UNIVERSITY OF CALIFORNIA, LOS ANGELES UCLA BERKELEY • DAVIS • IRVINE • LOS ANGELES • MERCED • RIVERSIDE • SAN DIEGO • SAN FRANCISCO

SANTA BARBARA • SANTA CRUZ

OFFICE OF CONTRACT AND GRANT ADMINISTRATION BOX 951406

11000 KINROSS, SUITE 211 LOS ANGELES, CALIFORNIA 90095-1406

PHONE: (310) 794-0259

FAX: (310) 943-1657

www. r es ea r ch . u c la . edu / oc ga

December 18, 2015 Benita Lynn Horn Metropolitan Water District of Southern California 700 North Alameda Street, 10th Floor – Room 320 Los Angeles, CA 90012 waterforum@mxdh2o.com UCLA PI: Professor Jennifer A. Jay Period of Performance: April 1, 2016 – March 31, 2017 Total Amount Requested: $10,000 UCLA Cost Share: $ 2,500 Project Title: IPARChEd: Inactivation of Pathogens and Antibiotic Resistance:

Choices and EDucation Dear Ms. Horn: On behalf of The Regents of the University of California, through its Los Angeles campus (UCLA) and Professor Jennifer A. Jay, I am pleased to submit the attached materials to your Agency in response to the Metropolitan Water District of Southern California 2015-2017 World Water Forum College Grant Program Request for Grant Proposals. We are submitting this application in anticipation of a contract subject to mutually agreeable terms and conditions. Future awards related to this proposal should be issued to The Regents of the University of California and forwarded to this office at awards@research.ucla.edu. Questions regarding the technical components of this proposal can be directed to Professor Jay at jjay@seas.ucla.edu. Questions concerning contractual, financial, and administrative matters should be referred to me at flora.obrien@research.ucla.edu or by telephone at (310) 206-0807. Sincerely, Flora O’Brien Contract and Grant Officer (UCLA Internal Reference No. 20162519)

University of California

Department of Civil and Environmental Engineering

IPARChEd: Inactivation of Pathogens and Antibiotic Resistance: CHoices and EDucation

2015 Jay Lab L to R: Dr. Saeedreza Hafeznezami, Wayne Tran, Victoria Whitener, Cristina Echeverria, Dr. Vanessa Thulsiraj, Dr. Amy Zimmer-Faust, Niru Senthilkumar,

PI Jennifer Jay, Kevin Ho

Faculty:

PI: Jennifer Jay Co-PI: Donald Kendall

Co-PI: Shaily Mahendra Co-PI: Michael Stenstrom

Ph.D. Students:

Cristina Echeverria Victoria Whitener

Project Strand: Local

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PROJECT SUMMARY

This proposed work investigates technology to reduce pathogen persistence and antibiotic resistance in order to advance the safe use of recycled water as a solution to water and food security.

1: A) For a suite of reclaimed water facilities with a range of disinfection and other treatment processes, we will evaluate levels of pathogens, antibiotic resistance genes (ARGs), and other parameters through distribution; B) We will conduct experiments comparing inactivation of FIB and ARGs by various disinfection schemes.

2: We will provide a web-based educational resource center on the relationships between food, environmental sustainability, and human health.

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CONTACT INFORMATION

College Regents of the University of California, Los Angeles Department Civil and Environmental Engineering (CEE) Make Check Payable To:

UC Regents

A.

Application Strand Select One LOCAL Project Name I PARChEd: Inactivation of Pathogens and Antibiotic

Resistance: CHoices and EDucation

GLOBAL Project Name B.

Faculty Project Manager Jennifer A. Jay Title Professor

Department Civil and Environmental Engineering Campus Address UCLA Civil & Envir Engr

BOX 951593, 5732H Boelter Hall Los Angeles, CA 90095-1593

Telephone / Email Address 310-267-5365 jjay@seas.ucla.edu Note: The faculty member must be a full-time or part-time employee of the applicant’s college. This person will serve as the project liaison and signatory on the contract between Metropolitan and the college / university. C.

Student Project Manager Victoria Whitener and Cristina Echeverria Undergraduate or Graduate Graduate Department Civil and Environmental Engineering

Cell Phone / Email Address 858-663-7683 310-818-9421

858-663-7683 310-818-9421

D.

Contracts Manager / Officer Flora O’Brien Title Contract and Grant Officer Department Office of Contract and Grant Administration Campus Address UCLA ORA - OCGA

BOX 951406, 11000 Kinross Bldg. Ste 211 Los Angeles, CA 90095-1406

Telephone / Email Address 310-206-0807 Flora.obrien@research.ucla.edu

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E. PROJECT MANAGEMENT TEAM Identify the team members of the project (i.e., budget, research, technology etc.). Add rows, as needed.

NAME TITLE / ORGANIZATION ADDRESS PHONE & EMAIL 1 Prof. Stenstrom UCLA CEE 5731 Boelter Hall LA,

CA 90095 310-825-1408

2 Prof. Mahendra UCLA CEE 5731 Boelter Hall LA, CA 90095

310-784-9850

3 Prof. Kendall UCLA CEE 5731 Boelter Hall LA, CA 90095

310-825-9819

E. MEMBER AGENCY(IES) / LOCAL WATER AGENCY(IES)

NAME TITLE / ORGANIZATION ADDRESS PHONE & EMAIL 1 Sam Unger California Regional Water

Quality Control Board 320 West 4th St. Suite 200 LA, CA 90013

sunger@waterboards.ca.gov

2 3

Letters of Support are included in the Appendix. Local Agency Letter will be submitted after the due date with approval from MWD External Affairs.

