Progress 11/21/13 to 09/30/18
Outputs
Target Audience:1. Small-scale and limited-resource farmers. 2. Minority farmers. 3. Public Health professionals. 4. Small- and medium-scale food processors. 5. KY stakeholders at large. 6. Farmers Markets Administrators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students received training in developing surveys as well as in their administration.These students received training in development of program brochures and farmer recruitment. They received hands-on training in microbiological protocols in sampling of soil, water, and produce and in-lab processing, which includes standard and advance protocols in growth, isolation, and identification of pathogenic bacteria, along with PCR for identification of genetic markers for antibiotic resistance. Two undergraduate students received training in development of program brochures and farmer recruitment. They received hands-on training in microbiological protocols in sampling of soil, water, and produce and in-lab processing, which includes standard and advanced protocols in growth, isolation and identification of pathogenic bacteria. How have the results been disseminated to communities of interest?Oral presentations were made at local, state, and international meetings. A training session on "Good Agricultural Practices (GAP) for Produce Growers" was offered for participating and non-participating farmers through the Small Farms Program of KSU via the KSU Third Thursday Thing sustainable agriculture workshop series in May 2017. A presentation titled "The quality of irrigation water and its impact on the food safety aspects along with its relevance in the Food Safety Modernization Act" was made at the 20th Anniversary of the Third Thursday Thing Program, held on July 20, 2017. Two publications have been published in peer-reviews journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Participating conventional, certified, and non-certified organic small-scale and limited-resource farmers were contacted and one new farmer was recruited through a mailing list from Kentucky State University's current outreach and Extension programs, including participants from the Small Farm Program, Third Thursday Thing (KSU's sustainable agriculture workshop series), the Socially Disadvantaged Farmer Outreach Project, and the Organic Association of Kentucky. The new farmer was given a survey about details regarding certification status, fertilization practices (such as the type/types of manure, compost, and/or chemicals used; age of the manure or compost and the time of application), the source of irrigation water, surrounding land use, handling practices during harvesting and post-harvesting, and handling practices, such as washing, packaging, and storage. In all, 23 small-scale and limited-resource farmers, comprising 16 certified and non-certified organic farmers and 5 conventional farmers were recruited and consented to offer access to soil, water and produce samples for analysis. A total of 12 farms were visited twice from Oct. 2016 to September 2017. In all, 76 produce samples, 14 water samples, and 66 soil samples were collected for microbial analysis. Microbial Analysis: Microbial analyses were started within 24 hours of receiving the samples. Using stringent aseptic conditions, ten grams of sample was mixed with 100 mL of lauryl sulfate tryptose (LST) broth in a stomacher bag for 5 min, at 230 RPM. The mixture was then placed in 9 mL of LST, in serial dilutions up to 10x-4. One mL of the serial dilution of 10x-3 was plated onto E. coli, Coliform, and Listeria Petrifilm plates (3M Company, Maplewood, Minn.) and 100 microliters of the serial dilution of 10x-3 was plated onto Eosin Methylene Blue (EMB) agar plates in duplicate and placed into the incubator at 37°C for 48 hours. For water samples, an advanced IDEXX system was used to analyze the microbiological quality. Coliform count was determined by the most-probable-number (MPN) system. After inoculation of the water sample, the reagent pre-treated bags were incubated for 24 hours at 37°C, and another reagent was added. Wells with coliform bacteria were visualized under UV light and the readings were recorded. The readings were then compared with the MPN table and final readings were recorded. In all, out of 14 water samples that were tested, 3 water samples had an MPN count of more than 1 cfu/100ml. Antibiotic Resistance/Sensitivity Testing: The antimicrobial susceptibility testing was performed by the standard CLSI (formerly known as NCCLS) using the Kirby Bauer disk diffusion technique with Mueller-Hinton agar (Fisher Scientific, IL). The antibiotics used in this study were amikacin (30 μg), amoxicillin (30 μg), ampicillin (10 μg), cefoxitin (30 μg), ceftiofur (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamycin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim (5 μg). Bacterial cultures were grown in 5 mL of nutrient broth at 37°C for 24 h. Each overnight culture was spread evenly onto Mueller-Hinton agar and incubated at 37°C for 48 h and the zones of inhibition were measured. The isolated strains of Enterobacteriaceae were identified using biochemical identification assays, such as API; the procedure for conducting confirmation of antibiotic resistant genes from the isolates using PCR are being standardized. Statistical Analysis: On the Petrifilm plates, colonies with color and gas were recorded as positive for each respective type. These results, as well as the data from the survey, were recorded in Microsoft Excel and analyzed using IBM SPSS statistical software using One-Results: A total of 16 certified and non-certified organic and 6 conventional farmers participated in the study by providing samples and answering questions about their management practices. Among the organic farms, two were certified by Kentucky Department of Agriculture and fourteen were non-certified, but reported using organic practices. Two of the six conventional farmers reported using composted manure in addition to their chemical fertilizer and commercial pesticide, while the remaining conventional farmers only used commercial fertilizer. Of the sixteen organic farmers, seven reported using aged or composted animal manure