VALIDATION OF USE OF WHOLE-CELL REPETITIVE EXTRAGENIC PALINDROMIC SEQUENCE-BASED PCR (REP-PCR) FOR TYPING STRAINS BELONGING TO THE ACINETOBACTER-CALCOACETICUS ACINETOBACTER-BAUMANNII COMPLEX AND APPLICATION OFTHE METHOD TO THE INVESTIGATION OF A HOSPITAL OUTBREAK

Acinetobacter spp. are being reported with increasing frequency as cau ses of nosocomial infection. In order to identify reservoirs of infect ion as quickly as possible, a rapid typing method that can differentia te epidemic strains from environmental and nonepidemic strains is need ed, In 1993, a cluster of Acinetobacter baumannii isolates from five p atients in the adult intensive therapy unit of our tertiary-care teach ing hospital led us to develop and optimize a rapid repetitive extrage nic palindromic sequence-based PCR (REP-PCR) typing protocol for membe rs of the Acinetobacter calcoaceticus-A. baumannii complex that uses b oiled colonies and consensus primers aimed at repetitive extragenic pa lindromic sequences, Four of the five patient isolates gave the same R EP-PCR typing pattern as isolates of A. baumannii obtained from the te mperature probe of a Bennett humidifier; the fifth isolate had a uniqu e profile, Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessit ating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the di scriminatory power (D) of REP-PCR for typing members of the A. calcoac eticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included repr esentatives of six DNA groups within the complex, Ribotyping using Eco RI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PC R patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than ribotyp ing. Our results indicate that REP-PCR typing using boiled colonies is a simple, rapid, and effective means of typing members of the A. calc oaceticus-A. baumannii complex.