Isolation and identification of P. aeruginosa strains.

The isolation methods for P. aeruginosa were described previously (22). Several methods were applied for the identification of P. aeruginosa. Selective agar culture media were used for the identification of P. aeruginosa at the initial stage. Suspected colonies that showed the characteristic appearance and color on nalidixic acid cetrimide agar plates were inoculated onto cetrimide kanamycin nalidixic acid agar (26) and incubated at 42°C overnight. Isolates that showed growth at 42°C were primarily identified as P. aeruginosa. Colonies were transferred to other selective agar plates and incubated at 4°C for 3 to 4 days. No colonies appeared at 4°C. Sequences of the 16S rRNA gene were determined by direct sequencing using the primers 27f and 1492r (28). The PCR products were purified with an EXOSAP-IT kit (USB Corp., Cleveland, OH), sequenced using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster city, CA), and run on an ABI 3100 genetic analyzer (Applied Biosystems). BLAST programs were used to find the most closely related sequences (2). The isolates were also identified by the BD (Becton, Dickinson and Company, MD) Phoenix automated microbiology system according to the method previously described by Khan et al. (22). It is necessary to perform DNA-DNA hybridization to judge whether a novel isolate belongs to a species when the isolate and species representatives share more than 97% 16S rRNA gene sequence similarity (14, 48). Thus, two marine strains were randomly selected for DNA-DNA hybridization. P. aeruginosa JCM 5962^T and P. fluorescence JCM 5963^T were selected as the same species and a closely related species, respectively. DNA-DNA hybridization was determined by using photobiotin-labeled probes in a polystyrene 96-well plate (Nunc Maxisorp) as described by Ezaki et al. (11), utilizing Bacteroides sp. strain IAM 15343 as a negative control.

Culture of isolates and preparation of chromosomal DNA.

Bacterial strains were maintained at −80°C in 40% (vol/vol) glycerol in nutrient broth, streaked to single colonies, and cultured on Mueller-Hinton agar at 37°C under aerobic conditions. Chromosomal DNA was extracted from 1-ml cultures of strains grown overnight in nutrient broth by using a Qiagen DNeasy tissue extraction kit (Valencia, CA).

Locus selection.

The seven genes acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE (see Table ​Table3)3) were selected according to the MLST scheme for P. aeruginosa (http://pubmlst.org/paeruginosa/). The criteria governing locus selection included biological role (comprising genes with diverse central housekeeping roles, such as mismatch repair, DNA replication, and amino acid biosynthesis), size (~600 bp), location (i.e., a minimum of 6 kbp upstream or downstream from known virulence factors, lysogenic phages, or insertion sequence elements), and suitability for nested primer design and sequence diversity (5). All phylogenetic analyses were performed with the same criteria.

Amplification of the loci.

The seven housekeeping genes were amplified with Z-Taq polymerase (Takara Bio, Inc., Shiga, Japan), using the primers described by Curran et al. (5). The primers had variable melting temperatures. The 25-μl amplification reaction mixture comprised 0.5 to 1.0 μl of chromosomal DNA (final concentration, 4 to 8 ng/μl), 0.5 μl each of the 10-pmol primers, 2.5 μl 10× Z-Taq buffer, 0.25 μl of Takara Z-Taq (2.5 units/μl), and 2 μl of deoxynucleoside triphosphate mixture (2.5 mM) and was brought up to the correct reaction volume with sterilized distilled water. The following PCR protocol was used: 1 s of denaturing at 98°C and 30 to 33 cycles (depending on the gene) of 1 s at 98°C (denaturing), 10 s at 57°C to 60°C (primer annealing), and 20 s at 72°C (polymerase extension). Reactions were performed with a PTC-100 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA).

BigDye reaction and sequencing.

Two microliters of ExoSAP-IT was added directly to the 7-μl reaction products following PCR. ExoSAP-IT was inactivated at 80°C for 15 min, and the treated PCR products were ready for subsequent analysis in BigDye reactions, which required the DNA to be free of excess primers and nucleotides. BigDye reactions were carried out using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA). DNA sequencing was performed with an ABI 3100 genetic analyzer (Applied Biosystems) with a 50-cm array. Both forward and reverse sequences were obtained by using the PCR primer or internal sequence primers (specifically for MLST sequences) for each locus.

