Grantee Research Project Results
Final Report: Development of a Universal Microbial Collector (UMC) for Enteric Pathogens in Water and its Application for the Detection of Contaminant Candidate List Organisms in Water
EPA Grant Number: R833009Title: Development of a Universal Microbial Collector (UMC) for Enteric Pathogens in Water and its Application for the Detection of Contaminant Candidate List Organisms in Water
Investigators: Bright, Kelly R. , Gerba, Charles P.
Institution: University of Arizona
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2006 through July 31, 2009 (Extended to July 31, 2010)
Project Amount: $466,817
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Water , Drinking Water
Objective:
While numerous methods (e.g. polymerase chain reaction - PCR, immunochemical) have been developed to detect small numbers of pathogens, their application to the detection of pathogens in water has been limited for a number of reasons including: Small assay volumes are required (usually 10 to 100 microliters), the presence of interfering substances in concentrates, the inability to determine viability, the cost for processing large volumes, low efficiency, variability of concentration methods, cost of filters, and time for concentration.
No matter which method is used to detect enteric pathogens in water, a concentration step is essential. The ultimate goal of this project was to develop a simple, efficient, low cost method for the concentration of microorganisms from water that yields a concentrate of minimal volume and with minimal substances that would interfere with the microbial detection methodology.
Specific Objectives:
- Evaluate several commercially available nanofiber or other surface-modified carbons (the collector) for the retention of enteric viruses, bacteria and protozoa.
- Assess various methods for the optimal recovery of the microorganisms from the collector. The goal is to select methods that yield the greatest recovery of viable organisms and contain the least amount of substances that may interfere with detection methodologies (e.g., PCR based methods).
- Evaluate the recovery methods with waters of varying physical and chemical quality to assess variability, accuracy, robustness, and precision of the method.
- Evaluate the method with a number of different viruses, bacteria and protozoa.
- Use the collector to assess the occurrence of adenoviruses and microsporidia in distribution systems and groundwater.
Summary/Accomplishments (Outputs/Outcomes):
Experimental Methods:
Tucson municipal tap water was dechlorinated via passage through an Amway activated carbon block filter (Amway, Ada, MI) (see Table 1). 20 L of water was added to a stainless steel pressure vessel and inoculated with approximately 108 PFU or 108 TCID50 of each virus (in separate experiments). To determine the influent titer, three 15-ml samples were collected from the 20 L volume for assay. Positive pressure was applied to the vessel using N2 gas at approximately 2.5 pounds per square inch (psi) to achieve a flow rate of 2.5 L/min for the seeded water through the filters. Effluent samples (1 L) were collected after the passage of 5, 10, and 15 liters of the test water through the filter to determine the amount of virus retained by the filter.
The test eluting solution was added to the housing unit until the filter was completely immersed. The unit was then re-sealed and inverted ten times, followed by a hold time of 15 minutes at room temperature. The unit was inverted ten additional times, followed by another 15 minute hold at room temperature. After inverting the unit another 10 times, the eluting solution was passed through the filter under positive pressure (N2 gas) into a sterile polypropylene beaker. The eluent was then added again to the filter housing unit with a one-minute hold time, then passed through the filter and collected in the same beaker. The pH of the final eluent was immediately adjusted to neutral (if necessary) using 1M HCl.
Detection Methods:
Bacterial Assays: Bacterial samples were serially diluted in phosphate buffered saline and the surviving bacteria were enumerated in duplicate using the spread plate method on trypticase soy agar medium. The plates were then incubated at 37°C for 24 hours, after which, the bacterial colonies were enumerated to determine the number of bacterial colony-forming units/ml.
Viral Assays: All non-viral contaminants were removed using a pre-blocked (with 2 ml of 1.5% beef extract) syringe filter with a pore size of 0.22 µm. The samples were then aliquoted into 1.5-ml volumes in cryogenic vials and stored at either -80°C (human viruses) or 4°C (MS2 coliphage) until quantitative infectivity assays were performed The MS2 bacteriophage (ATCC# 15597-B1) was assayed on tryptic soy agar plates using ten-fold serial dilutions and the double overlay plaque forming method (Adams 1959). Subsequent plaques (clearing) in lawns of its host bacterial strain, Escherichia coli (ATCC# 15597), were enumerated to determine the number of plaque-forming units of MS2 recovered.
