Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Integrative Biology

Major Professor

Valerie J. Harwood, Ph.D.

Committee Member

Kathleen Scott, Ph.D.

Committee Member

Camilo Zalamea, Ph.D.

Committee Member

John Lisle, Ph.D.

Keywords

water quality, fecal indicators, reclaimed water, anthropogenic pollution

Abstract

Fecal pollution in recreational waters can introduce pathogens that cause increased human health risk, where exposure can occur during recreational activities such as swimming, diving, surfing, and fishing. Furthermore, human fecal pollution (i.e., sewage) can introduce nutrients and other harmful chemicals that may negatively affect the environment and aquatic species. Generally, sewage is considered the highest risk to human health compared to animal fecal pollution because it typically contains a high diversity of pathogens (including human specific viruses), antibiotic resistant bacteria and genes associated with antibiotic resistance. However, pathogens are difficult to detect due to their low concentrations in the environment, and there are too many to test every possible target. The use of surrogates that indicate the presence of pathogens is a standard approach that is utilized in recreational water quality studies. The most common surrogates are fecal indicator bacteria (FIB, e.g. enterococci and Escherichia coli) and their concentrations in water are used to estimate human health risk from contact with surface waters. One drawback to using FIB is that they do not provide information on the source of fecal contamination since humans and multiple animals can contribute to their concentration in surface waters.

Microbial source tracking (MST) DNA markers, which are frequently measured by quantitative PCR (qPCR) can alleviate limitations that arise from FIB methods. HF183, a DNA marker associated with sewage, can be measured in recreational waters to distinguish human sewage from other sources of fecal pollution by targeting a host-specific gene (i.e., HF183) in bacteria strongly associated with sewage. However, recreational water quality studies that rely on qPCR alone lack the ability to distinguish viable intact cells from dead cells and extracellular DNA in surface waters. This limitation can lead to an overestimation of human health risk in recreational water quality studies that rely on analysis of DNA. This issue is particularly acute in cases when treated, disinfected sewage (e.g., recycled water), which is known to contain microbial DNA, is directly discharged into surface waters and could be incorrectly identified as untreated sewage contamination.

This dissertation aims to address the following knowledge gaps, i) the effectiveness of different levels of wastewater treatment on the reduction of DNA in recycled water, ii) the decay rate of DNA from recycled water vs. untreated sewage in recreational waters, and the usefulness of culturable EcH8 as a viable marker of sewage pollution, and iii) the extent to which recreational water quality methods approved by regulatory agencies such as the U.S. Environmental Protection Agency capture extracellular DNA. In Chapter Two, we examined the effect of recycled water discharge on DNA marker levels in a Florida stream and tested the persistence of sewage-associated markers (i.e., HF183, H8, and CPQ_056) from wastewater treatment facilities that have two different levels of treatment. Recycled water from an advanced wastewater treatment (AWT) facility was discharged into a Florida stream and increased concentrations of the sewage-associated HF183 marker 1000-fold. Persistence of sewage-associated microorganisms was compared by qPCR in untreated sewage and recycled water from conventional wastewater treatment (CWT) and AWT facilities in Tampa and St. Petersburg, Florida. Multivariate analysis found that the persistence of sewage-associated DNA markers (HF183 and crAssphage CPQ_056) were significantly greater following CWT compared to AWT. Differential decay of DNA markers was found in recycled water samples where bacterial markers HF183 and EC23S857 were significantly correlated with each other but were not correlated to the viral marker CPQ_056. We tested to see if culturable EcH8 can be used to distinguish untreated sewage from recycled water and examined the proportion of total E. coli that carry the H8 gene. The proportion of total E. coli that carried the sewage associated H8 gene (culturable EcH8) in untreated sewage ranged from 8 – 18%, while culturable E. coli were below the limit of detection (< 1 CFU/L) in all recycled water samples. Therefore, culturable EcH8 has potential to confirm the presence of untreated sewage in surface waters that also contain DNA from recycled water.

In Chapter Three, the persistence of sewage-associated DNA markers (HF183 and CPQ_056) in outdoor freshwater mesocosms that were spiked with recycled water or untreated sewage and sampled over a five-day period were compared by qPCR. The persistence of culturable EcH8 was also measured to assess how it compared to sewage-associated DNA markers and to determine if it would be a useful target for detecting sewage over time. Experiments were conducted on three separate trials in a shaded environment to simulate a Florida stream. On day 5, median log10 reduction of sewage-associated DNA markers in the recycled water treatment were 0.68 (HF183) and 0.44 (CPQ_056), and were 2.83 (HF183), and 1.0 (CPQ_056) in the sewage treatment. The persistence of DNA markers assessed by multivariate analysis was significantly greater in the recycled water treatment compared to the untreated sewage treatment. The relationship between light intensity and decay rate of microbial variables was significant. In the sewage treatment, culturable EcH8 was detected in 40 to 60% of samples after five days across the three trials but was undetectable in recycled water. These results demonstrate the environmental persistence of DNA from recycled water and support the usefulness of culturable EcH8 for detecting untreated sewage in recreational waters that are also impacted by recycled water and other disinfected discharges of wastewater.

The objective for Chapter Four was to use standard recreational water quality methods to determine the proportion of DNA from intact cells and extracellular DNA (exDNA) that can be captured on filters and subsequently detected by qPCR, while exploring the usefulness of DNase I to eliminate extracellular DNA on membranes. Intact cells and extracellular DNA (obtained by boil lysis) from pure cultures of the FIB E. coli or Enterococcus faecalis, and exDNA from Natronomonas pharaonis (used as an exogenous control for river water and recycled water) were concentrated by membrane filtration. DNA was extracted from the filter and the proportion captured was measured with qPCR genetic markers EC23S857 (E. coli), Entero1A (Ent. faecalis), and NPgyrA (N. pharaonis). For intact cells, the proportion of gene copies captured on membranes ranged from 80 – 86% and were not significantly different among EC23S857 and Entero1A markers, DNase I treatment did not negatively affect gene copy estimates for intact cells. For solutions of exDNA, the mean percentage of EC23S857 gene copies captured on membranes was 1.4% and was significantly greater than Entero1A (0.5%). For river water and recycled water spiked with exDNA from N. pharaonis, the mean percentage of gene copies captured was 0.62% and 1.32%, respectively. DNase I treatment of membranes significantly reduced exDNA in all sample types by ~ 2 log10. These data demonstrate that a low percentage (< 2%) of exDNA in environmental water can be captured by standard recreational water quality methods; however, high concentrations of exDNA in water samples could complicate interpretations of data based on DNA measurements. DNase I treatment of membranes is a useful strategy to alleviate exDNA interference and could be used to improve estimates of human health risk in recreational waters.

This work will benefit human and ecosystem health by providing information and tools that could improve identification of untreated sewage pollution in recreational waters. Knowledge gained from this research can expand the recreational water quality field by highlighting important limitations to standard methods and help prevent overestimations of human health risk.

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