Grantee Research Project Results
Final Report: Direct One-step Culture Detection of Vibrio cholera in Drinking Water in Low-Resource and Disaster Settings
EPA Grant Number: SU836116Title: Direct One-step Culture Detection of Vibrio cholera in Drinking Water in Low-Resource and Disaster Settings
Investigators: Sobsey, Mark D.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Page, Angela
Phase: I
Project Period: September 1, 2015 through August 31, 2016
Project Amount: $14,569
RFA: P3 Awards: A National Student Design Competition for Sustainability Focusing on People, Prosperity and the Planet (2015) RFA Text | Recipients Lists
Research Category: P3 Awards , Pollution Prevention/Sustainable Development , Sustainable and Healthy Communities , P3 Challenge Area - Safe and Sustainable Water Resources
Objective:
The objective is to develop and evaluate a new quantitative, culture-based test to detect and quantify Vibrio cholerae bacteria in drinking water and its sources by adapting the Compartment Bag Test for use with a new, defined substrate, chromogenic liquid culture medium that will be created, thereby resulting in a lab-independent, field-portable and self-contained test system.
Summary/Accomplishments (Outputs/Outcomes):
Phase I of this P3 Project aimed to develop a single-step, chromogenic bacteriological broth culture medium for the detection of Vibrio cholerae compatible for use in the Compartment Bag Test. The results from these experiments can provide proof of concept for the adoption of Tellurite Taurocholate Gelatin Broth (TTGB) for detection and quantification of Vibrio cholera in water. Several strains of toxigenic V. cholerae cause high mortality and outbreaks in both endemic and epidemic regions. A series of trials were done to evaluate performance of the candidate medium with some of these different strains including: O1, Non-01 Strain (ATCC 35971), Serotype O1 - El Tor Ogawa (ATCC BAA-2163), and Serotype O139 (ATCC 9-51395). Separate assays trials were performed for each strain via enrichment in Alkaline Peptone Water at 37 degrees C for 24 hours. The enrichment was diluted in phosphate buffer and then assayed in a multiple tube test with TTGB to determine bacteria concentration. The enrichment also was plated on Luria Broth Agar, Thiosulfate Citrate Bile Sucrose (TCBS) agar, and TTGA for comparison.
The concentrations determined from the TTGB Multiple Tube Test were compared with the concentrations from each of the standard media plates, for each V. cholera strain tested, using the Wilcoxon Rank Sum Test. There was no significant difference between the paired most probable number (MPN) concentrations and the CFU data from the Luria Broth Agar (LBA), TTGA, and Thiosulfate Citrate Bile Sucrose (TCBS) plates for any of the strains (p > 0.05). The results of these experiments suggest that the Tellurite Taurocholate Gelatin Broth is as sensitive to Vibrio cholerae detection and quantification as other standard detection media and can provide similar results to a non-selective medium. These results suggest proof of concept for the performance of the TTGB in a model system of buffered water containing no other kinds of bacteria. However, the results presented here are insufficient and not robust enough to draw conclusions about effectiveness in practical use in ambient waters. This is because they do not indicate if other bacteria in natural waters will interfere with the ability to detect and quantify Vibrio cholera or if they will cause false positive results. The modified broth medium is sensitive to multiple strains of Vibrio cholerae. As such, this project should continue to evaluate the medium for specificity, sensitivity and then use with the Compartment Bag Test format.
Goals and Limitations: The results from this project so far are consistent with expectations outlined in the Phase I goals. The team was able to adapt existing microbiological media to culture-based detection of Vibrio cholerae. The results document the sensitivity of the TTGB candidate medium to multiple strains of Vibrio cholerae in a standard buffer as an optimal test water. During this phase, we were not able to test the media with other test waters or with other microorganisms due to various shipping delays. Experiments are in progress to test the selectivity of the candidate medium against strains of Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio mimicus, and other non-Vibrio chloerae background microorganisms that could potentially to provide false positive results. In addition, these experiments will be also be done using natural surface waters that are more representative of those in target areas for the use of the proposed Cholera Compartment Bag Test.
Conclusions:
This project is driven by the motivation to improve cholera monitoring for surveillance and early outbreak detection in low-resource and disaster settings. The results of this research have great potential to overcome the current burdensome practices that rely on laboratory evaluation for the detection of cholera in water. By optimizing a low-cost, field-portable test for detection of Vibrio cholerae, we can empower communities to monitor the quality of their own drinking water, respond to cholera outbreaks, and protect vulnerable water resources. If the results of this quantitative and careful laboratory study document that TTGB is effective in detecting and quantifying Vibrio cholerae in ambient waters with the Compartment Bag Test, there is justification for field testing, product development, and global deployment.
References:
- "Cholera." World Health Organization. World Health Organization, n.d. Web
- Monsur, Kazi Abul. "A highly selective gelatin-taurocholate-tellurite medium for the isolation of Vibrio cholerae." Transactions of the Royal Society of Tropical Medicine and Hygiene 55.5 (1961): 440-442.
- O'Brien, M. A. R. K., and R. I. T. A. Colwell. "Modified taurocholate-tellurite-gelatin agar for improved differentiation of Vibrio species." Journal of clinical microbiology 22.6 (1985): 1011-1013.
Journal Articles:
No journal articles submitted with this report: View all 3 publications for this projectSupplemental Keywords:
Cholera, diarrheal disease, Vibrio cholerae, drinking water, point-of-use, detection, microbiologyRelevant Websites:
UNC Gillings School of Global Public Health News Exit
Carolina Connection Radio Journal Exit
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.