For poliovirus 1, echovirus 1, and Coxsackievirus B5, the viruses were quantified using the ten-fold serial dilution plaque-forming assay described by Bidawid et al. (2003) on BGM cells (Buffalo green monkey kidney; obtained from Don Dahling at the United States Environmental Protection Agency, Cincinnati, OH). Host cell monolayers in 6-well tissue culture plates were inoculated with 0.1-ml volumes of 10-fold serial dilutions (in duplicate) of the virus sample and incubated at 37°C for one hour to allow for virus adsorption to the cells. Following this incubation period, 3 ml of a molten solution of MEM containing 1.5% Bacto-agar, 2% fetal bovine serum, 1 M HEPES buffer, 7.5% sodium bicarbonate, 10 mg/ml kanamycin, 100X antimycotic (HyClone Laboratories), and 200 mM glutamine was added as an overlay to each well and allowed to solidify. The plates were then incubated at 35°C with 5% CO2 for two days for poliovirus 1 and echovirus 1, and five days for Coxsackievirus B5. Following this incubation, the agar overlays were removed and the cell monolayers were stained with 0.5% (w/v) crystal violet mixed 1:1 with 95% ethanol. The plaques (clearings in the cell monolayer) were counted to enumerate infectious viruses.
Adenovirus 2 enumerations were performed using the Reed-Muench method (Payment and Trudel 1993) to determine the tissue culture infectious dose that affected 50% of the wells (TCID50). Serial 10-fold dilutions of the virus sample were assayed in 96-well tissue culture plates containing monolayers of PLC/PRF/5 (primary liver carcinoma; ATCC# CRL-8024) cells and 100 ml of MEM containing 2% calf serum with incubation at 35°C with 5% CO2. Ten wells were inoculated with 50 ml of each dilution. Maintenance medium (25 µl of fresh medium) was added to each well every third day for the duration of the assay to maintain the integrity of the monolayer. Each well was checked every day for 12 days for viral cytopathogenic effects (CPE). The greatest dilution in which more than 50% of the wells were positive was used to determine the virus TCID50/ml.
In a separate set of experiments, one-step reverse transcriptase polymerase chain reaction (RT-PCR) followed by nested PCR as described by Rodriguez et al. (2008) was used to determine if the primary concentrate and the secondary concentrate contained any PCR-inhibiting substances. The PCR products (bands of 195 and 105 bp) were visualized via gel electrophoresis.
Summary of Findings:
Specific Objective 1 - Evaluate potential universal microbial collectors (UMC):
During the first two years, we focused primarily on the evaluation of two innovative nanofiber filtration technologies for the retention of microorganisms from water.
The first type of filter we have worked with is a proprietary charge-modified granular carbon nanofiber available from CUNO, Inc. (Merdian, CT). The surface of this material has been modified to be highly positively charged. The enhanced retention of microorganisms on these filters is due to the combination of a large surface area (through the use of carbon nanofibers) and an enhanced cationic charge. The charge-modified carbon nanofibers allow passage of water through a small amount of material (47 mm diameter ´ 5 mm width) at high flow rates (2 to 10 liters per minute).
The second filter type (NanoCeram) is comprised of nano alumina fibers on a microglass fiber matrix that is available from Argonide Corporation (Sanford, FL). The final composition has an average pore size of 2 mm and may be either pleated (allowing faster flow rates) or used in multiple layer discs (for greater retention of smaller particles such as viruses). This material is also highly electropositive and has a large surface area, particularly in the pleated form. Unlike other filters with similar pore sizes, the Argonide filters are capable of much faster flow rates.
In addition to these nanofiber filters, we also tested new sodocalcic glass wool filters supplied by Dr. Mark Borchardt of the Marshfield Clinic Research Foundation (Marshfield, WI) for the retention of microorganisms from water. Microbial retention was evaluated using dechlorinated municipal tap water (Table 1).
Table 1. Dechlorinated tap water quality.
Parameter Average Hardness (mg/L CaCO3) 140 Temperature (˚C) 27.2 Total Dissolved Solids (mg/L) 317 Conductivity (mg/L) 355 pH 7.8
Carbon Nanofibers Results: For our evaluation of the charge-modified carbon nanofiber material, we tested various classes of microorganisms (Table 2). Microbial retention was evaluated with waters of differing physical/chemical water quality (see footnote of Table 2). Test volumes of 10 to 125 liters were evaluated. The carbon nanofiber material was very effective at retaining multiple classes of microorganisms, with > 99.99 % retention from large volumes of water (10 to 125 liters) (Table 2). This material was capable of retaining several types of phages and human enteric viruses of different sizes and surface properties (i.e., isoelectric points, hydrophobicity). The ability of the filters to capture microorganisms was not affected by high turbidity (30 NTU), high or low salt concentrations (up to 1,500 mg/L), pH (6.0 through 9.0), or high dissolved organic levels (10 mg/L).
