Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic escherichia coli in sprouts

Authors: ROTUNDO, LUCA - University Of Urbino; AMAGLIANI, GIULIA - University Of Urbino; CARLONI, ELISA - University Of Urbino; OMICCIOLI, ENRICA - Diatheva Srl; MAGNANI, MAURO - University Of Urbino; Paoli, George

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2018
Publication Date: 11/13/2018
Citation: Rotundo, L., Amagliani, G., Carloni, E., Omiccioli, E., Magnani, M., Paoli, G. 2018. Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic escherichia coli in sprouts. International Journal of Food Microbiology. 291:59-64. https://doi.org/10.1016/j.ijfoodmicro.2018.11.011.
DOI: https://doi.org/10.1016/j.ijfoodmicro.2018.11.011

Interpretive Summary: Foodborne illness is a serious public health concern in the US and worldwide. Food regulatory agencies and public health agencies routinely monitor foods for the presence of known pathogens and track reportable human illnesses in their efforts to prevent and identify outbreaks of foodborne illness. In 2011, contaminated sprouts were identified as the cause of an outbreak of foodborne illness that sickened nearly 4000 people in Germany and other European countries, resulting in 855 cases of severe kidney disease and 53 deaths. This outbreak was caused by a previously unidentified and particularly virulent type of E. coli. Here we report a rapid, sensitive, and specific DNA-based method for the detection of this deadly pathogen from contaminated sprouts. The described method will be useful for researchers, the food industry, and regulatory agencies to determine the distribution of this new deadly type of E.coli in sprouts and other foods, as well as a for the detection of the pathogen to prevent distribution and consumption of contaminated food.

Technical Abstract: In this study real-time PCR assays were evaluated for the detection of EAHEC O104:H4 in artificially contaminated mung bean and alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting aggR/aaiC, stx/eae, and wzxO104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75-80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen’s kappa value between 0.61-1). In addition, the real-time PCR assay targeting the aggR and aaiC genes, indicative of EAggEC, was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts.