Distribution and Abundance of Human Specific Bacteroides and Relation to Traditional Indicators in an Urban Tropical Catchment, case of Kranji Reservoir Catchment, Singapore

1. Distribution and Abundance of Human
Specific Bacteroides and Relation to Traditional
Indicators in an Urban Tropical Catchment
Kranji Reservoir Catchment Singapore
Nshimyimana J. P.1 , Shanahan P.1 , Ekklesia E.2, Chua L.2 and Thompson J. R.1
AGU Fall Meeting, December 2012
1. Department of Civil and Environmental Engineering
Massachusetts Institute of Technology
2. Department of Civil and Environmental Engineering
1
Nanyang Technological University

2. Kranji Catchment, Singapore
Singapore Island
Percentage
5%
76%
2
19% South East Asia

3. Description of Kranji Catchment
Residential Area Farming Area
Drainage
Undeveloped Area Kranji Reservoir3

4. Research Rationale
MacRitchie Reservoir
Single sample maximum for Kranji Reservoir
and Catchment (NTU 2008) ?
Motivation: Kranji Reservoir
Determine the source and distribution of human sewage contamination
in drainages and water bodies in the Kranji catchment to prioritize
management targets for protecting reservoir water quality. 4

5. Strategies for Monitoring Water Quality
• Direct detection
• Indicators
Indicator Criteria E. coli/Coliforms
1. Consistently present in sewage and at higher
+
concentrations than pathogens
2. Should not multiply in the environment +/-
3. Should be as resistant or more resistant to
+
disinfection than the pathogens
4. Can be assayed by a simple and reliable test +
5. Concentrations in water correlates to a health
+
hazard
5

6. Physiology and Ecology of indicators
6

7. Strategies for Monitoring Water Quality
• Direct detection
• Indicators
Indicator Criteria E. coli/Coliforms HF183
1. Consistently present in sewage and at
+ +
higher concentrations than pathogens
2. Should not multiply in the environment +/- +
3. Should be as resistant or more resistant
+ +
to disinfection than the pathogens
4. Can be assayed by a simple and reliable
+ +
test
5. Concentrations in water correlates to a
+ TBD
health hazard
7

8. Study Goals
1. Characterize the distribution of the molecular sewage
indicator (HF183) in the Kranji Reservoir and Catchment
2. Evaluate the similarity of sequences recovered with the
HF183 assay to previous studies to validate its use in
Singapore
3. Compare the abundance of traditional fecal indicator bacteria
(FIB) and the HF183 marker across different land uses and
dates
4. Identify candidate sources of human sewage contamination
in the Kranji Reservoir catchment 8

9. Sample Collection and Processing
Land Use Samples
Farming (F) 19
Residential (R) 37
Undeveloped (U) 9
Kranji Reservoir (K) 16
Sampling a drainage
in residential area
Biomass Filtration
9

10. 1. Presence/Absence – Ubiquity of HF Marker
January 2009 July 2009
Challenge: Since HF is widespread, we cannot pinpoint sources
Need quantitative method
Benefit: Use results to confirm sequence similarity to previous studies
10

11. 2. HF183 Sequences Match Previous Studies
& Bacteroides dorei
Sequences from
sites in France,
USA, and Australia
Sequences
from this study
Sequences
from sites in USA and
Japan
Neighbor-joining phylogenetic tree of the 16S rRNA gene sequences from
sites within Kranji reservoir and catchment amplified by the HF183 Assay
11
(PCR primers HF183F-708R)

12. HF183 Quantitative PCR Assay Optimization
I. DNA extraction/QPCR
Test tube
Bacteria + DNA
?
Extraction
efficiency
DNA
1 um
Log10 HF marker/100ml
II. Quantification of target
DNA in environmental
samples
Sample Types 12

13. Results: DNA Extraction Efficiencies for
HF Marker Quantification by qPCR
Recovery
Number of
Samples and Sample Volume Efficiency
Samples
± s.d. (%)
River Water (200 ml) spiked with B. dorei
6 55.8% ± 20.8%
cells (1 ml)
River Water (200 ml) spiked with 1:100
6 60.6% ± 29.3%
dilution of B. dorei cells (1 ml)
All spiked samples 12 58.2% ± 24.4%
HF183 marker copies detected by QPCR were converted to cell
equivalents (CE) based on a measured DNA extraction efficiency
of 58.2% ± 24.4% for B. dorei suspended in freshwater.
13

