Antimicrobial Resistance Monitoring Of Zoonotic And Indicator Bacteria In Tennessee Local Foods And Agricultural Lands

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.