|ROTUNDO, LUCA - Collaborator|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/15/2018
Publication Date: 7/9/2018
Citation: Rotundo, L., Paoli, G. 2018. Evaluation of PCR-based methods for the identification of EAggEC in Sprouts. Meeting Abstract. abstract.
Technical Abstract: Introduction: Enteroaggregative E. coli (EAggEC) and Shiga toxin-producing E. coli (STEC) have been recognized worldwide as causes of foodborne gastroenteritis for the past three decades. In 2011 a hemorrhagic EAggEC (EAHEC)O104:H4 strain, having acquired a gene encoding Shiga toxin 2, caused an outbreak originating from contaminated fenugreek seeds in Germany resulting in 4,000 cases of gastroenteritis, 855 cases of haemolytic uremic syndrome and 53 deaths. The EFSA Scientific Opinion 2013 and EU Regulation 209/2013 suggest STEC and EAggEC molecular detection criteria and zero tolerance for the pathogens in seeds and sprouts, respectively. Purpose: This study was conducted to evaluate a real-time PCR method for detecting EAggEC contamination in sprouts. Methods: Inclusivity tests were conducted using a real-time PCR kit targeting aggR and aaiC that was provided by Diatheva Srl (Italy). Twenty five grams of mung bean and alfalfa sprouts were artificially contaminated at 1, 10, and 100 CFU (20, 10, and 2 replicates respectively) of EAHEC O104:H4. Samples were enriched as described in the FDA-BAM, Chapter 4A. Culture enrichments were tested using real-time PCR kits, provided by Diatheva, detecting aggR/aaiC, stx/eae, and wzxO104. For bacterial isolation enrichment cultures were subjected to IMS using anti-O104 magnetic beads (Abraxis, USA) and plated on mRBA and CHROMagar STEC. Presumptive O104 colonies were confirmed by latex agglutination (Abraxis, USA) and end-point PCR (stx, aggR, and wzxO104). Results: - The inclusivity and exclusivity was 100% using a panel of 49 strains. - Using the commercial real-time PCR kits, the artificially contaminated samples were 60-80% positive when contaminated with 1 CFU, and 100% at 10-100 CFU. Microbiological detection and confirmation by latex agglutination and PCR gave similar results (Cohen’s kappa value between 0.8-1). Significance: This study demonstrates the efficacy of the real-time PCR method for the specific and sensitive detection of EAHEC from spouts.