Grantee Research Project Results
2008 Progress Report: Development of a Virulence Factor Biochip and its Validation for Microbial Risk Assessment in Drinking Water
EPA Grant Number: R831628Title: Development of a Virulence Factor Biochip and its Validation for Microbial Risk Assessment in Drinking Water
Investigators: Rose, Joan B. , Whittam, Thomas S. , Gulari, Erdogan , Hashsham, Syed
Institution: Michigan State University , University of Michigan
EPA Project Officer: Packard, Benjamin H
Project Period: November 1, 2004 through October 31, 2007 (Extended to April 30, 2009)
Project Period Covered by this Report: November 1, 2007 through October 31,2008
Project Amount: $600,000
RFA: Microbial Risk in Drinking Water (2003) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The concept of using genetic databases for identifying microbial risks in water, coined as Virulence-Factor Activity Relationships (VFARs), was first developed by the Committee on Drinking Water Contaminants, National Research Council as an approach to screen microorganisms for their occurrence in water and/or their ability to cause harm and waterborne disease. MSU Water and Health teams have developed a waterborne genomics pilot program for the assessment of VFARs and have developed biochips that addressed bacteria and virus species, as well as pathogenicity and potential risk.
In our original proposal, we had proposed the development and validation of a high-density biochip for some 15 groups or genera/species of targeted bacteria and viruses, some of which are on the Contaminant Candidate List and others that are important for the assessment of the microbial safety of water. We targeted traditional indicator organisms, pathogenic bacterial strains and viruses, as well as key virulence factors. Our specific goals were to: (1) select gene targets to encompass the microorganisms of interest to water safety; (2) design probes to uniquely identify each of the above microorganisms and provide reliable detection; (3) synthesize microfluidic biochips containing the above set of probes in replicate with positive and negative controls (biochip synthesis); (4) validate and field test the synthesized biochips using standard individual targets, appropriate target mixtures, and field samples (validation and field testing); and (5) undertake a pilot risk analysis of a water system(s), testing a variety of computational approaches for interpreting the results of the biochip (analysis).
In this report, we summarize our final results and accomplishments: (1) Development of a Chip-based screening system for host-specific gene sequence signatures indicative of fecal contamination; and (2) Oligonucleotide Microarrays and application for detection of circulating enteric viruses in sewage .
Progress Summary:
Key Findings
- Using whole genome amplification, a pollution biochip has been developed and used for screening fecal pollution and sources.
- A viral microarray with a total of 780 unique probes targeting 27 different groups of both DNA and RNA viruses has been developed and used to monitor sewage for over 1 year, showed more RNA viral infections than DNA and more infections in general during the winter months.
- Methods for use and validation of microarrays as well as a compliation of genetic sequences for screening waters associated with virulence and pathogenic microorganisms have been produced as part of this research program.
Chip-based screening of host-specific gene sequence signatures indicative of fecal contamination:
To improve the identification of host-specific gene sequence signatures indicative of fecal contamination, a second version of the fecal indicator biochip was designed using an extensive set of functional genes as genetic markers. These included mainly genes linked to virulence, antibiotic resistance, and cellulose degradation. Multiple samples were collected, processed, and amplified using whole genome amplification to validate the indicator biochip. Eight samples were selected for initial validation of the biochip including the following: 1) chicken feces from a MSU poultry farm, 2) sand laden cow manure from a dairy in Northern Ingham County, 3) water collected from Butternut Creek (next to poultry farm), 4) Kellogg Biological Station Bird Sanctuary to act as a negative control, 5) chicken feces from a poultry farm in Genesee County, 6) East Lansing raw waste water, 7) surface water from a cow pasture in Genesee County, and 8) stored swine slurry from an outreach farm.
To cope with samples that yielded either low amounts of DNA or DNA of low purity, all samples were processed with whole-genome amplification (WGA) using rolling-circle amplification. Results comparing samples processed with and without whole genome amplification show a large increase in hybridized signal intensity (figure 1). The increase in signal could be due to both the increased concentration of DNA and the reduction of other materials that can inhibit labeling and hybridization.
Figure 1. Signal to noise ratio of the same sample hybridized with and without whole genome amplification.