14. 3. Variation in HF Abundance Across Sites
January 2009 July 2009
Questions:
3a. Does HF vary with date or
land use?
3b. How does HF compare to
Fecal Indicator Bacteria?
14

15. 3a. HF Marker Abundance Varies by Land Use
but Not Date
HF marker
Land Use Sites ANOVA
Geometric
HSD
F=8.80; p<0.0001 Sampled mean
HF
(CE/100ml)
Farming (F) 19 4.28 x104 A
Residential (R) 37 2.51 x103 B
Undeveloped (U) 9 2.73 x103 B
Kranji Reservoir 16 5.22 x103 B
(K)
• No variation with date (F=0.699; p=0.41)
• No variation with interaction of date and land
use (F=1.32; p = 0.27) 15

16. 3a. Correlation between HF EC TC
HF and E. coli or TC 1
Significant positive correlations were
observed between HF and E. coli or
TC in the farming areas (green) 2
Farming-only (n=19) 3
HF x EC R=0.59, p=0.0077 4
HF x TC R=0.47, p=0.042
5
6
7
8
9
Hierarchical clustering
Ward’s method
implemented in JMP.

17. 3a. Correlation between HF EC TC
HF and E. coli or TC 1
In non-agricultural sites correlations
between HF and E. coli or total
coliform were weak and not 2
statistically significant (p>0.05).
3
In the total dataset HF marker was 4
weakly correlated to E. coli (R=0.34,
p<0.0014). 5
6
7
8
9
Hierarchical clustering
Ward’s method
implemented in JMP.

18. 4. Potential Sources of Contaminations in
Farming Area
Reservoir (n=16)
Undeveloped (n=9)
Residential (n=37)
Farming (n=19)
Fish Pond-2
Fish Pond-1
Effluent-4
Effluent-3
Effluent-2
Effluent-1
Raw Sewage
1.00 3.00 5.00 7.00 9.00
Log10 CE/100 ml 18

19. Conclusions
• HF marker was ubiquitous in Kranji Reservoir Catchment
• HF marker was validated as a potential complimentary bacteria
indicator for Singapore
• Moderate correlation (HF marker & E. coli) was observed in the
farming areas
• HF marker concentration varied from high to low across these
land uses: Farming, Residential, Kranji Reservoir and
Undeveloped
• Major potential sources of HF marker found in farming areas
include septic tanks
19

20. Future Work
Applying HF Marker (HF183)
for Human Health Risk
Assessment
Freshwater closed Freshwater open to
for recreational activities recreational activities
Kranji Kranji
Reservoir Catchment
HF Marker (HF183) Concentrations
above Ashbolt et al. (2010) adapted 87.5% 74%
threshold (1.5x103CE/100ml)
Challenges : Future research should explore the use of epidemiologic data to
assess health risks using HF marker (HF183) quantification
20

21. Acknowledgements
Funding support:
• National Research Foundation Singapore
• SMART-CENSAM
Sampling Access:
• Public Utilities Board, Singapore
• Nanyang Technological University
DNA Sequencing:
MIT Biomicrocenter through the Center for
Environmental Health Science (CEHS)
Study :
• PI: Professor Janelle Thompson MIT-CEE
• Student Teams MIT-CEE M.Eng. (2009 and 2010)
Singapore Project
• Members of MIT Janelle Thompson Lab
• Eveline Ekklesia, NTU PhD student
• Dr. Samodha Fernando (MIT postdoc, now Professor
at U. Nebraska)
Project collaborators
• Dr. Peter Shanahan (MIT)
• Dr. Lloyd Chua (NTU)
• Dr. Karina Yew-Hoong Gin and research group
21
(NUS)

22. Thank you!
Questions?
Correspondence: Prof. Janelle Thompson
jthompson@mit.edu
22