1. © Copyright National University of Singapore. All Rights Reserved. CHARACTERIZING ANTIBIOTIC RESISTANCE IN SINGAPORE WATER BODIES AND TRIBUTARIES Name: Chen Hongjie Supervisor: A/Prof. Karina Gin Yew-Hoong Department of Civil and Environmental Engineering

2. © Copyright National University of Singapore. All Rights Reserved. OUTLINE -Background Information -Objective -Methodology -Results and Discussions -Conclusion

3. © Copyright National University of Singapore. All Rights Reserved. Background Information

4. © Copyright National University of Singapore. All Rights Reserved. BACKGROUND INFORMATION Downloaded from http://www.mirror.co.uk/news/technology-science/science/humans- facing-antibiotic-apocalypse-papercuts-6862186

5. © Copyright National University of Singapore. All Rights Reserved. BACKGROUND INFORMATION Download from http://www.nature.com/news/2011/110216/full/news.2011.46.html# B1

6. © Copyright National University of Singapore. All Rights Reserved. BACKGROUND INFORMATION Bacteria posing high health risk/s -Carbapenem resistant Enterobacteriaceae (CRE) -Extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae -Vancomycin resistant Enterococcus (VRE)

7. © Copyright National University of Singapore. All Rights Reserved. BACKGROUND INFORMATION Credits to Google Maps Singapore -“The Little Red Dot” Monsoon Season -Northeast monsoon (wetter) -Southwest monsoon (dryer)

8. © Copyright National University of Singapore. All Rights Reserved. BACKGROUND INFORMATION Singapore -Has catchment area of two-thirds of Singapore area -Water structures built to keep in fresh water -to be supplied as drinking water -Important to maintain good water quality standard -Monitor levels of antibiotic resistance

9. © Copyright National University of Singapore. All Rights Reserved. Objective and Methodology

10. © Copyright National University of Singapore. All Rights Reserved. OBJECTIVE -Investigate the abundance and percentage resistance of ARB -Investigate if there are spatial and temporal effects

11. © Copyright National University of Singapore. All Rights Reserved. METHODOLOGY Sampling Details -4 water bodies -4 tributaries -Across 4 months (Oct, Dec ‘15, Jan, Mar ‘16)

12. © Copyright National University of Singapore. All Rights Reserved. METHODOLOGY W1W2W3W4 T1T2T3T4 -High level of anthropogenic activity -Near residential and commercial area -W2 surrounded by heavy industries -Tributaries surrounded by vegetable and animal farms -Zoo near W3 -Largely forested and protected -Generally least impacted by human -Largely forested and protected

13. © Copyright National University of Singapore. All Rights Reserved. METHODOLOGY 1 Filter 100 mL through 0.45 µm cellulose nitrate membrane 2 Place membrane on selective agar 3 Inoculation and enumeration

14. © Copyright National University of Singapore. All Rights Reserved. METHODOLOGY No Antibiotics Amikacin Ceftazidime Ciprofloxacin Co-trimoxazole Meropenem E. coli (TBX) Klebsiella pnuemoniae (Klebsiella selective agar) Pseudomoas aeruginosa (Cetrimide agar) Salmonella spp. (SS agar) No Antibiotics Ciprofloxacin Meropenem Vancomycin Enterococcus spp. (Slanetz and Bartley agar) (2015 Clinical and Laboratory Standards Institute performance standards for Antimicrobial Susceptibility Test)

15. © Copyright National University of Singapore. All Rights Reserved. Results and Discussion

16. © Copyright National University of Singapore. All Rights Reserved. RESULTS AND DISCUSSION Taxonomical Correlation (Abundance) ARB concentrations based on each taxa and resistance -K. pneumoniae had the highest overall resistance -Resistance to Co- trimoxazole and Ciprofloxacin were the highest -Resistance to Amikacin was generally low except for P. aeruginosa

17. © Copyright National University of Singapore. All Rights Reserved. RESULTS AND DISCUSSION Taxonomical Correlation (Percentage Resistance) Percentage resistance based on each taxa and resistance -Observational pattern similar between % resistance and abundance for E. coli -Salmonella spp. had the highest percentage resistance against Meropenem

18. © Copyright National University of Singapore. All Rights Reserved. RESULTS AND DISCUSSION Temporal Correlation Total ARB concentration based on sampling months and average monthly rainfall -No significant difference across the months (p > 0.05) -Need more time points

19. © Copyright National University of Singapore. All Rights Reserved. RESULTS AND DISCUSSION Spatial Correlation Total ARB concentration based on tributary locations Water Bodies -WB1 is significantly higher than other WB (p < 0.05) -High level of anthropogenic activity in WB1 -Insignificant among WB2, WB3, WB4 (p >0.05)

20. © Copyright National University of Singapore. All Rights Reserved. RESULTS AND DISCUSSION Spatial Correlation Total ARB concentration based on tributary locations Tributaries -Significantly higher than WB (p < 0.05) -Difference between T1,T2 and T3,T4 insignificant (p > 0.05)

21. © Copyright National University of Singapore. All Rights Reserved. RESULTS AND DISCUSSION Principle Component Analysis

22. © Copyright National University of Singapore. All Rights Reserved. CONCLUSION -Amikacin can still be a relevant antibiotic clinically -K. pneumoniae had the highest resistance in terms of abundance -Salmonella spp. had significantly high percentage resistance against Meropenem -Tributaries had higher abundance of ARB than water bodies. WB1 had higher abundance than other WB -Land uses could be a major factor for abundance of ARB in water

23. © Copyright National University of Singapore. All Rights Reserved. ACKNOWLEDGEMENT A/Prof. Karina Gin Dr. Ng Shi Min Charmaine Dr. Le Thai Hoang Dr. Laurence Haller Dr. Yi Xin Zhu Fang Da Nurhizam Hakim This research grant is supported by the Singapore National Research Foundation under its Environmental & Water Technologies Strategic Research Programme and administered by PUB (Project ID: 1102-IRIS-12-02)