Antibiotic Resistance as a Measurement Tool

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Chapter 2. Goals and Objectives

The overall project goal was to monitor and evaluate twenty one stream locations in the Occoquan Basin identified as impaired due to high E. coli densities. One site on each of eight streams, two sites on each of five streams, and three sites on the remaining stream were chosen for E. coli monitoring and microbial source tracking

The project objectives are:

  1. The categories of fecal sources that lead to bacterial impairment will be determined using antibiotic resistance analysis (ARA).
  2. The known source library (KSL) to be used with ARA will be designed in a manner that best represents the major fecal sources (human, pets, livestock, wildlife) in the watershed in order to best identify the sources present in the environmental isolates. This will include performing two challenge sets against the KSL. This will serve as an assessment of the ability of the KSL to classify known-source isolates not already included in the library.
  3. Fluorometric analysis (FA) of optical brighteners will be utilized as an indicator of human wastewater. Data will be collected during the same collection period as for ARA. The FA data will be compared with the ARA results in order to assess the capability of FA to serve as a secondary indicator of human contamination

Chapter 3. Materials and Methods

Defining the Study Location

The Occoquan Basin (OcB) of the Middle Potomac-Anacostia-Occoquan watershed within Prince William County (PWC) (Appendix I) served as the region for the project. Encompassing an area of approximately 1528 km2, the OcB serves as a headwater for a Potomac River tributary, which eventually discharges into the Chesapeake Bay. Over the past several decades the OcB has transitioned from a traditionally rural farming region to one of the fastest growing regions in America. This change has affected the relationship between humans and animals by increasing the proportion of shared living spaces. Fewer open spaces forces animals into parks, refuges, and neighborhoods, causing over-crowding and increased competition for resources. An additional consequence is an increase in the concentration of fecal waste deposited near waterways. An increased concentration of animal waste can become a serious issue for communities as the waste is carried into waters designated for recreational uses.
The vast majority of animal waste for this project was collected from recreational and state parks located throughout PWC, such as Leesylvannia State Park. The animal sources included deer (Odocoileus virginianus), Canada geese (Branta Canadensis and Anser domesticus), various gull species (Larus sp.), horses (Equus caballus), dogs (Canis familiaris), and cows (Bos taurus and Bos indicus). Human (Homo sapiens) samples were collected from the H.L. Mooney Water Reclamation Facility in Woodbridge, VA

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Environmental Water Sample Locations

Environmental water samples were collected from 18 quarterly (once every three months) sites and three monthly sites. With the exception of sites Q4 (Cow Branch at Montgomery Avenue) and Q18 (North Fork of Lake Manassas), the 18 quarterly sites were a continuation of earlier efforts. The three monthly sites were first-time locations. Sampling of the quarterly sites commenced in June 2006 and happened once every three months until the final sampling in June 2007. Sampling of the monthly sites began in July 2006 and took place once every month (simultaneously with quarterly sampling when applicable) until the final collection in June 2007. Additionally, sediment samples were collected along with environmental samples during the June 2007 collection period for purposes of comparison and quality control. In general, the sites were surrounded by wooded buffer zones and dense undergrowth in the summer months. Individual site descriptions are provided below

Quarterly Site 1 (Q1): Neabsco Creek, Lindendale Road – N38°38.7273′ W077°21.9542′

Although located in a commercial district of the county, this site is surrounded by a green buffer zone. The sampling occurred where the creek runs under a two lane bridge, over which moderate traffic patterns were commonly observed. People were observed walking their dogs in the immediate vicinity, and evidence of dog scat was apparent on numerous occasions. No immediate or nearby construction or development was observed over the course of the project, but the water was quite muddy on several occasions, most likely the result of a recent rain event and/or upstream construction

Quarterly Site 2 (Q2): Neabsco Creek, Benita Fitzgerald Road – N38°37.5141’ W077°18.8082’

This location is typical of the many highly developed residential areas of PWC. An apartment complex has been constructed within the last couple years at this site, but a green buffer zone has helped to reduce the impact of the suburban sprawl. Sampling took place where the creek runs under a four lane bridge, and at least one large storm drain was observed leading directly from the road into the creek. No immediate or nearby construction or new development was observed

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Quarterly Site 3 (Q3): Neabsco Creek, Neabsco Mills Road & Route 1 – N38°36.6421′ W077°17.4307′

Sampling was conducted where the creek goes under a heavily-trafficked four lane highway. During the 2007 summer months, road construction was observed in the immediate vicinity. This site is classified as a non-residential, commercial area of the county

Quarterly Site 4 (Q4): Cow Branch, Montgomery Avenue – N38°38.1860′ W077°16.6908′

Sampling in this relatively quiet residential area took place where the creek runs under a two lane bridge. On several occasions the water possessed a reddish tint. This was a result of a heavy iron presence and is characteristic of urban and suburban streams, due to runoff from roads, sidewalks, parking lots, rooftops, and other impervious surfaces. A golf course is located nearby. No immediate or nearby construction was observed

Quarterly Site 5 (Q5): Cow Branch, Rippon Landing Park – N38°37.0715′ W077°16.4505′

Environmental samples were collected at a four lane divided-highway bridge, marked by heavy traffic. During the summer months, multiple construction jobs were observed. Construction was being performed at the bridge to install concrete pipes, and development of a large apartment complex was occurring nearby. The water had a reddish tint during multiple collections

Chapter 1. Literature Review
I. Project History and Justification
II. History and Development of Antibiotic Resistance Analysis
A. Selecting an Appropriate Indicator Organism
B. Antibiotic Resistance as a Measurement Tool
III. ARA in the Field.
IV. The Future of ARA and MST.
A. Understanding the Limits of ARA
B. ARA vs. Other MST Methods
C. ARA Remains in the Primary Literature.
D. Performance-based Criteria for MST Projects
V. Summary of Study Design
A. Antibiotic Resistance Analysis (ARA)
B. Fluorometric Analysis
VI. References
Chapter 2. Goals and Objectives
The project objectives are
Chapter 3. Materials and Methods.
I. Defining the Study Location
II. Environmental Water Sample Locations
III. Sample Processing
IV. Statistical Analysis
V. References
Chapter 4. Results and Discussion
I. Monitoring Results (Tables 5-6 and Figures 3-4)
II. The Known Source Library (Tables 7-9)
III. Environmental Water Isolates (Tables 10-34)
V. Real Value of Analysis
VIII. References
Appendix.
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