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Rapid Detection, Molecular Characterization, and Antimicrobial Resistance of Foodborne Pathogens


The objectives of the study are to develop rapid, sensitive, and high-throughput methodology for the simultaneous detection of Campylobacter jejuni/coli, pathogenic Escherichia coli, Listeria monocytogenes, and Salmonella in foods, and characterize genotypes of these foodborne bacterial pathogens in respective commodities from Louisiana by molecular methods, and to investigate antimicrobial resistance occurring in these pathogens.

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NON-TECHNICAL SUMMARY: Bacteria that cause human diseases can be transmitted by food we consume. Major foodborne organisms, such as Campylobacter, Salmonella, pathogenic Escherichia coli, and Listeria monocytogenes are responsible for millions of illnesses and thousands of deaths annually in the United States. Antibiotic treatment may be life-saving for patients of high risk groups or with severe infections. However, resistances have emerged. In addition, it has been increasingly recognized that our food production and distribution system is vulnerable to the introduction of pathogens and toxins through natural processes, global commerce, and by intentional means. Rapid and sensitive detection methods with considerable ease of use and low cost are needed for biodefense purpose. In this study, we will develop and validate a multi-analyte detection method for these major foodborne pathogens, which could potentially be used for biodefense purpose. We will also gain valuable scientific data on the contamination level of multiple foodborne pathogens in the retail food supply in Louisiana, their genotypes, and the extent of antimicrobial resistance. This will be an invaluable source of information for analyzing population biology and trends of antibiotic resistance among these foodborne bacteria and the likelihood of human exposure more pertinent to our region.


APPROACH: Experiments on detection methods development will combine immunofluorescent nanoparticles and flow cytometry. Four genera will be included in the assay development: E. coli, Salmonella, Campylobacter, and Listeria. Assay will first be developed by testing in a model system (bacterial broth media), then a liquid food system (milk and bottled water). Studies on the prevalence, genotype, and antimicrobial resistance will be based on a carefully planned and executed sampling scheme. Following sample collection, bacteria will be isolated, characterized, and tested for susceptibilities against different panels of antimicrobials. Data will then be analyzed and compared with data reported from national surveillance systems.


PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: A rapid, sensitive, and specific molecular-based detection assay, loop-mediated isothermal amplification (LAMP), was developed to detect Vibrio vulnificus in oysters. The assay characteristics including sensitivity, specificity, and applicability in raw oysters were evaluated. A survey examining the prevalence and antimicrobial susceptibility of Campylobacter and Salmonella in retail chickens in Baton Rouge, Louisiana was conducted over a one-year period. A strain collection database was established. The pulsed-field gel electrophoresis technique was used for molecular typing of Salmonella isolates from retail chickens. DNA fingerprinting profiles of these isolates were collected. Three graduate students, two M.S. and one Ph.D., were trained with technologies and skills including molecular detection assay for foodborne pathogens, PFGE typing technique, isolation and identification of foodborne pathogens from food products, antimicrobial susceptibility testing by broth microdilution or agar dilution, as well as planning experiments, conducting experiments, analyzing data, and writing up reports and manuscripts. <BR>PARTICIPANTS: Beilei Ge, Assistant Professor, Department of Food Science, Louisiana State University Agricultural Center. Feifei Han, PhD student, Department of Food Science, Louisiana State University Agricultural Center. Shofiyah I. Lestari, MS student, Department of Food Science, Louisiana State University Agricultural Center. Shuaihua Pu, MS student, Department of Food Science, Louisiana State University Agricultural Center. <BR>TARGET AUDIENCES: Food safety researchers, educators, and consumers. Food safety regulatory agencies and public health officials.
IMPACT: 2007/01 TO 2007/12<BR>
After 5 h enrichment, LAMP was capable of detecting 7 cells of V. vulnificus per gram of oyster tissue without lengthy DNA extraction steps. LAMP can be used for rapid, sensitive, and specific identification of V. vulnificus from raw oysters. Because of its isothermal format and unique amplicon detection technique via unaided eye, the LAMP assay holds potential for future field applications. Among 204 chicken samples collected for the survey, the prevalence rate for Campylobacter and Salmonella was 42.6% and 22.1%, respectively. No significant difference in prevalence of either Campylobacter or Salmonella was observed among chicken types, either conventional or organic. A total of 181 Campylobacter were isolated, consisting of 120 Campylobacter jejuni, 28 Campylobacter coli, and 33 other Campylobacter. Among four antimicrobial agents tested, the highest resistance rate was observed for tetracycline (32.0%), followed by erythromycin (28.2%), and ciprofloxacin (4.4%). No resistance to gentamicin was observed. Organic chickens had lower resistance rates for Campylobacter than conventional chickens. Among 135 Salmonella isolates tested against 17 antimicrobial agents, resistance to sulfisoxazole (93.3%) was the most common, followed by streptomycin (50.4%), tetracycline (46.6%), ampicillin (18.5%), ceftiofur (17.7%), amoxicillin/clavulanic acid (15.5%), and cefoxitin (15.5%). All Salmonella isolates were susceptible to amikacin, ceftriaxone, and ciprofloxacin. The resistance rates to the vast majority of antimicrobials were significantly higher among Salmonella isolates from conventional chicken compared to those from organic chicken. Our study indicated a relatively low prevalence of Campylobacter and Salmonella among Louisiana retail chickens, and organic chicken samples carry less antimicrobial-resistant Campylobacter and Salmonella. Analyzing PFGE profiles of Salmonella found that isolates within a chicken sample generally carry the same pattern, indicating single colonization. This knowledge will facilitate the selection of suitable isolates for Salmonella serotyping.

Ge, Beilei
Louisiana State University
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