ANTIMICROBIAL RESISTANCE MONITORING OF ZOONOTIC AND INDICATOR BACTERIA IN TENNESSEE LOCAL FOODS AND AGRICULTURAL LANDS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1004863
• Project No.: TENX-1507-FS
• Project Start Date: Dec 23, 2014
• Project End Date: Sep 30, 2017

Project Director
Kilonzo, AG.

Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209

Performing Department
Family and Consumer Sciences

Non Technical Summary
Antibiotic use in food animals for treatment, disease prevention and growth promotion permits resistant bacteria and resistance genes to spread from food animals to humans through the food-chain. The exploitation of antibiotics has resulted in the development and spread of antibiotic resistance. Despite the significant efforts that have been made to understand the many different facets of antibiotic resistance in the food chain and interventions needed to meet the challenge, strategies should focus more on agricultural farm lands as potential hotspot source for antimicrobial resistance (AMR) and antimicrobial resistant genes (AR). To mitigate antimicrobial resistance in the food chain, quality data on the profiles and patterns of both pathogenic and commensal bacteria and their resistance, categories of antibiotics used in agriculture is urgently needed. The overall goal of the proposed project is to identify environmental reservoirs of antimicrobial resistance and determine antibiotic susceptibility of zoonotic and indicator bacteria isolated from Tennessee farmlands and Farmers' Market foods. Promotion of knowledge and research, and advocacy and communication to raise awareness of antibiotic resistance in the food chain will be the main focus of this project. Monitoring the prevalence of antibiotic resistant bacteria in Tennessee agricultural lands and foods is an essential step for developing an increased understanding of antimicrobial resistance. The extent of antibiotic use in Tennessee agriculture will be determined through surveys on which background information on knowledge and actual activities carried from animal and produce farms will be assessed. Farm samples: produce, soil, animal, and water from Tennessee water sheds will be collected and analyzed for antimicrobial resistance of zoonotic and indicator bacteria. Farm produce from Farmers' markets will also be analyzed of antimicrobial resistance. Data on profiles and antibiotic resistant patterns of bacteria, and usage of antibiotics collected in the proposed study will be disseminated to the stockholders. An educated Tennessee community will allow informed practical decisions thereby forming a solid basis for broadly accepting policy changes aimed at limiting antibiotic resistant bacteria in the environment and ultimately in the food chain.

Animal Health Component

0%

Research Effort Categories

Basic

20%

Applied

70%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1103	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1103 - Other microbiology;

Keywords

antimicrobial resistance

zoonotic

indicator bacteria

agricultural lands

Goals / Objectives
The overall goal of the proposed project is to identify environmental reservoirs of antimicrobial resistance and determine antibiotic susceptibility of zoonotic and indicator bacteria in Tennessee farmlands and Farmers' Market foods.Knowledge of the prevalence bacterial isolates and their resistance patterns in Tennessee will help the policy makers and veterinarians to formulate the guidelines for antibiotic therapy in agricultural production. Consumers should have access to accurate information on antibiotic resistance in the food system. Objectives:Determine the extent of antibiotic use in Tennessee agricultureDetermine zoonotic and indicator bacteria in growing crops, soil, animal manure and feed, water sources, and locally produced foods retailed in farmers marketsDetermine the resistance of Salmonella, Shigella, Escherichia coli, Enterococcus, and Listeria to cephalosporin, carbapenems, aminoglycosides, fluoroquinolones, macrolides, and tetracyclines.Education and outreach on judicious use of antibiotics

