Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.A eromonas spp. are highly adapted to aquatic environments and have been described as pathogenic to humans and animals. The genus Aeromonas consists of more than 20 valid species, of which A. hydrophila, A. caviae (synonymous with A. punctata), A. media, A. veronii bv. sobria, and A. veronii bv. veronii are of particular clinical significance, because they can cause gastroenteritis, wound and soft tissue infections, and septicemia (1). Aeromonas spp. may produce an array of virulence factors (e.g., cytolytic toxins with hemolytic activity and enterotoxins). Recent reviews suggested that only a subset of Aeromonas spp. are truly pathogenic and may be transmitted by hitherto unknown routes, and they proposed that further epidemiological and molecular studies are needed (2, 3). Aeromonas species identification has traditionally been performed by a combination of different biochemical tests. However, these are not always conclusive, since some Aeromonas species display heterogeneous biochemical properties; compared to molecular methods, the correct identification rate with biochemical tests has been shown to be very low (4, 5). Molecular species identification has been exploited by the 16S rRNA gene, either by restriction fragment length polymorphism (RFLP) (6-9) or direct sequencing (10, 11). However, due to insufficient interspecies sequence variation and heterogeneity among copies of ribosomal RNA operons in the same bacteria (10, 12, 13), this gene may not be an optimal target. A number of studies have shown that the sequences of several different housekeeping genes are able to differentiate this tight taxonomic group of organisms. These genes include an RNA polymerase B subunit (rpoB), an RNA polymerase D subunit (rpoD), and a DNA gyrase B subunit (gyrB) (10, 14-16). The objective of the present study was to identify species of Aeromonas by partial gyrB and rpoB sequencing in order to develop a multiplex PCR (mPCR) that targets the four most prevalent and clinically relevant species identified by sequence analysis on a Danish strain collection.The strain collection used in this study was composed of 51 Aeromonas spp. collected from diarrheagenic patients during the period of 2005 to 2010. Each stool specimen was grown on enteric medium (Statens Serum Institut, Hillerød, Denmark) (17), and Aeromonas was identified by its distinct colony morphology, while further delineation of clinically relevant species was performed manually according to their biochemical characteristics, including Voges-Proskauer test results and lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, glucose (gas), esculin, and acid production fr...