# Presentation: 614738

## IMS/ATP Rapid Method for the Determination of E. coli Concentrations in Recreational Waters

- IMS/ATP Rapid Method for the Determination of *E. coli* Concentrations in Recreational Waters

- Rebecca Bushon
- U.S. Geological Survey
- Columbus, Ohio

- May 9, 2006

## IMS/ATP Definition

- Immunomagnetic separation (IMS)
- Method of capturing microorganisms
- Uses antibody-coated paramagnetic beads which bind to antigens present on the surface of cells

- Adenosine triphosphate (ATP)
- ATP is present in all cells
- ATP transports chemical energy within cells

## Concentrate bacteria by filtration, if needed

Add magnetic beads that are coated with antibodies

- Add magnetic beads that are coated with antibodies

- IMS/ATP Rapid Method

**Notes:**
Describe variations of the method:Double filtration
Single filtration
Direct analysis

## IMS/ATP Rapid Method

- Separate bacteria from the sample by IMS
- Rupture bacterial cells
- Measure ATP using luciferin-luciferase
- Results in Relative Light Units (RLUs)

**Notes:**
The OWML is currently working on developing new beads and a new eluting solution for this assay. The new beads have a stronger bond between the beads and antibodies. With these changes, we hope to decrease some of the variability seen by this method and improve our correlations to the standard method.

## IMS/ATP Rapid Method

- Time to obtain results = ~1 hour
- Measures viable cells
- Supplies cost per sample = ~$15-20
- Equipment costs = ~$4,000 - 6,000
- Field portable

**Notes:**
Equipment cost depends on what the laboratory already has....pipettors, etc.

Entire setup can be hooked up to a battery and run in the field.

## IMS/ATP Research

- Method developed by researchers at University of Michigan (Lee and Deininger; *Luminescence,2004*)
- IMS/ATP methods were developed for *E. coli* and enterococci
- Limited field studies:
- University of Michigan – Lake Michigan and Huron River
- USGS – Cuyahoga Valley National Park, Ohio
- USGS – Lake Erie beaches, Ohio
- SCCWRP – Rapid methods comparison study, California

**Notes:**
Southern California Coastal Water Research Project (SCCWRP) 

## Field Testing of IMS/ATP Method

- Cuyahoga River, Cuyahoga Valley National Park
- In cooperation with the National Park Service
- Project duration - 2004-2006
- Results will be published this year

**Notes:**
Most data is from Old Portage and Jaite – only presenting on these two sites

## Approach

- Samples collected 7 days per week from May-August in 2004; 5 days per week in 2005
- Ancillary data collected: turbidity, rainfall, and streamflow
- Statistical analyses:
- Correlations
- Simple-linear regression
- Multiple-linear regression

**Notes:**
Ran correlations to determine significance of the relations between E. coli and other variables. Based on these results, variables were selected for use in regression models.

Simple-linear regressions were done using E. coli RLUs to predict the E. coli concentrations. MLRs were done to see if we could improve our predictions by adding in the other variables.

## MLR results for E. coli

- 2004-2005
- Model variables:
- log RLUs          (direct analysis)
- log turbidity
- Rainfall yesterday

**Notes:**
MLR models to get predictions
Actual count for today – get results tomorrow
Predicted E. coli based on MLR

Single-sample standard = 298 CFU/100 mL (primary contact recreation)

R2 of the model, the coefficient of determination (fraction of the variation in E. coli that is explained by the model).  

Developed models for each site, but we also combined the sites to have a river specific model:
Old Portage/Jaite combined: R2 = 0.501

## MLR results for E. coli

- 2004-2005
- Model variables:
- log RLUs         (direct analysis)
- log turbidity
- Rainfall yesterday

## MLR versus yesterday’s E. coli

|  | Correct Responses | False Positive | False Negative |
| --- | --- | --- | --- |
| Old Portage |  |  |  |
| MLR | 69.1% (47) | 20.6% (14) | 10.3% (7) |
| Yesterday’s count | 82.7% (48) | 10.3% (6) | 6.9% (4) |
| Jaite |  |  |  |
| MLR | 72.0% (54) | 20.0% (15) | 8.0% (6) |
| Yesterday’s count | 69.0% (49) | 12.7% (9) | 18.3% (13) |

**Notes:**
Old Portage % exceeded = 66.7% of samples collected
Jaite % exceeded = 50% of samples collected

## SLR results for enterococci

- Model variable:
- log RLUs
- (direct analysis)

**Notes:**
Smaller # of data points

## SLR vs yesterday’s enterococci

|  | Correct Responses |  | False Positive | False Negative |
| --- | --- | --- | --- | --- |
| Jaite/Old Portage |  |  |  |  |
| SLR |  | 73.1% (19) | 19.2% (5) | 7.7% (2) |
| Yesterday’s count |  | 76.9% (20) | 11.5% (3) | 11.5% (3) |

**Notes:**
80.8% of samples exceeded the USEPA criteria of 78 colonies per 100 mL

## Field Testing of IMS/ATP Method

- Lake Erie beaches — Edgewater and Villa Angela, Cleveland, Ohio

- Preliminary study in 2005
-  USGS and Northeast Ohio Regional Sewer District

**Notes:**
Add approach:
Samples 5 days per week from July-September 2005
We did not collect ancillary data for this study

## SLR results for E. coli

- 2005
- Model variable:
- log RLUs
- (single filtration)

## SLR results for E. coli

- 2005
- Model variable:
- log RLUs
- (single filtration)

## SLR versus yesterday’s E. coli

|  | Correct Responses | False Positive | False Negative |
| --- | --- | --- | --- |
| Edgewater |  |  |  |
| SLR | 88.0% (21) | 0% (0) | 13.0% (3) |
| Yesterday’s count | 73.9% (17) | 13.0% (3) | 13.0% (3) |
| Villa Angela |  |  |  |
| SLR | 83.0% (20) | 0% (0) | 17.0% (4) |
| Yesterday’s count | 73.9% (17) | 13.0% (3) | 13.0% (3) |

**Notes:**
Edgewater % exceeded = 12.5% of samples collected
Villa Angela % exceeded = 25% of samples collected

## 2005 IMS/ATP results - Edgewater

- False positive

- Correct exceedance

- False negative

- Correct nonexceedance

**Notes:**
Another way to view results:

Simple linear regression was used to develop an equation to predict E. coli concentrations from rapid-method results. Output from model was the probability of exceeding the bathing water standard. Threshold was set at 33% to minimize false negative responses and maximize correct responses.

## Next steps

- Continue optimization of method
- Test new antibody-bead complex
- Bacteriophage to release ATP
- 2006-2008 study at Lake Erie beaches
- Develop MLR models
- Include measurements for chlorophyll
- 2006 study at Cuyahoga Valley National Park
- Test MLR models

**Notes:**
Naturally occurring algae is known to be bioluminescent and can cause problems with measuring relative light units in some applications. Chlorophyll will be measured electrometrically to determine whether algae is a problem and whether the effects can be accounted for in predictive models.

## Acknowledgements

- Meg Plona, National Park Service
- Lester Stumpe, Mark Citriglia, Eva Hatvani, Rose Kieliszek, and Ben Tedrick, Northeast Ohio Regional Sewer District
- Student interns – Jim Cireddu, Suzanna Dzejachok, and Anthony Kinyo
- Funding provided by the USGS/NPS Water Quality Monitoring and Assessment Partnership and the Ohio Department of Health