CICEET Progress Report for the period 3/16/06 Through 9/15/06

Project Title: Microbial source tracking using F-specific coliphages and quantitative PCR
Principal Investigator(s): David C. Smith

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

We chose to utilize F⁺-specific RNA coliphages (viruses that infect coliform bacteria) to address microbial source tracking. Coliform bacteria reside in the guts of humans and animals. This family of viruses (Leviviridae) contains four subgroups. Two of the subgroups are found in human waste while the other two are found in animal waste. By knowing the ratio of the four subgroups, estimates of the relative contributions of animal waste and human waste can be determined. F⁺-specific coliphages have been used in efforts to track the source of microbial contamination of surface waters in the past. These attempts rely on propagating the viruses in a host cell (Escherichia coli) in order to determine the ratio of the four subgroups. This method is severely hampered by inherent biases in the replication rates of the four viral subgroups.

We developed a non-culture based approach to quantifying F⁺-specific coliphages from water samples that maintains the in situ ratio of the viruses and therefore provides a more accurate estimate of the relative contributions of the sources of fecal contamination. We used real-time, reverse transcriptase polymerase chain reaction (real-time RT-PCR) to quantify the number of viruses belonging to each subgroup. For this effort we designed four molecular beacons in order to perform multiplexing reactions (i.e. all four subgroups are quantified in the same reaction tube). Molecular beacons are fluorescent DNA probes designed to fold back on themselves in the absence of a target thereby masking the fluorescence. In the presence of a target, in this case the virus they are designed for, the probe emits a fluorescent signal that is quantified.

We demonstrated that the molecular beacons are very specific and can be used to determine the coliphage abundance in samples spanning six orders of magnitude. The ability to quantitatively estimate the abundance of all four subgroups makes the use of Fspecific coliphages a viable option for microbial source tracking. This method is quite sensitive as we are able to detect as little as 50 individual coliphages in a sample. The use of real-time RT-PCR is also more rapid than the culture-based method using Fspecific coliphages although this point is minor compared to the quantitative advantages afforded by the new method.

We have also demonstrated the ability of the molecular beacons to discriminate among the four subgroups and have expanded the database by typing coliphage in fecal samples from animal types that had not previously been tested. The method for the quantification of the coliphages is complete and we have a manuscript accepted for publication in Applied and Environmental Microbiology (Kirs and Smith Accepted). This project funded the bulk of Marek Kirs’ PhD dissertation at the University of Rhode Island (Kirs 2005). The remaining task is to complete the virus collection and recovery from field samples.

This method will be useful for water quality managers dealing with highly impacted areas. Because this method relies on sophisticated instrumentation and highly skilled technical staff, it is not meant for routine monitoring of surface waters. Areas that are chronically impacted by high concentrations of fecal bacteria are of particular interest.

This method can be applied to determine the source of the contamination prior to implementing an expense remediation scheme (e.g. sewering neighborhoods).

The cost of applying the method is estimated in the range of $200 - $300 per sample but will depend greatly on the number of samples analyzed. The costs include the consumables, instrumentation and technical support. The main instrument (real time PCR machine) does not have unusual maintenance requirements. The method is fairly rapid with a 24-hour turnaround time if needed, otherwise a 48-hour turnaround time is easily achieved. Because this method is not for routine monitoring, a two-day turnaround time is more than adequate. The main drawback of the method is that it is not particularly user-friendly, due to the relatively high skill level required of the technical staff. It is not envisioned that see assay will be performed by the end user, rather, it is more likely that a centralized facilities will conduct the assay on samples supplied by the end users.