regularly as the main source of fertilizer. The remaining organic farmers did not apply fertilizer of any sort to their fields. When testing for E. coli, the soil amendments were categorized in three groups based on the type of the fertilizer used: farms that used non-commercial fertilizers (including compost, animal manures, and compost mixtures), farms that used no additional fertilizer on their fields, and farms that used only commercial fertilizers on their fields. In all, 76 produce samples, 14 water samples, and 66 soil samples were collected for microbial analysis. .No Escherichia coli, Salmonella spp, or Listeria were detected in the produce samples analyzed for fecal contamination. However, 26 of 76 produce samples were contaminated with members of the Enterobacteriaceae family. The top five members detected in the produce samples were Enterobacter cloacae, Stenotrophomonas maltophilia, Pantoea spp 2, Pseudomonas luteola, and Serratia. These members of the Enterobacteriaceae family exhibited drug resistance to one or more antibiotics when tested for their antimicrobial profile using Kirby-Bauer Technique. Enterobacter clocae was resistant to 9 different antibiotics: Chloramphenicol, Kanamycin, Cefoxitin, Trimethoprim, Amoxicillin, Ampicillin, Nalidixic acid, Gentamycin and Amikacin. Stenotrophomonas maltophilia was resistant to 9 different antibiotics: Amoxicillin, Cefoxitin, Ceftriaxone, Ciprofloxacin, Tetracycline, Ampicillin, Kanamycin, Chloramphenicol, and Trimethoprim. Pseudomonas luteola was resistant to 5 antibiotics: Amoxicillin, Cefoxitin, Ceftrixone, Ampicillin, and Trimethoprim. Pantoea spp was resistant to Cefoxitin and Kanamycin while Serratia marcescens was resistant to Cefoxitin and Trimethoprim. Top five antibiotics most isolates were resistant to were Cefoxitin (21), Amoxicillin (14), Ampicillin (13), Trimethoprim (9), and Ceftriaxone (5).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Finger-Gray, M. and Tope, A. (2017). Comparative risk evaluation of commercially available organic and conventional fertilizers for potential bacterial pathogens. Annual Meeting of Kentucky Academy of Science, Murray State University, Murray, KY, Nov. 3-4, 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Patel, S. V. and Tope, A. (2017). Evaluation of microbial quality of agricultural water from small farms in Kentucky. Annual Meeting of Kentucky Academy of Science, Murray State University, Murray, KY, Nov. 3-4, 2017.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Tope, A., Rogers, P. and Hitter, A. (2014). Evaluation of Good Agricultural Practices (GAP) Compliance by Small Farmers in Kentucky: Assessing Microbial Quality of Produce. Journal of Agriculture and Environmental Sciences. 3(4) 29-49.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Marshall, T. and Tope, A. (2018). Evaluation of drug resistant Enterobacteriaceae from produce from small farms. Annual Meeting of the Kentucky Academy of Sciences, Bowling Green, Nov. 2-3, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Tope, A., Patel, S. V. and Finger-Gray, M. (2018). Evaluation of Coliforms, Potential Pathogenic Bacteria and their Antibiotic Resistance Profile in Small Organic and Conventional Farms in Kentucky. 7th Annual World Congress of Food and Nutrition, Singapore, Nov. 13-15, 2018.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:1. Small-scale and limited-resource farmers. 2. Minority farmers. 3. Public Health professionals. 4. Small- and medium-scale food processors. 5. KY stakeholders at large. 6. Farmers market administrators. Changes/Problems:1. Due to the appointment of a new RA and the associated training, some aspects of the project were delayed. 2. Despite our consistent efforts to recruit through all possible avenues, recruiting a greater number of farmer participants has been a challenge. What opportunities for training and professional development has the project provided?Graduate Student: 1. Received training in developing surveys as well as in their administration. 2. Received training in development of a program brochure and farmer recruitment. 3. Received hands-on training in microbiological protocols in sampling of soil, water, and produce, in lab processing including standard and advance protocols in growth, isolation, and identification of pathogenic bacteria along with PCR for identification of genetic markers for antibiotic resistance. Undergraduate Student: 1. Received training in development of a program brochure and farmer recruitment. 2. Received hands-on training in microbiological protocols in sampling of soil, water, and produce, in lab processing including standard and advanced protocols in growth, isolation, and identification of pathogenic bacteria. How have the results been disseminated to communities of interest?1. A training session was offered for the participating and non-participating farmers called "Good Agricultural Practices (GAP) for Produce Growers" through the Small Farms Program of KSU at the Third Thursday Thing Sustainable Agriculture Workshop series in May 2017. 2. A presentation titled "The quality of irrigation water and its impact on the food safety aspects along with its relevance in the Food Safety Modernization Act" was made in the 20th Anniversary of the Third Thursday Thing Program, held on July 20th, 2017 on KSU campus. 3. An abstract for a graduate student oral presentation titled "Comparative risk evaluation of commercially available organic and conventional fertilizers for potential bacterial pathogens was presented at the Annual Meeting of Kentucky Academy of Science held at Murray State University, in Murray, KY, Nov. 3-4, 2017. 4. An abstract for an oral presentation titled "Evaluation of microbial quality of agricultural water from small farms in Kentucky" was presented at the Annual Meeting of Kentucky Academy of Science, held at Murray State University, in Murray, KY, Nov. 3-4, 2017. What do you plan to do during the next reporting period to accomplish the goals?1. Continue recruiting farmers. Using various avenues mentioned earlier, we will continue our efforts to recruit more consenting farmers. 2. We also are currently studying the antibiotic resistant genes from the isolates using PCR.
