrointestinal illness, ,, but these studies were all conducted at point source impacted beaches.…”

Comparison of Transcription-Mediated Amplification and Growth-Based Methods for the Quantitation of Enterococcus Bacteria in Environmental Waters

“…The optimization and development of the TMA method for the detection of Enterococcus deliberately spiked into seawater have been described previously (3), and the present study expands on this research to apply the method to a wide range of ambient environmental water samples. The goal of this study was to compare the results obtained with the TMA assay to results obtained with two of the most commonly used reference methods for quantitation of Enterococcus bacteria in water samples, Enterolert (Idexx Laboratories, Inc., Westbrook, ME) and U.S. EPA method 1600 (6), in estuarine, brackish, and storm water samples.…”

“…The TMA assay has been reported previously to have a good analytical specificity and does not cross-react with the species that are known to cause false positives in the U.S. EPA method. The main advantages of the TMA assay are the rapid time to result (approximately 4 h) and the high level of analytical specificity (3). The data presented here show that TMAbased methods hold promise for use in the future to accurately quantify fecal indicator bacteria in recreational waters.…”

“…This assay allows highly sensitive and specific quantitation using a standard curve to derive the equivalent of CFU-per-100-ml values. The assay does not detect nonenterococcal organisms that are known to cause false-positive results in some of the U.S. EPAapproved growth-based methods (2) or Enterococcus species that are underrepresented in human feces (3).…”

Investigation of transcription-mediated amplification as a rapid test method for Enterococci in recreational water

“…Several studies 8,9,10 have demonstrated that not all ''positive'' isolates from growth-based methods are Enterococci and that such methods are positive for all Enterococcus species. By contrast, it is known that this TMA method is more specific 15 and therefore it might be expected that it would yield a lower rate of positive results than less specific methods in situations where a wide spectrum of microorganisms exists. It is known that storm drains discharge a complex mixture of chemicals and microorganisms and one might anticipate greater variability in the results of such samples.…”