Grantee Research Project Results

Final Report: Development and Evaluation of an Innovative System for the Concentration and Quantitative Detection of CCL Pathogens in Drinking Water

EPA Grant Number: R833003
Title: Development and Evaluation of an Innovative System for the Concentration and Quantitative Detection of CCL Pathogens in Drinking Water
Investigators: Tzipori, Saul , Zuckermann, Udi , Walt, David
Institution: Tufts University
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2006 through August 1, 2009 (Extended to July 31, 2011)
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Water

Objective:

The purpose of this project was to develop, evaluate and validate an integrated rapid method for the quantitative assessment of pathogens and indicator organisms from large volumes of source and drinking water. One of the unique aspects of this project was the integration of technologies for concentration, purification, detection and testing for viability/infectivity into a universal simplified economical and user-friendly method. This project was built on earlier STAR research on a portable continuous flow centrifugation (CFC) method that concentrates oocysts, cysts, and spores from large volumes of water, and for continuous monitoring of their presence in water, as opposed to one-time sampling of existing methods. In this project, CFC was automated, and attempts were made to combine it with fiber optic bead microarray for rapid and accurate detection of waterborne pathogens.

The major tasks involved include: (1) integration of the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure; and (2) selection of sequences, development of assays and preparation of bead microarrays and testing in both synthetic and spiked samples to detect and quantitatively identify them.

The specific objectives of this project were to:

(1) Optimize the parameters (flow rate, centrifugal force, various bowls, elusion buffers, etc.) to recover representative pathogens, which include Escherichia coli for enteric bacteria, Cryptosporidium parvum for enteric protozoa, avirulent Anthrax for spores, and MS2 bacteriophages for viruses. In this objective, the team systematically optimized the concentration methodology for each representative pathogen with a view to generate reproducibly robust data.

(2) Integrate the concentration of protozoa (validated in the previous award), bacteria, spores and viruses from water into a single concentration procedure. The CFC then was fine-tuned for its ability to simultaneously concentrate representative pathogens from each group of the Contaminant Candidate List (CCL).

(3) Focus on the detection and quantitative identification of CCL pathogens in spiked samples, using multiplex miniaturized fiber optic bead microarrays coupled with a compact confocal-type imaging

system and comparing it with EPA-approved methods.

Summary/Accomplishments (Outputs/Outcomes):

Equipment

The Grafton and Boston Tufts laboratories are impressively equipped to perform this work. They were able to work with the company Haemonetics to make a new automated centrifuge, with remote control and offline capabilities. Sadly, while the final product is truly impressive, in terms of sensitivity time-efficiency and sensitivity, multiplex capability and cost-effectiveness, there does not seem to be any commercial interest in this brand of the CFC, but there is commercial development of the portable microarray detection by Ahura Scientific. No new equipment was purchased with project funds.

Personnel

After the completion of the 3 year funding period, a request for no-cost extension was requested and was approved for the years 2009-2011. The following individuals continued to work on the project: the PI Saul Tzipori (Grafton campus), David Walt (Boston campus), Ryan Haymand, Shonda Gaylord and Curtis Rich.

Results of over the award period summary and outcomes:

The Concentration System

So far, the Grafton laboratory has concentrated on the detection of DNA isolated from E. coli as a complete model, demonstrating the detection of PCR amplicons from three virulence genes using multiplexed bead-based microarrays. A prototype automated pathogen concentrator was designed and constructed with a commercial blood-banking company (Haemonetics), including modification of its hardware and of the disposable tubing kits. The new automated CFC methodology employs centrifugal force to spin out the protozoa and bacteria inside the bowl with minimal clogging problems. A new bowl for simultaneous concentration of multiple pathogens was designed. The 45 lb portable device is capable of simultaneous concentration of protozoa (Cryptosporidium), bacterial spores (B. anthracis) and MS2 bacteriophages from volumes of 10 L to 50 L at recovery rates that are still less than 50%, but consistent across volumes.

Pathogen	Spike	Recovery	Recovery
	dose	(%) for 10L N = 12	(%) for 50L N = 2

protozoa	100±1	40±0.06	~ 40
spores	50±5	34±0.14	~ 30

MS2 105 43±0.3 ~ 50

Once the detection platform was complete, the automated CFC spiked concentrates were applied and quantified (see below). Some of the pathogens were compared with currently approved standard methods in our laboratory. Below is a summary of a set of experiments:

Table 1. Recovery of C, parvum oocysts and B. antlracis (avirulent laboratory spores) from 10 L tap water samples using the CFC with a Standard HS bowel.