Glass Wool Filter Results: Retention rates for the glass wool filters were highly variable for MS2 bacteriophage, ranging from 25.0% to 86.0%. The results for poliovirus 1 were similar with retention ranging between 26.0% and 95.1%. The test organisms where added to the volume of water indicated. Two types of water were used with identical results. Average case water = turbidity = 0.1 NTU; pH = 7.5; total dissolved solids = 200-300 mg/L; total organic carbon = <1 mg/L. Worst case test water = turbidity = 30 NTU; pH = 5.0 and 9.0; total dissolved solids = 1500 mg/L; total organic carbon = 10 mg/L. The water was passed through a 2 inch by 4 inch cartridge at 10 liters per minute. Alumina Ceramic Nanofiber Results: For our initial evaluation of the alumina ceramic nanofiber pleated filters, we used the bacteriophage MS2, poliovirus 1, and Escherichia coli. The alumina ceramic nanofiber pleated filters fit in a standard filter housing (62 mm diameter ´ 127 mm length). Test volumes of 20 liters seeded with approximately 108 total viruses were evaluated at a flow rate of 2.5 liters per minute.
The pleated alumina ceramic nanofiber filters were very effective at retaining MS2 bacteriophage, with retention of the virus on the filters ranging from 97.4% to > 99.99% for 31 filters (average % retention = 99.8%, standard deviation = 0.59). Likewise, these filters were very effective at retaining poliovirus 1 (average retention of 99.92%, standard deviation = 0.01) and at capturing the bacterium Escherichia coli (average of 99.996% retention). These results are similar to those of more traditional filtering methods such as the use of 1MDS filters for viruses; however: "> In separate experiments with poliovirus 1 using a higher flow rate (5.7 L/min) and a higher volume of dechlorinated tap water (100 L), retention efficiencies were slightly lower (96% ± 2%, n = 4). Table 3. Virus retention efficiencies for the NanoCeram filters.
Virus # of trials Virus retention (% ± SD) Echovirus 1 5 > 99.98 ± 0.00 Coxsackievirus B5 4 > 99.991 ± 0.00 Adenovirus 2 4 > 99.997 ± 0.00 Specific Objectives 2 & 4 – Assessment of various methods for the optimal recovery of various microorganisms from the collector: Recovery of Organisms from the “Collector” (Primary Concentration): While the use of these filters will remove virtually all of the various types of pathogens from water, it is unrealistic to expect that all the organisms will be recovered with equal efficiency or with the same eluent. The goal is to develop a method with greater reproducibility, precision, and robustness that will be available at a lower cost than current methods. It is essential that the microorganisms on the filters be recovered in a viable form. Microbial retention on the “collector” is due to a combination of electrostatic and hydrophobic forces. The relative importance of these factors depends upon the nature of the surface of the adsorbent and the specific microorganism. The organisms were eluted from the “collector” using various eluents to break down hydrophobic interactions and/or electrostatic interactions (Table 4). Table 4. Types of Eluents Evaluated
Adsorption forces Eluent Hydrophobic interactions Surfactants (Tween 80) Chaotropic agents (e.g., Cl3CCO2Na) Electrostatic interactions High pH (beef extract, glycine) Cationic detergents Salt solutions Sodium polyphosphate (highly negatively charged)
The glass wool filters were not included in further studies due to the variability in their virus retention efficiencies. The variability observed with these filters is not well understood; however, these filters are hand-packed and thus there are likely small differences between filters. In addition, these filters require refrigeration and have a shelf-life of a few months. Therefore, differences in the age of the filter being used could also lead to some variability between experiments. MS2 coliphage and E. coli were used to evaluate various eluting solutions for their ability to recover viruses and bacteria from the various filters. The recovery of microorganisms from the carbon nanofiber filters was extremely poor, with efficiencies of <2%. It is believed that the microorganisms strongly adhere to the carbon nanofiber surfaces and are therefore extremely difficult to recover. Since none of the organisms tested could be recovered from these filters, they were also eliminated from subsequent analyses. The recovery of MS2 coliphage from the NanoCeram alumina nanofiber filters is shown in Table 5. We were able to elute the virus with efficiencies similar to existing methods using eluents such as phosphate buffer, glycine, and sodium polyphosphate. It appears that the phosphate groups in the eluting buffers may be interacting with the oxygen/hydroxyl groups on the alumina of the filter. Nevertheless, we were not able to recover E. coli from the NanoCeram filters with acceptable efficiencies (range of 1.8% to 6.4% recovery). It is likely that due to the larger size, the bacteria become entrapped within