COMPARISON OF ENTEROCOCCUS MEASUREMENTS IN MARINE
BEACH AND BAY SAMPLES BY QUANTITATIVE (REAL-TIME)
POLYMERASE CHAIN REACTION, MEMBRANE FILTRATION AND
ENTEROLERT
REPORT EXECUTIVE SUMMARY
1. INTRODUCTION
l Time or quantitative Polymerase Chain Reaction (qPCR) is a technique used to amplify or copy
a specific region of DNA. PCR's power comes from the fact that every animal, bacteria or virus
possesses sequences (or portions of their bodies' building blocks called DNA) that are unique and present
only in its own species. The development of this Nobel Prize winning technique by Kary Mullis in the
1980's resulted in the application of many new techniques in molecular biology. Automation of PCR
through the use of Thermal Cyclers has been the key to the rapid use and multiple applications of this
technology throughout the science community.
2. BACKGROUND
^•Previous health and epidemiological studies by US
Environmental Protection Agency (USEPA) have
demonstrated that the amount of the bacterial genus
Enterococcus in both marine and freshwater samples
is directly correlated with gastroenteritis illness rates
in swimmers exposed to these waters.
^- USEPA requires that recreational waters across the
United States be monitored routinely for Enterococcus
spp. and/or Escherichia coll. While neither of these
organisms is pathogenic, both are considered to be indicator organisms for the presence of the bacterial
and viral pathogens found in fecal material and/or the intestines of warm blooded animals.
>• Currently, approved methods for measuring concentrations eft Enterococcus and E. coli in recreational
waters include membrane filtration and most probable number tests. Although many of these methods
have been used for decades, results are typically not available for 24 hours.
>• At best, a decision regarding safe beach usage is made using one day old information or is not made
until after a confirmation test which can take up to 72 hours after the initial sample is collected. Most
qPCR tests can provide results in as little as 4 hours. Because microbial water quality can change
rapidly, testing based on indicator organisms (that requires 24 hours to obtain results), may result in
unnecessary beach closings or exposure of swimmers to water of poor microbial quality.
^•Congress passed an amendment to the Clean Water Act
called the Beaches Environmental Assessment and
Coastal Health (BEACH) Act. The BEACH Act requires
the USEPA to conduct research in support of new
recreational water criteria. Because qPCR methods
provide a faster assessment of water quality, they have
the potential to improve decision making for county and
state personnel involved in beach management decisions.
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3. qPCR COMPARSION STUDY IN MARINE BEACHES AND BAYS, 2007
^•In 2007, USEPA Region 2, New Jersey Department of Environmental Protection (NJDEP), and Ocean
and Monmouth County Health Departments collaborated on a comparison study using qPCR and
conventional microbiology methods at 20 beaches in Ocean and MonmouthCounties.
^-Ocean and bay samples with varying levels (based on historical data) of
expected microbial densities were sampled 10 times between June 18
and August 20, 2007.
^- Samples were collected in thigh deep water approximately 0.3 meters
from the surface.
densities of the fecal pollution indicator genus, Enterococcus, were
determined by qPCR, as well as two conventional 24 hour test methods
(membrane filtration [MF] and Enterolert). Over 1000 samples were
analyzed.
4. RESULTS
geometric means ranged from 1.3 to 38 calibrator cell equivalents (CCE) by qPCR analysis
compared to 5.2 to 64.9 colony forming units (CPU) by MF analysis in Monmouth
County Beach/Bay Samples (100 mL samples).
geometric means from the samples collected in Ocean County were 1.3 to
357 CCE by qPCR compared to 6.2 to 150 CPU by Enterolert (N=200).
^- In general, when Enterococcus concentrations were low using MF and
Enterolert, qPCR results followed the same trend. qPCR concentrations changed relative to changes in MF
or Enterolert.
^•Regression analysis of these results showed a significant positive correlation between qPCR and
MF/Enterolert methods with an overall correlation coefficient of 0.79.
5. CONCLUSIONS
results of the study indicate that qPCR shows promise as a beach monitoring tool which can provide
results in as little as 4 hours.
>• There was a strong correlation between qPCR results and conventional enterococci methods.
> Additional data are needed to further refine qPCR technology for routine use at marine bathing beaches,
including an interlaboratory method validation study and an epidemiological study using qPCR water
quality data. USEPA Region 2, NJDEP, and the Ocean and Monmouth County Health Departments are
planning a follow up study in 2008 to further delineate spatial and temporal trends of enterococci
measurements using qPCR in Ocean and Monmouth County beaches.
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