An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Enterococcus fecal indicator bacteria in environmental water samples. The results generated by this and two growth-based methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the three methods are in good qualitative agreement when tested against a range of water samples taken from different locations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection to result) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods

Blackwood, A. D.

Buttigieg, N. D.

Hogan, J. J.

Kirs, M.

Morgan, R. R.

Morris, C. A.

Noble, R. T.

Weeks, I.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2008, p. 3319–3320 Vol. 74, No. 100099-2240/08/$08.000 doi:10.1128/AEM.02623-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.Comparison of Transcription-Mediated Amplification and Growth-BasedMethods for the Quantitation of Enterococcus Bacteriain Environmental WatersCeri A. Morris,1* A. Denene Blackwood,2 Marek Kirs,2 Neil D. Buttigieg,1 Rhian R. Morgan,1James J. Hogan,3 Ian Weeks,1 and Rachel T. Noble2MLT Research Ltd., 5 Chiltern Close, Cardiff CF14 5DL, United Kingdom1; University of North Carolina at Chapel Hill,Institute of Marine Sciences, Morehead City, North Carolina 285572; and Gen-Probe Incorporated,10210 Genetic Center Drive, San Diego, California 921213Received 20 November 2007/Accepted 23 March 2008An assay based on transcription-mediated amplification (TMA) technology was used to quantitate Entero-coccus fecal indicator bacteria in environmental water samples. The results generated by this and two growth-based methods relative to the 104 most-probable-number or CFU-per-100-ml threshold show that the threemethods are in good qualitative agreement when tested against a range of water samples taken from differentlocations. The results demonstrate sensitive and rapid detection (approximately 4 h from sample collection toresult) and quantitation of Enterococcus bacteria compared to the results with the growth-based methods.Enterococcus is the recommended fecal indicator bacteriumfor monitoring the presence of fecal contamination in recre-ational waters (5). Standards for the maximum acceptable levelof Enterococcus bacteria in recreational waters have been es-tablished by the U.S. EPA (U.S. Environmental ProtectionAgency). The most common single-sample standard used formanaging recreational waters is 104 most probable number(MPN) or CFU per 100 ml. Presently, most regulatory agenciesworldwide use traditional growth-based tests, such as mem-brane filtration and multiple-tube fermentation, for the detec-tion and quantitation of fecal indicator bacteria in water sam-ples. These tests typically require 18 to 24 h to yield results.This report describes a rapid test for the quantitation ofEnterococcus rRNA that yields a result within 4 h. The methodinvolves filtration of the water sample, lysis of the target or-ganism, and purification of rRNA using magnetic-particle tar-get capture, followed by amplification of the purified rRNAusing transcription-mediated amplification (TMA) technology.Homogeneous, real-time detection of amplicons is achieved byusing a fluorescent oligonucleotide probe.TMA technology is both isothermal and rapid. This assayallows highly sensitive and specific quantitation using a stan-dard curve to derive the equivalent of CFU-per-100-ml values.The assay does not detect nonenterococcal organisms that areknown to cause false-positive results in some of the U.S. EPA-approved growth-based methods (2) or Enterococcus speciesthat are underrepresented in human feces (3).The optimization and development of the TMA method forthe detection of Enterococcus deliberately spiked into seawaterhave been described previously (3), and the present study ex-pands on this research to apply the method to a wide range ofambient environmental water samples. The goal of this studywas to compare the results obtained with the TMA assay toresults obtained with two of the most commonly used referencemethods for quantitation of Enterococcus bacteria in watersamples, Enterolert (Idexx Laboratories, Inc., Westbrook,ME) and