Determination of Pollutant Loading and Ambient E'coli and Enterococci Levels in Fresh Water Environm

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Determination of Pollutant Loading and Ambient
E.coli and Enterococci Levels in Fresh Water
Environments by QPCR Jennifer S. Lavender1,2 and
Julie L. Kinzelman1,2 1City of Racine Health
Department Laboratory, Racine, WI 53403,
2UW-Parkside, Kenosha, WI 53141
CONTACT INFORMATION Julie Kinzelman 730
Washington Avenue Racine, WI 53403 PH
262-636-9501 / FAX [phone] E/M
julie.kinzelman_at_cityofracine.org
Abstract Real-time testing of water quality
would be beneficial to public health agencies. Of
methods available for real-time testing of
surface waters via bacterial indicators,
quantitative polymerase chain reaction (QPCR)
demonstrates the best correlation between
organism density in recreational water and
adverse health outcomes. However, outstanding
questions remain regarding DNA in fresh water
environments. For example, average DNA loading
from potential pollutant sources such as waste
water effluent/storm water and persistence of
ambient background DNA levels. Effluent samples
taken from the Racine, WI waste water treatment
plant (WWTP) were analyzed using QPCR, mEI agar
and IDEXX Colilert-18. Low, but positive
correlation was seen between viable cell counts
and cell equivalents (CE) by QPCR (R2 0.50).
This is expected as viable cell counts detect
only cells capable of growth while QPCR detects
all cellular DNA regardless of viability.
Log-converted data demonstrated an average three
log decrease in CE in final effluent versus raw
influent. This is similar as to what is seen with
viable organism counts, it appears the WWTP
process is successful in decreasing levels of DNA
discharged to the environment. For ambient
background levels, surface water samples were
collected from two bathing beaches. QPCR showed
significantly fewer CE in surface water versus
final effluent (p 0.004). When viable organism
counts equaled zero in recreational water samples
QPCR detected 150 measured cells on average.
Therefore, a baseline level of background DNA in
the environment may be necessary when
establishing water quality criteria using QPCR,
especially when accounting for pollutant sources
such as WWTPs.
Introduction Of the methods currently available
for real-time testing of surface and source
waters for bacterial indicators, QPCR has been
the only one to successfully correlate elevated
indicator organism density in recreational water
to increases in adverse health outcomes in
exposed individuals. While the prospect of having
this method approved for ambient water quality
testing is promising outstanding questions must
be answered prior to this action taking place.
While recent health effects studies were
conducted at beaches in proximity to known
sources of human fecal contamination they only
assessed the efficacy of QPCR in determining
ambient water quality. The actual
characterization of real-time QPCR products
throughout the wastewater treatment process was
not assessed. The routine use of QPCR for the
analysis of Enterococci and E. coli will likely
require that this method be acceptable or all
Clean Water Act purposes hence the comparison of
DNA present in treated wastewater effluent (as
followed through the treatment train), bypasses,
storm water, and surface run-off in conjunction
with analyses of viable organism counts using
approved agar-based and chemical detection
techniques may prove valuable. Once average or
expected DNA loading from pollutant sources is
ascertained, the next step in moving forward to
adopt this technology for environmental
monitoring is to determine an ambient background
level of DNA in surface water environments (and
its standard deviation) and aid in defining
limits for health advisories. This study
explores such additional investigations conducted
in Racine, WI.
Maximizing DNA Recovery Evaluating Microbial,
Soil, and Fecal DNA Extraction Kits
Six different commercially available DNA
extraction kits were tested on samples collected
from the Racine, WI WWTP to determine which would
provide a higher yield of DNA and ascertain
whether untreated sewage most closely resembles
a microbial, fecal, or soil sample.

• DNA Extraction Kits Tested
• MoBio Fecal 4. MoBio
  Soil
• Zymo Fecal 5. Zymo
  Soil
• MoBio Microbial 6. MoBio
  Powersoil

Results The extraction kit that resulted in the
best yield of DNA based on a lower FAM CT value
was the Zymo Fecal DNA extraction kit (Figures 1
and 2). This kit gave FAM CT values ranging from
24.10 (Raw Effluent) to 30.69 (Primary
Effluent). The MoBio microbial DNA extraction kit
was a close second but had additional factors
that made it more difficult to use in this
situation such as smaller extraction tube volume
and lower recovery volume.
Methods Wastewater samples were filtered through
0.45 µM polycarbonate filters and treated as
either a soil, fecal, or microbial sample
according to the kit tested. Provided directions
for each kit were followed. The final extracted
DNA samples were analyzed using QPCR to detect
for Enterococcal DNA.
Pollutant Loading and Ambient Background Levels
of E.coli/Enterococci DNA
Goals 1.Determine if a correlation exists
between standard cell enumeration and QPCR
methods. 2. Determine if the WWTP reduces the
amount of DNA that is discharged to the
environment. 3. Identify to what an extent a
baseline ambient background DNA level exists in
surface waters

• Methods
• Wastewater samples were aseptically obtained and
  analyzed using IDEXX Colilert-18 (E.coli) and
  mEI agar (USEPA Method 1600, Enterococci).
• Samples were filtered onto 0.45 µM polycarbonate
  filters and analyzed using QPCR (USEPA proposed
  Method 1606 1607) to detect the presence of
  E.coli or Enterococci.
• Statistical analysis of the results was done to
  determine the relationship between these samples.

Figure 2 FAM CT values determining DNA recovery
through the use of soil DNA extraction kits.
Figure 1 FAM CT values determining DNA recovery
through the use of fecal and microbial DNA
extraction kits. Blue Pink Fecal Purple
Microbial
Table 1 Log converted data showing the amount
of DNA and number of viable cells passed through
the treatment train.

• Results
• There was only a small degree of correlation
  between QPCR CEs and Enterococci colony counts
  (Figure 3).
• There was a positive correlation between QPCR CEs
  and Colilert-18 MPN of E.coli (Figure 4).

• The wastewater treatment process is successful at
  reducing the number of viable cells and amount of
  DNA present in the final effluent as evident by
  log-converted data (Table 1) which demonstrated
  an average three log decrease in CE/CFU in final
  effluent versus raw influent.
• QPCR detects a higher number of CEs than
  Colilert-18 MPN of E.coli in approximately 50
  of sampling instances (Figure 6).

Figure 3 Correlation of QPCR cell equivalents
to mEI agar colony counts.
Figure 4 Correlation of QPCR cell equivalents
to Colilert-18 MPN of cells.

• Conclusions
• Evaluating maximum DNA recovery from WWTP samples
  indicated that samples taken from the Racine, WI
  WWTP are best treated as a fecal sample. Further
  more the best pre-treatment approach for these
  samples was the Zymo Fecal DNA Extraction kit.
• When determining a correlation between currently
  approved monitoring methods and QPCR, only a
  small degree of positive correlation was seen
  between viable Enterococci cell counts and cell
  equivalents (CE) as determined by QPCR. This may
  be explained by the fact that methods targeting
  viable cell counts detect only cells capable of
  growth while QPCR detects all cellular DNA
  regardless of viability. However, there was a
  stronger positive correlation between viable E.
  coli cell counts as determined by Colilert-18
  MPN and QPCR CEs.
• Log-converted data demonstrated an average three
  log decrease in CEs in final effluent versus raw
  influent. This is similar as to what is seen with
  viable organism counts. It appears that the WWTP
  process, utilizing UV as the final step, is
  successful in decreasing levels of viable cells
  and DNA discharged to the environment.
• Regarding ambient background DNA levels in
  surface water samples, QPCR showed significantly
  fewer CE in surface water versus final effluent.
  When viable organism counts equaled zero in
  recreational water samples QPCR detected 150
  measured cells on average. Therefore, a baseline
  level of background DNA in the environment may be
  necessary when establishing water quality
  criteria using QPCR, especially when accounting
  for pollutant sources such as WWTPs.

Figure 5 Evaluation of an ambient DNA
background level at North Beach Racine, WI. A
comparison of QPCR cell equivalents to
Colilert-18 MPN of cells at four routine
monitoring sites.
References 1 Haugland, R., Siefring, S.,
Wymer, L., Brenner, K., and Dufour, A. Comparison
of Enterococcus measurements in freshwater at two
recreational beaches by quantitative polymerase
chain reaction and membrane filter culture
analysis. Water Research (2005) 559 568. 2
Holland, P., Abramson, R., Watson, R., and
Gelfand, D. Detection of specific polymerase
chain reaction product by utilizing the 5 ? 3
exonuclease activity of Thermus aquaticus DNA
polymerase. Proceedings of the National Academy
of Science (1991) 88, 7276 7280. 3 Santo
Domingo, J., Siefring, S., and Haugland, R.
Real-time PCR method to detect Enterococcus
faecalis in water. (2003) 25, 261 265. 4
Ludwig, W., and Schleifer, K. How Quantitative is
Quantitative PCR with Respect to Cell Counts?.
(2000) 23, 556 562.
Acknowledgments These projects were funded by
grants from the Wisconsin Coastal Management
Program and Wisconsin Department of Natural
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