Molecular and phenotypic approaches to identify Pseudomonas strains
Mohammad Abdul Bakir, Shinji Sakata and Yoshimi Benno
＊Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
The purpose of this study was to determine the taxonomic status of the Pseudomonas strains that were deposited in the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center by Dr. R. Hugh, School of Medicine, George Washington University. A total of 49 strains were studied by partial 16S rRNA gene sequence analysis, ribotyping, and biochemical characterization. All the studied strains belonged to the genus Pseudomonas except two strains, which belonged to Shewanella algae. Among the Pseudomonas strains, 64% (30/47) were closely related to the following species, Pseudomonas aeruginosa, P. pseudoalcaligenes, P. stutzeri and P. alcaliphila. The remaining 36% (17/47) of strains could not be assigned to any validly published species of Pseudomonas. Although the 16S rRNA gene sequence analysis of the strains demonstrated very close similarities (>99%) with more than one validly published Pseudomonas species, pheno- typic characterizations and ribotyping of these strains produced various results. A reliable method of identifying these microorganisms needs to be developed.
Key words: Pseudomonas, biochemical, 16S rRNA, ribotyping
INTRODUCTION
The genus Pseudomonas Migula 1894 served as a repository for straight, strictly aerobic, Gram- negative rods that were motile by one or several polar flagella (Tayeb et al., 2005). The original classi- fication of the genus Pseudomonas into five rRNA homology groups (Palleroni et al., 1973) has under- gone extensive revision, resulting in the reclassifica- tion of many Pseudomonas species into separate genera. These genera include Acidovorax, Burkho
lderia, Brevundimonas, Comamonas, Methylobacte
rium, Ralstonia, Shewanella, Stenotrophomonas and Sphingomonas. (Kersters et al., 1996). The genus Pseudomonas (sensu stricto) comprises the type spe- cies Pseudomonas aeruginosa and related species (rRNA homology group 1) (Palleroni, 1984).
Characterizations of Pseudomonas must resolve the intrageneric subdivisions of Pseudomonas (sensu stricto) into species. A complicating factor for such analyses is that species of the genus Pseudomonas and other pseudomonads are well known for exchanging genetic material (Moore et al., 2005).
Questions as to whether observed traits are stable, reliable and truly characteristic of a given organism
become relevant for taxonomic and identification purposes. The genus Pseudomonas comprises a group of species for which there are varying amounts of information available. While some spe- cies of the genus Pseudomonas (e.g., P. aeruginosa) are homogeneous taxonomic units that are easily dif- ferentiated and identified, other species include com- plex subdivisions, such as the P. fluorescens and P.
putida biovars, the P. syringae and P. marginalis pathovars, or the P. stutzeri genomovars. Such internal organization reflects the heterogeneity of the genus Pseudomonas. Discrepancies between phenotypic and genotypic properties have made it difficult to solve the taxonomic problem. The basic morphological characteristics of Pseudomonas are common to other genera and so are of little value in the positive identification or diagnosis of the mem- bers of the genus. Analyses of 16S rRNA gene seq- uence similarities of bacteria have been essential for elucidating their intergeneric relationships. How- ever, the resolution between individual sequences of the species has decreased and the branching orders have become less discernible with the increase of Pseudomonas species. Overall, 16S rRNA gene sequence similarities among the species of Pseudo
monas range from approximately 93% to 99.9%.
Such observations led to a significant problem in using 16S rRNA gene sequences for species identifi-
＊Corresponding author E-mail: [email protected] Accepted: September 30, 2008
Fig. 1 Ribopatterns of studied Pseudomonas strains Note: -, Unknown
cation (Moore et al., 2005).
Members of the genus Pseudomonas are found as phytopathogens and human or other animal patho- gens. Some are involved in the denitrification pro- cess in soils. Pseudomonas species are often meta- bolically versatile because they have the ability to degrade numerous low molecular weight organic compounds, including aromatic compounds, haloge- nated derivatives and various recalcitrant organic residues. Several also carry catabolic plasmids that code for enzymes involved in the catabolism of vari- ous aromatic compounds. P. aeruginosa has excep- tionally high metabolic versatility and adaptability.