U.S. EPA method 1600 (6), in estuarine, brackish,and storm water samples.Different types of water samples were collected to obtainsamples with various Enterococcus concentrations, environ-mental parameters, ionic characteristics, and high-molecular-weight organic matter levels. In total, 77 water samples werecollected from a variety of sites in North Carolina, the UnitedStates, during different seasons. Water samples were collectedfrom several geographic locations and depths on the NeuseRiver Estuary, along with water samples from nearby creeksand storm drains (n  55). Storm water samples (n  22) wereincluded to have samples with expected higher Enterococcusconcentrations.Samples were collected and filtered as prescribed by U.S.EPA method 1600 (6). For the TMA assay, each water sample(100 ml) was filtered and extracted in triplicate and each ex-tract was subsequently amplified in triplicate, yielding nineresults per original water sample (3). The Enterolert assay(results reported in MPN per 100 ml) and U.S. EPA method1600 (reported in CFU per 100 ml) were performed accordingto the manufacturer’s instructions (4) and according to U.S.EPA guidelines (6), respectively. The sensitivities and specific-ities of the TMA assay and the Enterolert assay were estimatedby comparing the test results to the U.S. EPA method 1600results. In this study, the U.S. EPA-recommended threshold of104 MPN or CFU per 100 ml of water (1) for a single samplewas used to define a sample as positive or negative, though themethod is applicable to any threshold determined by a givenauthority.A series of solutions of purified Enterococcus faecalis rRNA(1 to 1,000,000 fg) were assayed in triplicate with the TMAassay with every experiment to create a standard curve. Inter-* Corresponding author. Mailing address: Research and Develop-ment, MLT Research Ltd., 5 Chiltern Close, Cardiff Industrial Park,Cardiff CF14 5DL, United Kingdom. Phone: 44 (0)29 20747033. Fax:44 (0)29 20747118. E-mail: ceri.morris@mltresearch.com. Published ahead of print on 31 March 2008.3319assay regression parameters and precision data for 12 curvesare shown in Table 1. The standard curve for each experimentwas used to estimate the amount of rRNA per amplificationreaction. In the first instance, environmental samples werecollected from Neuse creek (n  8) and measured using TMA,Enterolert, and U.S. EPA method 1600. Where Enterolert andthe U.S. EPA method gave the same qualitative results (pos-itive or negative against the 104 CFU threshold), the data wereused to calculate a conversion factor. One CFU per 100 ml ofEnterococcus bacteria corresponded to 5 fg of rRNA per re-action, which was in reasonable agreement with the conversionfactor previously generated from laboratory-generated sam-ples (1 CFU per 100 ml corresponded to 3 fg rRNA perreaction) (3). This factor was subsequently used to convertfemtograms to CFU per 100 ml. TMA and U.S. EPA method1600 results of 10 CFU were assigned a value of 10 to beconsistent with the stated lower limit of quantitation of theEnterolert assay (when using the manufacturer-recommended1:10 dilution).In the 55 water samples from estuarine and brackish waters,the Enterococcus concentrations ranged from 10 to 2,867 CFUper 100 ml with a mean value of 92 CFU per 100 ml (asdetermined by U.S. EPA method 1600). In the 22 storm watersamples, the Enterococcus concentrations ranged from 10 to57,000 CFU per 100 ml with a mean value of 12,659 CFU per100 ml.The U.S. EPA method 1600, TMA assay, and Enterolertassay qualitative results are summarized in Table 2. The sen-sitivity and specificity of the TMA assay were 91.7% (22/24)and 96.2% (51/53), respectively, giving an overall accuracy of94.8% (73/77) (relative to the results of U.S. EPA method1600). Enterolert demonstrated a sensitivity of 83.3% (20/24)and a specificity of 96.2% (51/53), giving an overall accuracy of92.2% (71/77).The data were tested for normality using the D’Agostino andPearson omnibus test and found to be nonparametric. Directquantitative comparison of the TMA assay results to the En-terolert assay and U.S. EPA