Source: LINCOLN UNIVERSITY submitted to
NOVEL ESCHERICHIA COLI GENETIC MARKERS FOR WATER SAFETY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0226305
Grant No.
2011-38821-31033
Project No.
MOX-ZHENG2
Proposal No.
2011-02539
Multistate No.
(N/A)
Program Code
EQ
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2015
Grant Year
2011
Project Director
Zheng, G.
Recipient Organization
LINCOLN UNIVERSITY
(N/A)
JEFFERSON CITY,MO 65101
Performing Department
Agriculture
Non Technical Summary
The goal is to reduce human illness caused by the direct body-contact (e.g. in recreation water) or consumption (e.g. drinking water) of feces-polluted water, through developing deoxyribonucleic acid (DNA)-based methods for real-time detection of fecal pollution for its sources and amount. Currently, detection of the concentration (amount) of bacterium Escherichia coli (E. coli) is used by the US Environmental Protection Agency (EPA) to measure fecal pollution in fresh water. However, current EPA-approved methods provide no information regarding to the host sources (human vs. nonhuman) of a fecal contamination, although the source information is essential for elimination or remediation of the pollution sources to restore quality of polluted waters. In addition, the EPA-approved methods include a step of incubation of E. coli and thus, are time consuming and cannot present water quality at real time. Result of our preliminary study and others' research has demonstrated that different DNA signature sequences (genetic markers) of E. coli are associated with different host species. These sequences, with additional genetic markers identified in this project, will be used to develop DNA-based methods for identification of the sources of fecal pollution in water, especially in recreation water. Furthermore, by eliminating the lengthy incubation step in the EPA-approved methods, a DNA-based method will be developed for detection of E. coli concentration at real time. The resulting methods, for the fecal source detection and the E. coli amount measurement respectively, will be evaluated at selected feces-polluted sites at the Lake of the Ozarks in Missouri for their usefulness. Through the multidisciplinary research and partnership with an 1862 land-grant university, a local business, and a local environmental group, this project is to address the important issues of water safety that Missouri and the nation face.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1330210110020%
1330210108050%
1330210208030%
Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0210 - Water resources;

Field Of Science
1080 - Genetics; 2080 - Mathematics and computer sciences; 1100 - Bacteriology;
Goals / Objectives
The goal is to reduce human illness caused by the direct body-contact (e.g. in recreation water) or consumption (e.g. drinking water) of feces-polluted water, through developing deoxyribonucleic acid (DNA)-based methods for real-time detection of fecal pollution for its sources and amount. The broad objective of this project is to overcome the limitations associated with current methods for the detection of fecal pollution for its sources and amount in water. The specific aims of this project are: 1) in-silico and experimental identification of E. coli genetic markers unique to humans, domestic animals and wildlife, respectively; 2) evaluation of the specificity and prevalence of the putative host-specific genetic markers; 3) development of real-time PCR assays for the measure of total viable fecal E. coli and host-specific E. coli in water samples; 4) field test of the resulting real-time PCR assays in comparison with EPA-approved methods for evaluation the usefulness of the resulting methods.
Project Methods
1)Potential E.coli genetic markers unique to humans, domestic animals, or wildlife will first be indentified in-silico using the published E.coli DNA sequences, through data mining with bioinformatics approaches, and then determined by experiments. Alternative, metagenomic methods will be used to do this identifications. 2)The specificity and prevalence of the putative host-specific genetic markers will be evaluated by PCR assays using fecal DNAs extracted from target and non-target animal fecal samples; 3)Propidium monoazide real-time PCR assays will be developed for quantification of total viable E. coli cells and for determination of the sources of E. coli in feces-polluted water, respectively; 4)The ability of the resulting products in real-time enumeration of E. coli and real-time determination of the sources of fecal pollution in water will be tested using water samples from selected fecal-polluted sites at the Lake of the Ozarks in Missouri.