Hence, Pseudomonas species play a crucial role in the carbon cycle in nature and consequently have attracted the interest of molecular biologists and bio-
technologists (Kersters et al., 1996). Culture collec- tion centers are responsible for assigning bacterial isolates into appropriate taxonomic status according to current knowledge. Based on the significance of Pseudomonas, 49 putatively identified Pseudomonas strains deposited in the Japan Collection of Microorganisms (JCM) were studied for identifica- tion using partial 16S rRNA gene sequence, ribotyp- ing, physiological and biochemical analyses.
MATERIALS AND METHODS Bacterial strains and cultivation
The bacterial strains used in this study were obtained from Professor Robert Hugh (George Washington University) and deposited in JCM as Pseudomonas species. The strains were isolated Fig. 2 Neighbor-joining phylogenetic tree showing the clustering of studied strains with related
Pseudomonas species. Bootstrap values (>50%) based on 1000 replications are listed at the branching point.
from clinical, environmental and animal sources (Fig.
1). All strains were cultivated on Trypticase Soy Agar medium (Difco) with or without addition of 5%
horse blood at 30℃ for 48 hours and maintained in glycerol-nutrient agar broth as deep-frozen (-80℃) stock cultures.
PCR and sequencing of 16S rRNA gene
The 16S rRNA gene of the strains was amplified by PCR (Biometra) and partial sequences were determined. The 16S rRNA gene was amplified with universal primers 27F (5′-AGAGTTTGATCC- TGGCTCAG-3′) and 520R (5′-GCCAGCAGCCGCG GT -3′) according to the following program: 95℃ for 3 min, followed by 30 cycles at 95℃ for 3 sec, 60℃ for 30 sec and 72℃ for 1.5 min with a final extension period at 72℃ for 10 min. PCR products were puri- fied by Montage PCR96 filter plate (Millipore) and sequenced directly by the dideoxynucleotide chain- termination method using a DNA sequencer ABI PRISM 3100 (Applied Biosystems/Hitachi) with a BigDye Terminator, version 3.1, cycle sequencing RR-100 kit (Applied Biosystems) according to the manufacturer’s instructions. The 16S rRNA gene sequences of about 500 bases (corresponding to Escherichia coli positions 27 to 520) were obtained.
Nucleotide sequence accession number
A total of 49 16S rRNA gene sequences were determined in this study and deposited in the DDBJ/EMBL/GenBank database under accession numbers AB247210, AB247213 to AB247234 and AB247237 (Fig. 2).
Phylogenetic analysis
All 16S rRNA gene sequences of validly published Pseudomonas species (a total of 103 species at the time of writing this manuscript) available from the database were retrieved. Sequences were aligned with the studied strains using Clustal X version 1.81 (Thompson et al., 1997). The neighbor-joining tree (Saitou & Nei, 1987) was inferred according to the model of Jukes and Cantor (1969). Topology of phy- logenetic trees was evaluated by the bootstrap re- sampling method of Felsenstein (1985) with 1000 replicates (data not shown). The tree was re-con- structed using the selected sequences of the type strains of Pseudomonas species that appeared as neighbors with the studied strains following the same method as described above (Fig. 1).
Ribotyping
Ribotyping was carried out using an automated RiboPrinter microbial characterization system (DuPont-Qualicon) as described previously (Sakata et al., 2006). Standard reagents were used in all analy- sis steps according to the manufacturer’s instruc- tions. Colonies were picked from solid medium, sus- pended in sample buffer and treated by heat. The lysing agent was added and the samples were trans- ferred to the automated RiboPrinter system.
Restriction endonuclease digestion (EcoRI), gel sepa- ration, transfer and hybridization with a chemilumi- nescence-labeled DNA probe containing the ribo- somal RNA operon from Escherichia coli were car- ried out by the automated instrument in 8 h. Gel images were exported in TIFF format and numeri- cal analysis of ribopatterns was performed using the GelCompar system version 4.0 (Applied Maths) which normalizes fragment pattern data for band intensity and relative band position compared with molecular weight marker. UPGMA (Unweighted Pair Group Method using Arithmetic means) was used for determining the similarity and constructing the dendrogram.
