Impact/Purpose:

This proposal will develop, evaluate and validate a rapid method for the quantitative assessment of pathogens/indicator organisms from large volumes of source and drinking water.  Microorganisms of concern will include protozoa (Cryptosporidium, Giardia, Microsporidium), bacteria (E. coli, Legionella, Helicobacter pylori, Aeromonas hydrophila, Micobacterium avium intracellular), algae (Cyanobacteria), and viruses (Adenoviruses, Caliciviruses, Coaxackieviruses, Echoviruses).  We propose integrating the technologies of concentration, purification, detection and testing for viability/infectivity into a universal simplified economic and user friendly methodology.

Description:

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:

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.

URLs/Downloads:

2007 Progress Report

Final Progress Report

2008 Progress Report

Record Details:

Record Type:PROJECT( ABSTRACT )

Start Date:08/01/2006

Completion Date:08/01/2009

Record ID: 169335

Show Additional Record Data

Related Organizations:

Role :OWNER

Organization Name :TUFTS UNIVERSITY

Citation :Medford

State :MA

Zip Code :2155

Project Information:

Approach :

Concentration of the microorganisms will be by continuous flow centrifugation (CFC) and the detection of a small number of microorganisms from large volumes of water will be done using multiplex miniaturized fiber bead microarrays.  In a previous EPA STAR award we developed, optimized and validated a novel method, the CFC, for the concentration of three protozoa (Cryptosporidium spp. Giardia, Microsporidia) from large volumes of water (1000L).  In this award we propose to expand the CFC approach to include selected Contaminant Candidate List (CCL) pathogens, including bacteria, algae and viruses. The proposed method will integrate the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure using a continuous flow and compare it with conventional methods.  We will focus on detection and quantitative identification of CCL pathogens in water using multiplex miniaturized fiber optic DNA bead microarrays coupled with a compact confocal-type imaging system.  The proposed detection technique will be rapid, cost-effective, portable, robust and employ simple procedures.

The major tasks involved with this approach include:  (1) integrate the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure; (2) select sequences, develop assays, prepare bead microarrays, and test both synthetic and spiked samples; and (3) validate the combined method with complex water matrices.

Cost :$600,000.00

Research Component :Drinking Water

Risk Paradigm :RISK MANAGEMENT

Approach :

Concentration of the microorganisms will be by continuous flow centrifugation (CFC) and the detection of a small number of microorganisms from large volumes of water will be done using multiplex miniaturized fiber bead microarrays.  In a previous EPA STAR award we developed, optimized and validated a novel method, the CFC, for the concentration of three protozoa (Cryptosporidium spp. Giardia, Microsporidia) from large volumes of water (1000L).  In this award we propose to expand the CFC approach to include selected Contaminant Candidate List (CCL) pathogens, including bacteria, algae and viruses. The proposed method will integrate the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure using a continuous flow and compare it with conventional methods.  We will focus on detection and quantitative identification of CCL pathogens in water using multiplex miniaturized fiber optic DNA bead microarrays coupled with a compact confocal-type imaging system.  The proposed detection technique will be rapid, cost-effective, portable, robust and employ simple procedures.

The major tasks involved with this approach include:  (1) integrate the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure; (2) select sequences, develop assays, prepare bead microarrays, and test both synthetic and spiked samples; and (3) validate the combined method with complex water matrices.

Cost :$600,000.00

Research Component :OTHER

Risk Paradigm :RISK MANAGEMENT

Project IDs:

ID Code :R833003

Project type :EPA Grant