the filter and are not able to be recovered. Protozoa, which are even larger in size, are therefore also not likely to be adequately recovered from these types of microporous filters. Therefore, these filters appear to be only effective for the recovery and concentration of viruses. These filters are available at a much lower cost than the filters typically used to concentrate viruses from large volumes of water [$40 (alumina nanofiber) versus ~$150 (1MDS) per filter]. The most effective eluting solution containing sodium polyphosphate, phosphate buffer and glycine at a pH of 9.3 was then used to recover several human viruses from the filters (Table 6). Greater elution efficiency was observed when lower influent titers of MS2 coliphage were used. The number of viruses present in real water samples (from various sources) would likely be more reflective of this lower influent titer. Table 5. Recovery of MS2 from NanoCeram filters using various eluting solutions able 6. Virus elution efficiencies from NanoCeram filters Table 6. Virus elution efficiencies from NanoCeram filters
Eluting Solution pH Elution efficiency (% ± SD) 3% beef extract 9.3 34 ± 18 Glycine 9.3 0.4 ± 0.5 3% beef extract + Glycine 9.3 12 ± 1 Phosphate Buffer + Glycine 9.3 26 ± 5 Phosphate Buffer + Glycine 7.5 24 ± 7 Phosphate Buffer + Glycine + 0.3% Tween80 9.3 37 ± 2 Sodium Polyphosphate + Phosphate Buffer + Glycine (108 virus titer)
9.3 57 ± 3 Sodium Polyphosphate + Phosphate Buffer + Glycine (108 virus titer)
7.5 26 ± 4 Sodium Polyphosphate + Phosphate Buffer + Glycine (104 virus titer)
9.3 86 ± 9 0.6 M NaI + Phosphate Buffer + Glycine 9.3 3 ± 2 Table 6. Virus elution efficiencies from NanoCeram filters
Virus Elution efficiency (% ± SD) MS2 coliphage (108 PFU) 57 ± 3 MS2 coliphage (104 PFU) 86 ± 9 Poliovirus 1 69 ± 8 Echovirus 1 134 ± 27 Coxsackievirus B5 72 ± 13 Adenovirus 2 39 ± 13
Secondary Concentration of the Recovered Viruses: An added goal was to minimize the volume of eluent and the amount of substances that might interact with detection methods such as cell culture and polymerase chain reaction (PCR) assays. Our method results in an eluate of approximately 450 mL from the alumina ceramic nanofiber filters. Centricon Plus-70 ultrafilters (30 kDa cut-off; Millipore, Billerica, MA) were utilized to further concentrate the eluted viruses. The process of organic flocculation used for concentrating beef extract (proteinaceous) eluents typically results in secondary concentrate volumes ranging from 15 to 40 ml. The average volume of the secondary concentrates in this study measured 3.3 ± 0.3 ml. The results for the optimized secondary concentration step are shown in Table 7. Table 7. Virus secondary concentration efficiencies using Centricon ultrafilters
Virus Secondary concentration efficiency (% ± SD) MS2 coliphage (108 PFU) 75 ± 21 MS2 coliphage (104 PFU) 65 ± 6 Poliovirus 1 95 ± 5 Echovirus 1 61 ± 18 Coxsackievirus B5 109 ± 11 Adenovirus 2 33 ± 14
The overall method efficiencies for the recovery of each virus were determined by comparing the virus titers measured in the secondary concentrates to the numbers of viruses originally used to seed the dechlorinated tap water. Concentration efficiencies of 66% to 83% were achieved for all of the human viruses tested with the exception of adenovirus 2 (14%). Similar method efficiencies were found for both high seed titer (108 PFU) and low seed titer (104 PFU) trials with MS2 coliphage.
Specific Objective 3 – Evaluate the recovery methods with waters of varying quality:
Inhibition of Detection Methods for Recovered Viruses: The concentrated human viruses were enumerated in all cases using cell culture assay methods. The secondary concentrates of adenovirus 2 required a de-salting step (to remove ~99% of salts) due to toxicity observed with the PLC cell line (this was not necessary for any of the other viruses tested). Following the de-salting step, no further problems were observed with the adenovirus 2 assays.
Although the primary poliovirus concentrate in the eluting solution did inhibit PCR, this inhibition was eliminated during the secondary concentration step. In addition, the semi-nested PCR product was observed even with an initial virus titer of only 10 PFU/L of eluting solution.
We also compared the NanoCeram and 1MDS electropositive filters for their ability to concentrate poliovirus in 40L of secondary wastewater effluents (Table 8). The 1MDS filters were eluted with beef extract followed by secondary concentration via organic flocculation following Standard Methods. The poliovirus was recovered from the NanoCeram filters using the methods established during this project involving elution with a sodium polyphosphate-based solution followed by secondary concentration via Centricon ultrafilters The mean retention of poliovirus 1 by the NanoCeram and 1MDS filters were >96.7% ± 0.6% and >90.8% ± 4.6%, respectively. The overall method efficiency for the NanoCeram and 1MDS filters were 54.3% ± 12% and 22.0% ± 6%, respectively.