Biochemical and physiological characterization The API 20 NE kit for identifying non-fastidious Gram-negative rods, which consists of 8 conventional and 12 carbohydrate assimilation tests, was used as described by the manufacturer (bioMérieux). Each strain was inoculated into 0.85% NaCl and turbidity was adjusted to the 0.5 MacFarland standard. The inoculum was distributed into test strips, which were incubated at 30 ℃ and read at 24 and 48 h, according to the manufacturer’s instructions.
Biochemical reactions were read as positive or nega- tive. The strains that appeared as P. aeruginosa in the blastn search were grown on King’s A and King’s B media (King et al., 1954) for examining the produc- tion of pigments. All the studied strains were sub- jected to examination of growth at 4℃ and 41℃.
Seventeen strains were selected and tested for their ability to grow in the presence of 5% NaCl.
RESULTS AND DISCUSSION
Blastn searches were conducted using about 500 bp of 16S rRNA gene sequence for initial identifica- tion of the studied Pseudomonas species. Database searches showed that all the studied strains (49 strains) belong under the genera Pseudomonas and Shewanella. Two strains (JCM 13988 and JCM
13989) demonstrated 99% sequence similarities with Shewanella algae and did not show ≥97% sequence similarity with any other species under the genus Shewanella (Stackebrandt & Goebel, 1994). Most of the Pseudomonas strains (68%, 32/47) showed >98%
similarities with more than one species present in the database, except the strains that appeared to be P. aeruginosa and S. algae (35%, 17/49).
Pseudomonas strains showed high levels of sequence similarities (<0.4% sequence divergence) with more than one Pseudomonas species and were difficult to place under a single species. 16S rRNA gene sequence analysis is widely used in evolution- ary, taxonomic and ecological studies and the prob- lem is species identification rather than genus identi- fication. 16S rRNA gene sequences can be used rou- tinely to distinguish and establish relationships between genera and well-resolved species, however, diverged species may not be recognizable (Fox et al., 1992).
Phylogenetic analysis
The studied strains were subjected to phylogenet-
ic analysis to determine their taxonomic relation- ships with validly published Pseudomonas species.
The resulting phylogroups are shown in Fig. 2.
Fifteen strains were grouped with P. aeruginosa LMG 1242T in the tree. Twelve strains showed simi- larities with P. pseudoalcaligenes. Strain RH 2814 demonstrated similarity with P. stutzeri. The two strains JCM 13959 and JCM 13960 were similar to P. alcaliphila. Strains JCM 13963, JCM 13964 and JCM 13965 were grouped with P. plecoglossica in the tree. Strain JCM 13969 had P. mosselii as a phylogenetic neighbor. Strain JCM 13961 demon- strated similarity with P. nitroreducens. Strains JCM 13968 and JCM 13971 appeared to be P. mon
teilii and JCM 13987 to be P. extremorientalis.
The six strains JCM 13209, JCM 13962, JCM 13966, JCM 13967, JCM 13970 and JCM 13972 did not show affinities with any Pseudomonas species in the phylogenetic analysis.