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ORGANIZATIONAL BACKGROUND We are an academic laboratory in the Civil and Environmental Engineering Department at UCLA. Our primary goal is the education and training of students at the B.S., M.S., and Ph.D. level. Through our own community-based research as well as our participation in various programs at UCLA, we also provide educational opportunities for community college, elementary, middle, and high school students. We conduct research in environmental engineering that has direct applications to public health. The research we pursue centers around three major areas:

1) Rapid detection and identification of sources of fecal contamination in natural

waters. Coastal waters in Southern California are impaired with respect to bacteria, and swimming near sources has been shown to result in increased risk of illness. Mitigation of beaches with water quality problems relies on accurate and rapid source identification protocols, as well as an understanding of the persistence of pathogens and indicator organisms in the environment.

2) Fate and transport of antibiotic resistant bacteria (ARB) and antibiotic

resistance genes (ARG). We are studying the prevalence and persistence of ARB and ARB in soils, water, and air. Specifically, we are examining the influence of farming practices and the presence of metals that may co-select of resistance.

3) The environmental fate of Hg and As. Toxic elements pose a serious threat to

humans and wildlife worldwide. We are specifically addressing the cycling of mercury and arsenic in surface and groundwaters. We use an approach that integrates experimental work in defined systems in the laboratory with field observations and geochemical modeling to eludicate mechanisms for the transformation and partitioning of these toxic elements in the environment.

We have a level of interaction with the community that is very rare for an academic laboratory. We have developed a novel community-based research program where UCLA students work collaboratively with elementary, middle, and high school students. UCLA student enrolled in a designated Service Learning (taught by Jay) interact with K-12 students to generate hypotheses having to do with environmental impairment at local sites. With the assistance of Jay lab researchers, the students test their hypotheses, and make posters of the results. The project culminates each year in a poster session at UCLA where the K-12 students present their results. Our research team is very diverse. Over the past ten years, approximately three-fourths of the doctoral students and more than half of the MS students have been female. Our diversity originates in part through our active participation in UCLA programs that provide research experiences for UCLA students with diverse backgrounds. We conduct work across international boundaries to contribute to the solution of significant environmental problems. Our recent project in Bangladesh is an investigation of

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the mobilization of arsenic to groundwater at a village with highly contaminated groundwater (400 ppm). Five UCLA students and PI Jay have traveled to Bangladesh on four separate trips, and we actively collaborate with our colleagues at the Bangladesh University of Engineering and Technology. We also have a field site south of Tijuana, Mexico, where we are investigated coastal water pollution. We have completed several successful field campaigns at this location.

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CERTIFICATE OF ATTENDANCE

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PROJECT DESCRIPTION

Introduction

Water sustainability, food security, and increasing global antibiotic resistance are issues of paramount importance for humanity, linked in complex ways and cast on a landscape of changing climate and rising population. Over the last century, major advances in water quality, food production, and treatment of diseases with antibiotics have improved life immeasurably. Again in parallel, each now faces issues of sustainability that if left unaddressed, could seriously threaten the gains we have enjoyed (WHO 2014).

Changes in our approach to how we consume water offer tremendous synergistic benefits. Far from being waste products, alternative waters are virtually untapped sources of freshwater, nutrients, and even energy. The numerous and far-reaching benefits of reuse include incentivizing higher levels of treatment, reducing water pollution, increasing water supply, minimizing stress on water supplying healthy ecosystems, mitigating seawater intrusion, and lowering costs compared to other water supplies that may need to travel long distances. In the face of altered water cycles and rising population, significantly increased use of recycled water will be critical to achieving water and food sustainability across the globe. Indeed in California, home to some of the most productive agricultural regions in the world, recycled water is already a crucial buffer against severe drought.

New knowledge is needed both to inform safe application of this resource and to address the stigma frequently associated with its use. Issues of particular concern for human health are the differential inactivation of ARGs and pathogens in various treatment schemes, and the ability of free DNA to transform into competent cells in downstream environmental compartments.

ARGs are unique emerging contaminants in that they are frequently observed to increase rather than decrease through the stages of water treatment, and their levels may increase through distribution systems and at field sites due to horizontal gene transfer (HGT) and co-selective pressures. Due to the seriousness of the risks of ARG proliferation, the relative paucity of data on ARGs in reclaimed water for irrigation, and the necessity for increased water reuse, a detailed study in this area is warranted.

We propose here a locally-focused, technology-based project to inform safe use of reclaimed water while addressing the worldwide concern of increasing antibiotic resistance. Specific Aims

This proposed work addresses at bench, pilot and field scales concerns of pathogen persistence and antibiotic resistance to advance the safe use of recycled water as a solution to water and food security.

Objective 1: We will investigate dynamics of pathogens and ARGs through both low and high technology treatment options and distribution systems with differing disinfection schemes. A) For a suite of reclaimed water facilities with a range of disinfection and other treatment processes, we will evaluate levels of pathogens (bacterial and viral), ARGs, nutrients, FIB, and selected metals in several reclaimed waters at the point of entry to the distribution system and the point of use. B) We will conduct controlled experiments comparing inactivation of FIB, ARB, and ARGs by disinfection by ultraviolet (UV) radiation, free available chlorine (FAC), monochloramine, and solar inactivation, which is relevant internationally.

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Objective 2: We will provide a web-based resource center including course module materials (including both hands on and quantitative activities) for elementary, middle and high school, and college educators. Background

Antibiotic resistance and impacts on human health. Rising antibiotic resistance is a serious threat to public health worldwide. Development of new antibiotics has slowed dramatically in recent decades; thus it is critical to extend the useful life of the drugs on which we currently rely. Certain once-lifesaving drugs are now thought to be “worthless” (Woolhouse and Farrar, 2012). Global total antibiotic has increased dramatically, and deeper understanding of the complex determinants of this proliferation is urgently needed due to scale and gravity of the problem.

