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Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd, NE, MS-D11, Atlanta, GA 30333, USA
Correspondence
Jay E. Gee
JGee1{at}cdc.gov
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains C6786T, C7532, C7533, E0147 and ATCC 23343T are DQ108388, DQ108389, DQ108390, DQ108391 and DQ108392, respectively.
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; Yabuuchi et al., 1992b). Burkholderia pseudomallei, a saprophytic soil bacterium that is the aetiological agent of melioidosis in animals and humans (White, 2003), has been designated a category B select agent by the CDC, due to its potential use for biological terrorism (Rotz et al., 2002).
B. pseudomallei is typically seen in South-East Asia and northern Australia, but is occasionally reported in tropical areas around the world. Most melioidosis cases reported in other regions arise from travel to areas endemic for B. pseudomallei. In the USA, case reports of locally acquired melioidosis in the states of Oklahoma and Georgia have been documented (McCormick et al., 1977; Nussbaum et al., 1980). It is important to establish whether B. pseudomallei is native to the USA and to characterize further the B. pseudomallei-like organisms that were recovered from these cases.
The Oklahoma strain of Pseudomonas (now Burkholderia) pseudomallei was first isolated in 1973 from a 27-year-old farmer with a deep leg wound heavily contaminated with soil as a result of a farming accident in Oklahoma, USA (McCormick et al., 1977). The clinical isolate C6786T was identified by using standard microbiological procedures. Environmental isolates C7532 and C7533 were recovered from the soil near the accident site and were similar to C6786T. These three isolates comprise the original Oklahoma strain. The initial study indicated that the Oklahoma strain may represent a variant of B. pseudomallei or potentially a novel species based on the results of biochemical assays, guinea pig inoculations, fatty acid analysis and a fluorescent antibody test (McCormick et al., 1977). Subsequent studies have produced conflicting interpretations regarding the identification of the Oklahoma strain as a strain of B. pseudomallei (Godoy et al., 2003; Tomaso et al., 2004, 2005; Yabuuchi et al., 1992a). Yabuuchi et al. (1992a) performed a variety of standard microbiological tests as well as DNA–DNA hybridization and 16S rRNA gene sequencing with the clinical isolate of the Oklahoma strain and concluded that it was a strain of B. pseudomallei. Recently, Godoy et al. (2003) completed a study using multilocus sequence typing (MLST) that indicated that the Oklahoma isolates were divergent from all other B. pseudomallei isolates in their study panel and suggested that the Oklahoma strain may be a novel species. Interestingly, they observed that B. mallei, also a category B select agent and the pathogen responsible for the disease glanders, appeared to be a clone of B. pseudomallei (Godoy et al., 2003). PCR assays recently developed by Tomaso et al. (2004, 2005) for detecting B. pseudomallei and B. mallei do not detect the Oklahoma strain.
An additional isolate recovered from an automobile accident victim was also evaluated. This isolate was recovered in 1977 from a 21-year-old male from Georgia, USA, who was involved in an automobile accident that propelled him into a clay embankment. His left eye was packed with soil and developed ulcers in the days after the accident. An organism assigned as B. pseudomallei (E0147, the Georgia strain) was isolated 8 weeks after the accident and identified by using biochemical assays (Nussbaum et al., 1980).
Because molecular studies of the Oklahoma strain suggested that it may be a separate species, we decided to re-examine and characterize the Oklahoma strain, as well as the Georgia strain, by using standard microbiological procedures, 16S rRNA gene sequencing, MLST and DNA–DNA hybridization to determine whether they were strains of B. pseudomallei. We determined that these isolates comprise a novel Burkholderia species and therefore propose the name Burkholderia oklahomensis sp. nov.
We amplified the 16S rRNA gene for strains C6786T, C7532, C7533, E0147 and ATCC 23343T (the type strain of B. pseudomallei). Bacterial culture and DNA template preparation were performed as described previously (Gee et al., 2003). Amplification of the 16S rRNA gene was performed by using a protocol and primers F229 (5'-CGCAAGCGAAAGTATCAAGA-3') and R1908 (5'-TTTACAGCCGATAAGCGTGAG-3'), described previously (Gee et al., 2003). The 16S rRNA gene sequences were obtained by using a previously described protocol and set of primers (Gee et al., 2003). We were able to determine the nearly complete sequence (corresponding to positions 26–1514 in the Escherichia coli numbering system) of the 16S rRNA gene for these five isolates.