Ribotyping
The studied Pseudomonas strains and 5 type strains of related Pseudomonas species were charac- Table 1 Characteristics of the strains that were grouped with P. aeruginosa in the phylogenetic tree
Parameter
P. aeruginosaa P. aeruginosa JCM 5962T JCM 13199 JCM 13200 JCM 13201 JCM 13202 JCM 13203 JCM 13204 JCM 13205 JCM 13206 JCM 13956 JCM 13957 JCM 13958 JCM 13207 JCM 13208 JCM 14235 JCM 14236
Pigment on King’s medium A + + + + + + + + + + + + + + + + +
Pigment on King’s medium B + + + + + - + + - + + + + + + + +
Growth at 4℃ ND - - - -
Growth at 41℃ + + + + + + + + + + + + + + + + +
Oxidation 98 + + + + + + + + + + + + + + + +
Nitrate reduction 96 - - - -
Indole production 0 - - - -
D-glucose fermentation 0 - - - -
Arginine dihydrolase 80 + W + W W W + + + + + + W + + +
Urease 20 W W + - W W W W - + W - W W W -
Esculin hydrolysis 1 - - - W - W W - - - -
Gelatin hydrolysis 92 + + + + + + + + + + + + + + + +
b-galactosidase 1 - - - W - - W - - - -
Glucose 99 + + + + + + + + + + + + + + + +
Arabinose 1 - - - -
Mannose 1 - - - -
Mannitol 89 + + + + + + + + + + + + + - + +
N-Acetyl-glucosamine 84 + + + + + + W + + + + W + W + +
Maltose 1 - - - -
Potassium gluconate 97 + + + + + + + + + + + + + - - +
Capric acid 98 + + + + + + + + + + + + + + + +
Adipic acid 91 + + + + + + + + + + - + + + + +
Malate 98 + + + + + + + + + + + + + + + +
Trisodium citrate 99 + + + + + + + + + + + + + + + +
Phenylacetic acid 1 - - - -
a % Positive reaction after 24-48 hrs, Identification table, API 20 NE, V6.0; +, positive; -, negative; W, weak
terized by automated ribotyping. The resulting ribogroups are shown in Fig. 2. Ribotyping was highly discriminative below the species level and the Pseudomonas strains were divided into 19 ribo- groups. The DuPont identification pattern database (version 6.5, release 12.2) contained 21 Pseudomonas species and 24% of strains (11/46) could be assigned at the species level, however, 2 strains (JCM 13973 and JCM 13974) of P. pseudoalcaligenes appeared to be Salinivibrio costicola subsp. costicola and Aerococcus viridans, respectively. The DuPont iden- tification pattern database was able to identify 47%
(7/15) of P. aeruginosa strains as demonstrated by phylogenetic analysis and API 20 NE. The strains that appeared to be P. aeruginosa by phylogenetic analysis were divided into 3 ribogroups (clusters I, III and XIII). In the previous study, a comparison of the patterns classified 81 P. aeruginosa isolates into 32 ribotypes (Grundmann et al., 1995). Most of the P. aeruginosa strains (80%, 12/15 strains) showed similar ribopatterns to that of P. aeruginosa JCM 5962T. The strains that appeared to be P. pseudoal
caligenes by 16S rRNA gene sequence analysis were grouped into 4 clusters (clusters II, VI, VII, VIII and XV). However, P. pseudoalcaligenes JCM 5968T clus- tered separately (cluster XIX). Variability among the isolates of P. pseudoalcaligenes was also observed when analyzed by rpoB gene (Tayeb et al., 2005). In general, this study did not show any corre- lation between the source of isolation of the strains and ribopattern. P. pseudoalcaligenes strains that were isolated from the feces of patients with diar- rhea demonstrated similar ribopatterns (cluster VIII). Two strains that showed phylogenetic rela- tionships with P. alcaliphila showed similar ribopat- terns to that of P. alcaliphila JCM 10630T and were grouped in cluster VI. The strains that showed phylogenetic similarities with P. monteilii, P. moss
elii/P. nitroreducens and P. taetrolens in the phylo- genetic tree demonstrated similar ribopatterns (clus- ters VII and XVII). Heterogenic ribopatterns (clus- ters V, XI and XVI) were observed for the strains that were grouped with P. plecoglossicida in the tree.