Table 8. Wastewater characteristics
Characteristics Measurements range Range Mean pH 6.74 - 6.89 6.83 Temperature (°C) 24.4 - 25.7 24 Total Suspended Solids (mg/L) 0.44 - 0.45 0.45 Turbidity (NTU) 2 - 3 2 Free chlorine (mg/L) 0.5 - 0.6 0.5
Specific Objective 5 – Use the collector to assess the occurrence of adenoviruses and microsporidia in water:
Since the NanoCeram filters and elution methods developed during the project period were found to be inadequate for the concentration and recovery of large microorganisms such as microsporidia and also were not very efficient at recovering adenoviruses from water, this specific objective was not pursued.
Conclusions:
None of the filtration methods studied during the course of this project appear to be suitable for the concentration and recovery of all classes of microorganisms. Although the carbon nanofiber filters were extremely capable of concentrating bacteria, viruses, and protozoa, none of these organisms could be effectively recovered from the filters. Therefore, this type of filter would not be useful in situations in which one needs to be able to detect and/or enumerate the captured microorganisms.
The glass wool filters show promise as a new approach to concentrating multiple classes of microorganisms; however, until the variability in the filter efficiency is reduced, this method is not reliable enough for routine use. If the variability is due to the presumably inconsistent method of production of the filters, perhaps in the future with a more standardized production method, these filters may be reconsidered.
As with the carbon nanofiber filters, the NanoCeram filters are able to capture and concentrate all classes of microorganisms with high efficiencies. Also similar to the carbon nanofiber filters, the recovery of the captured microorganisms from the filters is inadequate for larger microorganisms such as bacteria and protozoa. Nevertheless, the NanoCeram filters are able to recover viral pathogens from water with fairly high efficiencies.
Since the development of pleated, positively charged filters for the concentration of viruses from large volumes of water in the 1980’s, there has been no significant improvement in concentration technologies for viruses from water. The NanoCeram filters, along with the elution and secondary concentration methods developed in the present study are able to recover viral pathogens from water with efficiencies at least equal to currently available methods used for pleated microporous filters, and with much lower secondary concentrate volumes. These filters are also less expensive than the 1MDS filters that are typically used to concentrate viruses from water. This lower volume should help to increase the efficacy of current detection methods by allowing for the assay of very small volumes. In addition, this method appears to be compatible with both cell culture and PCR detection methods. Currently used methods which utilize organic flocculation as a secondary concentration step can interfere with molecular detection methods such as PCR.
In the current study, the NanoCeram alumina nanofiber filters were found to be effective for the concentration of viruses from dechlorinated tap water and secondary wastewater. Other researchers have also demonstrated the effectiveness of these filters for virus detection in source waters and seawater (Gibbons et al. 2010). These filters are therefore a viable alternative to currently used methods and may be used to detect viruses in a variety of water matrices. In addition, the lower cost of these filters may allow for virus monitoring under circumstances that were previously cost-prohibitive
References:
Adams, M. H. 1959. Bacteriophages. Interscience Publishers, Inc., New York, NY.
Bidawid, S., N. Malik, O. Adegbunrin, S. S. Sattar, and J. M. Farber. 2003. A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus. J. Virol. Methods 107: 163-167.
Gibbons, C. D., R. A. Rodriguez, L. Tallon, and M. D. Sobsey. 2010. Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses, adenoviruses, and male-specific coliphages from seawater. J. Appl. Microbiol. Epub ahead of print: 1-7.
Payment, P., and M. Trudel. 1993. Isolation and Identification of Viruses, p. 32-33. In P. Payment, and M. Trudel (ed.), Methods and Techniques in Virology. Marcel Dekker, Inc., New York, New York.
Rodriguez, R. A., P. M. Gundy, and C. P. Gerba. 2008. Comparison of BGM and PLC/PRC/5 cell lines for total culturable viral assay of treated sewage. Appl. Environ. Microbiol. 74:2583-2587.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this projectSupplemental Keywords:
viruses, water, concentration, filter, elution,, RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Environmental Monitoring, Drinking Water, Microorganisms, enteric viruses, aquatic organisms, bacteria, CCL, viruses, drinking water monitoring, activated carbon, parasites, contaminant removal, drinking water contaminants, drinking water treatment, contaminant candidate listProgress 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.