CICEET Progress Report for the period 9/02/07 Through 3/01/08

Project Title: Rapid concentration and quantification of bacteria and viruses from marine waters
Principal Investigator(s): Valerie J. Harwood and Daniel V. Lim
Project Start Date: May 1 2007

Tables

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

2. Confirm the ability to recover culturable Enterococcus spp. cells from the ultrafiltration/RAPTOR™ system.

3. Quantify Enterococcus spp. cells using the ultrafiltration/RAPTOR system in samples collected concurrently with the SCCWRP epidemiology study.

4. Elute cells from RAPTOR™ waveguides followed by enumeration of total Enterococcus and enrichment/PCR quantification of Ent. faecium carrying the esp gene.

5. Quantify human polyomaviruses (viral indicators of human sewage) in samples collected concurrently with the SCCWRP epidemiology study by a) elution from hollow fiber filters and b) conventional filtration of 600 ml samples.

Tasks to meet objectives
Samples received from the SCCWRP California epidemiology study over the last several months have been completely processed (see below). The first set of samples received were proficiency samples that were intentionally seeded with fecal material (Table 1). All subsequent samples were unseeded environmental water samples (Table 2). In addition, a Taqman® QPCR was developed and optimized and archived DNA from this study will be analyzed using the developed assay.

Progress on Tasks
Human Polyomaviruses (HPyVs) QPCR assay
We have developed and optimized a Taqman® quantitative PCR (QPCR) assay based on the conserved T-antigen to quantify as well as simultaneously detect two HPyVs; JCV and BKV. The assay was able to consistently detect >10 viruses. Specificity was demonstrated by negative assay results from DNA extracted from 74 animal fecal samples. To assess the prevalence and quantity of HPyVs in human waste, samples were collected from wastewater treatment plants (n=39), lift stations (n=2), and septic tank pump trucks (n=5) and analyzed for HPyVs, which resulted in detection of HPyVs in 100% of the human waste samples. HPyVs concentrations ranged from 0.12-8.01x10⁴ viruses per ml.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal? Please explain.
No

Dissemination activities during this reporting period (please include the number of participants where applicable).
Posters at scientific meetings:
S. D. Leskinen and D. V. Lim. 2007. Rapid ultrafiltration concentration and biosensor detection of enterococci from large volumes of Southern California coastal marine waters. Annual Meeting of the Southeastern Branch of the American Society for Microbiology, Auburn, AL.

Student activity (e.g. theses, dissertations, etc.) on the project (please identify students as graduate or undergraduate).
Shannon McQuaig (graduate student) presented an oral presentation of the data at the Southeastern Branch ASM during November, 2007 in Auburn, Alabama. Ms. McQuaig worked throughout the fall and spring semesters on this project, and will continue into the summer semester.

S. McQuaig, T. M. Scott, and V. J. Harwood. 2007. Blinded evaluation of alternative indicator methods for use in an epidemiology study at Doheny Beach, California. Annual Meeting of the Southeastern Branch of the American Society for Microbiology, Auburn, AL. Nov 1 ­ 3.

Difficulties
None of note encountered.

Data Generated to date
The HPyVs conventional PCR assay was among the most accurate of the microbial source tracking tool utilized for the proficiency samples. Of the sewage-seeded samples, 71.4% were positive for HPyVs. None of the control water samples or the guano-seeded samples were positive for HPyVs.

Table 5 outlines the results to date of surface water (beach) samples received over the course of the study. Note, on June 3 the method blank for HPyVs was positive, indicating contamination, therefore the results are negated and are indicated with an asterix (*). The esp analysis was only performed on samples that exceeded enterococci standards based on RAPTOR results. RecDec filters were not received for the July 1, 2007 sampling. At the Avalon location, only sites A, B and C were sampled with the RecDec apparatus.

Concentration of HPyVs by Hollow Fiber Filtration
The ability of hollow fiber filtration to concentrate HPyVs was assessed by seeding either purified viral particles grown in culture (BK virus) or raw sewage into 10 L of PBS (0.01M). The seeded levels were ~2.5 X 10⁴ viral particles or 5 ml sewage. These levels correspond to 2.5 viral particles/ml in the 10L volume for the seeded culture, and a 2,000-fold dilution for the sewage sample. HPyVs were readily quantified by QPCR from either seeded viruses or sewage (Table 6). QPCR results were confirmed by conventional PCR (observation of an amplicon of the correct molecular weight).

Assessment of viability of enterococci adsorbed to waveguides after the RAPTOR assay.
Objective: Assess the ability of viable enterococci to be cultured from waveguides following biosensor analysis.

Methods: Each sample waveguide (2 replicates) and each control waveguide from randomly selected sample coupons were utilized for viability testing following sensor analysis. The coupons were used for sensor analysis from May 28 to September 3 at Doheny State Beach or Avalon. Waveguides were aseptically removed from the coupons as previously described by Simpson and Lim (1). The waveguides were incubated at 37°C for 24 h in 3 ml of Enterococcosel broth (Becton Dickinson, Franklin Lakes, NJ). Positive and negative media controls were incubated along with the waveguides in broth. Presence of viable Enterococcus spp. was confirmed by blackening of the medium, which signified cleavage of the esculin present in the broth.

Results: Viable enterococci were detected for each sample coupon on at least one sample waveguide, while half of the coupons were positive from both sample waveguides. Control waveguides performed as expected, as did the media controls.

Project Objectives for Next Reporting Period

Objectives
Analyze data in the context of the epidemiology results to determine correlation of our results with public health outcomes.
Finish objective 5.

Work Plan to Meet Objectives
Archived DNA from selected samples collected from both Doheny and Avalon Beaches will be analyzed using the Taqman QPCR to quantify HPyVs.

Dissemination Objectives for next reporting period
Ms. McQuaig will be presenting the developed HPyVs QPCR assay at the American Society for Microbiology General Meeting in June 2008. Dr. Stephaney Leskinen (postdoc) will present a poster on the biosensor work at ASM General Meeting. In addition, two manuscripts are in preparation.

S.M. McQuaig, T.M. Scott, J.O. Lukasik and V.J. Harwood. Development and Validation of a Sensitive TaqmanŽ Real Time PCR Assay for the Quantification of Two Human Polyomaviruses (JCV and BKV) in Human-Associated Waste Samples. American Society for Microbiology General Meeting, Boston Mass. June 1-5.

S. D. Leskinen and D. V. Lim. 2008. Rapid assessment of enterococci in Southern California coastal marine water by ultrafiltration concentration coupled to biosensor detection. 108th General Meeting of the American Society for Microbiology, Boston, MA (accepted, Meeting: June 1-5).

Overall Project Timeline Update
The timeline is largely unchanged from the original, except for a two-month extension of the timeline for validation and data analysis (allowing completion of objectives 2, 4 and 5).

Expenditures
Yes, expenditures are in the range anticipated for the work accomplished to date