Project Development
Abstract
Microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Water quality managers are hampered in providing effective remediation strategies by the inability to routinely identify the source of microbial contamination. Efforts to identify the source of the fecal contamination (animal waste vs. human waste) falls under the term ‘microbial source tracking’ or MST. One promising method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F⁺-specific RNA coliphages in impacted water samples. This family (Leviviridae) of viruses contains four subgroups. Two of the subgroups are found in human waste while the other two are found in animal waste. By knowing the ratio of the four subgroups, estimates of the relative contributions of animal waste and human waste can be determined. Because of typically low concentrations of these viruses in the environment, enrichment assays are typically performed prior to detection in order to increase the sensitivity of the assay. Our data indicate that due to differential replication rates of the viruses during the enrichment assay, this approach is not valid for MST efforts. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment thus obscuring the initial ratio. To overcome this bias, we developed a cultureindependent multiplex real-time RT-PCR protocol for the simultaneous quantification of all four subgroups of F⁺-specific RNA coliphages using novel primer sets and molecular beacons. This assay is extremely sensitive; achieving detection with as few as ten copies of isolated coliphage RNA and is linear for a minimum of six orders of magnitude. Because coliphages quickly loose their ability to infect the host cells after exposure to seawater, we compared the decay rates of infectivity and the decay rates of detection by RT-PCR and found that the real-time RT-PCR technique was able to quantify coliphages in seawater when culture based assays failed. More importantly, while infectivity was lost at different rates at the subgroup level, decay constants in seawater did not vary among subgroups when analyzed using the RT-PCR method. This ensure that the initial in situ ratio of the four subgroups is maintained over time and therefore is an accurate representation of the relative contributions of organisms to the fecal contamination. The accurate determination of the in situ concentration of F⁺-specific RNA coliphages using this method will facilitate more effective remediation strategies for impacted environments.

Introduction
Although it is well documented that microbial contamination of coastal waters poses a risk to human health through recreational exposure and consumption of shellfish, the source of the contamination may play a role in dictating the level of actual risk. Protocols to identify microbial contamination have been developed and standards set on allowable concentrations of different groups of bacteria: E.coli, fecal coliforms, enterococci (Clesceri et al. 1998; Usepa 2000b) in surface waters. Other organisms including Shigella spp., Klebsiella spp., Salmonella spp., Yersinia spp., Clostridium perferingens and Fspecific RNA coliphages (Leviviridae) have been suggested as indicator organisms for marine and/or freshwater systems (Griffin et al. 2001). Each of the indicators available has its own advantages and disadvantages, and the challenge is to select the appropriate indicator, or combination of indicators, for the particular purpose of water quality assessment (WHO 2000). Currently, about 40% of the U.S. waters (fresh and marine) are too polluted for swimming, fishing, and supporting aquatic life (U.S. EPA 2000b). There were at >13,000 beach closings and advisories issued in the U.S. during 2001 (Dorfman 2002). During this time, 87% of the closings and advisories were due to monitoring that revealed the presence of bacteria associated with fecal contamination yet 54% of these incidences were attributed to unknown sources of fecal contamination.

Several methods have been proposed to discriminate human from non-human fecal contamination in surface waters using different microorganisms or chemical parameters. One of the first and most commonly applied approaches was to use the fecal coliform to fecal streptococci ratio (Geldreich and Kenner 1969). This method was soon discarded due to variable survival rates of fecal streptococci, variable detection methods and sensitivity (Clesceri et al. 1998; Pourcher et al. 1991). Since then, other organisms have been proposed as indictors: Bifidobacterium (Mara and Oragui 1983), Escherichia coli (Wiggins 1996), Clostridium perferingens (Fujioka and Shizumura 1985), bovine enterovirus (Ley et al. 2002), Bifidobacterium phages (Tartera et al. 1989), F-specific RNA coliphages (Osawa et al. 1981). Several studies have targeted specific human enteric viruses instead of indicator organisms (Griffin et al. 1999; Jiang et al. 2001; Lee and Kim 2002). E. coli, as an indicator organism, has been most extensively studied. Different phenetic and molecular methods have been developed for source tracking using this organism: multiple antibiotic resistance patterns (Krumperman 1983), immunological assays (Parveen et al. 2001), pulse field gel electrophoresis (Simmons et al. 2000), denaturing gradient gel electrophoresis (Buchan et al. 2001; Farnleitner et al. 2000), repetitive element PCR (Carson et al. 2001; Dombek et al. 2000), amplified fragment length polymorphism (Guan et al. 2002), ribotyping (Parveen et al. 1999) and length heterogeneity PCR and terminal restriction fragment length polymorphism (Bernhard and Field 2000). While some of these methods have been relatively successfully applied in field studies, many concerns have been lately expressed: especially concerning reference database issues (which is limited and location specific - regional differences have been reported), changes in subpopulations during transition from primary (host) to secondary (outside the host) habitat, variations in methodology, reproducibility concerns, requirement for cultivation, cost and time issues (Guan et al. 2002; Simpson et al. 2002). Even more, Gordon (Gordon 2001), stated based on extensive genetic diversity studies that any program attempting to identify source of fecal contamination focusing on commensal E. coli appear to have very limited value. A similar opinion was shared by Dykes (Dykes 2002) suggesting that tracking other bacteria than E. coli may be more valid.

