Improving Detection of Shiga Toxin-Producing Escherichia coli by Molecular Methods by Reducing the Interference of Free Shiga Toxin-Encoding Bacteriophages

摘要

Detection of Shiga toxin-producing Escherichia coli (STEC) by culture methods is advisable to identify the pathogen, but recovery of the strain responsible for the disease is not always possible. The use of DNA-based methods (PCR, quantitative PCR [qPCR], or genomics) targeting virulence genes offers fast and robust alternatives. However, detection of stx is not always indicative of STEC because stx can be located in the genome of temperate phages found in the samples as free particles; this could explain the numerous reports of positive stx detection without successful STEC isolation. An approach based on filtration through low-protein-binding membranes and additional washing steps was applied to reduce free Stx phages without reducing detection of STEC bacteria. River water, food, and stool samples were spiked with suspensions of phage 933W and, as a STEC surrogate, a lysogen harboring a recombinant Stx phage in which stx was replaced by gfp. Bacteria were tested either by culture or by qPCR for gfp while phages were tested using qPCR targeting stx in phage DNA. The procedure reduces phage particles by 3.3 log₁₀ units without affecting the recovery of the STEC population (culturable or assessed by qPCR). The method is applicable regardless of phage and bacteria densities and is useful in different matrices (liquid or solid). This approach eliminates or considerably reduces the interference of Stx phages in the detection of STEC by molecular methods. The reduction of possible interference would increase the efficiency and reliability of genomics for STEC detection when the method is applied routinely in diagnosis and food analysis.