Project Methods
Determine the extent of antibiotic use in Tennessee agriculture. Prior to collection of samples in the farms, a focus group will be formed to provide the opportunity to make necessary plans to achieve the set objectives. On-farm visits of the same participants of focus group in addition to other farmers will be conducted to gather detailed information on actual activities carried in animal and fresh produce production. Determine zoonotic and indicator bacteria in growing crops, soil, animal manure and feed, water sources, and locally produced foods retailed in farmers markets: Sample collection: Locally purchased fresh produce and meats will be used in this study. Environmental samples such as animal feed, animal manure, irrigation water, soil, locally produced meats; chicken (n=100); pork ((n=100); beef (n=100); leafy produce (n=100)); fruits ((n=200) will be collected and transported immediately on ice for analyses.Bacteriological analysis: Generally, approximately 25 g of each farm and farmer's markets products samples will be added into 225 ml of buffered peptone water (BPW) in stomacher bags and homogenized.Detection of Salmonella spp.: Briefly, from the original 250 ml BPW dilution, the homogenized mixture will be incubated at 37°C for 24 hrs. Each homogenized mixture (1ml) will be transferred into 9 ml of tetrathionate broth and Rappaport-Vassiliadis broth and incubated at 42°C for 24 hrs. Then a loop of tetrathionate broth and Rappaport-Vassiliadis broth will be streaked onto Xylose-Lysine-Desoxycholate selective Agar and CHROMagar Salmonella plates respectively for Salmonella. After incubation at 37°C for 24 hrs, colonies red-yellow with black centers on XLD agar and mauve (rose to purple) on CHROMagar will be identified as Salmonella spp.Salmonella in water. Briefly, 1 L of water will be concentrated via filtration (0.45 μm pore size, 45 mm nitrocellulose filter). The filter will then be placed in 100 ml buffered peptone water and enriched at 37 °C overnight. Twenty milliliters of the enrichment culture will be diluted 1:1 in selective media. The enrichment will be plated on XLD and Salmonella Chromogenic agar for Salmonella.Detection of E. coli O157:H7: Briefly, 25g of samples tested will be added to TSB broth with novobiocin, homogenized for 2 min, and incubated for 20 h at 36 °C. Following incubation, 120 μl of the sample will be added to the sample port of the reveal test device, equilibrated to room temperature. Reveal devices will be left at room temperature for 15 min, after which results will be recorded.Detection of Shigella. The method used will be modified from FDA Bacteriological Analytical Manual (BAM) methods. Each homogenized mixture (1ml) will first be enriched in 9ml of Shigella supplement with 0.5 μg/ml of novobiocin, under anaerobic conditions incubated at 42°C for 20 hrs. Sample will then be streaked on MacConkey agar and incubated at 37°C for 20hrs. Colonies slightly pink and translucent on MacConkey agar will be identified as Shigella. Typical colonies will be inoculated onto a triple sugar iron agar slant followed by Enterobactericiae MICRO-ID for analysis of biochemical reactions.Detection of Listeria: For enrichment of Listeria, 10 mL of homogenized sample will be added to 90 mL of Modified Listeria Enrichment Broth. Enrichment bottles will be incubated at 37°C for 48 h, and then the broth will be streaked (10 μL) onto Listeria selective agar and Chromogenic Listeria Agar (CM1084; supplement SR0226 or SR0244) with incubation at 37ºC for 48hDetection and enumeration of indicator bacteria: Enumeration of aerobic microorganisms: Briefly, homogenized samples from above will be serially diluted in BPW. Dilutions will be aseptically surface plated on tryptic soy agar, incubated at 37 °C for 48 h, and colonies enumerated. For aerobic plate counts (APC) and coliform counts where microbiological counts will fall