Instead of using E. coli or other indicator bacteria we have been working to further develop an alternative approach based on detecting F⁺-specific RNA coliphage. These single stranded RNA containing viral particles (~ 26 ­ 27 nm diameter) infect coliform bacteria via F (fertility) pili (Zinder 1975). The figure at the right is a transmission electron micrograph showing E. coli with attached coliphage, the inset is a close of a pilus with attached coliphage (Figure 1 - photo by M. Kirs). An important characteristic of these phages is that they can not replicate once released into most environments from the animal intestines (Woody and Cliver 1995) as F pili are not produced below 25°C and maximally produced at 37°C (Novotny and Lavin 1971).

Another important feature of F⁺-specific RNA coliphages is their similarity to enteric viruses. Enteric viruses are major agents of waterborne outbreaks of diseases such as gastroenteritis and infectious hepatitis (Hedberg and Osterholm 1993; Moore et al. 1993). F⁺-specific RNA coliphages are similar to them in structure, morphology, origin, release and tolerance to environmental conditions and thus have been suggested as useful proxy for human enteric viruses (Grabow 1990; Havelaar et al. 1993). The absence of F⁺- specific RNA coliphages offers a meaningful indication of the absence of human enteric viruses in the sample (Havelaar and Nieuwstad 1985), an important conclusion that can not be drawn from any phenetic or molecular bacterial study. Consistent similar seasonal trend between Norwalk-like viruses, F⁺-specific RNA coliphages and incidences of oyster-associated gastroenteritis was documented (Dore et al. 2000).

F⁺-specific RNA coliphages constitute the taxonomically distinct family Leviviridae (Murphy et al. 1995). This family contains two genera: Allolevivirus and Levivirus which both contain two distinct subgroups based upon serological cross-reactivity (Furuse 1987). Levivirus contains subgroup I and II and Allolevivirus contains subgroup III and IV. The key trait on which F-specific RNA coliphage approach of source tracking is based on, is that subgroup I and IV are predominantly isolated from non-human feces while subgroup II and III are predominantly isolated from human feces (Furuse 1987; Griffin et al. 2000; Havelaar et al. 1990; Hayward 1999; Hsu et al. 1995; Uys 1999). This phenomena was first observed by the group at the Keyo University (Osawa et al. 1981). As original serogrouping methodology using phage antisera was expensive and time consuming, hybridization techniques using dioxigenin-labeled probes (Hsu et al. 1995) and/or radioactive probes (Beekwilder et al. 1996) were introduced. More recently biotin labeled probes have been used for genotyping of F-specific RNA coliphages (Griffin et al. 2000). We have developed molecular beacon based methodology for genotyping F⁺- specific RNA coliphages that allows for in solution hybridization.

While this method is very promising, several aspects need to be addressed prior to it becoming an effective tool for water quality management. All current hybridization techniques of genotyping F-specific RNA coliphages require cultivation. There is no consensus on which host bacterium must be used (Leclerc et al. 2000) and problems with host stability have been reported (Leclerc et al. 2000). In addition, there is no consensus on how to concentrate phages from environmental samples (Furuse 1987; Grabow 2001; Leclerc et al. 2000). It has been claimed that methods in use are not reproducible (Leclerc et al. 2000). In addition to these concerns, our preliminary experiments have shown that common enrichment assay (Griffin et al. 2000; U.S. EPA 2000a) is biased.

There is a critical need at local, regional and national levels for a method that allows reliable discrimination of human and non-human microbial contamination (microbial source tracking). We are convinced that F⁺-specific RNA coliphage approach has many significant advantages to become a successful method for distinguishing between human and non-human fecal contamination in any water system.