method 1600 data yielded Spear-man’s rank correlation coefficient (r) values of 0.82 and 0.88,respectively. The comparison of the Enterolert assay and U.S.EPA method 1600 data gave an r value of 0.92 (Prism version4.03).Conclusion. Comparison of the results generated by thethree methods relative to the 104 MPN or CFU-per-100-mlthreshold shows that the three methods are in good qualitativeagreement when tested against a range of environmental watersample types varying in salinity, depth, organic-matter constit-uents, and total suspended solids.The data generated in this study were compared to the U.S.EPA method 1600 as the current gold-standard method; how-ever, this method is reported to have between 6 and 26%false-positive results (2, 6), with Streptococceae species, such asS. bovis, and Aerococcus species appearing identical to Entero-coccus on membrane-Enterococcus indoxyl-D-glucoside agar(6). The TMA assay has been reported previously to have agood analytical specificity and does not cross-react with thespecies that are known to cause false positives in the U.S. EPAmethod. The main advantages of the TMA assay are the rapidtime to result (approximately 4 h) and the high level of ana-lytical specificity (3). The data presented here show that TMA-based methods hold promise for use in the future to accuratelyquantify fecal indicator bacteria in recreational waters.This research was funded by a grant from Molecular Light Technol-ogy Research Limited, Cardiff, Wales, United Kingdom. MLT Re-search Ltd. is a subsidiary of Gen-Probe Incorporated.All probes and primers were generously donated by Gen-ProbeIncorporated, San Diego, CA.REFERENCES1. California Department of Health Services. 1999. Assembly Bill 411, Statutesof 1997, Chapter 765. California Department of Health Services, Sacramento,CA. http://www.dhs.ca.gov/ps/ddwem/beaches/ab411_1999report.htm.2. Ferguson, D. M., D. F. Moore, M. A. Getrich, and M. H. Zhowandai. 2005.Enumeration and specification of enterococci found in marine and intertidalsediments and coastal water in southern California. J. Appl. Microbiol. 99:598–608.3. Morgan, R. R., C. A. Morris, K. Livzey, J. Hogan, N. D. Buttigieg, R. Pollner,D. Kacian, and I. Weeks. 2007. Rapid tests for detection and quantitation ofEnterococcus contamination in recreational waters. J. Environ. Monit. 9:424–426.4. Noble, R. T., S. B. Weisberg, M. K. Leecaster, C. D. McGee, K. Ritter, K. O.Walker, and P. M. Vainik. 2003. Comparison of beach bacterial water qualityindicator measurement methods. Environ. Monit. Assess. 81:301–312.5. U.S. Environmental Protection Agency. 1986. Ambient water quality criteriafor bacteria. Office of Water, EPA 440/5-84-002. Environmental ProtectionAgency, Washington, DC.6. U.S. Environmental Protection Agency. 2002. Method 1600: enterococci inwater by membrane filtration using membrane-enterococcus indoxyl-D-glu-coside agar (mEI). Office of Water, EPA-821-R-02-022. Environmental Pro-tection Agency, Washington, DC.TABLE 2. Summary of resultsaAssayTotalno. ofsamplesNo. of samples with resultPositive NegativeFalsenegative(%)Falsepositive(%)U.S. EPA method160077 24 53 NA NATMA 77 24 53 2 (2.6) 2 (2.6)Enterolert 77 22 55 4 (5.2) 2 (2.6)a The U.S. EPA-recommended threshold of 104 MPN or CFU per 100 ml ofwater for a single sample (1) was used to define a sample as positive (104 CFU)or negative (104 CFU). NA, not applicable.TABLE 1. Interassay regression parameters and precision data for12 TMA assay standard curvesaStandard curve(fg)Mean emergencetime abovethresholdStandarddeviation CV (%)0 65.9 6.9 10.41 62.1 8.3 13.410 53.3 6.8 12.7100 45.8 6.0 13.11,000 40.5 4.6 11.510,000 34.7 3.4 9.7100,000 28.4 3.4 12.11,000,000 23.4 3.6 15.3a Each rRNA concentration was assessed in triplicate with every experiment,and the mean of the results of 12 experiments was calculated. CV, coefficient ofvariation. r2, 0.98; x, 0.141; intercept, 9.89.3320 MORRIS ET AL. APPL. ENVIRON. MICROBIOL.

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

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

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