There are several routes by which an increased environmental reservoir of ARB and ARGs can impact human health (Huijbers et al., 2015). One way is through human exposure to AR zoonotic pathogens (not adapted for human-to-human transmission) (Chang, 2012; Travers & Barza, 2002), such as the millions of cases of food-borne gastrointestinal illnesses that result from pathogens including Salmonella and Campylobacter each year (Scallan et al., 2011). In contrast to these self-limiting cases, ARGs can also be transferred to commensal and pathogenic bacteria that are capable of human-to-human transmission. Once such bacteria cross the species barrier they can result in sustained illness in humans (Lipsitch et al. , 2002). However, even non-pathogenic, commensal bacteria originating in animals can be a source of ARG in the human microbiome that may be passed among species.

Importance of ARG in environmental compartments. Recent work has identified the important of environmental compartments in the movement of ARG. Aquatic systems (Baquero et al., 2008; Marti et al., 2014; Martins et al., 2014; Pruden et al., 2012; Storteboom et al., 2010), biofilms (Engemann et al., 2008; Schwartz et al., 2003), and aquatic sediments (Chen et al. 2013; Cummings et al., 2011; Yang et al., 2013) can all serve as important reservoirs for ARGs, especially near agriculture (McEachran et al., 2015; Pruden et al., 2012). ARBs and ARG can be transported via air (Chapin et al., 2005; Gibbs et al., 2006; Rule et al., 2008), and our recent work in this area is described below. Transport via air or water can mobilize ARGs to other areas where selective and co-selective processes have the potential to increase ARG levels.

Exposure pathways for ARB to humans are extremely complex. Currently acknowledged pathways include food, person-to-person contact, and direct contact with farm animals. Unexplored pathways include less direct occupational exposure such as farm work on fields with manure and/or reclaimed water applications as well as environmental exposures from air, soil, and water. ARGs are present in these matrices and more research is needed on exposure via environmental routes. Recently Completed Significant Activities

Antibiotic resistant bacteria and ARGs in air, water, and soil. Recently we completed a study analyzing the prevalence of ARB and ARGs in environmental compartments in southern California as well as the Central Valley of CA. 1200 bacterial isolates were purified from air samples collected downwind of organic and conventional cattle raising facilities and screened for resistance to six antibiotics. Levels of ARB and ARG were elevated in the vicinity of cattle farms where antibiotics are used relative to organic farms (Figure 1, Sanchez et al, to be submitted December 2015). Overall resistance was found to be consistently higher in feedlot-

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derived (labeled as “conventional”) samples than in samples derived from organic farms for some antibiotics. A similar study from our lab showed elevated airborne ARG levels downwind relative to upwind concentrated poultry facilities (Sanchez et al., submitted).

Figure 1. Bars depict the fraction of isolates showing no zone of inhibition for the disk diffusion method for each antibiotic. Isolates from three conventional (hollow bars) and three organic farms (filled bars) were tested.

ARGs in water, soil and air at public parks. We measured ARGs in air, water, and soil at 24 public parks (six from each of four cities: Los Angeles, San Diego, Bakersfield, and Fresno). For soils, three composite soil samples were collected within 10 m of each other. See Figure 2. Los Angeles parks had higher levels of sul1 in drinking water than parks in San Diego, Bakersfield and Fresno, while for blaSHV, San Diego parks had higher levels (Echeverria et al., to be submitted December 2015).

Technical Approach Objective 1: We will investigate dynamics of pathogens and ARGs through both low and high technology treatment options and distribution systems with differing disinfection schemes. A) For a suite of reclaimed water facilities with a range of disinfection and other treatment processes, we will evaluate levels of pathogens (bacterial and viral), ARGs, nutrients, FIB, and selected

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Figure 2: Levels of ARGs in water and soil samples from 24 public parks across CA. Error bars depict standard deviation of three composite soil samples.

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metals in several reclaimed waters at the point of entry to the distribution system and the point of use. B) We will conduct controlled experiments comparing inactivation of FIB, pathogens, ARB, and ARGs by disinfection by ultraviolet (UV) radiation, free available chlorine (FAC), and monochloramine.

Hypotheses for Objective 1: H1.1) Wastewater treated with various treatment strategies will contain a range of concentrations of pathogens, ARGs, nutrients, FIB, and metals. Specifically, certain disinfection processes may result in reclaimed water with fewer ARGs. H1.2) ARGs may increase in level through distribution systems. H1.3) Controlled experiments with local secondary effluent will show different ARG persistence and transformation activity depending on disinfection technique. Rationale:

Wastewater treatment plants as sources of ARGs. Wastewater treatment plants (WWTPs) can be an important reservoir for ARGs and can result in either large decreases (Chen et al., 2012) or increases in ARG levels throughout the treatment process, depending in part on design (Czekalski, et al., 2012; S. Kim et al., 2014; Kim et al., 2010; Rizzo et al., 2013). Some work has shown differential survival of ARGs through the treatment process, for example, stable levels of sul genes concurrent with decreasing levels of tet genes, normalized to 16S rDNA (Gao et al., 2012). Effluents from WWTPs contain significant levels of ARGs (Kim and Aga, 2007; Kim et al., 2010) and ARB (Odjadjare et al., 2012), and can affect downstream water bodies with respect to ARG levels (Lapara et al., 2011; Marti, et al., 2013). Hong et al. (2013) summarizes levels of a suite of ARGs in influent and effluent of wastewater treatment plants across the U.S. and internationally (Hong, et al., 2013). Recent work has shown that reclaimed water can also be a significant source of ARGs (Barker-Reid et al., 2010; Fahrenfeld et al., 2013) and disseminate them through irrigation.

