Rapid Protocol for Characterizing Klebsiella pneumoniae Isolates by Pulsed Field Gel Electrophoresis (PFGE) in Contour Clamped Homogeneous Electric Field (CHEF) Minigels

Abstract

Klebsiella pneumoniae strains 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 for K. pneumoniae subtyping and for the laboratory confirmation of outbreaks. We report the implementation of a rapid protocol for characterizing K. pneumoniae isolates 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 of K. pneumoniae DNA 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 further XbaI DNA 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 of K. pneumoniae and three of K. oxytoca. The XbaI DNA fingerprint analysis in CHEF mini gels was able to discriminate the seven isolates of K. pneumoniae that were epidemiologically related and the nineteen that were unrelated. The nonenzymatic DNA plugs preparation reported in this paper is a cost-effective alternative for K. pneumoniae PFGE subtyping in laboratories where the technique is already implemented.

Keywords:

• Bacterial subtyping
• contour clamped homogeneous electric field (CHEF) mini gel
• Klebsiella pneumoniae
• nonenzymatic DNA preparation
• pulsed-field gel electrophoresis (PFGE)

Acknowlegments

We thank Dr Humberto Barrios-Camacho for providing us with the K. pneumoniae strain with a hypervirulent and hypermucoviscous phenotype. We also gratefully acknowledge Mr. Alfredo Padilla for his assistance in composing the figures.

Additional information

Funding

This work was supported by the Science and Technology College of Autonomous University of Mexico City.