Objectives
The objective of this study is to develop a rapid method to discriminate human from nonhuman microbial contamination in aquatic systems. This capability would greatly enhance the decision-making process by focusing efforts on a more specific source of contamination.

a) Database extension. One of the limitations of F⁺-specific RNA coliphage approach is that these coliphages have been identified from relatively limited number of different animals. Phages from many common in temperate areas animals like deer, raccoon, squirrels, rats, mice, seagulls and other birds, etc., have not yet been identified. We used the molecular beacons developed in this study to type the newly isolated coliphages.

b) Real-time quantitative RT-PCR. Current hybridization methodologies all require cultivation in order to obtain sufficient amounts of viral RNA for detection. Because there is no consensus on which host bacterium to use and problems with host stability (Leclerc et al. 2000) an alternative method for amplifying the signal is desirable. These methods are relatively time consuming and the number of phages typed per sample is limited. We developed a real-time quantitative RT-PCR assay for genotyping F⁺-specific RNA coliphages thus eliminating the cultivation step and problems associated with host bacterium selection. It also allows us to type all coliphages present in the sample.

c) Coliphage Survival. Relatively little is known on the survival of four subgroups of F⁺- specific RNA coliphages. Appreciable variation appears to exist in the survival of different F⁺-specific RNA coliphage strains of the same subgroup (Brion et al. 2002). We conducted laboratory experiments to study survival of different subgroups in sterile seawater to compare the decay rates for infectivity versus the decay in the RT-PCR signal.

f) Field studies. We will apply our real-time quantitative RT-PCR methodology in the field to determine the source of fecal contamination. One of the problematic water bodies in the southern Rhode Island is the Pettaquamscutt Estuary (Narrow River) in which fecal coliform concentrations are high. The source of the contamination is unknown as recent sewering of the adjacent neighborhoods has not resulted in a significant reduction in the fecal contamination as evidenced by coliform counts. The Pettaquamscutt Estuary flows to the Narragansett Bay just at the north end of the Narragansett State Beach. We propose to apply our filtration and real-time quantitative PCR techniques to study the reasons of elevated fecal coliform concentrations on this beach.

Methods
a) database extension Fecal samples were collected in the field using sterile spatulas and placed into 8 ml BEGT in 15 ml polycarbonate tube and transported into the laboratory. Samples are vortexed and serially diluted. Phages will be isolated using double-agar overlay technique as described (Griffin et al. 2000) using E.coli FAMP as a host bacterium. After 12 hours 20 phages were isolated per sample using sterile pipette and suspended in 0.5 ml SM solution. Suspended material will be incubated at room temperature for 3-4 hours with periodic vortexing.

Isolated viruses were confirmed to be RNA phages by spotting phage solution (10 µl) individually on a top agar containing RNase (40 µl ml^-1). We analyzed only those phage isolates that did not form plaques on top agar containing RNase. Figure 3 shows coliphage plaques (clear) in a plate containing the host cells (red).

RNA was extracted from phage material using TRI Reagent LS according to manufacturer's protocol (Molecular Research Center, Inc.) and RNA pellet suspended in the hybridization buffer (100 mM TRIS-HCl pH 7.5, 100 mM NaCl, 3 mM MgCl₂). The RNA was subjected to the real-time RT PCR protocol in the presence of all four molecular beacons. The phages were classified on the basis of the response to the beacons.

b) Real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). All F⁺-specific coliphage sequences in the GenBank database were downloaded and analyzed for targets for primers and beacons. Molecular beacons become fluorescent when hybridized to their target nucleic acid (Figure 4). We identified targets in the RNA replicase gene for subgroups I, II and IV and the gene encoding for the coat protein for subgroup III. Because of the limited amount of sequence data in GenBank for these coliphage, particularly subgroup IV therefore, we sequenced the genome of our isolate of Fi. The genomes were analyzed and sites for molecular beacons were determined using mFold. PCR primers were designed to flank the sites identified for the molecular beacons.

c) Coliphage survival. The survival time of F⁺-specific coliphages after they are released into the water has significant implications the usefulness of these viruses in MST applications. The length of time in which the viruses can be detected is function of what to determine if the survival time differed. This was accomplished by adding phages to filtered seawater (0.45 µm filtered). These experiments were conducted in triplicate. The samples were incubated away from direct sunlight at ambient room temperature (~22 °C). Samples were assayed for infective phages using double agar overlay (DAL) technique and for total phage concentrations using the real-time RT-PCR technique at 24-hour intervals. Volumes tested by DAL technique varied; initially 1:100 dilutions were necessary. The image on the right is an example of quantifying infective phages (clear zones) using the DAL technique.