Notably, different methods of disinfection, which act through various mechanisms, span a range of effectiveness in their ability to inactivate ARGs (Dodd, 2012). It has long been known that the proportion of multiply antibiotic resistant (MAR) bacteria can increase through disinfection, even while the total bacterial counts decrease dramatically (Armstrong et al., 1982; Armstrong et al., 1981). More recent work using molecular techniques has confirmed that gene copies of ARGs normalized to 16S rDNA (indicating total bacterial concentration) increased throughout various types of treatment and increased again following disinfection (Xi et al., 2009). In fact, UV disinfection can even be selective for tetracycline resistance (Kim et al., 2010; Meckes, 1982).

Disinfection processes are traditionally compared with respect to decreases in live cells, and thus, almost all related literature on disinfection and antibiotic resistance has focused on decreases in levels of viable ARB. However, only very limited work has compared disinfection processes with respect to the ability of DNA fragments to confer antibiotic resistance traits to bacteria post-treatment. This process can occur through transformation, where free DNA is taken up by competent cells, and by transduction, if bacteriophages are still active after inactivation of their host ARB (Dodd, 2012).

In general, while FAC is very effective at oxidizing many organic molecules, it is much less reactive with double stranded DNA than would be expected due to the protection of reactive sites by hydrogen bonding (it is much more effective on denatured DNA) (Prütz, 1996, 1998). Monochloramine (NH2Cl) is a weaker oxidant than FAC, and is only slowly active against DNA and RNA. However, DNA’s ability to transform competent recipient cells has been shown to be

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decreased after NH2Cl treatment (Shih & Lederberg, 1976). UV has been shown to be effective at degrading pyrimidine and purine nucleobases, and has been generally considered well-suited to reducing levels of ARGs and other genes (McKinney & Pruden, 2012). However, field-scale tests of ARG inactivation by UV have shown little effect (Auerbach et al., 2007; Kim et al., 2010). In addition, the effectiveness of UV inactivation at bench-scale on four ARGs both as free DNA and in host ARB was recently compared (McKinney & Pruden, 2012). While doses of 10-20 mJ/cm2 UV resulted in a 4 to 5 log inactivation of ARB, a much higher dose, 200-400 mJ/cm2 was required for a 3 to 4 log reduction in ARGs.

Previous studies have shown potential for regrowth of some bacteria and increased in ARGs through the distribution system (Fahrenfeld et al., 2013; Ryu et al., 2005; Xi et al., 2009). For example, Fahrenfeld et al. (2013) observed some ARGs at the point of use that were below the detection limit at the point of entry to the distribution system. The role of distribution systems in ARG fate in water reclamation must be understood and characterized.

Anaerobic processes could be beneficial for water reclaimed for agricultural purposes, as higher fractions of nitrogen and phosphorus are retained (Hong et al., 2013). However, evidence on the fate of ARGs in anaerobic processes is conflicting; while some work shows a loss of ARGs during anaerobic processing (Rysz et al., 2013), other work shows an increase (Merlin et al., 2011). Research Design: In part A of this Objective, we will measure levels of ARGs, pathogens, and metals that may be co-selective in water systems with varying types of treatment, both before and after distribution, throughout Los Angeles County, with a particular focus on the Calleguas Creek Watershed. In part B, we will conduct controlled bench scale tests of the effectiveness of UV, FAC, monochloramine, and solar inactivation on levels and transformation ability of ARGs in waters from our local and remote field sites.

Most of our field work will center on the Calleguas Creek Watershed in Southern California, which is an ideal setting for a regional study of reclamation of water for agriculture. Farmers use combinations of three general categories of water for irrigation: groundwater, reclaimed water, and imported water. Patterns are seen for particular crops; for example, strawberries and berries typically use imported water, while avocado farmers frequently pump groundwater. There are over 20 water systems in the watershed. The imported water systems consist of the State Project Water and the Colorado River Water Projects. The Santa Clara River is diverted by the United Water Conservation District to spreading basins and then pumped primarily to agricultural users. The Cities of Santa Paula and Fillmore discharge water from their WWTP's upstream of the spreading basins. In addition, there are water deliveries for agriculture utilizing wastewater from Thousand Oaks (Hill Canyon WWTP). Some of the groundwater from the Upper Aquifer System is influenced from wastewater runoff, while groundwater from the Lower Aquifer System does not source recycled water. The groundwater from the Oxnard Plain has multiple influences. Various source waters available to farmers will be analyzed for ARGs, pathogens, as well as metals that may be co-selective. Analyses: Fecal Indicator Bacteria (FIB) Analysis. To obtain FIB concentrations, Total Coliform (TC), E. coli (EC), and enterococci (ENT) will be measured with Colilert-18TM and EnterolertTM (IDEXX, Westbrook ME) reagents and protocols to determine the most probable number (MPN) of cells per 100 ml sample.

Choice of ARGs. We will quantify genes conferring resistance to several classes of antibiotics. Specifically, we will quantify genes conferring resistance to β-lactams (blaTEM,

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blaCTX-M, blaSHV), tetracyclines (tet(O), tet(W), fluoroquinolones (qnrA, qnrB, qnrS), macrolides (erm(F), erm(B), and sulfonamides (sul1, sul(II)).