below the limit of detection, a value halfway between zero and the detection limit will be assigned (i.e. 5 colony forming units [CFU]/g or 0.7 log10 CFU/g).Detection of Fecal coliforms and Escherichia coli: Generic E. coli of fecal origin will be detected in collected samples. Briefly, from the original 250 ml BPW dilution, 50 ml will be removed and combined with 50 ml of 2 × E. coli broth. Following mixing, samples will be incubated at 45 °C for 24 h at which time a loopful of inoculum will be streaked onto EMB agar. From each EMB plate, three to five typical E. coli colonies will be transferred to EC Medium with 4-methylumbelliferyl-β-glucuronide, incubated at 45 °C for 24 to 48 h, and examined for gas production and fluorescence. Presumptive positive samples will be further streaked onto MacConkey agar, and confirmed by indole test and API 20E. Water samples will be filtered at appropriate dilutions and volumes onto sterile, gridded, nitrocellu, water samples will be filtered at appropriate dilutions and volumes onto sterile, gridded, nitrocellulose membranes (0.47 μm pore-size, 47 mm diameter). Membranes will be transferred to mFC agar in sterile, 50 mm diameter petri dishes and then incubated for 24 h at 44.5°C in a water bath. Colonies will be selected from mFC plates containing 30-50 sparsely distributed colonies. Only dark blue colonies were considered to be fecal coliforms. Coliforms will be transferred to transfer into EC broth and then incubated at 44.5 ?C for 24-48 h, in water bath. Culture from positive tubes will be streaked onto Eosin Methylene Blue agar. Colonies with a metallic green sheen will be considered to be E.coli.Detection of Enterococcus: A 0.5 ml portion from the original 250 ml BPW dilution the homogenized samples will be streaked to Slanetz and Bartley Agar and incubated for 24 ± 2 h at 37 ± 1 °C. Suspected colonies of Enterococcus spp. those with a maximum diameter of 1 mm, pink or dark red, with a narrow whitish border will be selected. Three of the suspected colonies, per sample, will be transferred to Tryptone Soya Agar and incubated for 24 ± 2 h at 37 ± 1 °C, and characterized by Gram stain and catalase production. The Enterococcus species will be identified through rapID STR performed only on Gram positive and catalase negative cocci.Animal manure processing: The manure samples will be thoroughly mixed in the plastic bags before 1 g of each sample will be removed and transferred to a diluent bottle containing 100 ml phosphate buffer for determining the microbial population size by membrane filtration and isolating the bacteria. The sample will be vortexed for 1 min before making serial dilutions for plating by membrane filtration. Fecal indicator bacteria will be enumerated via standard membrane filtration (47 mm nitrocellulose filters, 0.45 μm pore size). Membranes will be placed on m-Enterococcus agar and incubated for 48 h at 37 °C before counting red colonies. Colonies will be picked at random and streaked on Bile Esculin agar (Becton Dickinson) for confirmation as Enterococcus after 48 h at 37 °C.Antimicrobial resistance profiling: Pathogenic confirmed will be screened for antimicrobial resistance using 10 antibiotics and the Kirby-Bauer disc diffusion assay, according to the recommendations of NCCLS . Antimicrobial discs (μg) used in the panel will include: Fluroquinolones (ciprofloxacin 5, nalidix acid); macrolides (erythromycin 15, azithromycin); aminoglycosides (gentamicin 10, streptomycin 300); cephalosporins (cetiofur, cefoxitin); and tetracycline (oxytetracycline;)..This project will sort educational and outreach interventions to deliver science-based antibiotic resistance information to farmers, veterinarian practitioners, industry that profit from antimicrobial sales, scientists, public and the government. A one day workshop will be offered to educate the stakeholders on judicious use of antibiotics.

