A Novel Infield Metagenomic Approach to Evaluating Surface Water Quality in Lake Warner

In January 2010, a magnitude 7.0 earthquake devastated Haiti, one of the poorest countries in the Western Hemisphere. Haiti’s weak sanitation infrastructure and limitations in the public health system made the country susceptible to the spread of waterborne diseases. Following the earthquake, cholera rapidly spread through Haiti, killing 4,672 people in 5 months and leaving thousands hospitalized (MSNBC, 2010). Cholera is an infectious diarrheal disease caused by the pathogen, Vibrio cholerae, which results in severe dehydration with a high mortality risk. The source of the epidemic was traced to the Artibonite River, the island’s longest and most essential drinking water source (Encyclopaedia Britannica. n.d.). The origin of the contamination was later discovered to be unsanitary conditions left from United Nations peacekeepers from Nepal. Eight years later, cholera cases are still prevalent, although numbers have declined recently due to aid from private organizations (Dowell, S.F. et al 2011, Katz, J.M. 2013). However, with climate-related increases in ocean water temperatures, scientists expect hurricanes to intensify and increase damage to developing countries (Center for Climate and Energy Solutions. n.d.). Natural disasters promote the spread of waterborne illness by isolating people from safe drinking water and destroying public health infrastructure such as happened with the cholera outbreak in Haiti (Funari, E. et al 2013). To prevent future waterborne disease epidemics in such areas with limited resources, it would be beneficial to improve environmental surveillance through development of rapid, reliable, and portable detection methods for waterborne pathogens. The advent of high-throughput sequencing technologies has enabled the detection and characterization of microbial communities in their natural environments, an approach known as metagenomics. Metagenomic sequencing, unlike more traditional PCR methodologies, is capable of sequencing thousands of organisms in a sample. This metagenomic shotgun sequencing approach detects the abundance of microbes and bacterial diversity in the environment (Illumina, n.d.). The Oxford Nanopore MinION is a shotgun sequencing device that is optimal for portable, rapid detection of the microbial diversity in an environmental sample (Oxford MinION, n.d.). This handheld device has enormous potential for field use in emergency preparedness and disease response, particularly in developing countries where more advanced analytical equipment may be inaccessible due to lack of facilities or damaged infrastructure. Having access to quick, infield assessment technology for rapidly emerging outbreaks would be beneficial to a disease-specific public health response. Current protocols recommend that DNA is extracted from environmental samples as rapidly as possible after collection. If cooling is available with an insulated ice chest, samples may be transported/stored for periods ranging from 6 to 24 hours. The shorter timeframes minimize unwanted shifts in microbial structure (U.S. Geological Survey, 1997, WHO, n.d.). Access to cold storage in remote areas is unlikely, and the use of liquid preservation methods could assist in maintaining quality of DNA, and hence produce more accurate data in metagenomic analyses. In the absence of cold storage facilities, infield filtration coupled with preservation techniques are necessary to maintain samples integrity for transport to laboratory facilities. This thesis aimed to develop an infield filtration and sequencing protocol, coupled with the Oxford Nanopore MinION sequencing platform, to identify the potential bacteria, viruses, protozoa, fungi, antimicrobial resistance (AMR), pathogenic strains, and virulence associated genes for use in remote locations. Five locations across Lake Warner, Massachusetts were used for method development, coupled with Millipore Sterivex filters for field filtration to determine the most effective method for sample preparation in remote locations. Additionally, a chemical preservation method was assessed using dimethyl sulfoxide, disodium EDTA, and saturated NaCl (DESS). A study by Gray et al, found that liquid preservation methods (DNAgard, RNAlater, and DESS) outperformed the card-based preservatives (FTA cards and FTA Elute cards) in terms of bacterial recovery (Gray, M.A., et al 2013). DESS was selected for investigation in this thesis because of the low cost compared to the other liquid-based preservatives. Lake Warner in Hadley, Massachusetts, which is heavily used for fishing and boating activities, flows into the Connecticut River via the Mill River. Historically, the lake experienced high Escherichia coli (E. coli) levels due to pollution from primary effluent released in the 1950s from the Amherst Wastewater Treatment Plant (Johnson, J., 2015). Similar to Vibrio cholerae, E.coli spp is a waterborne bacteria caused by fecal contamination. Although most E.coli are natural inhabitants of the gastrointestinal tract, pathogenic serotypes can result in severe complications in vulnerable populations such as kidney failure in children and the elderly adults. (Todar, K., 2012). Lake Warner was chosen for the method development because of its history of E. coli pollution and recreational traffic as well as its general accessibility for study. Designing a methodology for rapid detection of pathogenic bacteria using a metagenomic approach could help improve surveillance for environmental pathogens that pose future epidemic risk. These tools are becoming increasingly important for prediction and response to waterborne diseases as climate impacts increase the frequency, intensity, and duration of extreme weather events that damage critical infrastructure for vulnerable populations (van Aalst, M.K. 2006)