d) Field samples. The culture-based, DAL method was used to confirm high concentrations of Fspecific RNA phages in a raw sewage sample from the Narragansett Wastewater Facility (Rhode Island, USA) and a chicken stool sample (Casey Farm, Saunderstown, Rhode Island, USA) diluted in 5 ml of filtered (0.2 µm) and autoclaved seawater. Twenty phage isolates, which did not form plaques in the presence of RNAase, were typed from each sample using the real-time RT-PCR protocol. In addition, F⁺- specific RNA coliphages were quantified directly from the mixed samples using the RTPCR assay. The slurries were vortexed and centrifuged at 3000 x g for 10 min to remove larger solids. Supernatant of both samples was mixed at ratios of 1:0, 0.75:0.25, 0.5:0.5, 0.25:0.75, 0:1 (vol:vol); followed by RNA extraction (as above) and real-time RT-PCR quantification. Initial phage concentrations were determined by using a serial dilution of 1 ml for DAL and 0.1 ml for RT-PCR, the results were normalized to phage per ml. PCR inhibition. Samples of different origin (commercial drinking water, freshwater pond, seawater, cat and cow feces (0.5 g wet weight/ ml)) were verified to contain no detectable levels of F⁺-specific RNA coliphages using DAL and real-time PCR technique; and subsequently seeded with a cocktail of coliphage stocks: 171.7, 114, 6.7 and 3.5 x104 copy/ml final of subgroup I, II, III and IV respectively. Phage stocks were mixed and diluted in SM buffer (28) parallel to other treatments and used as controls of initial concentrations. RNA was extracted from each treatment using TRIReagent® LS extraction protocol and quantified using real-time RT-PCR protocol as specified above. Quadruplicate experiments were conducted for each treatment.

Samples were collected from Mumford Brook (Wakefield, Rhode Island). This brook, part of the Pettaquamscutt Estuary, is chronically impacted with high coliform counts. Two houses near the impacted area are on septic systems and the area hosts abundant wildlife, therefore, assigning a source to the coliforms is not straightforward.

Results
a) Database extension. F⁺-coliphage present in fecal samples from animals collected in the watershed were propagated in the host cells and assayed with the four molecular beacon/PCR primer sets to identify. We were able to confirm the presence of coliphages belonging to subgroup I in fecal samples from cat, chicken, seagull and an unidentified bird. Samples collected at municipal wastewater treatment facility contained coliphage subgroups II and III. No F⁺-specific coliphages were detected in fecal samples collected from Canada geese, cow, coyote, deer, dog, goat horse, pigeon, rabbit or sheep. The new animals in the database confirm the overall segregation of the four subgroups of coliphages. The absence of F⁺-specific coliphages in some of the animal fecal samples indicates that either not all animals harbor the coliphage or that the phage detection was not efficient. The screening of additional samples from these animal groups will assist in answering this question.

b) Real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Four separate molecular beacon/PCR primer pairs were designed, one set specific for each subgroup of coliphages. The structures of the molecular becons are shown below. Becon I contains the fluorochrome FAM-6-carboxyfluorescein, II contains HEX-hexachloro-6- carboxyfluorescein, III contains CY-5 - Indodicarbocyanine, and IV contains Texas Red. The use of different fluorochromes allows for multiplexing reactions as the instrument is capable of quantifying all four fluorescent signals in the same reaction.

The specificity of the PCR primers was tested against RNA extracted from laboratory isolates of F⁺-specific coliphages. There was no cross reactivity as indicated in the figure of the agarose gel. Each primer set amplified only coliphages from one subgroup. This figure is from Kirs and Smith (accepted).