Progress 09/01/11 to 08/31/15

Outputs
Target Audience:1. Scientific and industrial communities in water safety 2. Federal and local water safety monitoring agencies 3. Total Maximum Daily Loads (TMDL) plan developers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. Three graduate students were supported and trained through this project. 2. One postdoctorial scientist was trained through this project 3. One technician was trained through this project How have the results been disseminated to communities of interest?1. The results have been published in the peer-reviewed journals including "Water Research" and "Science of the Total Environment." 2. The results have been presented in professional conferences and meetings, including the American Society for Microbiogy and the General Meeting and International Association for Food Protection annual Meeting 3. The results have been presented through two seminars to the local environmental group "Lake of the Ozarks Watershed Alliance". What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Escherichia coli (E. coli) is a fecal bacteria indicator. It is used in testing worldwide to determine water quality and manage water safety. E. coli is used because it is a part of the bacteria living in the intestines of warm-blooded animals. However, a significant part of the global E. coli population might come from outside the body. These "environmental" E. coli can be a factor in the high number of E. coli in water. This causes false alarms of fecal pollution. Thus, methods must be developed to tell the difference between enteric (in the intestine) E. coli and "environmental" E. coli. This project has created a rapid assay to do the differentiation, which can reduce unnecessary beach closures and other management procedures. Although the current E. coli testing methods recommended by the United States Environmental Protection Agency (EPA) can measure fecal contamination of water, they do not identify the sources (human vs. animal) of pollution, hindering the effective control or remediation of fecal pollution from its sources. This project has developed a rapid method to identify human fecal pollution. Human fecal pollution can spread human diseases and thus is the major concern of water safety. Using the method developed in this project can better protect people from diseases that result from human feces-polluted water. The project was a joint effort of scientists from Lincoln University (LU) in Missouri and University of Missouri-Columbia (UMC), in partnership with the Missouri State Parks, the Ameren UE (a local business)‎, and the local environmental group "Lake of the Ozarks Watershed Alliance". Overall, the proposed four objectives of this project have been achieved: 1) The objective 1: In-silico and experimental identification of E. coli genetic markers unique to humans, domestic animals and wildlife, respectively.For the in-silico identification, all E.coli gene sequences and their associated information (the isolation sources and hosts) available from the public GenBank database of the National Center for Biotechnology Information (NCBI) were used to compare DNA sequences among E. coli strains isolated from different host sources or different habits (human vs. animal and enteric vs. soil or water environment). As a result, several E. coli genetic sequences were identified to be highly associated with enteric E. coli population or with E. coli strains from feces of human. The aforementioned putative E. coli genetic sequences (genetic makers) were verified through the polymerase chain reaction assays (PCRs) using a panel of 367 E. coli strains isolated from feces of different host species, including human, cattle, chicken, deer, ducks, goats, geese, humans, raccoons, and swine. 2) Evaluation of the specificity and prevalence of the host-specific genetic markers. This was to determine each potential marker's ability to predict the presence of target fecal source. The novel E. coli genetic markers that were evaluated are the enteric-E. coli-associated and the human-feces-associated. The enteric E. coli marker discovered by this study was the putative glucosyltransferase gene (ycjM). Our study has further revealed that there are polymorphism among ycjM genes in E. coli population and that the polymorphism is host specific. Our data show that about 97% of E. coli strains isolated from animal feces are positive for gene ycjM and that about 50% of human-associated E. coli is the human-specific ycjM positive. 3) Development of real-time methods assays to measure total viable fecal E. coli and host-specific E. coli, respectively. The real-time methods refer to real-time PCR or quantitative PCR (qPCR) assays. Based on the DNA sequences of the aforementioned genetic markers, one qPCR assay each has been developed for quantifying enteric E. coli and human-associated E. coli in feces-polluted water, respectively, and a propidium monoazide (PMA) staining-real-time PCR method has been created for the measurement of viable enteric E. coli in environmental waters. ?4) Field test of the resulting real-time PCR assays in comparison with EPA's and corresponding benchmark methods. The field tests were conducted in the two public beaches at the Lake of the Ozarks, Missouri and in the Hinson Creek watershed in Columbia of Missouri. The study at the Lake of the Ozarks was performed using the human-associated qPCR assay in comparison with the EPA Method 1603. The qPCR method developed by this study demonstrates that human feces is not the major source of fecal pollution in the public beaches, while the EPA method provides not information of the source of pollution. The investigation of the Hinson Creek watershed was conducted using the enteric E. coli-associated qPCR assay in comparison with ß-D-glucuronidase-based PCR methods. Our PCR method can differentiate enteric E. coli from other ß-D-glucuronidase positive bacteria and appears to be superior to ß-D-glucuronidase-based PCR methods. Our data can help identify locations of the most feces-polluted within watersheds, prioritizing locations for remediation.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Zheng G, Deng D, Reed M. Human-specific E. coli was not a major E. coli population at the public beaches of the Lake of the Ozarks, Missouri. April 17-18, 2015 the Missouri Academy of Science Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Deng D, Zhang N, Xu D, Reed M, Liu F, Zheng G. Polymorphism of glucosyltransferase gene in Escherichia coli and its use in microbial source tracking. May 30-June 2, 2015. 115th General Meeting of American Society for Microbiology
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Deng D, Zhang N, Xu D, Reed M, Liu F, Zheng G. Polymorphism of the glucosyltransferase gene (ycjM) in Escherichia coli and its use for tracking human fecal pollution in water. Sci Total Environ. 2015 Dec 15;537:260-7.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Deng D, Zhang N, Mustapha A, Xu D, Wuliji T, Farley M, Yang J, Hua B, Liu F, Zheng G. Differentiating enteric Escherichia coli from environmental bacteria through the putative glucosyltransferase gene (ycjM). Water Res. 2014 Sep 15;61:224-31.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Daiyong Deng, Ning Zhang, Azlin Mustapha, Dong Xu, Tumen Wuliji, Mary Farley, John Yang, Bin Hua, Fengjing Liu,Guolu Zheng (2014) The ycjM gene is specific to enteric E. coli. The 114th ASM General Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Yuan Yuan, Guolu Zheng, Azlin Mustapha. Detection of Viable Escherichia coli in Environmental Water Using a Combined Propidium Monoazide Staining-real-time PCR. IAFP 2014 Annual Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Deng, D. and G. Zheng*. 2013. Identification of Escherichia coli genetic marker for tracking human fecal pollution in water. 1890 Association of Research Directors 17th Biennial Research Symposium, Abstracts and Programs.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lester, J. M. G. Zheng, B. Hua, and J. Yang. 2013. Using Escherichia coli and newly identified ecological indicators to assess the impacts of land uses on stream water quality. 1890 Association of Research Directors 17th Biennial Research Symposium, Abstracts and Programs.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Liu, Y., G. Zheng, A. Mustapha. 2013. Comparison of propidium monoazide real-time PCR and a conventional culture based method (EPA method 1603) for detection of viable Escherichia coli in water. Journal of Food Protection. 76
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Liu, F., D. Huang, J. Yang, G. Zheng, and B. Hua 2013. Land use change and stream water quality in Hinkson creek watershed. Abstracts of National Conference on Advances in Environmental Science and Technology


