title: 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 of the method to the investigation of a hospital outbreak.
abstract: Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique 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, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR 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 ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex.
authors.affiliation: Department of Microbiology, University of Leeds, United Kingdom.
authors.forename: A M
authors.initials: AM
authors.lastname: Snelling
authors.forename: P
authors.initials: P
authors.lastname: Gerner-Smidt
authors.forename: P M
authors.initials: PM
authors.lastname: Hawkey
authors.forename: J
authors.initials: J
authors.lastname: Heritage
authors.forename: P
authors.initials: P
authors.lastname: Parnell
authors.forename: C
authors.initials: C
authors.lastname: Porter
authors.forename: A R
authors.initials: AR
authors.lastname: Bodenham
authors.forename: T
authors.initials: T
authors.lastname: Inglis
mesh_terms: D000150:Acinetobacter / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D000151:Acinetobacter Infections / Q000453:epidemiology* / Q000382:microbiology*; D016954:Acinetobacter calcoaceticus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D000328:Adult; D015373:Bacterial Typing Techniques; D001483:Base Sequence; D003428:Cross Infection / Q000453:epidemiology* / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004196:Disease Outbreaks; D005069:Evaluation Studies as Topic; D006801:Humans; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial