Graduation Year

2022

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Valerie J. Harwood, Ph.D.

Committee Member

Mya Breitbart, Ph.D.

Committee Member

Kathleen Scott, Ph.D.

Committee Member

Andrew Kramer, Ph.D.

Committee Member

Kerry Hamilton, Ph.D.

Keywords

fecal indicator bacteria, method comparison, microbial source tracking, microbial transport, quantitative microbial risk assessment, tropical beach

Abstract

Fecal contamination at recreational beaches impacts the health of beachgoers, through the introduction of disease-causing microorganisms, and the well-being of communities dependent on income from recreational beach activities. Beach ecosystems are also impacted by sewage through the introduction of nutrients that can cause abnormal increases in autochthonous microorganisms which can impact the population of larger organisms in the ecosystem. Fecal contamination is introduced into sand via untreated sewage, direct deposition of human feces into sand, runoff, and deposition of animal feces into sand. The introduction of fecal contamination into sand exposes individuals to pathogens (disease causing microorganisms) which can result in gastrointestinal illness. While standard methods and regulatory guidelines exist for the monitoring of fecal contamination in water, none exist for sand despite the data that link recreational contact with sand to gastrointestinal illness.

The detection of fecal-associated pathogens in the environment is difficult due to their diversity and low concentration, therefore contamination is monitored using fecal indicator bacteria (FIB). FIB are present in the gastrointestinal tract of animals and provide an indication of the presence of fecal contamination. Escherichia coli (freshwater) and enterococci (fresh and salt water) are commonly used as FIB globally and in the United States. In sand, FIB are present at higher concentrations than in water, as sand protects FIB from environmental stressors such as UV or predation and provides easier access to nutrients. FIB that survive long-term and replicate in sand are termed “naturalized.” Naturalized populations can complicate the identification of recent fecal contamination, as FIB monitoring techniques cannot differentiate between naturalized FIB and those recently introduced through sources of fecal contamination. The ubiquity of FIB among animals and humans means it is also difficult to determine the source of contamination. Some sources of fecal contamination (human as compared to cow or bird fecal contamination) contain a higher number of pathogens that are likely to infect humans (adenovirus in humans or Cryptosporidium spp. from cows). Therefore, microbial source tracking (MST) was developed to include a suite of host-associated genes (markers) of fecal microorganisms that are specific to different animals. These markers can differentiate between sources of fecal contamination, supplementing FIB monitoring and providing a more accurate depiction of the fecal contamination picture. Few studies have investigated the presence of MST markers in sand or determined relationships between MST markers and FIB in sand.

The human health risk associated with exposure to fecal contamination can be assessed by epidemiological studies; however, these studies are typically expensive and require specialized teams that may not be available to organizations with limited resources. Quantitative microbial risk assessment (QMRA) is a mathematical modeling framework used to estimate human health risk (the likelihood someone becomes ill) from exposure to pathogens under different environmental scenarios. QMRA consists of four steps: Problem formulation, exposure assessment, dose-response modeling, and risk characterization. Problem formulation involves establishing the framework (reference pathogen identification, exposure pathway, sources of contamination) that helps target the risk management needs to be addressed. The exposure assessment then determines the concentration and frequency of exposure by individuals to the reference pathogen(s) and exposure pathway identified during the problem formulation step, also known as a dose. The dose is then compared to a dose-response model (typically sourced from the literature) to determine the probability that the estimated dose would lead to illness in an individual. Risk characterization then quantifies the level of risk based on the modeled data. Reverse QMRA, used in this experiment, estimates the probabilities of pathogen concentrations that correspond with a risk threshold that is defined by stakeholders.

Few QMRAs have been conducted in sand, therefore, this dissertation is focused on understanding the human health risk from exposure to pathogens at a tropical beach impacted by sewage. Jacó beach is a tropical beach located on the Pacific coast of Costa Rica and is a popular tourist destination. A water quality study determined the fecal contamination was severely impacting the water quality of the beach and placing individuals at risk of illness from exposure to contaminated waters. Understanding the human health risk from exposure to pathogens in beach sand, in addition to the human health risk from water, ultimately helps to improve beach management decisions and public health.

In chapter one, the analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus in sand was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus two direct extraction methods using PowerSoil™ or PowerMax Soil™ kits at a freshwater and saltwater beach in Tampa, Fl, USA. We found the slurry method had the lowest limit of detection at 20–80 gene copies g-1 (wet weight), recovered significantly more DNA, and was the only method that detected Enterococcus by qPCR in all samples; therefore, the slurry method was exclusively used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the human-associated microbial source tracking marker HF183 from marine and freshwater beach sand spiked with wastewater was 100.8% and 64.1%, respectively, but varied between dilutions, indicating that the mixing protocol needs improvement.

The objective of chapter two was to determine the extent of the influence a contaminated waterbody has on the concentration of microbes in beach sand. Upstream, downstream and ocean samples were collected on a transect in sand at 0 m (origin), 2.5 m and 5.0 m from the riverbank or swash zone. Samples were also collected in sediment. Samples were analyzed for the presence of the MST marker HF183 and FIB Enterococcus using the slurry method followed by DNA extraction and qPCR. Median concentrations of Enterococcus decreased as distance from the river or ocean increased. Enterococcus ranged from 1.46 x 104 to 8.11 x 103 gene copies 100 g-1 to the same distance. HF183 and Enterococcus were positively correlated at the riverbank/swash zone, but not at other subsites due to frequent failure to detect HF183. Sediment samples did not differ in HF183 or Enterococcus concentrations along the river and HF183 was not detected in the ocean sediment. The frequency of detection for HF183 was significantly greater among samples along the riverbank than samples collected at the 2.5 m and 5 m subsites. Polluted waterbodies can influence microbe concentrations in sand but the extent of the influence of a waterbody on MST markers in sand requires further study.

Chapter three employed a reverse QMRA in Copey River at Jacó Beach to estimate the varying probability that corresponds with meeting a risk target. The risk target was defined as 36 cases of gastrointestinal illness per 1000 people (36/1000), the health target set in the 2012 recreational water quality guidelines for the United States Environmental Protection Agency (USEPA). Data were used to calculate whether concentrations of Salmonella, Adenovirus, and Giardia exceeded or were below concentrations needed to meet a risk of 36/1000. Samples at two sites, one upstream and one downstream of where beachgoers tended to recreate at the riverbank were collected six times over three weeks. Samples were analyzed for the presence of enterococci (FIB), the human-host associated MST marker HF183 and sewage-associated MST marker PMMoV, and pathogens Salmonella, adenovirus, and Giardia. Sand samples were collected at the riverbank and analyzed for the fecal indicator bacteria enterococci, the human-host associated microbial source tracking marker genes HF183, sewage-associated marker pepper mild mottle virus (PMMoV), and pathogens Salmonella, Giardia, and human adenovirus. Enterococci and Salmonella were cultured using the slurry method followed by USEPA standard methods. HF183, PMMoV, and adenovirus were detected by (RT)-qPCR. Giardia was detected using microscopy following USEPA standard methodology. Enterococci were detected in all samples and had a geometric mean of 3.25 x 103 GC 100 g-1. Geometric means of the MST markers HF183 and PMMoV were 1.19 x 103 and 1.64 x103 GC 100 g-1 and were detected in 83% and 33% of samples, respectively. Salmonella was detected in 66.6% of samples and had a geometric mean of 2.45 x 102 CFU 100 g-1. Giardia and adenovirus were detected only once each at 45 oocysts 100 g-1 and 1.91 x 102 GC 100 g-1. QMRA analysis showed that adenovirus and Giardia concentrations exceeded the levels that coincide with the risk threshold of 36/1000 while Salmonella fell between the 50th and 75th % percentile probability of meeting the risk threshold. Exposure to sewage-borne pathogens in sand could contribute to the risk of illness for recreational users of Jacó Beach and should be analyzed in tandem with pathogens in water to implement the best strategy for the protection of public health. To implement a comprehensive beach monitoring strategy, it is important to understand all the contextual factors that affect public health at a beach. Recreation in sand has been linked to gastrointestinal illness, however there are no regulatory guidelines or standard methods for analysis of fecal microorganisms in sand. This research has shown that a comprehensive beach monitoring strategy should include sand monitoring strategies in tandem with water monitoring strategies.

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