The complete sequence of the genome of coliphage Fi was submitted to GenBank under accession number EF068134. The sequencing was accomplished by designing PCR primers at sites that would yield products of a size that could be completely sequenced. The agarose gel on the left contains the initial PCR products, gaps were filled in with PCR primers designed based on the initial sequence data (Figure 8).

c. coliphage survival. There is a significant difference among decay rates of the four subgroups of coliphages in seawater when analyzed by the culture dependent method (DAL) there is no difference amongst the groups when analyzed by real-time RTPCR. In addition, the signal from the viral RNA can be detected long after the viruses are no longer infective. The realtime PCR data are plotted with open symbols and the plaque forming units quantified via DAL are in the closed symbols. Figure 9 from Kirs and Smith (accepted)

d) field samples. The method was able to clearly distinguish waste collected at a municipal wastewater treatment facility and that from waste collected from a chicken farm. Mixtures of the two wastes at different ratios was used to determine if the real-time RT-PCR method could accurately reproduce the relative contributions of each type of waste in the mixture. The method accurately reproduced the ratios. The dark bars represent the percentage of municipal waste (100, 75, 50, 25, 0) and the open bars represent the percentage of waste from the chicken farm (0, 25, 50, 75, 100).

Sample collected at Mumford Brook were dominated by subgroup I which indicates that the high coliform counts are due to the wild life in the watershed, not the houses that abut the stream.

Utilization
To date, no end users have utilized this method. We have had inquires from the local EPA office as well as a community group in King County WA. We did test one chronically impacted site in the Pettasquascutt Estuary that has been identified by the Narrow River Preservation Society. Our results indicate that animal sources dominate the source of fecal contamination at this site.

I met several times with the intellectual property office at the University of Rhode Island. An initial filing of the idea was done prior to presenting the data at the 2005 meeting of the American Society for Microbiology. The intellectual property office pursued different avenues for commercialization but ultimately decided to not pursue a patent. We presented this method to a group of water quality managers at a CICEET workshop held in September 2006 in Durham, NH.

Next Steps to Application
The quantitative removal of coliphages from water samples has not yet been optimized. This work involves getting water samples from a variety of sources that encompass a wide range of salinities, organic matter, fecal loading etc. The quantitative removal of the coliphages from the water has been worked out. There are still some unresolved questions regarding the elution of the viruses from the filter prior to the RT real-time PCR step. We have demonstrated that the extraction efficiency varies among sample types. This is not important, what is critical is whether the extraction efficiency is consistent among the subgroups of coliphages regardless of the sample type. Our results to date suggest this is the case but the range of samples tested is too small to make any conclusions.

While the method is not intended to be used a routine monitoring tool, it would be helpful to streamline the method with the intent to increase sample through put. This would best be done by adapting a 96-well format for all stages of the procedure. Increasing the throughput would result in a reduction in the per sample cost of analysis.

Literature Cited

Beekwilder, J., R. Nieuwenhuizen, A. H. Havelaar, and J. Van Duin. 1996. An oligonucleotide hybridization assay for the identification and enumeration of Fspecific RNA phages in surface water. J. Appl. Bacteriol. 80: 179-186.

Bernhard, A. E., and K. G. Field. 2000. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66: 4571-4574.

Brion, G. M., J. S. Meschke, and M. D. Sobsey. 2002. F-specific RNA coliphages: occurrence, types, and survival in natural waters. Water Res. 36: 2419-2425.

Buchan, A., M. Alber, and R. E. Hodson. 2001. Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S intergenic spacer region. FEMS Microbiol. Ecol. 35: 313-321.

Carson, C. A., B. L. Shear, M. R. Ellersieck, and A. Asfaw. 2001. Identification of fecal Escherichia coli from humans and animals by ribotyping. Appl. Environ. Microbiol. 67: 1503-1507.

Clesceri, L. S., A. E. Greenberg, and A. D. Eaton. 1998. Standard Methods for examination of water and wastewater, 20th ed..

Dombek, P. E., L. K. Johnson, S. T. Zimmerly, and S. M. J. 2000. Use of repetitive DNA sequencies and the PCR to differentiate Eischerichia coli isolates from human and animal sources. Appl. Environ. Microbiol. 66: 2572-2577.

Dore, W. J., K. Henshilwood, and D. N. Lees. 2000. Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. Appl. Environ. Microbiol. 66: 1280-1285.

Dorfman, M. 2002. Testing the Waters XII: A Guide to Water Quality at Vacation Beaches., p. 211. NRDC.

