Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa Isolates from the ocean

Abstract

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.

FIG. 1.

Correlation between the geographical locations (sampling points) of P. aeruginosa strains and their positions in the phylogenetic trees. (A) Map showing the origins and sampling sites of clinical and coastal strains in Japan outside the Tokyo area. (B) Map showing the sampling sites in the Pacific Ocean, coastal, and freshwater environments in and around Tokyo, Japan. The figure shows the path of the Kuroshio Current as of the sampling time in 2003 during the Tansei Maru cruise in the Pacific Ocean. AB, Aburatsubo Bay; AK, Arakawa River; TL, Tama Lake; and ZP, Zenpukujii Pond. (C) Unrooted tree of the concatenated nucleotide sequences of seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) of P. aeruginosa obtained using the NJ method with Kimura 2 correction for distance calculations. The total length of the concatenated group of nucleotide sequences was 5,700 bp. Bootstrap percentages retrieved in 1,000 replications are shown at the nodes. Clusters and bootstrap values that were 50% or greater are indicated. The scale bar (0.001) indicates the number of nucleotide substitutions per site. (D) The tree was constructed using the MP method with the same data set. Bootstrap percentages retrieved in 1,000 replications are shown at the nodes. Clusters and bootstrap values that were 50% or greater are indicated.

FIG. 2.

Rooted tree of 16 rRNA gene sequence data of P. aeruginosa along with data on other Pseudomonas spp. obtained using the NJ method with Kimura 2 correction for distance calculations. Bootstrap percentages retrieved in 1,000 replications are shown at the nodes. Clusters and bootstrap values that were 50% or greater are indicated. The scale bar (0.005) indicates the number of nucleotide substitutions per site. Data for P. aeruginosa JCM 5962^T were collected from the DDBJ database, and data for all outgroup strains were collected from the NCBI GenBank database.

FIG. 3.

Rooted tree of the concatenated nucleotide sequences of seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) of P. aeruginosa obtained using the NJ method with Kimura 2 correction for distance calculations. The total length of the concatenated group of nucleotide sequences was 5,700 bp. Bootstrap percentages retrieved in 1,000 replications are shown at the nodes. Clusters and bootstrap values that were 50% or greater are indicated. The scale bar (0.02) indicates the number of nucleotide substitutions per site.

FIG. 4.

Unrooted and NJ radiation tree based on concatenated MLST sequences of seven gene loci. acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE gene sequences (2,873 bp) from 63 taxa (34 from the present study, 28 from the MLST database, and 1 P. aeruginosa PAO1 strain from the database) were concatenated and constructed by the MEGA 3.1 program. Sequence similarity was corrected for by using the Kimura 2 parameter. All nodes were supported by 1,000 bootstrap replications. The largest cluster was marked as open ocean, as all strains were from the ocean, and two other clusters were marked as coastal. The scale bar (0.001) indicates the number of nucleotide substitutions per site.