Using hydrochloric acid and bile resistance for optimized detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from sprouts

Abstract

Shiga toxin-producing Escherichia coli (STEC) in sprouts have caused large scale outbreaks in the past involving severe illness. The combination of this very diverse pathogen and a food matrix with high numbers of background microbiota poses a particular challenge for detection and isolation. An acid treatment of the enrichment before plating on agar has been shown to improve the recovery of STEC from sprouts. After enrichment in buffered peptone water (BPW) at 37 °C we applied an acid treatment, followed by plating on tryptone bile x-glucuronide (TBX) agar (acid bile method). An inter-laboratory study was organized with 21 laboratories taking part to evaluate the performance parameters and applicability of the acid bile method. A sample set of six sprout samples was prepared consisting of two uninoculated samples and four spiked samples, each containing one of two STEC strains at one of two concentrations (low and high). Analyzing a set of six samples at the National Reference Laboratory (NRL E. coli), we determined the relative abundance of STEC without, after acid-, after bile- and after acid-bile treatment using real-time PCR. The participating laboratories successfully applied the acid bile method and were better able to detect (sensitivity 92.9% vs. 70.0%) and isolate (sensitivity 87.5% vs. 31.3%) STEC from positive samples using the acid bile method compared to non-acid methods. The relative limit of detection (RLOD) after isolation using the acid bile method (vs. non-acid method) was <1 for both STEC strains used, BfR-EC-14434 O133:H25 (0.146) and BfR-EC-16015 O26:H11 (0.073). A collection of STEC (n = 71) of diverse type and characteristics was assessed for their resistance towards the acid bile treatment selection. The majority (n = 65) of STEC strains could be recovered after acid treatment on TBX plates. However, a few strains (n = 6), among them clinical isolates were (partly) sensitive. These results suggest that an acid bile method is a rapid and reasonable approach to improve the recovery of STEC from sprouts when used in combination with methods targeting other selection markers.