Progress 12/23/14 to 09/30/17

Outputs
Target Audience:The target audience were fresh produce and animal prodcerswhich included cattle and poutry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI went for training on metagenomics: Whole Genomic Sequencing Symposium Whole Genome Sequencing for Food Safety Symposium SEPTEMBER 28-30, 2016 Chicago Marriott Southwest at Burr Ridge Burr Ridge, Illinois The program has improved the communication with our stakeholders and were in position to share their challenges in both produce and animal production systems. Three students were also engaged to increase their participation in food safety research activities. PI attended and presented at International Association of Food Protection (IAFP) Annual Meeting, 2017, Tampa, Florida How have the results been disseminated to communities of interest?The results of this project have been disseminated to communitesthrough workshops, booklets, and fact sheets. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Monitoring antimicrobial resistant bacteria and identifying sources of resistance genes in farmland and local foods will contribute to the efforts of mitigating antimicrobial resistance in foods systems. Objective 1: Determine the extent of antibiotic use in Tennessee agriculture A questionnaire based survey was developed and conducted among 25 animal producers in different counties in Tennessee. The farmers were asked key questions including type of livestock and number on the farm, how/when were the antibiotics used, treated sick animals only, disease control plans, did the farm maintain written records for antibiotic treatments including medicated feeds, written plans for treating sick animals with antibiotics, was the veterinarian's advice sought before administering antibiotics, who determined when and which antibiotics to be used, any collaboration with a veterinarian and following administration of an antibiotic, was the course of treatment completed. The animal on the farms ranged from 3 to 200 per farm. The data collected was analyzed and 72% of animals kept were cattle, followed by chicken (16%), pigs (4%), goats (4%), and sheep (4%). The most surprising practice was that 52 % of the farmers indicated not to seek veterinarian's advice before administering antibiotics; only 40% indicated to collaborate with a veterinarian. Two farmers (8%) indicated not to have a veterinarian involved on their farms. Ten farms (56%) indicated that animals had at least developed some infections including mastitis, diarrhea, respiratory infections, and skin/foot and as a result the farmers administered antibiotics to the animals. A large number of animal producers dis not judiciously use antibiotics on their sick animals and indicated poor record keeping. The lack of knowledge on what pathogens cause specific diseases and education guidelines for antimicrobial use has led to overuse of antibiotics on farms. Objective 2: Determine zoonotic and indicator bacteria in growing crops, soil, animal manure and feed, water sources, and locally produced foods retailed in farmers markets Produce farms, animal farms and farmers' markets were contacted through the Tennessee State University extension program. At each visit, 3 samples per food type (produce, chicken, and eggs), environmental samples (water, soil, animal manure) were collected, transported in coolers with ice and analyzed within 24 h. Escherichia coli, Salmonella spp., Shigella spp. and other bacteria including, Enterobacter spp, and Klebsiella in the Enterobacteriaceae were isolated from local foods (produce, eggs, water from farms, animal manure, and soil. Objective 3: Determine the resistance of Salmonella, Shigella, Escherichia coli, Enterococcus, and Listeria to cephalosporin, carbapenems, aminoglycosides, fluoroquinolones, macrolides, and tetracyclines. Data on antibiotic resistant profiles of both pathogenic and commensal bacteria Escherichia coli, Salmonella spp., Shigella spp. and other bacteria including, Enterobacter spp, and Klebsiella were isolated from local foods, water from farms, animal manure, and soil. Pathogenic bacteria such as Shigella and Salmonella; and indicator bacteria including Escherichia coli, Klebsiella , and other showed resistance to ampicillin, cefpodoxime, tetracycline, erythromycin, cefotaxime, gentamicin, and other antibiotics used in human medicine. Escherichia coli which is an indicator bacteria showed the following antimicrobial resistance patterns. Salmonella which is also an indicator bacteria indicated several resistance patterns.. MAR index values indicate the usage of antibiotics in the environment. Cumulatively, the antibiotic resistance patterns showed MAR index values ranging from 0.08 to 0.70. The highest MAR index among the bacteria was for Escherichia coli (MAR Index=0.7). lation, identification, and classification of bacteria in local foods and environmental samples (soil, water, animal manure). We found a varying degree of antimicrobial resistance and ESBL- producing EC in poultry farms and retail meats. The prevalence of ESBL-producing E. coli in retail chicken (73.6%) The occurrence of ESBL-producingE. coli(82%) in chicken feces was significantly higher as compared to retail chicken, soil, and feed. We found ESBL-EC (42.3%) in soil within the vicinity of the poultry farms. The prevalence of ESBL-EC in feed (24%) was significantly lower as compared to other environmental samples. ESBL-EC isolates from poultry farms and retail chicken meat. The dominant resistance pattern was ERY-STR (36.5%; 42/115), followed by ERY-STR-KAN-NAL (9.6%, 