Quantitative PCR (qPCR). with reported detection levels of 1.4 × 10-15 g of DNA per reaction, will be used for monitoring pathogens Campylobacter, Salmonella, adenovirus, norovirus, ARG and 16S levels, which will allow for sensitive detection of the targeted genes and the quantification of their relative abundance, while eliminating the bias associated with cell culturing techniques. DNA will be extracted with a DNA-EZ ST1 Extraction kit (Generite, North Brunswick, NJ) according to manufacturer’s protocol, and the concentrations of total purified DNA as well as its quality will be determined spectrophotometrically (NanoDrop 2000) prior to analysis. Amplification will be performed in a StepOnePlus (Applied Biosystems) in 25 μL reaction mixtures on 96-well plates containing 1X SYBR® Green Master Mix.

Disinfection experiments. Disinfection experiments will be conducted at the bench-scale using a collimated beam apparatus (McKinney & Pruden, 2012) and a UVX Radiometer. Professor Nelson and Edwards will assist (see letters of support.) A suite of ten ARGs will be tested using the methods described above after disinfection by a range of doses of UV, monochloramination, and FAC addition. We will test the applicability of recent work showing that greater doses of UV are needed to inactivate ARG than ARB in our systems. We will test the impact of combining various disinfection procedures, which has been proposed by several recent authors as especially relevant for ARG removal (Dodd, 2012; Hong et al., 2013; A. Pruden, 2014). We will also test low technology methods of disinfection that are common in developing countries. For example, SODIS (solar disinfection) is used across the globe to provide safer drinking water; however, the fate of ARGs through this process is unknown. Also, waste stabilization ponds are widespread globally for passive solar treatment of wastewater. We have experience simulating DNA-based marker decay in natural systems and will use this setup to simulate the fate of ARGs in microcosms with various sources of soil and water, including side by side experiments using our local sites. Objective 2: We will provide a resource center including course module materials for college and high school educators as well as workshops for teachers on the relationships between food, environmental sustainability, and human health.

Seminar for development of curricular materials for K-12. PI Jay directs a freshman cluster course entitled, “Food: A Lens for Environment and Sustainability.” A seminar in K-12 curricular material development will be offered to the cluster students that will involve experiential learning, and interaction with K-12 students.

Web-based resource center for undergraduate courses. As part of this grant, a web-based resource center will be created containing slides, notes, quantitative experiential learning activities, hand-outs, and readings for a five-week, fifteen hour portion of an undergraduate college course covering water sustainability and food security (based on Jay’s cluster course).

Assessment of the curricular materials. The learning objectives for the K-12 students will be assessed through pre- and post-intervention surveys.

Feasibility Co-PI Michael Stenstrom has standing collaborations and extensive experience working with WWTPs treating water to various degrees. Also, Co-PI Donald Kendall is the former director of the Calleguas Water Management District and has extensive experience in watershed-scale water resource management. He has connections throughout the agricultural and water resources communities. Co-PIs Jay and Mahendra have extensive experience in qPCR detection of pathogens, DNA-based fecal markers and ARGs (Boehm et al., 2013; Ebentier et al.,

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2013), and development of qPCR assays (Lin et al., 2013). In addition, we have extensive experience in working with K-12 students and teachers. Jay has published together on the use of service learning to teach concepts to K-12 students (Jay & Mika, 2012; Mika et al., 2012). Impacts of the Proposed Work This work will make substantial contributions to our understanding of and ability to reuse water and address pathogens and antibiotic resistance in the environment. ARGs are emerging contaminants, and because they are not yet regulated, quantitative benefits of work are difficult to assess. The fate and transport of these contaminants in general are still little understood. This work would establish extent of ARG contamination in reclaimed water, informing a basis to begin monitoring quantities and trends, as well as possible regulation. In Calleguas Creek watershed, the largest potential impact of reclaimed water would be vegetable row crops. The 2013 Livestock and Crop Report of Ventura County valued the vegetable crop at $500 million. Concern over their proliferation is increasing, and this work will help us achieve water quality goals in the future. This work will provide educational opportunities for Victoria Whitener and Cristina Echeverria, both current UCLA Ph.D. students, as well as undergraduate and high-school students whose efforts will be funded through several programs at UCLA in which the PIs continuously participate. The goals of these programs include: i) increasing diversity in science, ii) encouraging high school students to attend UC, and iii) assisting community college students transferring to UCLA to become involved in research. Timeline

Task

Jun-Aug

Sept-Nov

Dec-Feb

Mar-May

Objective 1: A) Characterization of reclaimed water B) Disinfection experiments

X

X X

X

X Objective 2: A) Food cluster seminar course B) Web-based resource center

X

X

X X

Statistical Analysis of data set X X X X Communication of results X X X X

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CITED REFERENCES Armstrong, J. L., Calomiris, J. J., & Seidler, R. J. (1982). Selection of antibiotic-resistant standard plate count

bacteria during water treatment. Applied and Environmental Microbiology, 44(2), 308–316. Armstrong, J. L., Shigeno, D. S., Calomiris, J. O. N. J., & Seidler, R. J. (1981). Antibiotic-resistant bacteria in

drinking water. Applied and Environmental Microbiology, 42(2), 277–283. Auerbach, E. a., Seyfried, E. E., & McMahon, K. D. (2007). Tetracycline resistance genes in activated sludge

wastewater treatment plants. Water Research, 41(5), 1143–1151. http://doi.org/10.1016/j.watres.2006.11.045 Baker-Austin, C., Wright, M. S., Stepanauskas, R., & McArthur, J. V. (2006). Co-selection of antibiotic and metal

resistance. Trends in Microbiology, 14(4), 176–182. http://doi.org/10.1016/j.tim.2006.02.006 Baquero, F., Martínez, J.-L., & Cantón, R. (2008). Antibiotics and antibiotic resistance in water environments.

