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Journal of Clinical Microbiology, February 2003, p. 798-802, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.798-802.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Use of Multienzyme Multiplex PCR Amplified Fragment Length Polymorphism Typing in Analysis of Outbreaks of Multiresistant Klebsiella pneumoniae in an Intensive Care Unit

Anneke van der Zee,* Niels Steer, Eveline Thijssen, Jolande Nelson, Annemarie van't Veen, and Anton Buiting

Laboratory of Medical Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands

Received 3 May 2002/ Returned for modification 4 August 2002/ Accepted 31 October 2002

We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearson's product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit and various sporadic isolates of K. pneumoniae and Klebsiella oxytoca. Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of K. pneumoniae. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of Klebsiella spp. The method can be performed in every clinical microbiology laboratory.

* Corresponding author. Mailing address: Laboratory of Medical Microbiology, St. Elisabeth Hospital, Tilburg, The Netherlands. Phone: 31 13 549 2676. Fax: 31 13 549 1264. E-mail: a.vd.zee{at}elisabeth.nl.

Journal of Clinical Microbiology, February 2003, p. 798-802, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.798-802.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • van 't Veen, A., van der Zee, A., Nelson, J., Speelberg, B., Kluytmans, J. A. J. W., Buiting, A. G. M. (2005). Outbreak of Infection with a Multiresistant Klebsiella pneumoniae Strain Associated with Contaminated Roll Boards in Operating Rooms. J. Clin. Microbiol. 43: 4961-4967 [Abstract] [Full Text]  
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