11/115), ERY (7.0%, 8/115) and ERY-STR-KAN-TET-VAN-AMC (6.1%, 7/ 115). The least (0.9%; 1/115) resistance patterns were AMK-CIP-ERY-STR-CHL-KAN-NAL-TET-VAN-COL, ERY-CHL-STR-NOV-NAL-TET-VAN-COL-AMC, and among others (Table 2). Highly multi-resistant ESBL-EC (> 3 resistances) is displayed in our study. Among ESBL-producing E. coli isolates from feces, highest resistance against erythromycin (100 %), followed by streptomycin (100 %), tetracycline (95.1%), kanamycin (92.7%), and nalidixic acid (73.2%); moderate resistance rates were observed for chloramphenicol (68.3%), ampicillin (43.9%); and relatively low resistance rates were observed for , novobioxn (34.1%), vancomycin (31.7%), colistin and ciprofloxacin (12.2%), and amikacin (4.9%). Among ESBL-producing E. coli isolates from retail chicken, high resistance rates were observed for erythromycin (100.0%) streptomycin (, 92.3%), tetracycline 84.6% and nalidixic acid, (74.4%); moderate resistance rates were observed for kanamycin (51.3%), and relatively low resistance rates were observed for chloramphenicol, ampicillin, colistin, ciprofloxacin, and amikacin. Objective 4: Education and outreach on judicious use of antibiotics. Data from the project indicated farms were contaminated with antibiotic resistant bacteria. From these results, there is need to educate farmers on prudent use of antibiotics and record keeping. A workshop was conducted to educate the stakeholders on judicious use of antibiotics. During the workshop, there was discussion on management practices, dosing and treatment regiments for antibiotic and how antibiotics select for antibiotic resistance. During this workshop, produce growers and animal farmers were engaged in the learning process by asking them to comment on their own experiences. After the workshop, farmers understood the importance of judicious use of antibiotics. Booklets on judicious use of antibiotic were developed and delivered to farmers. Products, Results and Measurable Outcomes: (1) A questionnaire survey instrument on management practices, record keeping, dosing and treatment regiments on animal farms was developed and disseminated; (2) workshop on judicious use of antimicrobials in agriculture, aimed at delivering science based information on judicious use of antibiotics to agricultural commodity producers; (3) antimicrobial resistant data on both pathogenic and commensal bacteria from farms was collected to provide logical corridors to mitigate antibiotic-resistance in Tennessee food system; (4) 25 farmers, now part of educated community ready to accept policy changes aimed at mitigating antimicrobial resistance; (5) producers implementing 'Best Management Practices', or BMPs on their promises; (6) two journal articles are published; (7) three presentations made at the International Association of Food Protection, and Association of 1890 Research Directors (ARD), (8) three undergraduate students were engaged and trained on microbial analysis of environmental samples from animal and produce farms (9) one graduate thesis was produced. An educated agricultural community will allow informed practical decisions thereby forming a solid basis for broadly accepting policy changes aimed at limiting antibiotic resistant bacteria in the environment and ultimately in the food chain.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: A. Kilonzo-Nthenge, et al,. 2017. Extended-spectrum lactamase producing Escherichia coli in Small-scaled Poultry Farms and Retail Chicken. Submitted to Journal of Food Science and Engineering, September 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: A. Kilonzo-Nthenge, et al. 2017. Extended-spectrum ?-Lactamase Producing Escherichia coli in Feed, Manure, and Soil from the Poultry Farm Environment: P1-221, IAFP 2017
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2017 Citation: Joy Oshiorenua Igbafe, Agnes Kilonzo, Samuel Nahashon. 2017. Evaluating the efficacy of probiotic to reduce the colonization of Salmonella in chicken. ETD Collection for Tennessee State University. http://digitalscholarship.tnstate.edu/dissertations
• Type: Books Status: Other Year Published: 2017 Citation: Booklets: Prudent use of Antimicrobial Agent on Small-scaled Poultry and Cattle Farms(in the preparation)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: A. Kilonzo-Nthenge, S. N. Nahashon, S. Godwin, Siqin liu, and D. Long. 2016. Prevalence and Antimicrobial Resistance of Enterobacteriaceae in Shell Eggs from Small-Scale Poultry Farms and Farmers' Markets. Food Prot. 79: 2031-2037
• Type: Other Status: Published Year Published: 2016 Citation: Booklets on Egg Safety from Farm to Fork. 2016. TSU-16-0253 (A)-17090 were developed and distributed to farmers in Tennessee.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience were Tennessee farmers with 10 to 200 farm animals which included cattle, chicken, pigs, goats, and sheep. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI went for training on metagenomics: Whole Genomic Sequencing Symposium Whole Genome Sequencing for Food Safety Symposium SEPTEMBER 28-30, 2016 Chicago Marriott Southwest at Burr Ridge Burr Ridge, Illinois How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Determine antmicrobial resistance of foodborne pathogens isolated from local foods and environmental samples (soil, water, soil, and animal manure). The following antibiotics will be used in the resistance study: cephalosporin, carbapenems, aminoglycosides, fluoroquinolones, macrolides, and tetracycline. Conduct training workshop on judicious use of antibiotics to animal producers across Tennessee