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: 1. Scientific and industrial communities in water safety 2. Federal and local water safety monitoring agencies 3. Total Maximum Daily Loads (TMDL) plan developers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1. Two graduate students are supported and trained through this project. 2. One postdoctorial scientist is developing his skills for his career through this project How have the results been disseminated to communities of interest? 1. The results have been published in the peer-reviewed journal "Water Research" 2. The results have been presented in professional conferences and meetings, including the American Society for Microbiogy General Meeting and International Association for Food Protection annual Meeting. What do you plan to do during the next reporting period to accomplish the goals? To continueour research to identify novel E. coli genetic markersthat are associated with feces of wild animals (wild goose in particular) as described in our proposal.

Impacts
What was accomplished under these goals? The objectives of 1 to 4 have been achieved for detection of humam source of fecal pollution

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Daiyong Deng, Ning Zhang, Azlin Mustapha, Dong Xu, Tumen Wuliji, Mary Farley, John Yang, Bin Hua, Fengjing Liu, Guolu Zheng (2014) Differentiating enteric Escherichia coli from environmental bacteria through the putative glucosyltransferase gene (ycjM). Water Research 61: 224-231.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Daiyong Deng, Ning Zhang, Azlin Mustapha, Dong Xu, Tumen Wuliji, Mary Farley, John Yang, Bin Hua, Fengjing Liu, Guolu Zheng (2014) The ycjM gene is specific to enteric E. coli. The 114th ASM General Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Yuan Yuan, Guolu Zheng, Azlin Mustapha. Detection of Viable Escherichia coli in Environmental Water Using a Combined Propidium Monoazide Staining-real-time PCR. IAFP 2014 Annual Meeting


