Standardized Pulsed-Field Gel Electrophoresis of Shiga Toxin–Producing Escherichia coli: The PulseNet Europe Feasibility Study

Abstract

PulseNet USA, the American molecular subtyping network for foodborne infections, has since 1996 been highly successful combating infections caused by Shiga toxin-producing Escherichia coli (STEC) O157, Salmonella, Listeria, and Shigella spp. The PulseNet Europe feasibility study was initiated to ascertain the interest of public health and veterinary reference laboratories to establish a similar network, and to determine if it was possible to perform standardized pulsed-field gel electrophoresis (PFGE) typing of Salmonella, Listeria, and STEC on a large scale in Europe. The results of the STEC part of that study are presented here. Twenty-seven veterinary and public health laboratories participated in the study. The participants subtyped eight E. coli strains by PFGE using the restriction enzyme XbaI according to the PulseNet or a similar protocol, with strict adherence to the electrophoretic conditions stated in the former and submitted an image of their gel for centralized anonymous analysis. The quality of the gels was first graded visually as "good," "intermediate," and "unsatisfactory." The number of gels graded this way was 11, 14, and 2, respectively. All "good" and "intermediate" gels were also analysed and compared by computerized analysis to a reference gel. For gels graded "good," on average 5.6, 7.4, and 8 patterns out of 8 per gel were identified with a similarity of 100%, >95%, and >90%, respectively. The corresponding numbers for gels graded "intermediate" were 1.7, 4.9, and 7.4, respectively. The problems causing the grading to be "intermediate" was overloaded lanes, overexposed images, not optimally focused images and incomplete digestion, all problems that led to misinterpretation of the number of restriction fragments in the gel. These problems may be corrected by simple adjustments to the subtyping procedure. Thus, there seems to be little need for training of the participants in PulseNet Europe.