Grantee Research Project Results
2009 Progress Report: Detecting Pathogens in Water by Ultrafiltration and Microarray Analysis
EPA Grant Number: R833004Title: Detecting Pathogens in Water by Ultrafiltration and Microarray Analysis
Investigators: Lee, Anthea K. , Rochelle, Paul A. , DeLeon, Ricardo
Institution: Metropolitan Water District of Southern California
EPA Project Officer: Page, Angela
Project Period: July 21, 2006 through July 20, 2009 (Extended to July 20, 2011)
Project Period Covered by this Report: July 21, 2008 through July 20,2009
Project Amount: $594,106
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 ultimate objective of this project is to develop an innovative approach for detecting multiple waterborne bacterial, protozoan, and viral pathogens utilizing large volume ultrafiltration as a universal pathogen concentration technique, direct extraction of nucleic acids, whole sample genome amplification (WSGA), and hybridization to a multi-pathogen, water quality microarray. The most important aspects in applying molecular pathogen-detection techniques to water samples are the ‘front-end’ processes of sample concentration and nucleic acid extraction. The most sensitive and specific detection system available will be virtually useless if presented with low concentrations of poor quality DNA. This proposal addresses these critical issues by focusing on methods for concentrating pathogens from large volumes of water and extracting high quality nucleic acids so that the full potential of advanced molecular detection technologies can be realized. The specific objectives of this project are:
Objective 1: Optimize a universal ultrafiltration-based concentration method for all waterborne pathogens.
Objective 2: Improve nucleic acid extraction and recovery from concentrated water samples.
Objective 3: Design a custom microarray to detect waterborne protozoa, bacteria, and viruses.
Objective 4: Confirm infectivity of concentrated pathogens.
Progress Summary:
Year 3 of this project expanded on Objectives 1 and 2, using human adenovirus 2 as a model organism to optimize ultrafiltration recovery efficiencies and optimize DNA extraction techniques. Replicates of Cryptosporidium parvum ultrafiltration (started in Year 2) were completed. In addition infectivity assays confirmed that ultrafiltration does not change the ability of C. parvum to infect tissue culture cells (Objective 4). Ultrafiltration recovery efficiency for human adenovirus 2 using modified ultrafiltration protocols are listed in Table 1. Objective 3 was started, as a custom waterborne pathogen microarray is in the design stages.
Previous Results: Years 1 and 2 saw the completion of UF optimization for Escherichia coli and Salmonella typhimurium (70.38% and 58.4% recovery efficiency, respectively for 1000 organisms in 100 liters of water), and initiation of ultrafiltration optimization for C. parvum. DNA extraction post-ultrafiltration was optimized. In addition, several whole genome amplification kits were tested. Kits using Multiple Displacement Amplification technology were chosen for use.
Ultrafiltration: Average ultrafiltration recovery of C. parvum from 100 liters of water (3 repetitions) was 61% for 1000 organisms and 53.5% for 10,000 organisms. These results are consistent with previous results in the laboratory. To confirm that ultrafiltration does not affect infectivity of oocysts, ultrafiltration was repeated using 500 oocysts in 100 liters of water. Infectivity of recovered oocysts was comparable to positive controls (oocysts that had not undergone ultrafiltration). Infectivity was assessed by inoculating a monolayer of human intestinal adenocarcinoma HCT-8 tissue cultures cells with oocysts that had undergone ultrafiltration and isolation by immunomagnetic separation (IMS). Cells were incubated for 3 days to allow infectious foci to develop. Monolayers then were labeled with anti-sporozoite antibody followed by fluorescent secondary antibody, and infectious clusters were counted. Percent infectivity was calculated from number of infectious clusters from sample/average number of infectious clusters from control wells. Average infectivity post-ultrafiltration was 67.6%.
The ultrafiltration protocol optimized for E. coli (described in the 2007 Annual Progress report) was applied to human adenovirus 2. Adenovirus is quantified by plaque assay with A549 human carcinomic alveolar basal epithelial tissue culture cells. Modifications were needed to optimize virus ultrafiltration as the virus eluted poorly off the filter. Modifications tested include warming the elution buffer to 37°C, blocking the filter with 5% fetal bovine serum (FBS), and increasing the pH of the elution buffer to 9.5. Results of these modifications are listed in Table 1.
Custom Water Pathogen Microarray:
A custom microarray featuring bacterial, protozoan, and viral genes commonly found in water is under design. The design so far includes genes from the 3 test organisms: S. typhimurium, C.parvum, and human adenovirus 2, genes from E. coli and other waterborne pathogens including many from EPA Contaminant Candidate List 3 (CCL3), as well as some toxic cyanobacteria. Each represented organism on the microarray will have at least 3 genes and/or gene-regions thought to be unique to that organism, in addition to 16S or 18S ribosomal coding gene-regions where applicable. Gene and gene-region specificity are being assessed by using BLAST and other local alignment tools against genes in the Genbank database. The microarray will be designed to use 40 basepair probes across the chosen gene or unique gene region.
Future Activities:
The premise and goals of this project have not changed thus far. Ultrafiltration of human adenovirus 2 is not as efficient as other organisms, likely because the virus is known to be sticky and may be clinging to the filter. Pretreatment of the filters with FBS in combination with higher pH of the elution buffer may have an additive effect on recovery efficiency, and currently is being tested. Once the modifications for adenovirus have been optimized, concentration of virus (105, 104 and 103) in 100 L of water will be tested. The concentrated virus will have its DNA extracted and WSGA performed. The next step for ultrafiltration is to mix the three organisms together, and verify that recovery efficiency is as good as single spikes. Design and production of a custom microarray featuring genes from the three test organisms as well as many other waterborne pathogens will be finished during 2010. Testing of the custom microarray will begin as soon as feasible.Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
drinking water, risk assessment, safety, human health, pathogens, viruses, bacteria, parasites, monitoring, analytical, microbiology, water quality, innovative technology, EPA Regions 1 through 10, water industry, , POLLUTANTS/TOXICS, Water, Scientific Discipline, RFA, Drinking Water, Environmental Chemistry, Microorganisms, bacteria, drinking water contaminants, water quality, CCL, aquatic organisms, enteric viruses, viruses, contaminant removal, drinking water treatment, ultrafiltration, microarray analysis, contaminant candidate list, activated carbon, drinking water monitoring, RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Biochemistry, Drinking Water, Microorganisms, enteric viruses, aquatic organisms, bacteria, CCL, viruses, drinking water monitoring, microarray analysis, activated carbon, parasites, water quality, contaminant removal, drinking water contaminants, drinking water treatment, ultrafiltrationProgress and Final Reports:
Original AbstractThe 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.