Detecting Enterotoxigenic Escherichia coli in animal production: method development and validation

Abstract

Swine enteric colibacillosis is a disease characterized by an intestinal infection caused by the colonization of enterotoxigenic Escherichia coli (ETEC). This infection mostly causes illness or death in neonatal and weaned pigs making it responsible for significant economic losses worldwide [1,2]. Bacterial fimbriae (F4/F5/F6/F18) allow the adhesion of the bacteria to epithelial cells, and when both the immunological systems and the gut microbiota are poorly developed, ETEC colonizes and produces one or more enterotoxins (LT/STa/STb) that can have local and systemic effects [3,4]. Therefore, it is of prime importance to monitor and characterize ETEC in the swine industry to develop mitigation strategies. In this study, we aimed to isolate and characterize ETEC strains from faecal porcine swabs. As such, it was developed a methodology to detect the presence of ETEC and their major virulence factors (toxins/fimbriae). This procedure was divided into two phases, firstly the collected swabs were enriched and then then screened for the presence of genetic determinants of toxins (ST/LT/stx2) by real-time PCR (qPCR). Secondly, the positive enrichments were plated in Tryptone Bile X-glucuronide (TBX) agar and incubated at 37 ºC for 24 hours. Fifty characteristic E. coli colonies were then extensively screened for the presence of toxins (Sta/STb/LT/stx2e) and fimbriae (F4/F5/F6/F18/F41) by multiplex-PCR. The development of both qPCR/multiplex-PCR methods, as well as the optimization of the enrichment step, was done using ETEC controls harbouring the above-mentioned toxins/fimbriae. Nonselective and selective (with novobiocin) enrichment in TSB was performed by using ETEC inoculated faeces samples; with the 24h-selective enrichment providing higher ETEC recovery rates. Optimized qPCR conditions for toxins detection were as follow: 95 ºC for polymerase activation/denaturation, 60 ºC for annealing/extension during 40 cycles, and an internal control (pUC19 DNA) was used in each reaction. Multiplex-PCR was optimized through the conditions, 95 ºC initial denaturation and 35 cycles of 94 ºC denaturation, 60 ºC for annealing and 72 ºC for extension/final extension. In sum, this methodology has the potential to be adopted as a routine technique for the rapid detection of ETEC strains in livestock.