Sequence data analysis.

Both the forward and the reverse primer sequences of acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE were verified with the Chromas software program, version 2.32 (Technelysium Pty., Ltd.). Several representative DNA sequences were randomly selected from each group of isolates and converted into amino acid sequences, and multiple alignments of these sequences were calculated. These results and the published alignment data were used as templates to conduct multiple alignments of all DNA sequences determined (45). Positions in which gaps were present in any of the aligned sequences were excluded from the analysis. All sequences were confirmed by individual BLAST searches to determine whether the corresponding data matched well with the appropriate gene of P. aeruginosa. Both forward and reverse primer sequences were assembled by using ATGC, version 4.0 (Genetyx Corporation, Tokyo, Japan). The assembled sequences were saved with Genetyx-Win, version 5.0 (Genetyx Corporation, Tokyo, Japan) in Fasta format. The alignment was performed using a hierarchical method for multiple alignments implemented in the CLUSTAL-W tool in MEGA 3.1 (27, 51). Automatically aligned sequences were checked manually.

Allele and strain type assignment.

A number was assigned to each distinct allele within a locus according to the number available in the P. aeruginosa MLST database. Any allele that did not match with existing data was designated “new.” Each isolate was therefore given seven numbers that represented its strain type. Any strain type that did not match with the existing database was numbered as a “new” type. MLST analysis yielded alleles for individual loci of seven housekeeping genes. The alleles were numbered according to their entries in the MLST database. Alleles that did not exist in the database were termed “new.” A table was prepared (see Table ​Table4),4), and all the alleles were provided separate numbers. Strains having the same allele were given the same number.

Phylogenetic analysis.

Similarity and evolutionary distance were calculated with different software programs. Gene distance matrices were calculated from nucleotide sequences by the Jukes-Cantor method (20). Mann-Whitney's U test was employed for within-group and between-group mean distances, and significance levels were calculated (32). Dendrograms were generated by neighbor joining (NJ), maximum parsimony (MP), minimum evolution, the unweighted-pair group method with arithmetic means, and split decomposition (SD) with bootstrap analysis (1,000 replications) using MEGA (27) and PAUP* (version 4; D. L. Swofford, Sinauer Associates, Sunderland, MA). Analyses were performed on individual gene sequences as well as on the concatenated data set. Rooted and unrooted NJ trees of the concatenated seven-gene sequence data were generated with MEGA, version 3.1 (27), using the Kimura 2 parameter model (25) with gamma correction and 1,000 bootstrap replicates for all sequences. In NJ analyses, closely related pseudomonads were used as an outgroup. The data for outgroups were collected from the NCBI GenBank database. An MP tree was generated from the concatenated gene sequence data from which bootstrap percentages were retrieved for 1,000 replicates. The number of polymorphic nucleotide sites and the ratio of the number of nonsynonymous substitutions to the number of synonymous substitutions (the d_N/d_S ratio) were calculated with the MEGA and START programs (18, 19, 27, 35).

Nucleotide fragments and allelic profiles.

The fragment lengths for MLST data (obtained from MLST primers) and whole-gene sequence data (obtained from PCR amplification primers) for the seven housekeeping genes and their variable sites are given in Table ​Table3.3. The allelic sequences obtained were searched for in the P. aeruginosa MLST database. For aroE, all profiles were found in the existing database, whereas one (guaA and nuoD) to eight (mutL) new profiles were found for other genes (Table ​(Table4).4). Most of the oceanic and clinical isolates produced new allelic types in mutL gene sequences. We stipulated that if a strain possessed at least one new profile among the seven genes examined, it produced a new sequence type; among 34 strains, 16 were deemed to be new. Furthermore, even if all seven alleles were known, the combination of each profile sometimes represented new sequence types; 10 strains belonged to this category. The remaining eight strains matched with existing types.