Impacts
What was accomplished under these goals?
Participating conventional, certified and non-certified organic small-scale and limited-resource farmers were contacted and one new farmer was recruited through a mailing list from Kentucky State University's current outreach and Extension programs, including participants from the Small Farm Program, Third Thursday Thing Sustainable Agriculture Workshop series, the Socially Disadvantaged Farmer Outreach Project, and the Organic Association of Kentucky. The new farmer was given a survey that explored details regarding certification status; fertilization practices, such as the type(s) of manure or compost and/or chemicals, age of the manure or compost, and the time of application; the source of irrigation water; surrounding land use; handling practices during harvesting and post harvesting; and handling practices, such as washing, packaging, and storage. In all, 23 small-scale and limited-resource farmers (16 certified and non-certified organic farmers and 5 conventional farmers) were recruited and consented to allow access to soil, water, and produce samples for analysis. A total of 12 farms were each visited twice from Oct. 2016 to September 2017. In all, 76 produce samples, 14 water samples, and 66 soil samples were collected for microbial analysis. Microbial Analysis: Microbial analyses were started within 24 hours of receiving the samples. Using stringent aseptic conditions, ten grams of sample was mixed with 100 milliliter (mL) of lauryl sulfate tryptose broth (LST) in a stomacher bag for 5 min, at 230 RPM. The mixture was then placed in 9 mL of LST, in serial dilutions up to 10x-4. One mL of the serial dilution of 10x-3 was plated onto E. coli, Coliform, and Listeria Petrifilm plates (3M Company, Maplewood, MN) and 100 microliters of the serial dilution of 10x-3 was plated onto eosin methylene blue (EMB) agar plates in duplicate and placed into the incubator at 37°C for 48 hours. For water, an advanced IDEXX system was used to analyze the microbiological quality. Coliform count was determined by the most-probable-number (MPN) system. After inoculation of the water sample, the reagent pre-treated bags were incubated for 24 hours at 37°C and another reagent was added. Wells with coliform bacteria were then visualized under ultraviolet (UV) light and the readings were recorded. The readings were then compared with the MPN table and final readings were recorded. In all, of The 14 water samples that were tested, 3 water samples had an MPN count of more than 1 colony forming unit (cfu)/100 ml. Antibiotic Resistance/Sensitivity Testing: The antimicrobial susceptibility testing was performed by the standard Clinical and Laboratory Standards Institute (CLSI; formerly known as the National Committee for Clinical Laboratory Standards [NCCLS]) using the Kirby Bauer disk diffusion technique with Mueller-Hinton agar (Fisher Scientific, IL). The antibiotics used in this study were amikacin (30 μg), amoxicillin (30 μg), ampicillin (10 μg), cefoxitin (30 μg), ceftiofur (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamycin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim (5 μg). Bacterial cultures were grown in 5 mL of nutrient broth at 37°C for 24 h. Each overnight culture was spread evenly onto Mueller-Hinton agar and incubated at 37°C for 48 h and the zones of inhibition were measured. The isolated strains of Enterobacteriaceae were identified using biochemical identification assays, such as API and the procedure for conducting confirmation of antibiotic resistant genes from the isolates using PCR are being standardized. Statistical Analysis: On the Petrifilm plates, colonies with color and gas were recorded as positive for each respective type. These results, as well as the data from the survey, were recorded in Microsoft Excel and analyzed using IBM SPSS statistical software. Results: A total of 16 certified and non-certified organic and 6 conventional farmers participated in the study by providing samples and answering questions about their management practices. Among the organic farms, two were certified by Kentucky Department of Agriculture and fourteen were non-certified, but reported using organic practices. Two of the six conventional farmers reported using composted manure in addition to chemical fertilizers and commercial pesticides, while the remaining conventional farmers only used commercial fertilizers. Of the sixteen organic farmers, seven reported using aged or composted animal manure regularly as the main source of fertilizer. The remaining organic farmers did not apply fertilizer of any sort to their fields. When testing for E. coli, the soil amendments were categorized in three groups based on the type of the fertilizer used: farms that used non-commercial fertilizers (including compost, animal manures, and compost mixtures), farms that used no additional fertilizer on their fields, and farms that used only commercial fertilizers on their fields. In all, 76 produce samples, 14 water samples, and 66 soil samples were collected for microbial analysis. No Escherichia coli, Salmonella spp, or Listeria were detected in the produce samples analyzed for fecal contamination. However, 26 of 76 produce samples were contaminated with members of Enterobacteriaceae family. The top five members detected in the produce samples were Enterobacter cloacae, Stenotrophomonas maltophilia, Pantoea spp 2, Pseudomonas luteola, Serratia. These identified members of Enterobacteriaceae family exhibited drug resistance to one or more antibiotics when tested for their antimicrobial profile using the Kirby-Bauer Technique. Enterobacter clocae was resistant to 9 antibiotics: chloramphenicol, kanamycin, cefoxitin, trimethoprim, amoxicillin, ampicillin, nalidixic acid, gentamycin, and amikacin. Stenotrophomonas maltophilia was resistant to 9 antibiotics: amoxicillin, cefoxitin, ceftriaxone, ciprofloxacin, tetracycline, ampicillin, kanamycin, chloramphenicol, and trimethoprim. Pseudomonas luteola was resistant to 5 antibitoics: amoxicillin, cefoxitin, ceftrixone, ampicillin, and trimethoprim. Pantoea spp was resistant to cefoxitin and kanamycin while Serratia marcescens was resistant to cefoxitin and trimethoprim. The top five antibiotics most isolates were resistant to were cefoxitin (21), amoxicillin (14), ampicillin (13), trimethoprim (9), and ceftriaxone (5).