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Journal of Clinical Microbiology, August 2006, p. 2921-2932, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00619-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Acinetobacter Species and Genotyping of Acinetobacter baumannii by Multilocus PCR and Mass Spectrometry

Joseph A. Ecker,1 Christian Massire,1 Thomas A. Hall,1 Raymond Ranken,1 Thuy-Trang D. Pennella,1 Cristina Agasino Ivy,1 Lawrence B. Blyn,1 Steven A. Hofstadler,1 Timothy P. Endy,2 Paul T. Scott,2 Luther Lindler,2 Tacita Hamilton,2 Charla Gaddy,2 Kerry Snow,3 Marie Pe,3 Joel Fishbain,3 David Craft,3 Gregory Deye,4 Scott Riddell,4 Eric Milstrey,5 Bruno Petruccelli,5 Sylvain Brisse,6 Vanessa Harpin,1 Amy Schink,1 David J. Ecker,1 Rangarajan Sampath,1 and Mark W. Eshoo1*

Ibis Biosciences, Isis Pharmaceuticals, Inc., 1891 Rutherford Road, Carlsbad, California 92008,1 Walter Reed Army Institute for Research, Silver Spring, Maryland 20910,2 Walter Reed Army Medical Center, Washington, D.C. 20307,3 Landstuhl Regional Medical Center, Landstuhl, Germany,4 Aberdeen Proving Ground, Aberdeen, Maryland,5 Unité Biodiversité des Bactéries Pathogènes Emergentes (U389 INSERM), Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France6

Received 22 March 2006/ Returned for modification 11 May 2006/ Accepted 26 May 2006

Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.

* Corresponding author. Mailing address: Ibis Biosciences, Isis Pharmaceuticals, Inc., 1891 Rutherford Road, Carlsbad, CA 92008. Phone: (760) 603-3892. Fax: (760) 603-4653. E-mail: meshoo{at}isisph.com.

Journal of Clinical Microbiology, August 2006, p. 2921-2932, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00619-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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