Impacts
What was accomplished under these goals? Use of antibiotic in farm animals A questionnaire based survey was developed and conducted among 25 animal producers in different counties in Tennesee. The farmers were asked key questions including type of livestock and number on the farm, how/when were the antibiotics used, treated sick animals only, disease control plans, did the farm maintain written records for antibiotic treatments including medicated feeds, written plans for treating sick animals with antibiotics, was the veterinarian's advice sought before administering antibiotics, who determinedwhen and which antibiotics to be used, any collaboration with a veterinarianand following administration of an antibiotic, was the course of treatment completed. The animal on the farms ranged from 3 to 200 per farm. The data collected was analyzed and 72% of animals kept were cattle, followed by chicken (16%), pigs (4%), goats (4%), and sheep (4%). The most surprising practice was that 52 % of the farmers indicated not to seek veterinarian's advice before administering antibiotics; only 40% indicated to collaborate with a veterinarian. Two farmers (8%) indicated not to have a veterinarian involved on their farms. Tenfarms (56%) indicated that animals had at least developed some infections includingmastitis, diarrhea, respiratory infections, and skin/foot and as a result thye farmers administered antibiotics to the animals. Zoonotic and Indicator Bacteria in local foods and agricultural lands During the growing season of 2015 and 2016, produce farms and farmers' markets were visited 2 times per week, subject on the availability of the products. The farms were contacted through the Tennessee State University extension program. At each visit, 3 samples per food type (produce, chicken, eggs), environmental samples (water, soil, animal manure) were collected, transported in coolers with ice and analyzed within 24 h. Each sample was placed in a sterile sampling bag and labeled with identification letter, origin, and date of collection. The microbial quality of food, water, soil, manure was determined by standard quantitative, biochemical, and PCR techniques. Antimicrobial resistance of isolated bacteria was also determined. Amikacin (AMI); Amoxicillin/ clavulanic acid (AMO); Ampicillin (AMP); Azithromycin (AZI); Cefoxitin (FOX); Cefpodoxime (CPD); Chloramphenicol (CHL); Ciprofloxacin (CIP); Kanamycin (KAN); Nalidixic Acid (NAL); Streptomycin (STR); Tetracycline (TET). Erythromycin (ERY), Cefotaxime (CTX), Gentamicin (GEN) were some of the antibiotics used in this study. The antimicrobial susceptibilitywas determined using the Bauer and Kirby disk diffusion technique on Mueller-Hinton Agar Escherichia coli, Salmonella spp., Shigella spp. and other bacteria including, Enterobacter spp, and Klebsiella in the Enterobacteriaceae were isolated from local foods, water from farms, animal manure, and soil. Escherichia coli which is an indicator bacteria showed the following antimicrobial resistance patterns AMP-CPD-CIP- CHL ERY- KAN- NAL-TET, AMP-CPD-ERY-STR-TE, AMP- CTX- CHO-ERY- KAM- STY- VAN, AMP-CTX-CHO-ER-STY-VAN, AMP-CTX-CHO-ERY-KAM-VAN, AMP-ERY-KAM-STY-VAN, CTX-CHO- ERY-KAM-VAN, AMP-CTX- ERY-VAN, ERY-KAM-VAN, ERY-VAN. Salmonella which is also anindicator bacteria had the following resistance patterns AMP-CTX-CHO-ERY-KAM-STY-VAN, AMP- CHO-ERY-KAM-STY-VAN, AMP- ERY- KAM-VAN-AMP- ERY-VA, ERY-STY- VAN. Some of the other antimicrobial resistance patterns determined were AMP-CTX-CHO-ERY-KAM-STY-VAN, AMP- CHO-ERY-KAM-STY-VAN, AMP- ERY- KAM-VAN-AMP- ERY-VA, ERY-STY- VAN, , ERY-VAN, AMP-CTX-CHO-ERY-KAM-STY-VAN, AMP-CTX-CHO-ERY-STY-VAN , AMP-ERY-VAN, AMP-ERY-STY-VAN, AMP-ERY-STY-VAN, AMP-ERY-VAN, AMP- CTX-CHO-ERY-STY-VAN, and AMP-CTX-CHO-ERY- KAM-VAN. The MAR index values of isolated bacteria wasalso evaluated in this study. MAR index values indicate the usage of antibiotics in the environment. Cumulatively, the antibiotic resistance patterns showed MAR index values ranging from 0.08 to 0.70. The highest MARindex among thebacteria was for Escherichia coli (MAR Index=0.7) Students were also engaged to increase their participation in food safety research activities. Through this program two graduate and one undergraduate students were trained on isolation, identification, and classification of bacteria in local foods and environmental samples (soil, water, animal manure). Students were also trained onAntibiotic Sensitivity Testing.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: A. Kilonzo-Nthenge, S. N. Nahashon, S. Godwin, Siqin liu, and D. Long. 2016. Prevalence and Antimicrobial Resistance of Enterobacteriaceae in Shell Eggs from Small-Scale Poultry Farms and Farmers' Markets. Food Prot. 79: 2031-2037