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Kisanga, A., Tope, A., Patel, S. Evaluation of Potential Pathogenic Bacteria in Spices from Local Stores in Kentucky. Annual Meeting of Kentucky Academy of Science, Louisville, KY, November 4-5, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Davis, K., Patel, S., Tope, A. Evaluation of Drug-Resistant Enterobacteriaceae on Produce from Small Farms in KY. Annual Meeting of Kentucky Academy of Science, Louisville, KY, November 4-5, 2016.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:1. Small and limited-resource farmers. 2. Minority farmers. 3. Public health professionals. 4. Small and medium food processors. 5. KY stakeholders at large. 6. Farmers market administrators. Changes/Problems:1. With the appointment of a new research associate and completion of required training, some aspects of the project were delayed. 2. Despite our consistent efforts to recruit participants through all possible avenues, recruiting more farmers has been a challenge. What opportunities for training and professional development has the project provided?Graduate Student: 1. Received training in developing and administering surveys. 2. Received training in development of program brochures and farmer recruitment. 3. Received hands-on training in microbiological protocols for soil, water, and produce sampling and in-lab sample processing including standard and advance protocols in growth, isolation and identification of pathogenic bacteria. Undergraduate Student: 1.Received training in development of program brochures and farmer recruitment. 2. Received hands-on training in microbiological protocols for soil, water, and produce sampling and in-lab sample processing including standard and advanced protocols in growth, isolation and identification of pathogenic bacteria. How have the results been disseminated to communities of interest?1. A training session was offered for the participating and non-participating farmers on "Good Agricultural Practices (GAP) for Produce Growers" through the Small Farms Program of KSU as part of the Third Thursday Thing (a monthly sustainable agriculture workshop targeting small farmers, beginning farmers and agricultural professionals) in May 2016. 2. A research manuscript entitled Evaluation of Antimicrobial Resistance in Enterobacteriaceae and Coliforms Isolated on Farm, Packaged and Loose Vegetables in Kentucky was published in the Journal of Food: Microbiology, Safety & Hygiene, in August 2016. What do you plan to do during the next reporting period to accomplish the goals?1. Using various avenues mentioned earlier, we will continue our efforts to recruit more farmers. 2. The isolated strains of Enterobacteriaceae will be identified using biochemical identification assays such as API. 3. We also are currently studying the antibiotic resistant genes from the isolates using polymerase chain reaction- (PCR) based methods.
Impacts
What was accomplished under these goals?
Established conventional, certified and non-certified organic small and limited-resource farmers were contacted and one new farmer was recruited through a mailing list developed from Kentucky State University's current outreach and extension programs, including participants in the Small Farm Program, Third Thursday Thing (a monthly sustainable agriculture workshop targeting small farmers, beginning farmers and agricultural professionals), the Socially Disadvantaged Farmer Outreach Project and the Organic Association of Kentucky. The new farmer was given a survey that explored details regarding certification status, fertilization practices (such as the type(s) of manure, compost and/or chemicals, age of the manure/compost and the time of application), the source of irrigation water, surrounding land use, produce handling practices such as washing, packaging and storage during harvesting and post-harvesting. In all, 23 small and limited-resource farmers, comprised of 16 certified and non-certified organic farmers and 5 conventional farmers, who consented to offer access to soil, water and produce samples for analysis were recruited. A total of 10 farms were visited (twice each) from Oct. 2015 to September 2016. In all, 64 produces samples, 14 water samples and 66 soil samples were collected for microbial analysis. Microbial Analysis: Microbial analyses were started within 24 hours of receiving the samples. Using stringent aseptic conditions, ten grams of sample was mixed with 100 milliliter (mL) of lauryl sulfate tryptose broth (LST), in a stomacher bag for 5 min, at 230 RPM. The mixture was then placed in 9 mL of LST, in serial dilutions up to 10-4. One mL of the serial dilution of 10-3 was plated onto E. coli, Coliform and Listeria Petrifilm plates (3M Company, Maplewood, Minn.) and 100 microliters of the serial dilution of 10-3 was plated onto eosin methylene blue (EMB) agar plates in duplicate, and placed into the incubator at 37°C for 48 hours. For water analysis, an advanced IDEXX system was used. Coliform count was determined by the most-probable-number (MPN) system. After inoculation of the water sample, the reagent pre-treated bags were incubated for 24 hours at 37°C, and another reagent was added. Wells with coliform bacteria were then visualized under UV light and the readings were recorded, compared with the MPN table, and final counts were recorded. In all, of The 14 water samples tested, 3 water samples had an MPN count of more than 1 colony