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OWML: Completed Projects

Project Title: Testing of analytical methods for pathogens in finished drinking-water supplies.

Project chief:  Donna Francy

Project support:  Rebecca Bushon, Chris Kephart, Amie Brady, Erin Bertke, Erin Stelzer

Cooperators:  U.S. Environmental Protection Agency, National Homeland Security Research Center (USEPA NHSRC), Cincinnati, OH

Project duration:  January 2006 – September 2009

Introduction and problem: 
Certain microbiological pathogens are considered potential biological warfare agents and cause homeland security concerns, especially for drinking-water supplies. Traditional cultural and microscopic methods allow detection of these pathogens; however, the methods take days before any confirmatory answer is available. A quantitative polymerase chain reaction (QPCR) method can be used to obtain rapid analysis of pathogens from water samples by targeting the DNA of the pathogens. Little is known, however, about the ability of QPCR to detect pathogens in treated drinking water and the reliability of QPCR as compared to traditional methods.

Goals and objectives:
The goal of the proposed project is to collect data to assess the utility and performance of analytical methods for determination of pathogens in finished drinking waters. Specific objectives are the following:

  1. Test, optimize, and finalize protocols for analytical methods for six pathogens.

  2. Determine the efficiency and variability of recoveries of analytical methods for pathogens in drinking-water samples collected from public utilities. 

  3. Compare the performance of molecular detection methods to that of traditional methods.

  4. Document the effects of physical properties of water and concentrations of organic carbon in drinking-water supplies on the detection of six pathogens.

13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents—Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed.

For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or “Ct” values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results (including some replicate samples), F. tularensis and V. cholerae were detected in all samples after ultrafiltration, B. anthracis was detected in 13 and +/- detected in 1 sample, and C. parvum was detected in 9 and +/- detected in 4 samples. Bu. cepacia was detected in nine samples, +/- detected in two samples, and not detected in three samples (for two out of three samples not detected, a different strain was used). The qPCR assay for V. cholerae provided two false positive—but late—signals in one unseeded sample. Numbers found by qPCR after ultrafiltration were significantly or nearly significantly related to those found by traditional methods for B. anthracis, F. tularensis, and V. cholerae but not for Bu. cepacia and C. parvum. A qPCR assay for S. Typhi was not available.

The qPCR method can be used to rapidly detect B. anthracis, F. tularensis, and V. cholerae with some certainty in drinking-water samples, but additional work would be needed to optimize and test qPCR for Bu. cepacia and C. parvum and establish relations to traditional methods. The specificity for the V. cholerae assay needs to be further investigated. Evidence is provided that ultrafiltration and qPCR are promising methods to rapidly detect biological agents in the Nation’s drinking-water supplies and thus reduce the impact and consequences from intentional bioterrorist events. To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples.

Francy, D.S., Bushon, R.N., Kephart, C.M., Brady, A.M.G., Bertke, E.E., Stelzer, E., Likirdopulos, C.A., Schaefer III, F.W., and Lindquist, H.D.A., 2008, Detecting biological agents in drinking water using culture- or microscopic-based and molecular methods: In Proceedings of the AWWA Water Quality Technology Conference (WQTC), Nov 2008, Cincinnati, Ohio.

Francy, D.S., Bushon, R.N., Brady, A.M.G., Bertke, E.E., Kephart, C.M., Likirdopulos, C.A., Mailot, B.E., Schaefer III, F.W., and Lindquist, H.D.A., 2009a, Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration: Journal of Applied Microbiology – early release.

Francy, D.S., Bushon, R.N., Brady, A.M.G., Bertke, E.E., Kephart, C.M., Likirdopulos, C.A., Mailot, B.E., Schaefer III, F.W., and Lindquist, H.D.A., 2009b, Performance of traditional and molecular methods for detecting biological agents in drinking water: U.S. Geological Survey Scientific Investigations Report 2009-5097, 17 p.