Dykes, G. A. 2002. Tracing contamination and Escherichia coli diversity. Appl. Environ. Microbiol. 68: 4698.

Farnleitner, A. H., N. Kreuzinger, G. G. Kavka, S. Grillenberger, J. Rath, and R. L. Mach. 2000. Simultaneous detection and differentiation of Escherichia coli populations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene. Appl. Environ. Microbiol. 66: 1340- 1346.

Fujioka, R. S., and L. K. Shizumura. 1985. Clostridium perferingens: a reliable indicator of stream water quality. J. Water Pollut. Control Fed. 57: 986-992.

Furuse, K. 1987. Distribution of coliphages in environment.: General considerations. John Wiley & Sons.

Geldreich, E. E., and B. A. Kenner. 1969. Concepts of fecal streptococci in stream pollution. J. Water. Pollut. Control. Fed. 41: R336-R352.

Gordon, D. M. 2001. Geographical structure and host specificity in bacteria and the implications for tracing the source of coliform contamination. Microbiology 147: 1079-1085.

Grabow, W. O. K. 1990. Microbiology of drinking water treatment: reclaimed wastewater. Spinger Verlag. ---. 2001. Update on application as models for viruses in water. Water SA 27: 251-268.

Griffin, D. W., C. Gibson, Iii, E. K. Lipp, K. Riley, J. H. Paul, Iii, and J. B. Rose. 1999. Detection of viral pathogens by reverse transcriptase PCR and of microbial indicators by standard methods in the canals of the Florida Keys. Appl. Environ. Microbiol. 65: 4118-4125.

Griffin, D. W., E. Lipp, M. R. Mclaughlin, and J. B. Rose. 2001. Marine recreation and public health: Quest for the ideal indicator. Biosci. 51: 817-826.

Griffin, D. W., R. Stokes, J. B. Rose, and J. H. Paul, Iii. 2000. Bacterial indicator occurrence and the use of an F⁺ specific RNA coliphage assay to identify fecal sources in Homosassa Springs, Florida. Microb. Ecol. 39: 56-64.

Guan, S., R. Xu, S. Chen, J. Odumeru, and C. Gyles. 2002. Development of a procedure for discriminating among Escherichia coli isolates from animal and human sources. Appl. Environ. Microbiol. 68: 2690-2698.

Havelaar, A. H., and Nieuwstad T. J. 1985. Bacteriophages and faecal bacteria as indicators of chlorination efficency of biologically treated wastewater. J. Water Pollut. Control Fed. 57: 1084-1088.

Havelaar, A. H., W. M. Pot-Hogeboom, K. Furuse, R. Pot, and M. P. Hormann. 1990. Fspecific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin. J. Appl. Bacteriol. 69: 30-37.

Havelaar, A. H., M. Van Olphen, and Y. C. Drost. 1993. F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water. Appl. Environ. Microbiol. 59: 2956-2962.

Hayward, K. 1999. Editorial: Phages gain ground as water quality indicators. Water (Magazine of International Water Association) 21: 36-37.

Hedberg, C. W., and M. T. Osterholm. 1993. Outbreaks of food-borne and waterborne viral gastroenteritis. Clin Microbiol Rev 6: 199-210.

Hsu, F., Chih, Y. S. C. Shieh, J. Van Duin, M. J. Beekwilder, and D. Sobsey Mark. 1995. Genotyping male-specific RNA coliphages by hybridization with oligonucleotide probes. Appl. Environ. Microbiol. 61: 3960-3966.

Jiang, S., R. Noble, and W. Chu. 2001. Human adenoviruses and coliphages in urban runoff-impacted coastal waters of Southern California. Appl. Environ. Microbiol. 67: 179-184.

Kirs, M. 2005. Quantitative analysis of F-specific coliphage. Ph. D. Dissertation, University of Rhode Island.

Kirs, M., and D. C. Smith. Accepted. Multiplex quantitative real-time RT-PCR for the F⁺ specific RNA coliphages: A method for use in microbial source tracking. Applied and Environmental Microbiology.

Krumperman, P. H. 1983. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods. Appl. Environ. Microbiol. 46: 165-170.

Leclerc, H., S. Edberg, V. Pierzo, and J. M. Delattre. 2000. Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. J. Appl. Microbiol. 88: 5-21.