forming unit (cfu)/100 ml. Antibiotic Resistance/Sensitivity Testing: The antimicrobial susceptibility testing was performed by the standard Clinical and Laboratory Standards Institute (CLSI; formerly known as the National Committee on Clinical Laboratory Standards [NCCLS]) methods using the Kirby-Bauer disk diffusion technique with Mueller-Hinton agar (Fisher Scientific, IL). The antibiotics used in this study were amikacin (30 μg), amoxicillin (30 μg), ampicillin (10 μg), cefoxitin (30 μg), ceftiofur (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamycin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), streptomycin (10 μg), tetracycline (30 μg), and trimethoprim (5 μg). Bacterial cultures were grown in 5 mL of nutrient broth at 37ºC for 24 h. Each culture was spread evenly onto Mueller-Hinton agar, incubated at 37ºC for 48 h and the zones of inhibition were measured. Colonies on the plates with color and gas bubble were recorded as positive for each respective antibiotic. Statistical Analysis: Microbiology and survey results were recorded via Microsoft Excel and analyzed using IBM SPSS statistical software using one-way ANOVA and Pearson chi-squared statistical analyses. Results: A total of 6 conventional and 16 certified and non-certified organic farmers participated in the study by providing samples and answering questions about their management practices. Among the organic farms, two were certified by the Kentucky Department of Agriculture and fourteen were non-certified but reported using organic practices. Two of the six conventional farmers reported using composted manure in addition to chemical fertilizer and commercial pesticide, while the remaining four conventional farmers only used commercial fertilizer. Of the sixteen organic farmers, seven reported using aged or composted animal manure regularly as the main source of fertilizer. The remaining nine organic farmers did not apply fertilizer of any sort to their fields. When testing for E. coli, farms were categorized in three groups based on the type of the fertilizer used: farms that only used commercial fertilizers on their fields, farms that used non-commercial fertilizers (including compost, animal manures, and compost mixtures), and farms that used no additional fertilizer on their fields. Sixty-four produce samples, 14 water samples and 66 soil samples were collected for microbial analysis. No E. coli, Salmonella spp, or Listeria were detected in the produce samples analyzed for fecal contamination. However, 26 of 64 produce samples were contaminated with members of Enterobacteriaceae family. The five most common contaminants detected in the produce samples were: Enterobacter cloacae, Stenotrophomonas maltophilia, Pantoea spp 2, Pseudomonas luteola, and Serratia marcescens. These contaminants exhibited drug resistance to one or more antibiotics. Enterobacter cloacae was resistant to 9 different antibiotics. chloramphenicol, kanamycin, cefoxitin, trimethoprim, amoxicillin, ampicillin, nalidixic acid, gentamycin and amikacin. Stenotrophomonas maltophilia was resistant to 9 different antibiotics: amoxicillin, cefoxitin, ceftriaxone, ciprofloxacin, tetracycline, ampicillin, kanamycin, chloramphenicol, and trimethoprim. Pseudomonas luteola was resistant to 5 antibiotics: amoxicillin, cefoxitin, ceftriaxone, ampicillin, and trimethoprim. Pantoea spp was resistant to cefoxitin and kanamycin while Serratia marcescens was resistant to cefoxitin and trimethoprim. The five antibiotics with most resistant isolates were cefoxitin (21), amoxicillin (14), ampicillin (13), trimethoprim (9), and ceftriaxone (5).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Tope AM, Hitter AC, Patel SV (2016) Evaluation of antimicrobial resistance in Enterobacteriaceae and coliforms isolated on farm, packaged and loose vegetables in Kentucky. J Food Microbiol Saf Hyg 1:113. doi:10.4172/jfmsh.1000113
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:1. Small and limited resource farmers. 2. Minority farmers. 3. Public Health Professionals. 4. Small and medium food processors. 5. Kentucky stakeholders at large. 6. Farmers Markets Administrators. Changes/Problems:1. With the retirement of the Research Associate initially involved in the project, there was delay in appointing a new RA, which almost took 9 months. Thus, we could not aggressively recruit more farmers to participate in the study. 2. Challenges in greater farmer recruitment: Despite our consistent efforts to recruit through all possible avenues, recruiting a greater number of farmer participants has been a challenge. However, since the Third Thursday Thing event in May, 2015, we have added two more participants. What opportunities for training and professional development has the project provided?. Graduate Student: One received training in developing surveys as well as in their administration. Two received training in the development of a program brochure and farmer recruitment. Three received hands-on training in microbiological protocols in sampling of soil, water, and produce, in lab processing which includes standard and advance protocols in growth, isolation, and identification of pathogenic bacteria Undergraduate Student: One received training in the development of a program brochure and farmer recruitment. Two received hands-on training in microbiological protocols in sampling of soil, water, and produce, in lab processing which includes standard and advanced protocols in growth, isolation, and identification of pathogenic bacteria. How have the results been disseminated to communities of interest?1. A training session was offered for the participating and non-participating farmers on "Good Agricultural Practices (GAP) for Produce Growers" through the Small Farms Program of KSU under the "Third Thursday Thing" Program in May 2015. 