Vol. analyzed (L) (# replicates)	Spike dose Oocysts (Mean +/- SD)	Percent Recovery Oocysts (mean +/- SD)	Spike dose B. anth. Cfu (mean +/- SD)	Percent recovery B. anth CFU (mean +/- SD)
10 (5)	100 +/- 2.5	36.0 +/- 15.2	14.8 +/- 3.8	90.2 +/- 9.0

Detection System

David Walt’s laboratory (at the Medford campus) worked on the bioinformatics of the CCL list and the bead-based fiberoptic microarrays. Fiberoptic microarrays are made in a number of steps. First, DNA capture probes are immobilized to microspheres/beads and combined into a "library." Then, the beads are randomly placed onto the face of bundled optical fibers (thousands of individual fibers fused together such that each fiber retains its ability to transmit light independently) etched out with HCl so that each fiber end is now a well. Once dried, the beads are held firmly in the fiberoptic wells, optically "wiring" them to a fiber, and allowing specific interactions on each microsphere surface to be independently monitored. The fiber bundle can be put directly into test samples of cell lysates (no RNA purification needed) and fluorescently labeled rRNA signal probes. The combination of different fluorescent dyes enables encoding strategies for multispecies detection, essentially creating an optical bar code. The fiber optic microarrays then are "read" with a custom-built epifluorescence microscope, using a Hg lamp. The system is equipped with excitation and emission filter wheels, a dichroic housing, and a charge-coupled device (CCD) camera connected to a pertinently programmed computer and downloaded with appropriate bioinformatics. The fiberoptic microsphere-based biosensor is an impressively versatile platform that may ultimately enable rapid analysis at extremely low detection limits. Post-docs Ryan and Shonda demonstrated the fiberoptic bead multiplex miniaturized fiberoptic bead microarrays coupled with the compact confocal-type imaging system. Capture probes and PCR primers for the pathogens Campylobacter jejuni, E. coli (0157), Legionella pneumophila, Salmonella enterica, Shigella sonnei, and Vibrio cholerae were included in the microarray. The microspheres were obtained from Illumina, Inc.

We have concentrated on the detection of DNA isolated from E. coli cells as a model system. We have demonstrated the detection of PCR amplicons from three virulence genes using multiplexed bead-based microarrays. Research was focused on expanding our protocol and microarray to include all bacteria and viruses listed as CCL3 candidates in the following table:

Microbial Contaminant Information

Caliciviruses Virus (includes Norovirus) causing mild self-limiting gastrointestinal illness

Campylobacter jejuni Bacterium causing mild self-limiting gastrointestinal illness

Entamoeba histolytica Protozoan parasite which can cause short as well as long-lasting gastrointestinal illness

Escherichia coli (0157) Toxin-producing bacterium causing gastrointestinal illness and kidney failure

Helicobacter pylori Bacterium sometimes found in the environment capable of colonizing human gut that can cause ulcers and cancer

Hepatitis A virus Virus that causes a liver disease and jaundice

Legionella pneumophila Bacterium found in the environment including hot water systems causing lung diseases when inhaled

Naegleria fowleri Protozoan parasite found in shallow, warm surface and ground water causing primary amebic meningoencephalitis

Salmonella enterica Bacterium causing mild self-limiting gastrointestinal illness

Shigella sonnei Bacterium causing mild self-limiting gastrointestinal illness and bloody diarrhea

Vibrio cholerae Bacterium found in the environment causing gastrointestinal illness

We have designed additional capture probes and PCR primers for the remaining bacteria and viruses. Each microarray probe was matched with multiple potential PCR primer pairs that were tested with cultures prior to microarray hybridization experiments. The microarrays used were provided by Illumina, Inc.

Supplemental Keywords:

RFA, Scientific Discipline, Water, Environmental Chemistry, Drinking Water, Environmental Engineering, Environmental Monitoring, E. Coli, contaminant candidate list, analytical methods, contaminant removal, pathogens, cyanobacteria, drinking water contaminants, drinking water treatment, drinking water monitoring, Giardia, continuous flow centrifugation, CCL, cryptosporidium

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.