Novel, Rapid and Cost-effective Methods for Concentration, Detection and Monitoring of Waterborne Pathogens in Resource-Limited Settings

Citation

Wu, Xunyi (2021) Novel, Rapid and Cost-effective Methods for Concentration, Detection and Monitoring of Waterborne Pathogens in Resource-Limited Settings. Dissertation (Ph.D.), California Institute of Technology.. doi:10.7907/fwhf-a510. https://resolver.caltech.edu/CaltechTHESIS:06042021-184017139

Abstract

Waterborne pathogenic organisms including bacteria, viruses, protozoa and helminths, are responsible for a series of diseases which is a major public health concern worldwide. This issue is extremely severe in developing regions due to the scarcity of clean water supply and poor sanitation. Therefore, point-of-use (POU) detection and quantification processes as well as a monitoring program of waterborne pathogens are needed to ensure the safety of water and protect human health. However, the polymerase chain reaction (PCR) technology and its related detection platforms rely on complicated thermal cycling, centralized laboratory equipment and trained personnel, thus making PCR-based systems incapable of POU testing of environmental waters. In this dissertation, we develop a portable 3D-printed system with super-absorbent polymer (SAP) microspheres for sample enrichment, and a membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system for absolute quantification of pathogens. We also explored the interactions between microbial indicator of Escherichia coli (E. coli) and waterborne pathogen Vibrio Cholerae (V. Cholerae). The main results are as follows:

1. The application of detection and quantification methods is often hindered by the low pathogen concentrations in natural waters. Rapid and efficient sample concentration methods are urgently needed. Here we present a novel method to pre-concentrate microbial pathogens in water using a portable 3D-printed system with super-absorbent polymer (SAP) microspheres, which can effectively reduce the actual volume of water in a collected sample. The SAP microspheres absorb water while excluding bacteria and viruses by size exclusion and charge repulsion. The 3D-printed system with optimally-designed SAP microspheres could rapidly achieve a 10-fold increase in the concentration of E. coli and bacteriophage MS2 within 20 minutes with concentration efficiencies of 87% and 96%, respectively. Fold changes between concentrated and original samples from qPCR and RT-qPCR results were found to be 11.34-22.27 for E. coli with original concentrations of 10⁴-10⁶ cell·mL^-1; and 8.20-13.81 for MS2 with original concentrations of 10⁴-10⁶ PFU·mL^-1. Furthermore, SAP microspheres can be reused 20 times without performance loss thereby significantly decreasing the cost of our concentration system.

2. Following sample concentration, accurate quantification methods for waterborne pathogens are needed, especially at the point of sample collection. The surge of COVID-19 in late 2019 called for a more urgent need for a rapid and cost-effective quantification of SARS-CoV-2 in environmental waters. Quantification results contribute to wastewater-based epidemiology (WBE) which helps the monitoring of prevalent infections within a community and early detections of contamination. Here we demonstrated the usage of our portable membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system for absolute quantification of SARS CoV-2 in wastewater samples within a one-hour timeframe for point-of-use (POU) testing and data management. The limit of detection (LOD) of mgLAMP for SARS-CoV-2 quantification in Milli-Q water was observed to be down to 1 copy/mL, and that in surface water collected from Kathmandu, Nepal was down to 100 copies/mL. Both were 100-fold lower than that of RT-qPCR in corresponding matrices. Compared to alternative detection methods, our platform has a very high level of tolerance against inhibitors thanks to the restriction of the hydrogel matrix. This enables the highly sensitive detection in either clinical or environmental samples.

3. Regular environmental surveillance of waterborne pathogens is key to ensure the safety of water and protect public health. Due to the diversity of pathogenic bacteria in environmental waters, regular monitoring of so many pathogens for individuality is impractical. Therefore, microbial indicators are used to gauge the total pathogen concentration; and manage waterborne health risks. In this study, the interactions of V. cholerae, the etiologic agent of reemerging cholera, with E. coli, the most commonly used indicator for waterborne pathogens. Specifically, we investigated through evaluating the survival and growth of both bacteria under different temperature and nutrition deprivation using plate culturing and real-time polymerase chain reaction (qPCR). During co-growth, it was challenging for V. Cholerae to maintain initial population advantages as E. coli consumes nutrition more effectively. Whereas during co-existence, V. Cholerae soon fell into a viable-but–non-culturable state under environmental stress in 3-5 days while E. coli stay viable more than 14 days. We found that V. cholerae interacts with E. coli differently depending on the composition of the water that is sampled and analyzed. This suggests that bacterium-bacterium interactions influenced by the intrinsic chemical and biological parameters of ambient water will be a contributing mechanism in regulating the proliferation of V. cholerae.

In summary, two platforms for environmental sample concentration and detection have been developed and tested using ambient and engineered waters. In addition, interactions between a microbial indicator, E. coli, and the pathogenic bacteria, V. Cholerae, were studied. The chapters in this thesis describe in detail: (1) A hand-pressed 3D-printed system to produce SAP microspheres was developed with the goal of achieving efficient concentrations of environmental microorganisms for subsequent analysis. The simplified concentration procedure and can be easily integrated into various detection platforms; (2) A portable membrane-based in-gel loop-mediated isothermal amplification (mgLAMP) system was developed for absolute quantification of SARS-CoV-2 in environmental water samples within one hour, enabling a 100-fold lower detection limit compared to the gold-standard of RT-qPCR; and (3) Differences in bacterium-bacterium interactions of V. cholerae and E. coli under as a function of water composition indicated that environmental stress presented in ambient water matrices should be taken into consideration while using a microbial indicator such as E. coli to estimate the risk of waterborne pathogens. These collective advances allow for the rapid and ultrasensitive POU testing of waterborne pathogens that should provide for more effective monitoring strategies in terms of the use of indicator microorganisms.

Thesis Files