2. Overall, 25% of the produce sampled from the 22 participating farms was contaminated with Enterobacteriaceae. Five out of the six farms that indicated they used manure 90 days or less prior to planting had E. coli contamination on their produce samples, as well as those which recovered run-off water from upstream carrying animal feces, which indicates the link between Good Agricultural Practices (GAP) and the microbiological quality of fresh produce (Food and Drug Administration, 1998). After the second visit, farmers were given recommendations consistent with the GAP standards tailored toward their specific needs to improve the quality of their produce in the future. Farmers were enthusiastic about these suggestions, asked further questions, and started brainstorming cleaner options for irrigation and fertilization on their fields. 3. A research manuscript titled "Evaluation of Good Agricultural Practices (GAP) Compliance by Small Farmers in Kentucky: Assessing Microbial Quality of Produce" was published in the Journal of Agriculture and Environmental Studies, Vol. 3(4), Dec. 2014. 4. A research manuscript titled "Evaluation of antibiotic resistance in Enterobacteriacea isolated on farm, packaged and loose varieties of produce" is being prepared to be sent for publication in a peer reviewed, international online publication. What do you plan to do during the next reporting period to accomplish the goals?1. Continue Recruiting Farmers: Using various avenues mentioned earlier, we will continue our efforts to recruit more consenting farmers. 2. The isolated strains of E.coli will be serotyped using modern molecular biological techniques to know their pathogenic profile, as well as identify the other Enterobacteriaceae members using biochemical identification assays such as API. 3. We also are currently studying the antibiotic profiles of all contaminated samples for antibiotic resistance.
Impacts
What was accomplished under these goals?
Farmer Recruitment and Survey: Participating conventional, certified and non-certified organic small and limited-resource farmers were contacted and two new farmers were recruited through a mailing list from Kentucky State University's current outreach and extension programs, including participants from the Small Farm Program, Third Thursday Thing, the Socially Disadvantaged Farmer Outreach Project and the Organic Association of Kentucky. Each new farmer was given a survey that explored details regarding certification status, fertilization practices (such as the type(s) of manure or compost and/or chemicals, age of the manure or compost and the time of application), the source of irrigation water, surrounding land use, handling practices during harvesting, post harvesting and handling practices such as washing, packaging and storage. Further, the survey was targeted to help in ascertaining the needs and gaps in knowledge of the farmers in various aspects of 'on farm' food safety. The information collected from the survey was used to identify associations with the microbiological results.In all, 22 small and limited resource farmers, comprising 16 certified and non-certified organic farmers and 4 conventional farmers were recruited who consented to offer access to soil, water and produce samples for analysis. Microbial Analysis: Microbial analyses were started within 24 hours of receiving the samples. For lettuce, Japanese greens, Swiss chard, and cabbage, representative leaves from the exterior and interior sections were cut up and used to make 10 gram samples. Using stringent aseptic conditions, ten grams of sample was then mixed with 100 milliliter (mL) of Lauryl sulfate tryptose broth (LST), placed in a stomacher bag for 5 min, at 230 RPM. The mixture was then placed in 9 mL of LST, in serial dilutions up to 10x-4. One mL of the serial dilution of 10x-3 was plated onto E. coli, Coliform and Listeria Petrifilm plates (3M Company, Maplewood, Minn.) and 100 microliters of the serial dilution of 10x-3was plated onto Eosin Methylene Blue (EMB) agar plates in duplicate, and placed into the incubator at 37 C for 48 hours. For the water, the coliform count was determined by the three-tube most-probable-number (MPN) system using three 10-fold serial dilutions in double strength Lactose broth and was incubated for 48 hours at 37C. Lactose tubes showing growth and gas were plated onto Eosin Methylene Blue (EMB) plates in duplicate and incubated for an additional 48 hours. Five grams of each soil sample was mixed and manually stirred with 50 mL of sterile saline. One milliliter of this mixture was pipetted into 9 mL of sterile saline serial dilutions up to 10x-4 One milliliter of the serial dilution of 10-3 was plated in duplicate onto three different types of Petrifilm plates, namely E. coli, Coliform and Listeria, and incubated at 37C for 48 hours. Statistical Analysis: On the Petrifilm plates, colonies with color and gas were recorded as positive for each respective type. These results, as well as the data from the survey, were recorded using Microsoft Excel and analyzed using IBM SPSS statistical software using One-way ANOVA and Pearson Chi-Square statistics, when appropriate. Results: A total of 16 certified and non-certified organic and six conventional farmers participated in the study by providing samples and answering questions about their management practices. Among the organic farms, two were certified by Kentucky Department of Agriculture and fourteen were non-certified but reported using organic practices. Two out of the six conventional farmers reported using composted manure in addition to their chemical fertilizer and commercial pesticide, while the remaining conventional farmers only used commercial fertilizer. Of the sixteen organic farmers, seven reported using aged or composted animal manure regularly as the main source of fertilizer. The remaining organic farmers did not apply fertilizer of any sort to their fields. When testing for E. coli, the soil amendments were categorized in three groups