Table 2 Characteristics of the strains that were grouped with P. pseudoalcaligenes, P. stutzeri and P. alcaliphi- la in the phylogenetic tree
Parameter
P. pseuoalcaligenes JCM 5968T JCM 13973 JCM 13974 RH 619 JCM 13975 JCM 13976 JCM 13977 JCM 13978 JCM 13979 JCM 13980 JCM 13981 JCM 13982 JCM 13983 P. stutzeri JCM 5965T RH 2814 P. alcaliphila JCM 10630T JCM 13959 JCM 13960
Growth at 4℃ - - - -
Growth at 41℃ + + + + + + + + + + + + + + + + + +
Oxidation + + + + + + + + + + + + + + + + + +
Nitrate reduction + + + + + + + + + + + + + + + + + +
Indole production - - - -
D-glucose fermentation - - - -
Arginine dihydrolase + + + + - - + - + + + + - - - - + +
Urease + - - - -
Esculin hydrolysis - - - -
Gelatin hydrolysis - - - -
b-galactosidase - - - -
Glucose - - - + W + - -
Arabinose - - - -
Mannose - - - -
Mannitol - - - + - -
N-Acetyl-glucosamine - - - -
Maltose - - - + W - - -
Potassium gluconate - - + - + + + - + - + - - + + + - -
Capric acid + + + + + + + + + + + + + + + + + +
Adipic acid - - - -
Malate + + + + + + + + + + + + + + W + + +
Trisodium citrate + - + - - - + - - + + - + + W + + +
Phenylacetic acid - - - -
+, positive; -, negative; W, weak
Phenotypic and biochemical analysis
Biochemical characteristics of the Pseudomonas strains were determined using an API 20NE kit.
The results are listed in Tables 1 to 3. All the strains (15 strains) in the P. aeruginosa group by phylogenetic analysis produced pigments on King’s A medium and were able to grow at 41℃. None of the strains was able to grow at 4℃. All the strains produced pigment on King’s B medium except strains JCM 13202 and JCM 13205. All P. aerugino
sa strains were able to hydrolyze arginine. Overall, they showed similar biochemical properties with P.
aeruginosa JCM 5962T and P. aeruginosa characteris- tics present in the API 20 NE profile (Table 1).
Usually, the presence of blue-green pigment is suffi- cient for the diagnosis of P. aeruginosa. Oxidase positive, presence of pyocyanin or pyrubin, and growth at 42℃ are general characteristics for the identification of P. aeruginosa (Bergan, 1981). Twe-
lve strains that showed relationships with P. pseudo
alcaligenes in the phylogenetic tree also showed sim- ilar API 20 NE profiles with P. pseudoalcaligenes JCM 5968T (Table 2). However, the type strain of P.
pseudoalcaligenes JCM 5968T was urease-positive and all the tested strains in the P. pseudoalcaligenes group were urease-negative. Only 3% of P. pseudo
alcaligenes strains are able to hydrolyze urea (Kiska
& Gilligan, 2003). None of the examined strains used glucose and mannitol. Strain RH 2814 in the P.
stutzeri group of the phylogenetic tree showed simi- lar characteristics with P. stutzeri JCM 5965T (Table 2). The strain lacked arginine dihydrolase activity, a characteristic which separates this species from non- fluorescent P. mendocina (Kiska & Gilligan, 2003).
The two strains JCM 13959 and JCM 13960 that showed similarities with P. alcaliphila in the phylo- genetic tree also appeared to be P. alcaliphila based on biochemical properties (Table 2) (Yumoto et al., Table 3 Characteristics of the strains that were grouped with P. plecoglossicida, P. monteilii, P. mosselii, P.
taetrolens & P. extremorientalis in the phylogenetic tree
Parameter