We found that C6786T, C7532 and C7533 had identical 16S rRNA gene sequences, which we designated the OK 16S rRNA gene consensus sequence. A BLAST query of GenBank on 1 September 2005, using the OK 16S rRNA gene consensus sequence, did not yield an exact match to any 16S rRNA gene sequence in the database (Altschul et al., 1990). Although Yabuuchi et al. (1992a) stated that the 16S rRNA gene sequence for the Oklahoma strain was identical to the 16S rRNA gene sequence for B. pseudomallei, the sequence was not included in their paper, nor was an entry available in GenBank.
We performed an analysis with the Wisconsin package, ver. 10.3 (Accelrys), using BESTFIT. The 16S rRNA gene sequence for strain E0147, the Georgia strain, differed from the OK 16S rRNA gene consensus sequence at only two positions, yielding a match of 99.9 %. The OK 16S rRNA gene consensus sequence, as represented by the sequence derived from strain C6786T, was aligned with 16S rRNA gene sequences from related bacteria available in GenBank by using PILEUP. Gaps were removed for neighbour-joining analysis (Kimura two-parameter, 1000-step bootstrap) in MEGA 3 (Kumar et al., 2004) (Fig. 1).
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), also indicated a match of 98.5 %.
MLST of B. pseudomallei and closely related species is based on sequencing discrete segments of seven housekeeping genes and assigning sequence types (STs) based on sequence variations of the loci. Godoy et al. (2003) found that the three Oklahoma isolates were identical by MLST, and they assigned them to ST81. The alleles of the seven loci of the Oklahoma strain were not shared with either B. pseudomallei or B. thailandensis (Godoy et al., 2003).
We performed MLST on E0147 (the Georgia strain) and ATCC 23343T (the B. pseudomallei type strain). Bacterial culture and DNA template preparation were performed as described previously (Gee et al., 2003). We used the panel of primers described on http://www.mlst.net and by Godoy et al. (2003) for both amplification and sequencing. For amplification, we used the Expand Hi Fidelity PCR system (Roche). The amplification mix consisted of 1x buffer 2, 200 µM dNTP mix, 0.4 µM forward and reverse primers, 0.9 units Expand polymerase and 1 µl cell extract. PCR conditions consisted of an initial 4 min hold at 95 °C. This was followed by 30 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min with a final hold at 72 °C for 10 min. PCR products were purified with a QIAquick PCR purification kit (Qiagen). Sequencing was performed by using an Applied Biosystems (ABI) BigDye Terminator cycle sequencing ver. 3.0 kit as per the manufacturer's instructions, except that 3 µl BigDye was used instead of 8 µl (Applied BioSystems). Sequencing products were purified by using Centri-Sep spin columns (Princeton Separations) and were resolved by using an ABI model 3100 automated DNA sequencing system.
MLST analysis of E0147 revealed that four of the seven MLST loci (ace, gltB, lepA and lipA) of E0147 had alleles identical to the Oklahoma strain. Three loci were unique (gmhD, narK and ndh) and the resulting alleles did not match any of the alleles of the Oklahoma strain, B. pseudomallei or B. thailandensis. A comparison of these three alleles with the corresponding alleles of the Oklahoma strain using BESTFIT indicates 2/468 bp difference (99.6 % match) for gmhD, 2/561 bp difference (99.6 % match) for narK and 2/443 bp difference (99.6 % match) for ndh. The allelic profile of E0147 was submitted to http://www.mlst.net and was assigned as ST234. We also determined that ATCC 23343T is a match to ST51.
To compare STs directly, the sequences for the seven loci were combined or concatenated to form a sequence of 3999 bp as described by Godoy et al. (2003). Neighbour-joining analysis (Kimura two-parameter, 500-step bootstrap) of the concatenated allele sequences by using MEGA 3 indicated that E0147 clusters with the Oklahoma isolates (Fig. 2). A BESTFIT analysis indicated a match of 99.9 % between the concatenated sequences for the Oklahoma strain (ST81) and E0147 (ST234). A BESTFIT analysis comparing the concatenated sequences of ST81 with ST51, the ST of the type strain of B. pseudomallei, indicated a match of 95 %.
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performed DNA–DNA hybridization in their study, they concluded that the Oklahoma clinical isolate did not represent a novel species. However, they made this determination by using a lower threshold of species delineation (i.e. a 60 % threshold) (Yabuuchi et al., 1992a). We decided to perform DNA–DNA hybridization on the Oklahoma and Georgia strains. Cells were harvested and lysed and the chromosomal DNA was isolated and purified as described previously (Brenner et al., 1982). DNA from strain C6786T was labelled with [32P]dCTP by using a commercial nick-translation kit (Invitrogen Life Technologies) and tested for reassociation to unlabelled DNA from the same strain (homologous reaction), as well as to C7532, C7533, E0147 and the type or reference strains of Burkholderia cepacia (ATCC 25416T), B. thailandensis (strain E254) and B. pseudomallei (ATCC 23343T) (heterologous reactions). Relative binding ratios (RBR) and percentage divergence were calculated as described previously (Brenner et al., 1982). The G+C content of the Burkholderia-like strains was determined by the thermal-denaturation method of Mandel et al. (1970).
