Comparison of amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism fingerprinting for identification of Acinetobacter genomic species and typing of Acinetobacter baumannii

Abstract

Thirty-one strains of Acinetobacter species, including type strains of the 18 genomic species and 13 clinical isolates, were compared by amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA analysis (RAPD), and amplified fragment length polymorphism (AFLP) fingerprinting. ARDRA, performed with five different enzymes, showed low discriminatory power for differentiating Acinetobacter at the species and strain level. The standardized commercially available RAPD kit clearly enabled the discrimination of all Acinetobacter genomic species but showed great polymorphism between isolates of Acinetobacter baumannii. AFLP fingerprinting with radioactively as well as fluorescently labelled primers showed high discriminatory power for the identification of 18 Acinetobacter genomic species and typing of 13 clinical Acinetobacter isolates. Compared to radioactive AFLP, fluorescent AFLP was technically fast and simple to perform, and it permitted analysis with an automated DNA sequencer. Fluorescent AFLP seems particularly well suited for studying the epidemiology of nosocomial infections and outbreaks caused by Acinetobacter species.

FIG. 1

Digitized ARDRA patterns and dendrogram of 18 Acinetobacter genomic species (1 to 18) and 13 clinical Acinetobacter isolates (19* to 31*) obtained after restriction of the amplified 16S rRNA gene with five different enzymes (AluI, CfoI, MboI, MspI, and RsaI). For each strain, all restriction patterns have been combined into one single lane. The dendrogram was constructed with Gelcompar cluster analysis by UPGMA. Percentages of similarity and molecular weights are shown above the dendrogram. The strain code as presented in Table 1 is shown on the right.

FIG. 2

Digitized RAPD patterns and dendrogram of 18 Acinetobacter genomic species (1 to 18) and 13 clinical Acinetobacter isolates (19* to 31*) obtained after separate PCRs with six different primers. For each strain, all six RAPD patterns have been combined into one single lane (1 to 6). The dendrogram was constructed with Gelcompar cluster analysis by UPGMA. Percentages of similarity and molecular weights are shown above the dendrogram. The strain code as presented in Table 1 is shown on the right.

FIG. 3

Digitized radioactively labelled AFLP patterns and dendrogram of 18 Acinetobacter genomic species (1 to 18) and 13 clinical Acinetobacter isolates (19* to 31*) obtained after PCR on EcoA and MseC templates. The dendrogram was constructed with Gelcompar cluster analysis by UPGMA. Percentages of similarity and molecular weights are shown above the dendrogram. The strain code as presented in Table 1 is shown on the right.

FIG. 4

Fluorescently labelled AFLP patterns and dendrogram of 18 Acinetobacter genomic species (1 to 18) and 13 clinical Acinetobacter isolates (19* to 31*) obtained after PCR on EcoA and MseC templates. The dendrogram was constructed with Gelcompar cluster analysis by UPGMA. Percentages of similarity and molecular weights are shown above the dendrogram. The strain code as presented in Table 1 is shown on the right.