based on the type of the fertilizer used: farms that used non-commercial fertilizers (including compost, animal manures, and compost mixtures), farms that used no additional fertilizer on their fields, and farms that only used commercial fertilizers on their fields.Of the farms that used non-commercial fertilizers, 43% had Enterobacteriaceae contamination on their produce samples. Of the farms that did not use fertilizer at all, 41% had E. coli on produce samples. When comparing the group that did not use any type of fertilizer to the group that used non-commercial fertilizers, there was no statistical difference of E. coli prevalence. However, none of the farms that used commercial fertilizer had E. coli contamination either on their produce samples, and this was statistically significant when compared with the other two groups. Water samples were collected from the participating farms. The majority of farms used only one water source for irrigation, but some farms had as many as two sources and a few used no sources to collect (i.e., they just used rain water to irrigate their crops). In total, three types of water sources were collected in the study: city water, surface water, and well water. City water was the least contaminated irrigation source with a mean of 1.0 cfu/100 mL, and surface water had the highest average amount of Coliform count at 673.3 cfu/100 mL. Coliform bacteria were detected in 27% of all samples, and the overall average count in both organic and conventional produce was 1.6 x 104 cfu/g. No statistical significances were found when the coliform counts of the produce varieties were compared between the two groups of farms. E. coli was isolated from 25.42% of all fruits and vegetables analyzed, and the average count of those samples that tested positive was 3.5 cfu/g. The overall prevalence of E. coli was about the same in conventional and organic fruits and vegetables. However, statistically significant differences were observed in the distribution of E. coli, depending on whether the produce was grown above or below the soil surface. Produce that grew below ground or contacting the soil surface had a higher chance of being contaminated with E. coli than produce that grew above ground and not in contact with the soil, and this was statistically significant. In addition, isolation of E. coli O157:H7, using CHROMagar, will be performed on all E. coli positive samples. We also are currently studying the antibiotic profiles of all contaminated samples for antibiotic resistance.
Publications
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Progress 11/21/13 to 09/30/14
Outputs
Target Audience: Small and limited resource farmers. Minority farmers. Public Health Professionals. Small and medium food processors. KY stakeholders at large. Farmers Markets Administrators. Changes/Problems: Challenges in greater farmer recruitment: Despite our consistent efforts to recruit through all possible avenues, recruiting greater number of farmer participants has been a challenge. However, since the Third Thursday Thing event in May, 2014, we have added five more participants. What opportunities for training and professional development has the project provided? Graduate Student: 1. Received training in developing surveys as well as in their administration. 2. Received training in development of program brochure and farmer recruitment. 3. Received hands on training in microbiological protocols in sampling of soil, water, and produce, in lab processing which includes standard and advance protocols in growth, isolation and identification of pathogenic bacteria Undergraduate Student: 1.Received training in development of program brochure and farmer recruitment. 2. Received hands on training in microbiological protocols in sampling of soil, water, and produce, in lab processing which includes standard and advanced protocols in growth, isolation and identification of pathogenic bacteria. How have the results been disseminated to communities of interest? 1. A training session was offered for the participating and non-participating farmers on "Good Agricultural Practices (GAP) for Produce Growers" through the Small Farms Program of KSU under the Third Thursday Thing Program in May 2014. 2. Overall, 25% of the produce sampled from the 20 participating farms was contaminated with E. coli. Five out of the six farms that indicated they used manure 90 days or less prior to planting had E. coli contamination on their produce samples, as well as those which recovered run-off water from upstream carrying animal feces, which indicates the link between Good Agricultural Practices (GAP) and the microbiological quality of fresh produce (Food and Drug Administration, 1998). After the second visit, farmers were given recommendations consistent with the GAP standards tailored toward their specific needs to improve the quality of their produce in the future. Farmers were enthusiastic about these suggestions, asked further questions, and started brainstorming cleaner options for irrigation and fertilization on their fields. 3. A research manuscript titled "Evaluation of Good Agricultural Practices (GAP) Compliance by Small Farmers in Kentucky: Assessing Microbial Quality of Produce" is being prepared to be sent for a peer reviewed scientific journal. What do you plan to do during the next reporting period to accomplish the goals? 1. Continue Recruiting Farmers: Using various avenues mentioned earlier, we will continue our efforts to recruit more consenting farmers. 2. The isolated strains of E.coli will be serotyped using modern molecular biological techniques to know their pathogenic profile, as well as identify the other enterobacteriaceae members using biochemical identification assays such as API. 3. We also are currently studying the antibiotic profiles of all contaminated samples for antibiotic resistance.
Impacts
What was accomplished under these goals?