Current Opinion in Biotechnology, 19(3), 260–5. http://doi.org/10.1016/j.copbio.2008.05.006 Barker-Reid, F., Fox, E. M., & Faggian, R. (2010). Occurrence of antibiotic resistance genes in reclaimed water and

river water in the Werribee Basin, Australia. Journal of Water and Health, 8(3), 521–531. http://doi.org/10.2166/wh.2010.102

Beneberu, G., Mengistou, S., Eggermont, H., & Verschuren, D. (2014). Chironomid distribution along a pollution gradient in Ethiopian rivers, and their potential for biological water quality monitoring. African Journal of Aquatic Science, 39(January 2015), 45–56. http://doi.org/10.2989/16085914.2013.870525

Blute, N. K., Jay, J. a., Swartz, C. H., Brabander, D. J., & Hemond, H. F. (2009). Aqueous and solid phase arsenic speciation in the sediments of a contaminated wetland and riverbed. Applied Geochemistry, 24(2), 346–358. http://doi.org/10.1016/j.apgeochem.2008.10.012

Boehm, A. B., Werfhorst, L. C. Van De, Griffith, J. F., Holden, P. A., Jay, J. A., Shanks, O. C., … Weisberg, S. B. (2013). Performance of forty-one microbial source tracking methods : A twenty-seven lab evaluation study, 47(18):6812-6828.

Burke, M.P., T.S. Hogue, M. Ferreira, C.B. Mendez, B. Navarro, S. Lopez, J.A. Jay (2010) The effects of wildfire on soil mercury concentrations in Southern California watersheds. Water Air Soil Poll. 212:369-385.

Caille, O., Rossier, C., & Perron, K. (2007). A copper-activated two-component system interacts with zinc and imipenem resistance in Pseudomonas aeruginosa. Journal of Bacteriology, 189(13), 4561–8. http://doi.org/10.1128/JB.00095-07

Chakravorty, S., Helb, D., Burday, M., Connell, N., & Alland, D. (2007). A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. Journal of Microbiological Methods, 69(2), 330–9. http://doi.org/10.1016/j.mimet.2007.02.005

Chang, Q., Wang, W., Regev-Yochay, G., Lipsitch, M., & Hanage, W. P. (2014). Antibiotics in agriculture and the risk to human health: how worried should we be? Evolutionary Applications, (Levy 1997), n/a–n/a. http://doi.org/10.1111/eva.12185

Chapin, A., Rule, A., Gibson, K., Buckley, T., & Schwab, K. (2005). Airborne multidrug-resistant bacteria isolated from a concentrated swine feeding operation. Environmental Health Perspectives, 113(2), 137–142. http://doi.org/10.1289/ehp.7473

Chee-Sanford, J. C., Mackie, R. I., Koike, S., Krapac, I. G., Lin, Y.-F., Yannarell, A. C., … Aminov, R. I. (2009). Fate and transport of antibiotic residues and antibiotic resistance genes following land application of manure waste. Journal of Environmental Quality, 38(3), 1086–108. http://doi.org/10.2134/jeq2008.0128

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Chen, H., & Zhang, M. (2013). Occurrence and removal of antibiotic resistance genes in municipal wastewater and rural domestic sewage treatment systems in eastern China. Environment International, 55, 9–14. http://doi.org/10.1016/j.envint.2013.01.019

Chiang, S.-Y. D., Mora, R., Diguiseppi, W. H., Davis, G., Sublette, K., Gedalanga, P., & Mahendra, S. (2012). Characterizing the intrinsic bioremediation potential of 1,4-dioxane and trichloroethene using innovative environmental diagnostic tools. Journal of Environmental Monitoring : JEM, 14(9), 2317–26. http://doi.org/10.1039/c2em30358b

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Cummings, D. E., Archer, K. F., Arriola, D. J., Baker, P. A., Faucett, K. G., Laroya, J. B., Pfiel, K.L., Ryan, C.R., Ryan, K.R.U., Zuill, D. E. (2011). Broad dissemination of plasmid-mediated quinolone resistance genes in sediments of two urban coastal wetlands, 45(2), 447–454.

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Ebentier, D. L., K.T. Hanley, Y. Cao, B.D. Badgley, A.B. Boehm, J.S. Ervin, K.D. Goodwin, M. Gourmelon, J.F. Grffith, P.A. Holden, C.A. Kelty, S. Lozach, C. McGee, L.A. Peed, M. Raith, H. Ryu, M.J. Sadowsky, E.A. Scott, J. Santo Domingo, C.D. Sinigalliano, O.C. Shanks, L.C. Van De Werfhorst, D. Wang, S. Wuertz, Jay, J. A. (2013). Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods, 47(18):6839-6848.

Echeverria, C., Senthilkumar, N., Thulsiraj, V., Haro, H., Sun, F.F., Li, R., Zimmer-Faust, A., Ficara, E., Ericksen, E., Walpert, D., Melendez, I., Sanchez, M., Yuan, T., Tran, N., Jay, J.A. (2015) Regional differences in antibiotic resistance genes in soil and water from 27 public parks in California. To be Submitted November 2015.

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Flores, A.A., V. Thulsiraj, C. Echeverria, H. Sanchez, S. Mahendra, J.A. Jay (2015) Arsenic and Copper Pre-exposure Exerts Co-selective Pressure for Oxytetracycline Resistance in Escherichia coli O55. Submitted.

Gao, P., Munir, M., & Xagoraraki, I. (2012). Correlation of tetracycline and sulfonamide antibiotics with corresponding resistance genes and resistant bacteria in a conventional municipal wastewater treatment plant. The Science of the Total Environment, 421-422, 173–83. http://doi.org/10.1016/j.scitotenv.2012.01.061

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Jay, J. A., & Mika, K. B. (2012). Collaborative research with K-12 students on impacts of climate change on ecosystems in an environmental engineering service-learning course. World Transactions on Engineering and Technology Education, 10(2), 4–8.