P. plecoglossicida JCM 13297T JCM 13963 JCM 13964 JCM 13965 P. monteilii JCM 13968 JCM 13971 P. mosselii JCM 13969 P. nitroreducens JCM 2782T JCM 13961 P. taetrolens JCM 13984 JCM 13985 JCM 13986 P. extrmorientalis JCM 13987 JCM 13209 JCM 13962 JCM 13966 JCM 13967 JCM 13970 JCM 13972
Growth at 4℃ - - - + - - ND - + + + + + + - - - -
Growth at 41℃ - + + - - + + - - ND - - - + + + - + -
Growth at 5% NaCl + - + + - + + + + ND + ND ND + + - + +
Oxidation + + + + + + + + + + + + + + + + + + + + + + +
Nitrate reduction + - - - + - - - + - + - - - - - Indole production - - - ND - - - - D-glucose fermentation - - - ND - - - ND - - - - Arginine dihydrolase + + + + + + + + + + + ND + + + - + - + + + + +
Urease - - - - + - - ND - - - ND + + + ND - - - - + - -
Esculin hydrolysis - - - ND - - - ND - - - - Gelatin hydrolysis - - - V - - - -
b-galactosidase - - - ND - - - ND - - - -
Glucose + + + + + + + ND + + + + + + + - + + + + + + +
Arabinose - + - - + - - - ND + + + + + + + + + - +
Mannose - W + + - + + + + - + ND W W W + + + + + + + +
Mannitol - - - + - - - + - - - ND - W - + + + - - - - -
N-Acetyl-glucosamine - - - ND - - - + + - - - -
Maltose - - - ND - - - ND - - - + - + - - - - -
Potassium gluconate + + + + + + + + + + + ND + + + ND + + + + + + +
Capric acid + + + + + + + ND + + + ND + + + ND + + + + + + +
Adipic acid - - - V - - - ND - - - ND - - - -
Malate + + + + +/- + + ND + + + ND + + + ND + + + W + + +
Trisodium citrate + + + + + + + ND + + + ND + + + + + + + W + + + Phenylacetic acid + - + + + + + + + + W ND - - - + - Strains JCM 13209, JCM 13962, JCM 13966, JCM 13967, JCM 13970 and JCM 13972 did not show similarities with any Pseudomonas species in the tree. +, positive; -, negative; W, weak; V, variable; ND, no data. P. extremorientalis (Ivanova et al., 2002); P. monteilii (Elomari et al., 1997); P. mosselii (Dabboussi et al., 2002); P. taetrolens (Tompkin & Sharparis, 1972).
2001).
The 17 strains of Pseudomonas (36%, 17/47) showed different biochemical properties with the related species and could not be assigned under val- idly published Pseudomonas species (Table 3).
Three strains (JCM 13963, JCM 13964 and JCM 13965) in the P. plecoglossicida group of the phyloge- netic tree showed differences from P. plecoglossicida in their ability to reduce nitrate and utilize mannose.
Strains JCM 13968 and JCM 13971 showed phyloge- netic affinities with P. monteilii, but biochemical dif- ferences from P. monteilii in their ability to grow in the presence of 5% NaCl, and utilization of urease and mannose. Strain JCM 13969 had P. mosselii as a phylogenetic neighbor. The strain showed differ- ent characteristics from P. mosselii in the utilization of mannitol. Strain JCM 13961 differed from P.
nitroreducens in nitrate reduction and utilization of mannose.
The characteristics of the three strains JCM 13984, JCM 13985 and JCM 13986 could not be com- pared due to lack of data for P. taetrolens. Strain JCM 13987 demonstrated phylogenetic similarity with P. extremorientalis, although P. extremoriental
is was able to reduce nitrate whereas this strain could not.
Most of the Pseudomonas species are not included in the databases that are based on biochemical char- acteristics. The API 20NE identification table lists only five Pseudomonas species (API 20NE, V6.0).
Molecular techniques provide superior results, how- ever, the API 20NE kit is easy to use, less laborious and offers excellent identification of Gram-negative, oxidase positive rods (van Pelt et al., 1999;
Wellinghausen et al., 2005).
