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

Members of the genusAcinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotypeAcinetobacter 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 theAcinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE ,adk ,efp ,mutY ,fumC , andppa ) from 267 isolates ofAcinetobacter . 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 wereAcinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterizedA. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing differentAcinetobacter species, including 8 representatives ofAcinetobacter genomospecies 13TU and 13 representatives ofAcinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of thetrpE ,adk ,mutY ,fumC , andppa genes distinguished 47 of the 48A. baumannii genotypes identified by sequencing and identified at the species level at least 18Acinetobacter 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.