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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND PROTEOMIC ANALYSIS OF FOODBORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: Food safety concerns in the U.S. and research on Shiga Toxin-producing E. coli

Authors
item Fratamico, Pina
item Baranzoni, Gianmarco -

Submitted to: UJNR Food & Agricultural Panel Proceedings
Publication Type: Proceedings
Publication Acceptance Date: November 4, 2013
Publication Date: December 8, 2013
Citation: Fratamico, P.M., Baranzoni, G. 2013. Food safety concerns in the U.S. and research on Shiga Toxin-producing E. coli. UJNR Food & Agricultural Panel Proceedings. P.

Technical Abstract: In the U.S., there are several government agencies that deal with food safety. Under the Department of Health and Human Services, there are the Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC). The CDC collaborates with state agencies, private organizations, and other federal agencies to provide health information in the area of disease prevention, and it is also involved in surveillance and investigation of illnesses associated with food consumption. One of the missions of the FDA is to protect the public’s health by ensuring that the nation's food supply is safe, sanitary, wholesome, and honestly labeled, and that cosmetic products are safe and properly labeled. Agencies under the USDA include the Agricultural Marketing Service and the Food Safety and Inspection Service (FSIS), which is responsible for the safety, wholesomeness, and correct labeling of commercial meat, poultry, and egg products. The Agricultural Research Service is the research arm of the USDA, and one of the research areas is food safety. Scallan et al. (2011) estimate that 9.4 million cases of food-borne illness, 55, 961 hospitalizations, and 1,351 deaths occur each year in the U.S. due to 31 major pathogens. A large portion of the illnesses are due to infection with norovirus, followed by Salmonella, Clostridium perfringens, and Campylobacter. Salmonella, Toxoplasma gondii, Listeria monocytogenes, and norovirus were the leading causes of deaths. Shiga toxin-producing E. coli (STEC) are estimated to cause more than 265,000 illnesses each year in the U.S. with more than 3600 hospitalizations and 30 deaths. Most of the deaths and hospitalizations are estimated to be due to infection with STEC O157:H7. FoodNet is the principal food-borne disease component of the CDC’s Emerging Infections Program and provides the data necessary for measuring the progress in food-borne disease prevention. E. coli O157:H7 infections have been monitored through FoodNet since its inception in 1996, and surveillance for non-O157 STEC infections began in 2000 when they became nationally reportable. From 2000 to 2010, 7695 STEC cases were reported; 5688 were associated with the serogroup O157, and 83% of the other STEC belonged to serogroups O26, O45, 103, O111, O121 and O145 (Gould et al., 2013). However, other serogroups, including O91, O113, and O104 have also caused serious human illness. Prior to 2011, STEC serogroup O104 was not considered as an important STEC; although it caused an outbreak involving 11 cases in the U.S. and many sporadic human cases in Germany, the United Kingdom, Korea, France, Finland, Norway, Denmark, Belgium, Sweden, and Austria, and other countries. A large outbreak due to STEC O104:H4 linked to sprouts from fenugreek seeds occurred in Europe in 2011, and it involved close to 4000 cases of illness, 855 cases of hemolytic uremic syndrome, and over 50 deaths. The DNA sequence of the genome of the outbreak strain revealed that it carried virulence genes associated with both STEC (stx2, iha, lpfO26, lpfO113) and enteroaggregative E. coli (EAEC) (aggA, aggR, set1, pic, aap) (Bielaszewska et al., 2011). Studies confirmed that E. coli O104:H4 is an EAEC that had increased pathogenicity due to transfer of the gene encoding for Shiga toxin 2 (stx2) and antibiotic resistance genes. Similar to STEC O91 and O113, STEC O104 strains that have caused outbreaks and cases of human illness do not carry the eae gene that encodes for intimin; however, many carry the STEC enterohemolysin gene (ehxA). The objective of our research was to develop a methodology to enhance the ability to detect and isolate STEC O104 by incorporating immunomagnetic separation (IMS) for concentration of the target pathogen and latex agglutination to confirm presumptive positive colonies picked from selective-differential agars. The O104 IMS and latex agglutination kits were developed through a collaboration between our laboratory and Abraxis, LLC, and they are now commercially available. In addition, multiplex PCR assays were designed to target the E. coli O104:H4 enteroaggregative STEC strain that caused the outbreak associated with sprouts in 2011 in Europe, as well as STEC O104 strains. The assay for the enteroaggregative STEC O104:H4 targeted the stx2, wzx104, and aggR genes, and the PCR assay for STEC O104 targeted stx1-2, ehxA, and wzx104. The results obtained for sprouts inoculated at the different levels of contamination are shown in Table 1. All samples inoculated at the higher level (24 -160 CFU/25 g) were positive by the PCR assays, and presumptive positive colonies were identified as the target pathogen by latex agglutination and the PCR assays. Samples inoculated with lower levels (= 4 CFU/25 g) were also positive; however, a number of inoculated samples were negative by the PCR (Table 1). No mauve colonies were found on both of the selective agars from the PCR-negative samples. It is unclear why some inoculated samples and not others that were inoculated with low levels of the pathogens gave negative results; samples inoculated with 4 and 9 CFU of O104:H4 were positive by the PCR. It is possible that the incubation at 4°C for 2 days may have injured the bacteria in some of the samples, and they did not recover during enrichment. The limit of detection of the real-time multiplex real-time PCR assay for E. coli O104:H4 was =103 CFU/ml, and for E. coli O104:H7, the detection limit was =104 CFU/ml, although some samples tested showed detection limits that were 10-fold lower for both strains. The results were similar in samples consisting of enrichment broth to which the O104 strains were added and uninoculated sprout enrichments into which the E. coli strains were seeded at the different dilutions. Regardless of the PCR screening results, all sprouts samples underwent IMS, and presumptive positive mauve colonies were tested by latex agglutination and the multiplex PCR assays. The E. coli O104 strain was isolated on CHROMagar and/or mRBA, and the colonies were subsequently confirmed from all PCR-positive samples. Two multiplex PCR assays targeting STEC and enteroaggregative-STEC belonging to serogroup O104, as well as commercially available O104 IMS and latex agglutination reagents for detection, isolation, and identification of the pathogens from artificially contaminated sprouts were evaluated in this study. The multiplex PCR assays can be used to determine the presence of specific virulence genes of enteroaggregative-STEC and STEC O104, as well as an O104 serogroup-specific gene. The pathogens were detected and isolated in sprout samples inoculated with less than 1 CFU/g and subjected to a cold stress treatment for 2 days using a combination of enrichment in mBPWp + ACV for 18 h, the real-time multiplex PCR assays, IMS, and isolation on selective agars. The beads were able to concentrate the target bacteria and allow for removal of background organisms, and presumptive colonies were easily identified and confirmed on selective-differential agars using O104 latex particles and the multiplex PCR assays, notwithstanding a high level of background flora in the sprouts based on the aerobic plate counts. Thus, the IMS beads and the latex reagents linked with antibodies against E. coli O104 used in this study can enhance the ability to detect E. coli belonging to this serogroup.

Last Modified: 12/18/2014
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