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The overall objective of this study is to evaluate foodborne pathogens and antibiotic residues in imported and domestic seafood at retail sites on the Delmarva Peninsula. <P>The specific objectives are as follows: <br/>1. To examine the levels of total aerobic bacteria, total coliforms, E. coli, Salmonella, Campylobacter and Vibrio species in imported and domestic shrimp, tilapia and catfish. <br/>2. To determine the concentrations of antibiotic residues (chloramphenicol, nitrofuran, and tetracycline) in imported and domestic shrimp, tilapia and catfish. <br/>3. To determine the antibiotic resistance patterns of Vibrio, Salmonella and Campylobacter recovered from imported and domestic shrimp, tilapia, and catfish. <br/>4. To determine whether the antibiotic resistance patterns of pathogenic bacteria are correlated with antibiotic residues in imported and domestic shrimp, tilapia and catfish. <br/>5. To determine the pulsed-field gel electrophoretic patterns for Salmonella, Campylobacter, and Vibrio species recovered from imported and domestic shrimp, catfish, and tilapia. <br/>6. To develop outreach and extension programs for education and control of foodborne pathogens and antibiotic residues in shrimp, catfish, and tilapia. <P>Outputs are collection and analysis of samples; interpretation of results; presentation of research findings at regional, national and international professional meetings; publication of research findings in peer-reviewed journals; sharing the results with the seafood industry, regulatory agencies, public health agencies and risk assessors at local, regional, national, and international levels through presentations, websites and workshops; train students and a technician.
Non-Technical Summary:<br/>
The United States (U.S.) is the third leading seafood consuming nation in the world and its average seafood consumption grew from 12.5 pounds per capita in 1980 to 15.8 pounds in 2009. Catfish, canned tuna, salmon, shrimp, and tilapia are the most favored seafood products consumed in the U.S.. Due to population growth and increased per capita seafood consumption, the gross U.S. seafood supply has grown over 70% from 1980 to 2009. Seafood imports increased significantly from below 50% of the gross seafood consumption in 1980 to more than 80% in recent years to meet the deficit of the domestic production. Catfish, crab, ground fish, salmon, shrimp, squid and tuna were the favored imported species. The U.S. Centers for Disease Control and Prevention (CDC) (2012) stated that seafood has been a major group incriminated in foodborne outbreaks in the U.S. and a total of 39 seafood - related outbreaks with 2,348 illnesses were reported between 2005 and 2010. CDC also reported that 76.1, 21.3, and 2.6% of 188 seafood-associated outbreaks were caused by bacterial, viral, and parasitic agents, respectively. Bacterial agents of major concern in seafood include Vibrio, Salmonella, Campylobacter, Shigella, Listeria momocytogenes, Clostridium botulinum, and other toxin-forming bacteria. Several investigators reported that Salmonella was the most common contaminant isolated from imported seafood and Salmonella strains isolated from seafood were resistant to antimicrobials. The presence of Salmonella and Vibrio species were also reported in domestic seafood. To treat and prevent a wide variety of production-related diseases in aquaculture, antibiotics are used. Antibiotic residues in seafood may pose risks to consumers. To prevent this risk, USFDA sets antibiotic residue tolerance levels and inspects seafood for violation of these limits. Most of the violations were detected in seafood (catfish, shrimp and tilapia) imported from Asia. Currently, there is a lack of knowledge about the prevalence of foodborne pathogens and antibiotic residues in imported and domestic seafood. The overall of objective of this study is to address this data gap.
<br/>To achieve these objectives, imported and domestic seafood samples will be randomly collected in monthly intervals from retail stores on the Delmarva Peninsula for a period of two years, and analyzed for foodborne pathogens and antibiotic residues using standard methods. The project will provide valuable information about the microbial and chemical contamination of imported and domestic seafood. In addition, integration of research and extension efforts provide an opportunity to adapt and apply these techniques for the benefit of the seafood industry, regulatory agencies, public health agencies and risk assessors at local, regional, national, and international levels. The outcomes of this project also include professional exposure to minority graduate students at UMES. This will also enhance the national and international standing of UMES research programs, thereby enhancing student recruitment and the recognition of its microbiological research and outreach programs.
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Approach:<br/>
A total of 432 imported and domestic seafood samples will be collected in monthly intervals from three retail stores on the Delmarva Peninsula for a period of two years. A 25 g portion of each sample will be weighed and placed in a sterile plastic bag containing 225 ml of buffered peptone water. The contents will be mixed and transferred to individual 50-ml screw-cap tubes for subsequent analyses. Petrifilm will be used for enumeration of aerobic plate counts and indicator organisms. For isolation of Campylobacter, samples will be enriched in double-strength of Bolton broth under microaerophilic conditions. The culture will be inoculated onto modified charcoal cefoperazone deoxycholate agar and incubated. For isolation of Salmonella, samples will be enriched in tetrathionate broth, and the culture will be streaked onto xylose lysine Tergitol 4 agar and incubated. Vibrio spp. will be isolated using a standard method. Briefly, samples will be enriched in alkaline peptone water and incubated. The culture will be streaked onto thiosulfate citrate bile salts sucrose agar and modified cellobiose - polymycin B-colistin agar plates for the isolation of Vibrio parahaemolyticus and Vibrio vulnificus, respectively. Three to five presumptive colonies of Campylobacter, Salmonella, and Vibrio spp. will be confirmed using BAX PCR. Samples positive for Campylobacter, Salmonella, and Vibrio spp. will be analyzed by three tube most probable number (MPN) method for enumeration. A gas chromatographic method will be used for the detection of antibiotic residues in the samples. Standard disc diffusion assay will be used for antimicrobial susceptibility testing. Pulsed-field gel electrophoresis will be used to determine the clonal diversity of foodborne pathogens.
<br/>Statistical analysis: For quantitative measurement outcomes, such as total aerobic counts and total coliforms, statistical significance of observed differences will be assessed by t-tests or one-way ANOVA. For qualitative outcomes statistical significance of observed group differences will be assessed by the continuity corrected chi-squared statistic or Fishers exact test with mid-p adjustment. Correlation between the antibiotic resistance patterns of pathogenic bacteria and antibiotic residues will be assessed by logistic regression.The significance level of 0.05 will be considered for all analyses. To develop outreach and extension programs for control of foodborne pathogens and antibiotic residues in imported and domestic seafood, the results of this project will be shared with seafood industry and regulatory agencies through presentations, publications, workshops newsletters and websites. Results will be incorporated in existing Good Aquaculture Practices training courses conducted overseas in cooperation with the University of Maryland and U.S. FDA. An evaluation committee comprised of investigators and collaborators of the project will meet regularly to evaluate the progress of the projects and determine if any modification is needed. Samples will be promptly processed and the data will be tightly scrutinized so that timely adjustment can be made to procedure, if needed.