Progress 09/01/12 to 08/31/13

Outputs
Target Audience: 1. Scientific and industrial communities in water safety 2. Federal and local water safety monitoring agencies 3. Total Maximum Daily Loads (TMDL) plan developers Changes/Problems: While accurate detection of the sources of fecal pollution has made a great deal ofprogresses in the field,the differentiation of enteric E. coli and “naturalized” one remains a challenge. In the next period, we will also tackle this issue. What opportunities for training and professional development has the project provided? 1. Two graduate students are trained through this project. 2. One postdoctorial scientist is developing his skills for his career through this project How have the results been disseminated to communities of interest? 1. The results have been presented in professional conferences and meetings. 2. Two manuscripts are under development for publications in peer-reviewed and water-quality- related journals What do you plan to do during the next reporting period to accomplish the goals? To extend our research to wild animals, wild goose in particular, as described in our proposal.

Impacts
What was accomplished under these goals? The objectives of 1 to 4 have been achieved for detection of humam source of fecal pollution.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: 1. Deng, D. and G. Zheng*. 2013. Identification of Escherichia coli genetic marker for tracking human fecal pollution in water. 1890 Association of Research Directors 17th Biennial Research Symposium, Abstracts and Programs. P119. 2. Lester, J. M. G. Zheng, B. Hua, and J. Yang. 2013. Using Escherichia coli and newly identified ecological indicators to assess the impacts of land uses on stream water quality. 1890 Association of Research Directors 17th Biennial Research Symposium, Abstracts and Programs. P224. 3. Liu, Y., G. Zheng, A. Mustapha. 2013. Comparison of propidium monoazide real-time PCR and a conventional Culture-based method (EPA method 1603) for detection of viable Escherichia coli in water. Journal of Food Protection. 76 (supplement A): 76. 4. Liu, F., D. Huang, J. Yang, G. Zheng, and B. Hua 2013. Land use change and stream water quality in Hinkson creek watershed. Abstracts of National Conference on Advances in Environmental Science and Technology.


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: Activities: 1. Extraction of DNA from more than 500 strains of E.coli isolated from different host species, including human, cattle, swine, chicken, turkey, horse, deer, goose, duck, and raccoon; 2. Data mining of the GenBank to identify potential E. coli genetic markers that are associated with human or animals; 3. Verification of the host specificity of the potential E. coli genetic markers by polymerase chain reaction (PCR) assays; 4. Development, optimization and comparison of the EMA-quantitative PCR and PMA-quantitative PCR techniques for the detection of viable E.coli cells; and 5. Advertising and recruiting a Postdoc and a Master's degree candidate to participate in this project. 6. Collecting fecal samples of major animals that may cause fecal pollution to the Lakes of Ozark. Events: none Service: none Products: 1. a dozen of potentially host-specific E. coli genetic markers have been identified; 2. postdoc was hired, starting in June. Dissemination: none PARTICIPANTS: Paticipans; Zheng, G.: PI, coordinating all project activities and designing PCR primers. Xu, D.: co-PI, supervising the data mining of genomic data of E. coli. Mustapha, A.: co-PI, development, optimization and comparison of the EMA-quantitative PCR and PMA-quantitative PCR techniques for the detection of viable E.coli cells. Broz, R:co-PI,coordinating activities with Missouri Department of Nature Natural Resources for water sampling and with famers for fecal samples. Deng, D.: Postdoc, PCR verification of the host specificity of potential E. coli genetic markers Li, R.; temporary technician, DNA extraction and the PCR verification Zhang, C.; data mining of genomic data of E. coli TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: 1.E. coli strains isolated from human and pig are the most similar genome-wise 2.Detection limit of PMA-qPCR is lower than that of EMA-qPCR for the detection of viable E. coli cells. Change in actions: No applicable Change in conditions: 1.The activities of this report period contribute to Lincoln University's (LU's)newly established Master's degree program in Environmental Science and enhance this program's ability to address current environmental problems, particularly microbiological issues of water. 2.The activities of this report period enhance LU's outreach and interaction with a 1862 land-grant university (University of Missouri at Columbia)and Missouri Department of Natural Resources.

Publications

  • No publications reported this period