1. Patricia Weiss 1, Tiong Gim Aw 2, Joan B. Rose 2 1 School of Public Health, College of Human Medicine, Michigan State University, East Lansing, Michigan, 48824, United States. 2 Department of Fisheries and Wildlife, 3 Natural Resources, Michigan State University, East Lansing, Michigan, 48824, United States.

2. Introduction 783 million people lack access to safe water (United Nations, 2013) 2.5 billion people do not have improved sanitation (United Nations, 2013) 1.1 billion practice open defecation (World Health Organization, 2010) Most of these people live in rural villages such as Pueblo Nuevo, Nicaragua

3. Objectives of Study Test microbial water quality of hand dug wells in Pueblo Nuevo, Nicaragua Determine Sources of Contamination

4. Background on Nicaragua 1979 opposition to governmental manipulation—Marxist Sandinistas came into power (Central Intelligence Agency, n.d.) 1988 hurricane Joan caused more damage to infrastructure and economy (Central Intelligence Agency, n.d.). About 50% have access to improved sanitation (63% in urban areas and 37% in rural areas) (Central Intelligence Agency, n.d.) 20% without safe drinking water in Latin America (Pearce- Oroz, 2011) Government is slowly improving access to water and sanitation (Social Investments Funds)

5. Location Pueblo Nuevo is located on the Caribbean Coast

6. Cases of Illness in Pueblo Nuevo 2012 Water-related Diarrhea: 5,675 Cases Parasites: 2,850 Cases Pneumonia: 47 Cases Respiratory Infections: 8,750 Cases Injuries due to Weapons: 467 Cases Pregnancy Issues: 272 Cases *Provided by Dr.Alejandro Picado

7. Types of Wells Sampled Rope Pump Well Simple Well

9. Methods Compartment Bag Test (CBT) – E.coli Membrane Filtration & qPCR – Microbial source tracking markers: B.theta (human), M2/M3 (bovine) E. coli, enterococci

10. Compartment Bag Test

11. 8 key steps to perform CBT 1. Prepare dilutions 2. Collect and record 3. Mix water sample with growth medium 4. Open and fill CBT 5. Seal 6. Incubate 7. Score and record results on data sheets 8. MPN chart for quality rating

12. Last Step: Decontamination Each bucket=13.4L 5 ml bleach per bucket Put rope etc. in bucket for at least half hour 0.05g Sodium Thiosulfate

13. Membrane Filtration Filtered 1.4-1.8L of well water

14. DNA Extraction and qPCR DNA extracted using QIAamp DNA mini kit (Qiagen) Platform used: Roche LightCycler ® 480 Real-Time PCR System with 96 well block

15. Results and Discussion Results of Compartment Bag Test and qPCR

16. Compartment Bag Test Results 32 wells total 87.5% of wells contaminated with E. coli Contamination ranged from 3-404.5 MPN/100 ml Geometric mean 25.8/100 ml

17. Comprehensive Water Quality Rating

18. Average MPN of E. coli Relative to Depth and Elevation

19. Comparison of E. coli MPN between simple and rope-pump wells Geometric Mean t-Test, t=0.88, p = 0.19 Log-transformed t-Test t=-2.11, p = 0.02 64.1% Reduction in E. coli *Used MPN of 3 for lower confidence interval Similar to results seen in Gorter et al. (1998)—Using geometric mean overall 47% reduction of fecal coliforms seen in rope-pumps.

20. qPCR detection of molecular markers (No. sample tested: 31)

21. Conclusion Generally the well water was deemed unsafe in this small rural area in Nicaragua. The CBT made it possible to test in the field for E.coli contamination in a rural location where supplies and lab equipment was limited. qPCR analysis indicated bovine fecal pollution in the well water samples.

22. Acknowledgement Michigan State University School of Public Health and College of Human Medicine Thanks to Mark Sobsey for helping with Bag Test Regina Weiss, Sammia Forbes, Ashley Hodges, guide Don Tito, Director Don Miguel, and Dr.Alejandro Picado Fundacion para la Autonomia y el Desarollo De la Costa Atlantica de Nicaragua (FADCANIC)

23. Questions?