Lee, S.-H., and S.-J. Kim. 2002. Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea. Water Res. 36: 248-256.

Ley, V., J. Higgins, and R. Fayer. 2002. Bovine enteroviruses as indicators of fecal contamination. Appl Environ Microbiol 68: 3455-3461.

Mara, D. D., and J. I. Oragui. 1983. Sorbitol-fermenting bifidobacteria as specific indicators of human faecal pollution. J. Appl. Bacteriol. 55: 349-357.

Moore, A. C., B. L. Herwaldt, G. F. Craun, R. L. Calderon, A. K. Highsmith, and D. D. Juranek. 1993. Survelliance of waterborne disease outbreaks - United States, 1991-1992. Morbid. Mortal. Weekly Rep. CDC Surveill. Summ. 42: 1-22.

Murphy, F. A. and others 1995. Virus taxonomy. Classification and nomenclature of viruses. Sixth report of the International Committee on Taxonomy of Viruses. Arch. Virol. Supplement 10: 51-54.

Novotny, C. P., and K. Lavin. 1971. Some effects of temperature on the growth of F pili. J. Bacteriol. 107: 671-682.

Osawa, S., K. Furuse, and I. Watanabe. 1981. Distribution of ribonucleic acid coliphages in animals. Appl. Environ. Microbiol. 41: 164-168.

Parveen, S., N. C. Hodge, R. E. Stall, S. R. Farrah, and M. L. Tamplin. 2001. Phenotypic and genotypic characterization of human and non-human Escherichia coli. Water Res. 35: 379-386.

Parveen, S., K. M. Portier, K. Robinson, L. Edmiston, and M. L. Tamplin. 1999. Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution. Appl Environ Microbiol. 65: 3142-3147.

Pourcher A. M., Devriese L. A., Hernandez J. F., and D. J. M. 1991. Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters. J. Appl. Bacteriol. 70: 525- 530.

R.I. DEM 2003. State of Rhode Island and Providence Plantations. 2002 Section 305(b). State of The State's waters. Report., p. Department of Environmental Managaement. Office of Water Resources.

Simmons, G. M., D. F. Waye, S. Herbein, S. Myers, and E. Walker. 2000. Estimating nonpoint fecal coliform sources in Northern Virginia's four mile run watershed, p. 248-267. In T. Younos and J. Poff [eds.], Proceedings of the Virginia Water Research Symposium 2000.

Simpson, J. M., J. W. Santo Domingo, and D. J. Reasoner. 2002. Microbial source tracking: state of the science. Environ. Sci. Technol. 36: 5279-5288.

Springthorpe, S. V., C. L. Loh, W. J. Robertson, and S. A. Sattar. 1993. in situ survival of indicator bacteria, MS-2 phage and human pathogenic viruses in river water. Water Sci. Technol. 27: 413-420.

Tartera, C., F. Lucena, and J. Jofre. 1989. Human origin of Bacteroides fragilis bacteriophages present in the environment. Appl. Environ. Microbiol. 55: 2696- 2701.

U.S. EPA. 2000a. Method 1601: Male-specific (F⁺) and somatic coliphage in water by two step enrichment procedure (Draft), p. 1-25. United States Environmental Protection Agency, Office of Water.

---. 2000b. National Water Quality Inventory: 1998 Report to Congress, EPA841-S-00- 001,. United States Environmental Protection Agency Office of Water.

Uys, M. 1999. Molecular characterization of F-specific RNA phages in South Africa. M.S. Thesis. University of Pretoria.

WHO. 2000. WHO Guidelines for Drinking Water Quality Training Pack, Microbiological Aspects. World Health Organization (WHO).

Wiggins, B. A. 1996. Discriminant analysis of antibiotic resistance patterns in fecal streptococci, a method to differentiate human and animal sources of fecal pollution in natural waters. Appl. Environ. Microbiol. 62: 3997-4002.

Woody, M. A., and D. O. Cliver. 1995. Effects of Temperature and Host Cell Growth Phase on Replication of F-Specific RNA Coliphage QB. Appl. Environ. Microbiol. 61: 1520-1526.

Zinder, N. D. 1975. RNA Phages. Cold Spring Harbor Laboratory.