Jay, J. A., Murray, K. J., Gilmour, C. C., Mason, R. P., Morel, M. M., Roberts, A. L., & Hemond, H. F. (2002). Mercury Methylation by Desulfovibrio desulfuricans ND132 in the Presence of Polysulfides, 68(11), 5741–5745. http://doi.org/10.1128/AEM.68.11.5741

Jay, J., Blute, N., Lin, K., Senn, D., Hemond, H., & Durant, J. (2005). Controls on Arsenic Speciation and Solid-Phase Partitioning in the Sediments of a Two-Basin Lake. Environmental Science & Technology Technology, 39(23), 9174–9181.

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Ji, X., Shen, Q., Liu, F., Ma, J., Xu, G., Wang, Y., & Wu, M. (2012). Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures and agricultural soils adjacent to feedlots in Shanghai; China. Journal of Hazardous Materials, 235-236, 178–85. http://doi.org/10.1016/j.jhazmat.2012.07.040

Kampalath, R. a., Lin, C.-C., & Jay, J. a. (2013). Influences of Zero-Valent Sulfur on Mercury Methylation in Bacterial Cocultures. Water, Air, & Soil Pollution, 224(2), 1399. http://doi.org/10.1007/s11270-012-1399-7

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Lee, C. M., Lin, T. Y., Lin, C.-C., Kohbodi, G. a, Bhatt, A., Lee, R., & Jay, J. a. (2006). Persistence of fecal indicator bacteria in Santa Monica Bay beach sediments. Water Research, 40(14), 2593–602. http://doi.org/10.1016/j.watres.2006.04.032

Lin, C.-C., & Jay, J. a. (2007). Mercury methylation by planktonic and biofilm cultures of Desulfovibrio desulfuricans. Environmental Science & Technology, 41(19), 6691–7. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23634937

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Lin, T., Kampalath, R., Lin, C., Zhang, M., Chavarria, K., Lacson, J., & Jay, J. A. (2013). Investigation of Mercury Methylation Pathways in Biofilm versus Planktonic Cultures of Desulfovibrio desulfuricans. Environmental Science & Technology, 47(5695-5702). Retrieved from http://pubs.acs.org/doi/abs/10.1021/es400079n

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Mika, K. B., Imamura, C. Chang, V. Conway, G. Fernandez, J. Griffith, R.A. Kampalath, C. Lee, C.-C. Lin, R. Moreno, S. Thompson, R. Whitman, J.A. Jay (2009). Pilot- and bench-scale testing of faecal indicator bacteria survival in marine beach sand near point sources. Journal of Applied Microbiology, 107(1), 72–84. http://doi.org/10.1111/j.1365-2672.2009.04197.x

Mika, K. B., TY Lin, M Ferreria, J Lacson, CM Lee, C-C Lin, K O'Byrne, W Sandoval, V Thulsiraj, J.A. Jay (2012). Incorporating service learning in traditionally lecture-based environmental engineering courses through researching bacterial contamination at a local beach. Global Journal of Engineering Education, 14(2), 155–162.

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Reyes, V. C.; Spitzmiller, M. R.; Hong-Hermesdorf, A.; Kropat, J.; Damoiseaux, R.; Merchant, S. S.; Mahendra, S. Copper Status of Exposed Microorganisms Influences Susceptibility to Metallic Nanoparticles. (2015b) Environmental Toxicology & Chemistry (in press).

Reyes, V. C.; Tseng, N.; Gedalanga, P. B.; Van Nostrand, J. D.; Keely, S. P.; De Long, S. K.; Zhou, J.; Mahendra, S. (2015) Differential Sensitivity of Wetland Derived Nitrogen Cycling Microorganisms to Copper Nanoparticles. Submitted.

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Rothenberg, S.E., M.E. Kirby, M.B. DeRose, B,W. Bird, C.-C. Lin, R.F. Ambrose, J.A. Jay (2009) The impact of over 100 years of wildfires on mercury levels and accumulation rates in two lakes in Southern California, USA. Environ. Earth Sci. 10.1007/s12665-009-0238-7.

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Sanchez, H.M., A. Hornstra, Suzanne E. Paulson, J.A. Jay (2015) Airborne Antibiotic Resistant Genes Upwind and Downwind of Concentrated Animal Feeding Operations for Poultry. Submitted.

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Yang, S., & Carlson, K. (2003). Evolution of antibiotic occurrence in a river through pristine, urban and agricultural landscapes. Water Research, 37(19), 4645–56. http://doi.org/10.1016/S0043-1354(03)00399-3

Zhou, L.-J., Ying, G.-G., Zhao, J.-L., Yang, J.-F., Wang, L., Yang, B., & Liu, S. (2011). Trends in the occurrence of human and veterinary antibiotics in the sediments of the Yellow River, Hai River and Liao River in northern China. Environmental Pollution (Barking, Essex : 1987), 159(7), 1877–85. http://doi.org/10.1016/j.envpol.2011.03.034

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FINANCIAL CRITERIA Budget Overview