Of the 34 P. aeruginosa strains isolated from different sources, we found 24 distinct strain types. Among them, three (types 9, 155, and 179) existed in the MLST database (Table ​(Table4),4), while the other 21 were new. Three coastal strains (Coastal-54, Coastal-60, and Coastal-86) belonged to type 9. Three strains with this type had been deposited, one from an unknown source from France (strain 5757) and two from sputum samples from Canada (strains R1 and T1). The strain Dolphin-245, isolated from a dolphin in Otsuchi Bay, Iwate Prefecture, Japan, matched with type 155, which comprises two clinical strains from Canada (strains B5 and S3). The Arakawa River-1508 strain, from Arakawa River, matched with type 179, which includes five clinical strains from Canada (strains B4, E4SIB1, C1, W4, and C2). Other than for these three types, the strains did not match with any types available in the 409-strain database. Among ocean (9 strains) and coastal (12 strains) isolates, six and six new strain types, respectively, were found.

Genetic distances between the P. aeruginosa groups.

The average mean genetic distances between and within the designed P. aeruginosa groups were calculated by MEGA. Mann-Whitney's U test was carried out to see whether the differences within and between the group average means were statistically significant (32). In concatenated gene sequences (5,700 bp), the distances within groups were lower than the distances between groups (P < 0.05). The average mean distance between ocean-derived and other P. aeruginosa groups varied from 0.0075 to 0.0092, which was higher than the within-group average mean distance (P < 0.05) (Table ​(Table55).

Phylogenetic analysis and comparison with the geographical locations of the origins of the strains.

In order to assess the phylogenetic relationships between all the strains tested, PCR fragments for each gene, as well as the concatenated sequences of all PCR fragments, were analyzed to construct trees based on NJ, MP, minimum evolution, the unweighted-pair group method with arithmetic means, or SD. Thirty-nine strains were used, including P. aeruginosa PAO1, P. aeruginosa JCM 5962^T, and three outgroup species strains (Pseudomonas putida KT2440, P. fluorescens Pf-5, and Pseudomonas syringae B728a) obtained from the NCBI database. Because the most conservative segments of the housekeeping genes were used in this study, the distance to the outgroups was too great to display in the tree. Only the unrooted trees are shown (Fig. ​(Fig.11 and ​and4).4). Figure ​Figure33 shows the rooted NJ tree of the concatenated data set of seven housekeeping genes with the outgroup.

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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.

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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.

In this study, we present only the data derived by the NJ and MP methods, as other methods showed similar patterns for the trees, so they were eliminated. Figure ​Figure11 shows two unrooted trees constructed by NJ and MP methods from concatenated gene sequence data together with a map identifying the geographical locations of the isolates. All open-ocean strains showed a trend of forming separate clusters in the trees except two ocean P. aeruginosa strains. Except for strains Ocean-222 and Ocean-238, all ocean strains formed a single cluster, with one coastal strain (Coastal-1276) included. Although the freshwater strains did not show a distinct cluster, they were mostly separated from the other strains, with the exception of strains Freshwater-206 and -1559. Strains P. aeruginosa PAO1, Dolphin-245, and Coastal-963 were included in the freshwater group. Mixing of coastal strains with freshwater strains might indicate the influence of freshwater outfalls on coastal systems. Although the coastal strains are rather scattered in the tree, they were mostly close to each other and formed two separate clusters (one from the strains of Sagami Bay and one from those of Suruga Bay) that were always prominent. Four of the five clinical isolates also clustered together.

Phylogenetic analysis of individual loci.

The relationships between the 34 strains were further analyzed by constructing unrooted trees using the NJ method based on the nucleotide sequences of PCR fragments for each gene (data not shown). Ocean alleles were clustered with respect to acsA, aroE, guaA, ppsA, and trpE. This clustering was supported by bootstrap values higher than 50%, indicating their distinct positions in the trees.

We thank the Japanese Society for the Promotion of Sciences for sponsoring Nurul Huda Khan with a postdoctoral fellowship. The study was supported by Grants-in-Aid for Creative Basic Research, no. 12NP0201 (DOBIS) and no. 14208063, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

We are grateful to Yoshikazu Ishii, Department of Microbiology and Infectious Diseases, Toho University School of Medicine, for providing clinical P. aeruginosa strains. We remain indebted to Rita R. Colwell, University of Maryland, for her valuable comments and inspiration regarding this study. This publication made use of the P. aeruginosa MLST website (http://pubmlst.org/paeruginosa/), developed by Keith Jolley and located at the University of Oxford (18). The development of this site has been funded by the Wellcome Trust.