Most of the Pseudomonas strains that could not be assigned under species were isolated from clini- cal specimens (59%, 10/17), which included sputum, blood, urine and human feces (Fig. 1). Identification of deposited Pseudomonas strains is necessary to determine the level of biosafety. This study demon- strated limitations for the identification of Pseudomonas strains at the species level. We were able to assign 64% of Pseudomonas strains (30/47) based on phylogenetic, ribotyping, physiological and biochemical analyses as summarized in Table 4. A total of 35% strains (17/49) could not be discriminat- ed from closely related Pseudomonas species. The taxonomy of Pseudomonas has been changed and commercially available biochemical characterization kits contain only a few species. Descriptions of available Pseudomonas species were based on differ- Table 4 Identity of studied Pseudomonas strains based on phylogenetic, ribotyping and biochemical characteriza-
tion
Strain Phylogenetic
similarity Ribogroup API 20 NE Identified strains JCM 13199, JCM 13200, JCM 13201,
JCM 13202, JCM 13203, JCM 13204, JCM 13205, JCM 13206, JCM 13207, JCM 13208, JCM 13956, JCM 13957, JCM 13958, JCM 14235 & JCM 14236
P. aeruginosa I, III & XIII P. aeruginosa
JCM 13973, JCM 13974, RH 619, JCM 13975, JCM 13976, JCM 13977, JCM 13978, JCM 13979, JCM 13980, JCM 13981, JCM 13982 & JCM 13983
P. pseudoalcaligenes II, VI, VIII &
XV P. pseudoalcaligenes
RH 2814 P. stutzeri Not determined P. stutzeri
JCM 13959 & JCM 13960 P. alcaliphila VI P. alcaliphila Unidentified strains JCM 13963, JCM 13964 & JCM 13965 P. plecoglossicida V, XI & XVI Not identified JCM 13968 & JCM 13971 P. monteilii VII Not identified
JCM 13969 P. mosselii VII Not identified
JCM 13961 P. nitroreducens VII Not identified
JCM 13984, JCM 13985 & JCM 13986 P. taetrolens XVII Not identified
JCM 13987 P. extremorientalis VII Not identified
JCM 13967 & JCM 13972 No group X & XII Not identified JCM 13962, JCM 13966 & JCM 13970 No group IV, VI & X Not identified
JCM 13209 No group XIV Not identified
ent sets of biochemical tests. The inclusion of only type strains for comparison may not provide ade- quate characterization of the species. Further, the biochemical characteristics of Pseudomonas strains were observed to vary (55%) between duplicate tests (Wiedmann et al., 2000). Ribotyping showed a high level of discrimination and is useful for deter- mining intraspecies variability. Although not exten- sive, the phylogeny of the Pseudomonas species based on 16S rRNA, gyrB and rpoD genes was con- structed (Anzai et al., 2000; Yamamoto et al., 2000).
Recently, the phylogeny of Pseudomonas based on rpoB sequences showed three times higher resolu- tion compared with 16S rRNA gene sequences, how- ever, 6.2% of strains demonstrated uncertain affilia- tion (Tayeb et al., 2005). Other genes such as dnaJ showed higher divergence of the intragenus rela- tionship of Aeromonas species. Sequence similarity based on dnaJ was 89% compared with that of the 16S rRNA gene (99%) (Nhung et al., 2006). In this study, 16S rRNA gene sequence analysis clearly assigned all Pseudomonas strains at the genus level, but it must be noted that among the available data- bases, only the 16S rRNA gene sequence database is extensive. The complication of databases using genes that show higher resolutions may resolve the problem of identifying Pseudomonas species demon- strated in this study.
ACKNOWLEDGEMENTS
The authors are indebted to Prof. R. Hugh for the deposition of strains at JCM and to Prof. K.
Komagata and Dr. Y. Kosako, for their help in this regard. This work was supported by grants from the National BioResource Project (NBRP: Pathogenic microorganisms, Head: Profs. K. Nishimura and J.
Mikami).
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分子生物学的手法および表現型による Pseudomonas 属菌株同定の検討 モハマド アビドウル バキラ，坂田慎治，辨野義己
（独）理化学研究所バイオリソースセンター微生物材料開発室
米国ジョージワシントン大学医学部 R. Hugh 教授から理研バイオリソースセンター微生物材料開発室 (IJCM) に寄託され た未同定 Pseudomonas 属関連菌株の再同定を行った．臨床材料，動物および環境由来の未同定 49 菌株の 16S rRNA 遺伝子 による系統解析，リボタイビング解析および生理生化学的性状検査を行った．16S rRNA 遺伝子の系統解析では，
Shewanella algae に属する 2 株を除いて，Pseudomonas 属であることが認められ，64%（47 株中 30 株）を Pseudomonas aeruginosa, P. pseudoalcaligenes, P. stutzeri および P. alcaliphila に同定した．残り 36%（17 株）は既存菌種に同定するこ とは困難であった．これらのグループは 16S rRNA 遺伝子による系統解析で菌種間で 99% 以上の高い相同性を示すが，表 現性状およびリボタイピングにおいては菌株間で多様な性状を示した．以上の成績は Pseudomonas 属に属する菌株におけ る有効な菌種同定法の開発が急務であることを示唆している．
（担当編集委員：中川恭好）