DESCRIPTION AMOUNT NOTES GRANT FUNDS REQUESTED

$10,000

University State Funding

$2,500

PROJECT TOTAL $12,500

DIRECT COSTS COMPUTATION BUDGET ITEM DESCRIPTION PRICE/RATE UNIT QTY MWD/

WWF College TOTAL COST

SALARIES AND WAGES

Jay, Jeniifer $77.61 Hours 4.17 $ $323.85 $323.85

Kendall, Donald $61.92 Hours 4.18 $ $259.08 $259.08

Mahendra, Shaily $102.73 Hours 4.18 $ $429.26 $429.26

Stenstrom, Michael $130.04 Hours 3.13 $ $407.67 $407.67

Graduate Student 1 $21.27 Hours 29.34 $ $624.03 $624.03

Graduate Student 2 $21.27 Hours 20.88 $ $444.10 $444.10

Subtotal Subtotal $ $2,488.00 $2,488.00

SUPPLIES/MATERIALS - Describe all major types of supplies/materials, unit price, # of units, etc., to be used on this assisted activity. Master Mix $71.50 Per item 54 $3,861.00 $ $3,861.00 Extraction kits $493.00 Per item 8 $3,944.00 $ $3,944.00 qPCR plates $38.52 Per item 27 $1,040.00 $ $1,040.00 DNAse free water $31.00 Per item 1 $31.00 $ $31.00 Pipet tips $124.00 Per item 1 $124.00 $ $124.00 Technology Infrastructure Fee $12.00 Total 1 $ $12.00 $12.00

Subtotal Subtotal $9,000.00 $12.00 $9,012.00

TOTAL DIRECT COSTS: $9,000.00 $2,500.00 $11,500.00

INDIRECT COSTS - 10% Indirect Total Cost $1,000.00 $ $1,000.00

Subtotal $1,000.00 $ $1,000.00 TOTAL INDIRECT COSTS $1,000.00 $ $1,000.00 TOTAL ESTIMATED PROJECT/ACTIVITY COSTS:

$10,000.00 $2,500.00 $12,500.00

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APPENDIX

24

                                                               

1714 21st Street Santa Monica, California 90404 T 617 970 4833 F 310-828-5636 apatel@xrds.org

Jenny Jay, Ph.D., Professor UCLA Civil & Environmental Engineering 5732H Boelter Hall Los Angeles, CA 90095 October 9, 2015 Dear Jenny,

This letter is to express my strong support for your proposal entitled, “IPARChEd: Inactivation of Pathogens and Antibiotic Resistance—CHoices and EDucation.” In this proposal, you have included an educational component that will enhance and extend our existing collaboration on research opportunities for high school students.

Last fall, my student very much enjoyed being a part of your study of antibiotic resistance in air-borne bacteria downwind of agricultural facilities. She was excited to be a part of a UCLA research team and I have watched her interest in science increase dramatically through this participation. This year’s students are also excited about the prospect of working with your students on a project related to mercury in local fish.

The project proposed here is especially relevant for me as my doctoral work involved colonization of surfaces by biofilms in the marine environment. I will be happy to assist with imaging biofilms cultivated in the current proposal and community analysis of field biofilms as the research progresses to that stage.

Crossroads is extraordinary in its support of high school researchers. Each year HS students have the opportunity to spend an entire year on an individual project of his or her choosing. As director of this Independent Study Program at Crossroads, I look forward to continued collaboration with you and your research group on this project. Sincerely,

Anand Patel, Ph.D. Upper School Science Faculty and Chair of Science Department Crossroads School for Arts & Sciences

December 4, 2015 Dear Jenny, I am happy to provide this letter in support of your proposal entitled, "IPARChEd: Inactivation of Pathogens and Antibiotic Resistance--CHoices and EDucation." I understand the education portion of this project includes development of curricular materials on water sustainability, food security, and human health. I would be very interested in participating in a panel to develop course materials and in piloting them in my own classroom. The proposal also includes development of a teacher workshop. I am interested in helping with such a workshop. I have enjoyed working with you and your students in the past on hands on science activities. My students looked forward to interacting with your undergraduates each week and learned a great deal through the poster making process. They also really benefitted from getting to present their posters at UCLA. I am looking forward to collaborating with you again if funded by this grant. Sincerely,

Lindsay Light 5th grade teacher Roosevelt Elementary School    

  October 7, 2015 Dear Jenny, This letter is in support of your proposal, “I-PARChEd Water Reuse: Reducing Pathogen and Antibiotic Resistance--CHoices and EDucation.." Due to the very serious drought we are currently experiencing, I am excited about the prospect of introducing more material on this topic in my classroom. I would also be interested in helping to adapt the materials you are using in your course entitled "Food: A Lens for Environment and Sustainability" for younger audiences. A workshop to assist teachers in using these materials would be a great idea. Best,

Tammy Mackey Fifth grade teacher Roosevelt Elementary School A California Distinguished School      

December 10, 2015 Dear Jenny, I am pleased to write this letter of collaboration regarding the proposal entitled "IPARChEd: Inactivation of Pathogens and Antibiotic Resistance--CHoices and EDucation." I understand the education portion of this project includes development of K-12 curricular materials on water sustainability, food security, and human health. I would be very interested in participating in your grant’s Teacher Advisory Group to assist your students in developing course materials. I would also be happy to help your students pilot a two-hour learning module in my own classroom at Santa Monica High School. Currently, I am a science instructor at Santa Monica High School and at Santa Monica Community College, where I teach courses in Biology, Environmental Science and Marine Biology and advise Team Marine, a group of students passionately involved in ocean and environmental issues. I try to incorporate inquiry-based methods, hands-on labs, and field trips into my courses. I am particularly interested in helping with the high school curriculum component of this grant, which would increase opportunities for students to bolster their higher-order thinking and communication skills. I understand that your course entitled, “Food: A Lens for Environment and Sustainability" aligns well with several topics within the disciplines I teach and are of particular interest to me. I am happy to support this proposal and I look forward to collaborating with you. Sincerely,

Benjamin Kay