Biogeographical Analysis of Antibiotic Resistance Genes in United States Wastewater Público

Rainey, Andrew (Spring 2019)

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Antibiotic resistance is one of the biggest public health challenges of our time; accounting for about 2 million infected individuals and about 23,000 deaths from AR infections, costing about $55 billion annually in the United States. The purpose of this study is to provide data on the biogeographical distribution and concentrations of antibiotic resistance genes in United States influent wastewater samples. This project is a descriptive, hypothesis-driven study that will provide data from across the country that will help improve water quality, waste management and public health applications. Quantitative PCR assays of 10 clinically significant antibiotic resistant genes were conducted on influent wastewater samples that were collected from 49 wastewater treatment plants throughout the United States, chosen to represent influent wastewater from urban and rural areas. Urban samples had greater concentrations than rural for all targets except for sul1, KPC had the largest urban v. rural difference of 1.3074 Log10 copies/ml (p-value < 0.05). The difference of rural sul1 to urban sul1 levels was 3.2390 Log10 copies/ml (p-value < .0001). Regional differences were seen among the targets NDM, OXA-48, sul1 and tetW, and the Mountain and West regions were most frequently different among these targets. The mcr-1 gene is present in more states than clinical surveillance has discovered in the U.S. Urban areas are a major source of environmental ARGs and are more likely to provide positive samples. Clinically relevant ARGs are present at high quantities in U.S. wastewater and suggest urban areas and Mountain and Western states as major areas of interest.

Table of Contents

Chapter 1: Literature Review

1.1: Antibiotic Resistance History

1.2: Antibiotic Resistance Gene Background and Mechanisms

1.3: Mechanisms of Antibiotic Resistance Development

1.4: United States Policy on Antibiotic Resistance

1.5: Antibiotic Resistance Burden and Statistics

1.6: Surveillance of Antibiotic Resistance in the United States

1.7: Clinical Antibiotic Resistance

1.8: Environmental Antibiotic Resistance

Chapter 2: Methods

2.1: Purpose of Project

2.2: Sample Sites and Collection Methods

2.3: DNA Extraction

2.4: DNA Quantification and Dilution

2.5: PCR Reagents

2.6: Gblock and Standards

2.7: Inhibition Testing

2.8: Quantitative PCR

2.9: Statistical Analysis

Chapter 3: Results

3.1: Overall Antibiotic Resistance Gene Concentrations

3.2: Urban and Rural Comparisons

3.3: Regional Comparisons

Chapter 4: Discussion

4.1: Discussion

4.2: Limitations

4.3: Conclusion

4.4: Recommendations

Chapter 5: References

Chapter 6: Figures and Tables

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