Detection of Shiga toxin-producing Escherichia coli (STEC) in food: evaluation of culture enrichment conditions

Authors: AMAGLIANI, GIULIA - University Of Urbino; ROTUNDO, LUCA - University Of Urbino; CARLONI, ELISA - University Of Urbino; OMICCIOLI, ENRICA - Diatheva Srl; MAGNANI, MAURO - University Of Urbino; BRANDI, GIORGIO - University Of Urbino; Fratamico, Pina

Submitted to: Food Research International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/30/2017
Publication Date: 1/1/2018
Citation: Amagliani, G., Rotundo, L., Carloni, E., Omiccioli, E., Magnani, M., Brandi, G., Fratamico, P.M. 2018. Detection of Shiga toxin-producing Escherichia coli (STEC) in food: evaluation of culture enrichment conditions. Food Research International. 103:398-405.

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) infections are associated with severe diseases in humans, including hemorrhagic colitis and hemolytic uremic syndrome, and infection can result in death. Beef, produce, and other foods have been associated with foodborne outbreaks caused by various types of STEC, including serogroups O157, O26, O103, O111, O145, and O104. To prevent food contaminated with STEC from reaching the consumer, rapid and sensitive methods that can be used by regulatory agencies and the food industry to detect these important pathogens are needed. In this work, various types of growth media/broths and growth temperatures were tested for their ability to allow optimal growth of the STEC serogroups during what is referred to as culture enrichment prior to detection by the polymerase chain reaction. Overall, more rapid multiplication of the STEC serogroups occurred in a growth media referred to as mBPWp+CV and mTSB+N2 and at a growth temperature of 42 deg C or 37 deg C followed by 44 deg C, and the STEC could be detected in the food at a level as low as 1 – 10 bacteria per 25 grams of food. Thus, growth media and protocols should be adapted to the food being analyzed, since protocols provided in official reference methods may produce insufficient sensitivity. This work provides critical information to regulatory agencies and the food industry on the best methods to employ to ensure detection of low levels STEC in food.

Technical Abstract: The main purpose of this work was to evaluate culture enrichment conditions, with particular regard to those reported in ISO/TS 13136:2012, for STEC detection in food. The culture media evaluated included mTSB with novobiocin 0-16 mg/l (mTSB+N0-16) or acriflavin 12 mg/l (mTSB+A12); BPW; mBPWp with acriflavin 10 mg/l, cefsulodin 10 mg/l, vancomycin 8 mg/l (mBPWp+ACV); and mBPWp with cefsulodin 10 mg/l, vancomycin 8 mg/l (mBPWp+CV). They were used for the growth of STEC O157, O26, O103, O111, O145 and O104 in pure cultures or in artificially contaminated food matrices (ground beef, mung bean sprouts). STEC detection was accomplished using commercially available multiplex real-time PCR assays targeting stx1-stx2 and eae, and serogroup-associated genes. More rapid multiplication of STEC in pure cultures occurred in mBPWp+CV, while an inhibitory effect of novobiocin and acriflavin was observed for some STEC serogroups in media with these selective agents. mBPWp+CV allowed the detection of all serogroups in bean sprouts when inoculated at levels as low as 1 CFU/25 g. A reduced novobiocin concentration of 2 mg/L in mTSB was required for STEC detection in ground beef samples. A temperature of 42 deg C for the entire duration of the enrichment or 44 deg C after an initial phase of 6 h at 37 deg C was important to limit the multiplication of non-target bacteria. Results of this study suggest that media and protocols should be adapted to the food being analyzed, since protocols provided in official reference methods may produce insufficient sensitivity.