Transcript Slide 1

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
(732) 932-9800 ext. 6224
[email protected]
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
INTRODUCTION
 Bacteroidetes
 A group of anaerobic, Gram positive bacteria including the genus
Bacteroides.
 Are good indicator organisms because they do not replicate once released
into the environment.
 Significantly correlate with the presence of human enteric viruses.
 Normal inhabitants of the large intestines of all warm-blooded animals.
- Up to 1011 cells/gram of feces
- Comprise approx. 10% of fecal mass;
- 1,000 to 10,000 times more numerous than coliforms
 Studies have shown that there are host-specific strains in different animals.
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 4˚C until processing.
• All samples analyzed for a full nutrient series, TSS,
fecal coliforms and Microbial Source Tracking
• A total of 290 samples processed.
Standards
 Dilutions of plasmid DNA provided standard curves which were linear from 10 to 100,000 copies per µL.
b
Mike Marandola, Salem RCE, measuring
flow in Cohansey.
MST Assay
c
AllBac Standard Curve
40
40
35
30
• DNA extracted from total filtered biomass using a DNeasy® tissue kit
Brian Hulme (RCE of
(Qiagen).
y = -3.4221x + 36.679
R2 = 0.9997
30
25
25
20
20
15
15
10
Monmouth County)
filtering a sample.
HR1
1
2
3
4
5
6
7
8
25
20
15
10
0
10
0
1
2
Log copy number
3
4
5
6
7
8
0
1
2
Log copy number
3
4
5
6
7
CL-2
8
Log copy number
to a concentration of 1 µg/ml
• Used qPCR to measure the number of bacteroidetes present.
Total (AllBac)
Human (HuBac)
Bovine (BoBac)
•
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.
TaqMan®
based assay using Applied Biosystems reagents and standard
conditions on an Applied Biosystems 7300 Real-Time PCR system
• Copy number of each target was calculated by comparison to a standard
curve made with plasmids containing human- or bovine-sourced target 16S
RNA genes amplified with the primers Bac 32f and Bac 708r (Bernhard and
Field, 2000).
Cohansey River Watershed, NJ
CL-2
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.
AllBac
HuBac
BoBac
1.0E+07
8.0E+06
6/28/2006
6.0E+06
6.0E+06
4.0E+06
4.0E+06
2.0E+06
2.0E+06
0.0E+00
0.0E+00
C1
CL-1
C2
C3
C4
C5
C6
CL-1 CL-2 FR1
AllBac
HuBac
BoBac
1.0E+07
8.0E+06
HR1
7/12/2006
C1
C2
C3
C4
C5
Station
C6
CL-1 CL-2 FR1
HR1
Station
 Pollution sources could be determined by the frequency of detection of specific markers at a particular station.
C5
HR1 FR1
C3
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.
% of Samples Containing Target Sequence
C2
C1
Figure 1
A map of the Cohansey River watershed showing land use patterns and
the surface water quality monitoring locations.
C1
C2
C3
C4
C5
C6
CL-1
CL-2
FR1
HR1
AllBac
70
90
100
70
100
90
100
90
80
100
HuBac
40
30
20
30
10
10
10
10
0
30
BoBac
10
10
20
10
10
10
TSS
(mg/L)
TN
(mg/L)
FC
(cfu/100ml)
10
AllBac
(copies/100ml)
BD
2.0
5.89
6/28
1.59
62.0
3.24
.439
>600
7/12
0.14
5.3
5.20
<.025
20
6.5 X 106
7/26
0.00
6.7
4.68
.113
80
8.0 X 105
8/9
0.44
6.7
6.04
.034
2000
6.8 X 105
6/14
0.00
5.3
6.96
<.025
5
1.8 X 106
6/28
1.59
73.0
1.85
.431
>600
1.6 X 106
7/12
0.14
3.0
7.22
<.025
190
3.3 X 106
7/26
0.00
8.0
7.11
.078
250
1.4 X 106
8/9
0.44
5.7
8.25
.034
2000
4.8 X 106
6/14
0.00
4.7
5.10
<.025
10
1.6 X 107
6/28
1.59
39.0
1.65
.915
>601
BD
7/12
0.14
10.7
6.13
.061
100
8.4 X 106
7/26
0.00
8.7
2.16
<.025
370
2.1 X 106
8/9
0.44
5.0
6.30
<.025
50
1.8 X 106
BD
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.
Source Identification
C6
Precipitation
(in.)
30
Cycles
• 100 ml of sample filtered aseptically onto a membrane filter which was cut
into quarters using a sterile blade.
35
0.00
TP
(mg/L)
<.025
Station Date
C1
6/14
BoBac Standard Curve
35
y = -3.4986x + 37.187
R2 = 0.9998
2
R = 0.9997
d
HuBac Standard Curve
y = -3.4925x + 38.416
• All DNA quantified by spectroscopy and diluted in sterile water
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.
a
C4
 MST Assay
 qPCR used to quantify specific sources of fecal contamination
 Based on primers and probes developed by Layton et al. for the detection
and quantification of:
- All Bacteroidetes (AllBac)
- Human-specific Bacteroidetes (HuBac)
- Bovine-specific Bacteroidetes (BoBac)
 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.
Copy Number
 Watershed Characteristics
 Freshwater system covering 31 square miles of mixed-use land
 73% Agriculture
- Includes row crop, sod farms, and field and container nurseries
- Livestock, horse and chicken farms
 11% Forested Areas and 7% Wetlands
 7% Urban
 Includes older homes on septic systems
 All 10 stations exceed the fecal coliform water
quality standard more than 10% of the time.
RESULTS
Cycles
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 4˚C 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 costeffective and realistic solutions for microbial contamination in the watershed.
METHODS
Cycles
ABSTRACT
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10
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0
 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.
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. 66:4571-4574.
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).