Grantee Research Project Results
2011 Progress Report: Rapid Concentration, Detection, and Quantification of Pathogens in Drinking Water
EPA Grant Number: R833840Title: Rapid Concentration, Detection, and Quantification of Pathogens in Drinking Water
Investigators: Hu, Zhiqiang , Riley, Lela K. , Lin, Mengshi
Institution: University of Missouri - Columbia
EPA Project Officer: Aja, Hayley
Project Period: May 1, 2008 through April 30, 2011 (Extended to April 30, 2013)
Project Period Covered by this Report: May 1, 2011 through April 30,2012
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objectives of this research are to: (1) evaluate a lanthanum-based colloidal destabilization method to rapidly concentrate pathogens in water; (2) determine the efficiency of fluorescence-based oxygen microrespirometry in differentiating live/dead pathogens; (3) develop and validate a new surface-enhanced Raman spectroscopy (SERS)-based method for pathogen detection and quantification; and (4) improve pathogen detection using modified concentration and molecular detection methods and compare the detection efficiencies to the Environmental Protection Agency's (EPA) existing methods with seeded or unseeded drinking water samples.
Progress Summary:
We have completed our research tasks to: (1) evaluate a lanthanum-based colloidal destabilization method to rapidly concentrate pathogens in water, and (2) determine the efficiency of fluorescence-based oxygen microrespirometry in differentiating live/dead pathogens. The results have been published in Water Research (Water Research, 2010, 44, 3385-3392). Briefly, we have shown that compared with traditional flocculants, LaCl3 has higher relative concentration and recovery efficiencies and thus possesses the potential for microbial concentration in water samples (Zhang et al., 2010). In Task 2 (Fluorescence-Based Oxygen Microrespirometry for Pathogen Determination), we have successfully developed an oxygen based microrespirometric assay for bacterial detection. The time-resolved fluorescence (TR-F) from each water sample was monitored every 15 min over an approximate 48-h period by the microreader using standard sets of filters of 340 nm (excitation) and 642 nm (emission) (Zhang et al., 2010).
For Task 3 (SERS Coupled with SERS-Active Substrates for Pathogen Determination), we have demonstrated that SERS coupled with gold SERS-active substrates is reliable for identifying and differentiating different virus strains (Fan et al., 2010). In this study, a protocol of SERS coupled with internal-coated silver nanoparticles was established for detection of bacterial samples.
We continue to develop SERS-based rapid and sensitive methods to detect foodborne pathogens, with an emphasis on pathogenic bacteria (part of Task 3). Our recent study aimed to develop a new method to detect and discriminate Escherichia coli O157:H7 and Staphylococcus epidermidis by SERS coupled with silver nanomaterials. Our results indicate that internal coating with silver nanostructures for bacterial cells is a feasible and effective approach to conduct SERS measurement for bacterial samples. Coupled with internal-coated silver nanostructures, SERS may also rapidly detect different water- and foodborne bacteria. The experimental work on nanosilver-based nanostructure fabrication is more challenging than expected and its application in SERS remains to be carefully studied. The results have been published recently (Fan et al., 2011).
For the proposed Task 4 (Molecular Detection Methods for Cross-Checking and Method Improvement), we have tested a few bacterial species, including Helicobacter pylori. Lanthanum-based concentration coupled with real-time PCR could detect E. coli at a concentration of 22 CFU/mL in raw water (from Missouri River) and H. pylori at a concentration of about 2 CFU/mL in finished water (from the effluent of a water treatment plant before chlorination). By eliminating the membrane fouling and clogging problem often encountered in the direct filtration, the lanthanum-based chemical flocculation coupled with the use of qPCR is promising to detect specific bacteria or pathogens in water at low cell densities (Zhang et al., 2012).
The task using quantitative PCR assays to detect low-density human adenovirus takes a longer time than what we have expected. Currently, we are using RT-PCR methods to optimize concentration steps (such as ultrafiltration and chemical flocculation). The pathogen recovery efficiencies need to be compared with those of EPA's existing methods (e.g., 1MDS filter method) and the polyethylene glycol (PEG) precipitation method. Our preliminary results are encouraging. Chemical concentration using LaCl3 resulted in the highest recovery (73.4%) in water samples with RT-PCR enumeration, indicating that most viruses can be entrapped by flocs after LaCl3 treatment (Figure 1). After lanthanum-based chemical concentration, additional sample water volume reduction was achieved through the use of a two-step membrane filtration approach. In the first step of membrane filtration, selected membrane filters (including 1MDS electropositive filters and nitrocellulose electronegative filters, Millipore HATF filters) were used and compared to determine the virus recovery efficiency. The HATF membrane after magnesium chloride (MgCl2) modification had better performance on virus retention (more than 99.9%) with higher virus recovery (more than 80%). Centrifugal ultrafiltration through a 30 k Dalton filter was applied as the final step of membrane filtration resulting in an overall concentration by > 20,000-fold. Compared with direct cartridge filtration using Nanoceram and 1MDS, the lanthanum-based chemical concentration method coupled with modified membrane filtration has advantages in eliminating the use of expensive cartridge filters with improved virus detection efficiency. The new method is therefore promising for the determination of low-density viral particles in water (Zhang et al, manuscript in preparation).
Figure 1. Recovery of MS2 after lanthanum-based concentration based on infectivity assay (a) and RT-qPCR (b)
References:
Fan, C., Hu, Z., Mustapha, A., Lin, M., 2011. Rapid detection of food- and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates. Applied Microbiology and Biotechnology 92, 1053-1061.
Fan, C., Hu, Z.Q., Riley, L.K., Purdy, G.A., Mustapha, A., Lin, M., 2010. Detecting food- and waterborne viruses by surface-enhanced Raman spectroscopy. J. Food Sci. 75, M302-M307.
Zhang, Y., Riley, L.K., Lin, M., Hu, Z., 2010. Lanthanum-based concentration and microrespirometric detection of microbes in water. Water Research 44, 3385-3392.
Zhang, Y., Riley, L.K., Lin, M., Hu, Z., 2012. Determination of low-density Escherichia coli and Helicobacter pylori suspensions in water. Water Research 46, 2140-2148.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
| Other project views: | All 6 publications | 5 publications in selected types | All 5 journal articles |
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Fan C, Hu Z, Mustapha A, Lin M. Rapid detection of food-and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates. Applied Microbiology and Biotechnology 2011;92(5):1053-1061. |
R833840 (2010) R833840 (2011) R833840 (Final) |
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Zhang Y, Riley LK, Lin M, Hu Z. Determination of low-density Escherichia coli and Helicobacter pylori suspensions in water. Water Research 2012;46(7):2140-2148. |
R833840 (2010) R833840 (2011) R833840 (Final) |
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Supplemental Keywords:
Drinking water, exposure, waterborne pathogens, microbiology, monitoring, measurement methods, physical processes, health effects, field-based detection, lanthanum, respirometry, Surface Enhanced Raman Spectroscopy, nanotechnology, biotechnology, 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, virulence factor biochip, virulence factor activity relationships, microbiological organisms, detection, exposure and effects, 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.