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Developing Technologies for Tracking Drug Resistant Escherichia coli
Project Goal
To develop methods for the identification of genetic sequences of E. coli that will provide rapid, cost-effective, and specific source determinations of antibiotic resistant E. coli in estuarine waters and marine organisms. Learn more about the background information for this project »
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Abstract
Traditionally, microscope and antibody based techniques have been used to identify pathogens such as those selected for study in this project. However, neither of these methods can identify species that specifically infect humans. Such identification requires the use of molecular tools such as polymerase chain reaction (PCR) and gene sequencing. In addition, bacterial source tracking technology, such as antibiotic resistance analysis, is a basic tool in identifying human and nonhuman fecal sources from environmental samples. The project objectives which seek to transfer source tracking technology from CCEHBR Charleston to CCEHBR Oxford, develop new diagnostic techniques for the detection of the primary Cryptosporidium, Giardia, Toxoplasma, and Microsporidia species or genotypes that infect humans have essentially been accomplished. What remains is to validate recovery of these organisms in environmental samples, build a baseline of these pathogens in sediments, water, and shellfish in Chesapeake Bay and nationally, to ultimately develop an accurate quantitative methodology for pathogen detection, and transfer technology to targeted users.
Objectives
The objectives of this project are to:
- Identify potential conserved genes that have characteristics suitable for developing assays that uniquely identify large numbers of antibiotic-resistanct plasmids.
- Conduct initial cloning and sequencing to identify regions that would make potentially good capture probes for the Luminex system.
- Develop and test base of Luminex probes that will work with the conserved PCR amplification products.
Expected Results
The techniques developed through this project are expected to provide rapid, cost effective, and specific source determinations of antibiotic resistant E. coli in estuarine waters and marine organisms. Escherichia coli is being tested as a model species and a similar approach could be applied to other microbial species of interest. These techniques will:
- Aid in understanding the fate, transport, and load of antibiotic resistant E. coli or other microbes in coastal ecosystems; and
- Ultimately provide technology that environmental managers can apply to make timely decisions about the source of antibiotic resistant organisms.
For example, the methods could be used to determine if the source of antibiotic resistant E. coli, or other bacteria, was of a human or livestock origin. The information obtained could then form the basis for remediation strategies for reducing or mitigating pathogens of public health concern in coastal waters.
Accomplishments (This project received no OHHI funding for FY 2006) :
- Completed design and preliminary laboratory testing of PCR primers for Cryptosporidium, Giardia, and Toxoplasma. New PCR techniques detected 90 and 100% of samples spiked with 100 and 1000 Cryptosporidium oocysts respectively compared to 20 and 74% detection by the traditional Immnofluorescent Antibody Assay (IFA) method.
- Optimized recovery, DNA extraction and PCR techniques for pathogen detection in laboratory studies using spiked samples in water and sediment collected from Chesapeake Bay . Current PCR methods being tested are not quantitative, but based on a presence or absence of the pathogen in a sample. However, positive findings are believed to be infectious for humans based on successful detection of 5 to 100 oocysts (Cryptosporidium) and cysts (Giardia) in spiked samples. A single oocyst is believed capable of causing infections in some individuals and Okhuysen et. al (1999) reported a dose of approximately 87 oocysts as capable of causing infections in 50% of individuals exposed to Cryptosporidium.
- Initiated building DNA libraries for antibiotic resistance and bacterial source tracking protocols. Library currently includes resistance information for bacteria from four sewage treatment plants and approximately 20 wild and domesticated animals.
- Began field tests for detection of selected pathogens and fecal bacteria in environmental samples taken before and after tertiary treatment at sewage treatment plants and in sediments along transects up and downriver of outfalls of sewage treatment plants rated at low, moderate, and high levels of effluent discharge. Twenty stations sampled in three different river systems during May and June were evaluated for pathogen detection using conventional PCR and fecal bacterial source using antibiotic resistance analysis. Giardia duodenalis assemblage A is infectious for humans and other primates as well as cattle, sheep, deer, horses, pigs, and ferrets. Giardia assemblage A was detected in the effluent of 5 of 7 samples taken at treatment plants with moderate and high volume discharge. Cryptosporidium andersoni (primary source adult cattle) was detected in 3 of 4 samples from high volume discharge facilities. No Giardia or Cryptosporidium was detected from effluent at the low volume discharge site.
- Conducted first quarterly cruise to detect pathogens and antibiotic resistant bacteria in water, sediment, and shellfish at 13 locations around the Maryland portion of Chesapeake Bay. Samples were evaluated for pathogens using conventional PCR and fecal bacterial source using antibiotic resistance analysis. Giardia sp. (assemblage A) was found in water and sediment at one of 13 sites and in the digestive diverticula from a pool of 5 oysters at a second site. Both sites are in proximity to sewage treatment plants. Cryptosporidium was not detected at any of the 13 locations during the summer quarterly sampling effort.
Transfer of Results
When technologies are produced that prove to be reliable to local and state shellfish and public health testing laboratories, they will be disseminated through individual meetings and conference presentations. Officials in North Carolina have demonstrated an interest in using the approach.
Publications/Presentations:
A poster “Molecular Techniques to Detect Emerging Human Pathogens
In Coastal Ecosystems” was presented at the HML Center of Excellence principal investigators meeting, Charleston, S.C. January 2006
A presentation “NOAA's Oceans and Human Health Initiative - An overview of the Microbiology Program” was presented at the Interstate Shellfish Seminar, Ocean City, MD. October 2005
Public Information and Outreach:
This and similar projects that have a public health link to shellfish are of strong interest to the Interstate Shellfish Sanitation Conference (ISSC), shellfish managers, growers, local public health groups, and consumers. This project helps develop techniques that can be employed to assess the risk in consumption of raw shellfish from areas that may harbor or accumulate human pathogens. In the recent years the ISSC has been made aware of this work and has provided logistical support and contacts used in making collections for evaluation. This association is important to maintain toward the end goal of conducting a national baseline survey of protozoan pathogens and fecal bacteria in shellfish and growing areas. Project investigators continue to provide communication to annual meetings of the Interstate Shellfish Seminar and Northeast Shellfish Sanitation Association. Upon project completion technology transfer to users is planned through convening an ISSC workshop
For More Information
Contact: Wayne Litaker, (252)728.8791
Email: Wayne.Litaker@noaa.gov