Validation of samples consisted of hybridizing at 20oC for 18 hours and performing a nonequilibrium dissociation curve by scanning the chip at increasing 2oC intervals (as described in previous reports). Results have shown that probes are indeed specific to fecal host based on the hybridization profile. An example is provided showing comparative hybridization patterns (figure 2). As expected, testing of surface water samples resulted in fewer hybridized probes compared to fecal samples. Further analysis must be performed to determine if sample hybridization patterns can be clustered correctly based on fecal host. Hybridized probes (presence/absence and signal intensity) will be used to align samples. It is expected that samples 1,3, and 5 would cluster closely, and samples 2, and 7 would cluster together. The clustering analysis will be performed with the dissociation curve to determine the optimal temperature for differentiation between fecal hosts. To determine if the hybridization pattern has any indication on the presence of pathogens, these pathogens will also be tested (using qPCR) for presence of virulence genes.
Figure 2. Comparative plots showing hybridization profiles of cow fecal (plotted as negative values on both plots), chicken fecal (positive value on left panel), and a water sample taken from ButterNut Creek (positive value on right panel). Plots only show probes that had a signal-to-noise-ratio (SNR greater than 3). Dashed circle highlights probes specific to chicken fecal sample and dotted line highlights probes specific to cow fecal sample. Even though the water sample has much less hybridized probes compared to the two fecal samples, presence of cow fecal matter and absence of chicken fecal matter is easily discernable. Further analysis is underway to evaluate the statistical significance.
Examining The Presence And Prevalence Of Key Human Enteric Viruses In Environmental Samples Using Cultivation, Molecular Array-Based Tools For Detection
This study describes the novel use of a viral microarray to screen municipal wastewater for the presence of circulating human pathogenic viruses. A total of 780 unique probes targeting 27 different groups of both DNA and RNA viruses were designed and tested against laboratory strain viruses and environmental water samples (Table 1). Approximately thirty probes were used to target each viral group. Laboratory strains of poliovirus and adenovirus type 40 and 41 were evaluated initially and indicated that the probes were highly specific for their targets and that cross hybridization of target nucleic acid was minimal even when closely related virus species were mixed and co-hybridized on the array. During 13 months of sampling, RNA viruses were more frequently detected in a community wastewater compared to DNA viruses (Figure 1). Overall, more viruses were detected during the winter season compared to the summer months. This study also showcases the use of two pieces of biocomputing software – OligoArray 2.1 was used to design target specific probes for inclusion unto the array and DetectiV was used to visualize and analyze microarray hybridization data and assign probable identities to the pathogens present in the sample. Conclusion: Microarrays are capable of screening for a broad number of pathogenic viruses which may be circulating in the population and excreted in the community wastewater. This is the first demonstration of an environmental microarray for detection of viruses in water and could serve as an example for improved public health surveillance.
Figure 3. Viral Probes Positive in Sewage Samples from August 06 to Aug 07
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 14 publications | 5 publications in selected types | All 4 journal articles |
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Stedtfeld RD, Baushke SW, Tourlousse DM, Miller SM, Stedtfeld TM, Gulari E, Tiedje JM, Hashsham SA. Development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume PCR on the OpenArray platform. Applied and Environmental Microbiology 2008;74(12):3831-3838. |
R831628 (2008) R833010 (2008) R833010 (Final) |
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Tourlousse DM, Ahmad F, Stedtfeld RD, Seyrig G, Duran M, Alm EW, Hashsham SA. Detection and occurrence of indicator organisms and pathogens. Water Environment Research 2008;80(10):898-928. |
R831628 (2008) |
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Wong M, Kumar L, Jenkins TM, Xagoraraki I, Phanikumar MS, Rose JB. Evaluation of public health risks at recreational beaches in Lake Michigan via detection of enteric viruses and a human-specific bacteriological marker. Water Research 2009;43(4):1137-1149. |
R831628 (2008) |
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Supplemental Keywords:
RFA, Scientific Discipline, Health, PHYSICAL ASPECTS, Water, Ecosystem Protection/Environmental Exposure & Risk, Health Risk Assessment, Environmental Chemistry, Monitoring/Modeling, Risk Assessments, Physical Processes, Environmental Monitoring, Drinking Water, microbial contamination, monitoring, measurement , microbial risk assessment, biochip, microbiological organisms, detection, exposure and effects, virulence factor activity relationships, virulence factor biochip, bacteria monitoring, exposure, other - risk assessment, E. Coli, human exposure, microbial risk management, microorganism, measurement, assessment technology, drinking water contaminants, other - risk managementProgress 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.