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Journal of Clinical Microbiology, September 2005, p. 4328-4335, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4328-4335.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Standardization and Interlaboratory Reproducibility Assessment of Pulsed-Field Gel Electrophoresis-Generated Fingerprints of Acinetobacter baumannii

Harald Seifert,1* Lucilla Dolzani,2 Raffaela Bressan,2 Tanny van der Reijden,3 Beppie van Strijen,3 Danuta Stefanik,1 Herre Heersma,4 and Lenie Dijkshoorn3

Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany,1 Dipartimento di Scienze Biomediche, University of Trieste, Trieste, Italy,2 Leiden University Medical Center, Leiden,3 National Institute for Public Health and the Environment, Bilthoven, The Netherlands4

Received 21 February 2005/ Returned for modification 29 April 2005/ Accepted 1 June 2005

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

* Corresponding author. Mailing address: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany. Phone: 0049 221 4783009. Fax: 0049 221 4783979. E-mail: harald.seifert{at}uni-koeln.de.

Journal of Clinical Microbiology, September 2005, p. 4328-4335, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4328-4335.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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