Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii

Abstract

In this study a multilocus sequence typing (MLST) scheme for Acinetobacter baumannii was developed and evaluated by using 40 clinical A. baumannii isolates recovered from outbreaks in Spanish and German hospitals during the years 1990 to 2001, as well as isolates from other European hospitals and two DSMZ reference strains of A. baumannii. For comparison, two isolates of Acinetobacter species 13 (sensu Tjernberg and Ursing), two clinical isolates, and three DSMZ strains of A. calcoaceticus (both belonging to the A. calcoaceticus-A. baumannii complex) were also investigated. Primers were designed for conserved regions of housekeeping genes, and 305- to 513-bp internal fragments of seven such genes-gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD-were sequenced for all strains. The number of alleles at individual loci ranged from 6 to 12, and a total of 20 allelic profiles or sequence types were distinguished among the investigated A. baumannii strains. The MLST data were in high concordance with the epidemiologic typing results generated by pulsed-field gel electrophoresis and amplified fragment length polymorphism fingerprinting. The MLST scheme provides a high level of resolution and an excellent tool for studying the population structure and long-term epidemiology of A. baumannii.

FIG. 1.

Dendrogram constructed by UPGMA cluster analysis based on the nucleotide differences obtained by sequencing of gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD gene fragments. From left to right are indicated isolate number, species identification (A.b, A. baumannii; 13TU, Acinetobacter genomic species 13TU; A.c, A. calcoaceticus), PFGE type (boxed capital letters), ST, and allelic profile.

FIG. 2.

Relative location of the selected housekeeping genes used in the present study on a schematic map of the genome of Acinetobacter strain ADP1 (NC_005966.1). The A. baumannii cpn60 gene is homologous to chaperone Hsp60 from ADP1. Genes used in the present MLST scheme are underlined. The coordinates are given in bases, and the protein accession numbers are indicated.

FIG. 3.

Fluorescently labeled AFLP patterns and UPGMA/product-moment cluster analysis of the 49 Acinetobacter strains. Twenty-seven different patterns were obtained after PCR with EcoC and MseT templates among all isolates. Levels of correlation are expressed as percentages of similarity. The known PFGE types are indicated inside boxes in capital letters for comparison. AFLP types are indicated at the far right side.