COMPARISON OF qPCRBASED MICROBIAL SOURCE TRACKING DATA TO TRADITIONAL WATER QUALITY MEASUREMENTS IN

1
COMPARISON OF qPCR-BASED MICROBIAL SOURCE
TRACKING DATA TO TRADITIONAL WATER QUALITY
MEASUREMENTS IN THE UPPER COHANSEY RIVER
WATERSHED C. D. Phelps, K. A. Buckley, C. C.
Obropta, and L. Y. Young Rutgers, The State
University of New Jersey
Rutgers, The State University of New Jersey Dept.
of Environmental Sciences School of Environmental
and Biological Sciences 14 College Farm Rd., Cook
Campus New Brunswick, NJ 08901 [phone]
ext. 6224 phelps_at_envsci.rutgers.edu
ABSTRACT Background Various methods of
microbial source tracking (MST) have been
developed for identifying the sources of fecal
contamination in the environment. We examine the
usefulness of a quantitative PCR-based method
targeting host-specific Bacteroides sequences for
helping to develop a watershed restoration plan
in an impaired watershed (Upper Cohansey River).
Methods An extensive sampling plan involving 10
stations sampled biweekly from June through
November with additional events in July, August
and September was carried out. Samples were
analyzed for pH, DO, temperature, a full nutrient
series and fecal coliforms as well as MST. The
MST analysis was carried out on surface water
grab samples collected in sterile bottles and
held at 4C until processed. 100 ml of the
sample was filtered onto a membrane filter and
DNA was extracted from the total filtered
biomass. The number of bacteroidetes from all
sources (AllBac) along with human-specific
(HuBac) and bovine-specific (BoBac) sources was
determined by using quantitative, real-time PCR.
The qPCR method is a modification of the method
developed by Layton et al. (2006). The results
from the qPCR analysis are compared to the
results from a sanitary survey, fecal coliform
monitoring, and water quality measurements.
Results The initial qPCR results show that by
using bacteroidetes as a target, fecal
contamination can be detected and quantified
directly from surface water samples without the
use of pre-culturing. The amount of
contamination can be determined in terms of total
fecal load and the percentage from individual
sources (human, bovine and other). These values
provide a higher level of discrimination than the
other, traditional, measurements of water quality
or the sanitary survey. Conclusions MST based
on qPCR can be used to easily identify sources of
fecal contamination in watersheds. These results
will enable us to expand the use of MST to better
prioritize projects and therefore, produce more
cost-effective and realistic solutions for
microbial contamination in the watershed.

• METHODS
• Sampling
• 10 stations were sampled from June through
  September of 2006.
• In situ measurements of pH, DO, and Temperature
  made for all samples.
• Surface water grab samples collected in sterile
  bottles.
• Samples held at 4C until processing.
• All samples analyzed for a full nutrient series,
  TSS,
• fecal coliforms and Microbial Source Tracking
• A total of 290 samples processed.
• MST Assay
• 100 ml of sample filtered aseptically onto a
  membrane filter which was cut into quarters using
  a sterile blade.
• DNA extracted from total filtered biomass using a
  DNeasy tissue kit (Qiagen).
• All DNA quantified by spectroscopy and diluted in
  sterile water
• to a concentration of 1 µg/ml
• Used qPCR to measure the number of bacteroidetes
  present.
• Total (AllBac)

• RESULTS
• Standards
• Clonal libraries of 16S RNA genes generated from
  PCR of human and bovine feces yielded plasmids
  specific for HuBac and BoBac primer sets. These
  plasmids were quantified and used as standards
  for the qPCR assay.
• Dilutions of plasmid DNA provided standard curves
  which were linear from 10 to 100,000 copies per
  µL.

Table 2 Comparison of Bacteroidetes
measurements by qPCR to other measures of water
quality at 3 stations over 5 sampling dates. BD
below detection. These data show the highly
variable nature of all of the water quality
measures used.
Figure 2 Standard Curves for quantification
of Bacteroidetes Amplification plot of all three
standard curves (a), and the individual standard
curves plotting log copy number vs. threshold
cycle (Ct) for AllBac (b), Hubac (c), and BoBac
(d) primer sets.

• Quantitative Analysis
• Bacteroidetes were detectable in samples from all
  stations at various times.
• The number of human bacteroidetes was often as
  high as that of the total bacteroidetes and
  bovine bacteroidetes were rarely detected.

• CONCLUSIONS
• Bacteroidetes from all sources could be readily
  detected in 100ml surface water samples by using
  a qPCR assay.
• Human and Bovine contributions to fecal
  contamination could be distinguished from each
  other.
• Pollution sources could be determined by the
  frequency of detection of specific markers at
  particular stations over the course of the
  summer.
• Despite the lack of obvious correlations between
  total Bacteroidetes and fecal coliforms, or any
  of the other water quality measurements, we were
  able to gain useful data about the sources and
  extent of fecal contamination in the watershed.

Figure 3 Sample Data showing the numbers of
bacteroidetes detected by the three primer sets
on two days of sampling at all 10 stations.
There was 1.59 inches of rain on 6/28 and 0.14
inches on 7/12.
6/28/2006
7/12/2006
Copy Number
Station
Station

• Source Identification
• Pollution sources could be determined by the
  frequency of detection of specific markers at a
  particular station.

Table 1 Frequency of detection of AllBac,
HuBac or BoBac target sequences in samples taken
on 10 separate occasions. These data show that
certain stations have a higher incidence of
contamination with human (C-1, C-2, C-4 and HR1)
or bovine (C-3) feces.
References Layton, A., L. McKay, D. Williams, V.
Garret, R. Gentry, and G. Sayler. 2006.
Development of Bacteroides 16S rRNA Gene
TaqMan-Based, Real-Time PCR Assays for Estimation
of Total, Human, and Bovine Fecal Pollution.
Applied Environmental Microbiology
72(6)4214-4224. 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. 664571-4574.
Acknowledgements The authors would like to
acknowledge the laboratory contributions of Brian
Hulme and Ke Shi as well as undergraduate
students Thomas Wang and Nicole Lordan. This
project was funded in part by the New Jersey
Department of Environmental Protection 319(h)
Program. Project partners included the Cumberland
Salem Conservation District, Rutgers Cooperative
Extension of Salem County, and the Rutgers
Cooperative Extension Water Resources Program
(www.water.rutgers.edu).