The G+C content of strain C6786T was determined to be 66 mol%, so the hybridization reactions were done at 65 °C (optimum) and 80 °C (stringent). The results of DNA relatedness studies are given in Table 1. Isolates C7532, C7533 and E0147 exhibited >90 % relatedness (RBR) under both the optimum and stringent reassociation criteria and had divergence (D) of <5 % from the labelled reference (C6786T) strain. The reference strain was <65 % related to strains of B. cepacia (ATCC 25416T), B. thailandensis (E254) and B. pseudomallei (ATCC 23343T), with D values >5.5 % and the stringent RBR <40 %. Thus, we propose that the Oklahoma and the Georgia strains represent a single species, which we designate Burkholderia oklahomensis sp. nov., that is separate from the previously described Burkholderia species according to the molecular criteria for species-level relatedness established by Wayne et al. (1987). This assignment is supported strongly by the near identity of the concatenated sequences of the seven housekeeping (MLST) loci of the Oklahoma and Georgia isolates and their 
5 % divergence from those of B. pseudomallei.
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). Therefore, frontline diagnostic laboratories will probably continue to identify an isolate of B. oklahomensis presumptively as B. pseudomallei. Molecular-based methods described in this work performed in reference laboratories may then be used to aid in identifying the isolate. As these methods are not commonly used in frontline diagnostic laboratories, it is encouraging that PCR tests have recently been developed that exclude the Oklahoma strain, but detect B. pseudomallei and the closely related select agent B. mallei (Tomaso et al., 2004, 2005).
Although a previous study indicated that B. oklahomensis had a lower level of virulence than B. pseudomallei in an animal model, little is known about the virulence of B. oklahomensis in humans (McCormick et al., 1977; Nussbaum et al., 1980). More work is needed to assess the geographical prevalence and clinical significance of B. oklahomensis.
Description of Burkholderia oklahomensis sp. nov.
Burkholderia oklahomensis (ok.la.hom.en'sis. N.L. fem. adj. oklahomensis pertaining to Oklahoma, of Oklahoma).
The following description is based on the results of studies of four isolates. Cells are Gram-negative, bipolar-staining rods. The cells are aerobic and motile and grow well at 25 and 37 °C, but slowly at 42 °C. Colonies on 5 % sheep blood agar are irregular, white, smooth and show a diffuse lysis of red cells extending out from heavy growth with no action by individual colonies. They may become wrinkled or mucoid after 72 h incubation. Cells grow well on MacConkey agar. Cells are oxidase-, catalase-, arginine decarboxylase (dihydrolase)- and nitrate reduction-positive. Cells grow in nutrient broth with 0 % NaCl. The three Oklahoma strains (C6786T, C7532 and C7533) produce variable results for gas from nitrate. Both production of gas and of little to no gas have been observed (McCormick et al., 1977; Yabuuchi et al., 1992a). The Georgia strain (E0147) is negative for production of gas from nitrate. Tests for indole, urease, lysine decarboxylase, ornithine decarboxylase and hydrogen sulfide are negative. Cells show oxidative metabolism and, on triple-sugar iron agar, produce an acid reaction on the slant and no change in the butt of the medium. All strains are positive for assimilation of glucose, D-xylose, mannitol, lactose, sucrose and maltose. All are negative on minimal salts agar with 10 % arabinose (Wuthiekanun et al., 1995). Purple, wrinkled colonies are seen on Ashdown's agar after 1 week incubation at 37 °C (Ashdown, 1979). Differential tests are listed in Table 2.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Godoy, D., Randle, G., Simpson, A. J., Aanensen, D. M., Pitt, T. L., Kinoshita, R. & Spratt, B. G. (2003). Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei. J Clin Microbiol 41, 2068–2079.
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Tomaso, H., Scholz, H. C., Al Dahouk, S., Pitt, T. L., Treu, T. M. & Neubauer, H. (2004). Development of 5' nuclease real-time PCR assays for the rapid identification of the Burkholderia mallei//Burkholderia pseudomallei complex. Diagn Mol Pathol 13, 247–253.[CrossRef][Medline]
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Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I., Hotta, H., Hashimoto, Y., Ezaki, T. & Arakawa, M. (1992b). Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol Immunol 36, 1251–1275.[Medline]
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