Rapid Protocol For Characterizingklebsiella Pneumoniaeisolates By Pulsed Field Gel Electrophoresis (Pfge) In Contour Clamped Homogeneous Electric Field (Chef) Minigels

Klebsiella pneumoniaestrains may act as opportunistic pathogens infecting susceptible patients, or as hypervirulent germs infecting healthy people, or as multidrug-resistant organisms due to extended-spectrum beta-lactamase (ESBL)-production provoking infection outbreaks in hospital settings and communities. Consequently, it is a priority to encourage the development of simple, rapid and economical methods for the molecular subtyping of the outbreak strains and then to facilitate the infection control. Pulsed field gel electrophoresis (PFGE) remains a good choice forK. pneumoniaesubtyping and for the laboratory confirmation of outbreaks. We report the implementation of a rapid protocol for characterizingK. pneumoniaeisolates by contour clamped homogeneous electric field (CHEF) in mini gels in a total analysis time of less than 12 h. This approach is based on the preparation ofK. pneumoniaeDNA of PFGE-quality by a single incubation step of immobilized bacteria for 2 h with a nonenzymatic solution containing 4 M urea and nonionic detergents with furtherXbaIDNA fingerprint resolution in CHEF mini gels in 5.5 h. The DNA suitable for PFGE obtained by this procedure was approximately 91% of the total DNA in the plugs. This procedure enabled the reproducible subtyping of 26 isolates ofK. pneumoniaeand three ofK. oxytoca. TheXbaIDNA fingerprint analysis in CHEF mini gels was able to discriminate the seven isolates ofK. pneumoniaethat were epidemiologically related and the nineteen that were unrelated. The nonenzymatic DNA plugs preparation reported in this paper is a cost-effective alternative forK. pneumoniaePFGE subtyping in laboratories where the technique is already implemented.