Farmer Recruitment and Survey: Consenting conventional, certified and non-certified organic small and limited-resource farmers were contacted and recruited through a mailing list from Kentucky State University's current outreach and extension programs, including participants from the Small Farm Program, Third Thursday Thing, the Socially Disadvantaged Farmer Outreach Project and the Organic Association of Kentucky. Each farmer was given a survey that explored details regarding certification status, fertilization practices (such as the type(s) of manure or compost and/or chemicals, age of the manure or compost and the time of application), the source of irrigation water, surrounding land use, handling practices during harvesting, post harvesting and handling practices such as washing, packaging and storage. Further, the survey was targeted to help in ascertaining the needs and gaps in knowledge of the farmers in various aspects of 'on farm' food safety. The information collected from the survey was used to identify associations with the microbiological results. In all, 20 small and limited resource farmers, comprising 16 certified and non-certified organic farmers and 4 conventional farmers were recruited who consented to offer access to soil, water and produce samples for analysis. Microbial Analysis: Microbial analyses were started within 24 hours of receiving the samples. For lettuce, Japanese greens, Swiss chard, and cabbage, representative leaves from the exterior and interior sections were cut up and used to make 10 gram samples. Using stringent aseptic conditions, ten grams of sample was then mixed with 100 milliliter (mL) of Lauryl sulfate tryptose broth (LST), taken in a stomacher bag for 5 min, at 230 RPM. The mixture was then placed in 9 mL of LST, in serial dilutions up to 10x-4. One mL of the serial dilution of 10x-3 was plated onto E. coli, Coliform and Listeria Petrifilm plates (3M Company, Maplewood, Minn.) and 100 microliters of the serial dilution of 10x-3was plated onto Eosin Methylene Blue (EMB) agar plates in duplicate, and placed into the incubator at 37oC for 48 hours. For the water, the coliform count was determined by the three-tube most-probable-number (MPN) system using three 10-fold serial dilutions in double strength Lactose broth and was incubated for 48 hours at 37oC. Lactose tubes showing growth and gas were plated onto Eosin Methylene Blue (EMB) plates in duplicate and incubated for an additional 48 hours. Five grams of each soil sample was mixed and manually stirred with 50 mL of sterile saline. One milliliter of this mixture was pipetted into 9 mL of sterile saline serial dilutions up to 10x-4 One milliliter of the serial dilution of 10-3was plated in duplicate onto three different types of Petrifilm plates, namely E. coli, Coliform and Listeria, and incubated at 37oC for 48 hours. Statistical Analysis: On the Petrifilm plates, colonies with color and gas were recorded as positive for each respective type. These results, as well as the data from the survey, were recorded onto Microsoft Excel and analyzed using IBM SPSS statistical software using One-way ANOVA and Pearson Chi-Square statistics, when appropriate. Results: A total of 16 certified and non-certified organic and 4 conventional farmers participated in the study by providing samples and answering questions about their management practices. Among the organic farms, two were certified by Kentucky Department of Agriculture and fourteen were non-certified but reported using organic practices. Two out of the four conventional farmers reported using composted manure in addition to their chemical fertilizer and commercial pesticide, while the remaining conventional farmers only used commercial fertilizer. Of the sixteen organic farmers, seven reported using aged or composted animal manure regularly as the main source of fertilizer. The remaining nine organic farmers did not apply fertilizer of any sort to their fields. When testing for E. coli, the soil amendments were categorized in three groups based on the type of the fertilizer used: farms that used non-commercial fertilizers (including compost, animal manures, and compost mixtures), farms that used no additional fertilizer on their fields, and farms that only used commercial fertilizers on their fields. Of the farms that used non-commercial fertilizers, 43% had E. coli contamination on their produce samples. Of the farms that did not use fertilizer at all, 41% had E. coli on produce samples. When comparing the group that did not use any type of fertilizer to the group that used non-commercial fertilizers, there was no statistical difference of E. coli prevalence. However, none of the farms that used commercial fertilizer had E. coli contamination either on their produce samples, and this was statistically significant when compared with the other two groups. Water samples were collected from the participating farms. The majority of farms used only one water source for irrigation, but some farms had as many as two sources and as few as no sources to collect (i.e., they just used rain water to irrigate their crops). In total, three types of water sources were collected in the study: city water, surface water, and well water. City water was the least contaminated irrigation source with a mean of 1.0 cfu/100 mL, and surface water had the highest average amount of Coliform count at 673.3 cfu/100 mL. Coliform bacteria were detected in 25.42% of all samples, and the overall average count in both organic and conventional produce was 1.6 x 104 cfu/g. No statistical significances were found when the coliform counts of the produce varieties were compared between the two groups of farms. E. coli was isolated from 25.42% of all fruits and vegetables analyzed, and the average count of those samples that tested positive was 3.5 cfu/g. The overall prevalence of E. coli was about the same in conventional and organic fruits and vegetables. However, statistically significant differences were observed in the distribution of E. coli, depending on whether the produce was grown over or below the soil surface. Produce that grew below ground or contacting the soil surface had a higher chance of being contaminated with E. coli than produce that grew above ground and not in contact with the soil, and this was statistically significant. In addition, isolation of E. coli O157:H7, using CHROMagar, will be performed on all E. coli positive samples. We also are currently studying the antibiotic profiles of all contaminated samples for antibiotic resistance.
Publications
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