Molecular characterization of clinical isolates of Aeromonas species from Malaysia

Abstract

Background: Aeromonas species are common inhabitants of aquatic environments giving rise to infections in both fish and humans. Identification of aeromonads to the species level is problematic and complex due to their phenotypic and genotypic heterogeneity.

Methodology/principal findings: Aeromonas hydrophila or Aeromonas sp were genetically re-identified using a combination of previously published methods targeting GCAT, 16S rDNA and rpoD genes. Characterization based on the genus specific GCAT-PCR showed that 94 (96%) of the 98 strains belonged to the genus Aeromonas. Considering the patterns obtained for the 94 isolates with the 16S rDNA-RFLP identification method, 3 clusters were recognised, i.e. A. caviae (61%), A. hydrophila (17%) and an unknown group (22%) with atypical RFLP restriction patterns. However, the phylogenetic tree constructed with the obtained rpoD sequences showed that 47 strains (50%) clustered with the sequence of the type strain of A. aquariorum, 18 (19%) with A. caviae, 16 (17%) with A. hydrophila, 12 (13%) with A. veronii and one strain (1%) with the type strain of A. trota. PCR investigation revealed the presence of 10 virulence genes in the 94 isolates as: lip (91%), exu (87%), ela (86%), alt (79%), ser (77%), fla (74%), aer (72%), act (43%), aexT (24%) and ast (23%).

Conclusions/significance: This study emphasizes the importance of using more than one method for the correct identification of Aeromonas strains. The sequences of the rpoD gene enabled the unambiguous identication of the 94 Aeromonas isolates in accordance with results of other recent studies. Aeromonas aquariorum showed to be the most prevalent species (50%) containing an important subset of virulence genes lip/alt/ser/fla/aer. Different combinations of the virulence genes present in the isolates indicate their probable role in the pathogenesis of Aeromonas infections.

Figure 1. Polyacrylamide gel showing 16S rDNA-RFLP patterns (AluI and MboI).

L: pBR322 DNA/BsuRI marker (Fermentas, USA), Lane 1: typical pattern of A. hydrophila (JN 686656), Lane 2: typical pattern of A. caviae (JN 686668), Lane 3: atypical pattern of A. trota (JN 686649), Lanes 4–6: atypical pattern of A. veronii (JN 686665, JN 686691, JN 686739), Lanes 7–10: A. aquariorum (JN 686662, JN 686731, JN 686725, JN 686700).

Figure 2. Phylogenetic relationship of the rpoD sequences between 94 Aeromonas isolates and 9 references strains using neighbor-joining method.

Numbers next to nodes indicate percentage bootstrap values of 5000 replicates.