Progress 12/23/14 to 09/30/15

Outputs
Target Audience:The target audience reached were small to medium-sized fresh produce growers, small scale produce growers, farmers' markets, and graduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Data on antibiotic use on food animals will be collected from anima producers and analyzed for antimicrobial resistance. More samples from farms and water in agricultural lands will be analyzed for bacterial contamination and resistance.

Impacts
What was accomplished under these goals? As emerging food contaminant in the food chain, antibiotic resistant bacteria has become a public health concern. The potential threat to human health as a result of inappropriate antibiotic use in food animals is significant, as pathogenic-resistant organisms in these animals are poised to enter the food supply and could be broadly distributed in food products. Antibiotic use in food animals may represent a risk to human health and therefore, it's important to investigate the use of antimicrobials on farms. It is important to determine the level of antimicrobial use in food animal production and identify the environmental reservoirs of antimicrobial resistant bacteria in Tennessee agro-ecosystems. Objective 1: Determine the extent of antibiotic use in Tennessee agriculture: To acquire feedback on the usage of antimicrobial usage on anima farms, a survey was developed and distributed to animal reproducers. This objective will help examine the scope and nature of antibiotics used in food animals. The data obtained will be used for public discussions and give guidance to future directions for agricultural use of antibiotics. Currently, the suvveys on antimicrobial use on food animals are being collected from animal producers. Objective 2: Determine zoonotic and indicator bacteria in growing crops, soil, animal manure, feed, water sources, and locally produced foods retailed in farmers markets: Microbiological analyses of eggs from locals farms and farmers markets, water form agricultural lands, fresh produce was conducted to determine, coliforms, aerobic microbe contamination levels, generic E. coli, E. coli O157:H7, and Salmonella. Antimicrobial susceptibility testing was carried out on the isolated bacteria and the Kirby-Bauer disk diffusion method was used to determine the resistance. Microbial analyses indicated that shell eggs can harbor resistant foodborne and commensal bacteria and failure to properly handle raw eggs, positions a potential health hazard to consumers. Bacterial isolates from water in agricultural lands and fresh produce are being analyzed for antimicrobial resistance

Publications