Phylogenetic analysis of Ara+ and Ara- Burkholderia pseudomallei isolates and development of a multiplex PCR procedure for rapid discrimination between the two biotypes

Abstract

A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate L-arabinose. These Ara+ isolates are also less virulent than the Ara- isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara-. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara- and one Ara+) and nine soil isolates (five Ara- and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara- soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara- biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara- and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens.

FIG. 1

Specificity of the PCR primers. Comparison of nucleotide sequences of the 16S rRNA genes corresponding to primers Bps16S-42L (A) and Bps16S-427R (B). The nucleotide sequence of Ara⁻ B. pseudomallei isolates was compared with those of Ara⁺ B. pseudomallei isolates, other Burkholderia spp., H. seropedicae, R. eutropha, P. aeruginosa, P. putida, S. typhi, and E. coli. The sequences at the primer sites are underlined.

FIG. 2

Alignment of nucleotide sequences of 16S rRNA genes from four clinical isolates (K96243, K94050, S94004, and S94017) and five soil isolates (TRF684, TRF685, TRF688, TRF824, and TRF830) of Ara⁻ B. pseudomallei, and one clinical isolate (S95019) and four soil isolates (TRF681, TRF682, TRF683, and TRF686) of Ara⁺ B. pseudomallei (GenBank accession no. AF093047 to AF093060). The published sequences of B. pseudomallei 1026b (U91839) and B. thailandensis E264 (U91838) are also included for comparison.

FIG. 3

Phylogenetic tree of the 16S rRNA genes shows the relationship of the Ara⁻ and Ara⁺ B. pseudomallei isolates with other bacteria. The sequences used in this analysis are from the bacteria (1) Ara⁻ B. pseudomallei, (2) Ara⁺ B. pseudomallei, (3) B. plantarii, (4) B. glumae, (5) B. vietnamensis, (6) B. cepacia, (7) B. cocovenenans, (8) B. pyrrocinia, (9) B. glathei, (10) B. gladioli, (11) B. graminis, (12) B. phenazinium, (13) B. vandii, (14) B. caryophylli, (15) B. andropogonis, (16) B. caribiensis, (17) R. eutropha, (18) H. seropedicae, (19) B. bronchiseptica, (20) I. dechloratans, (21) B. holmesii, (22) Z. ramigera, (23) O. formigenes, (24) J. lividum, (25) P. syzygii, (26) denitrifying Fe-oxidizing bacteria, (27) D. zoogloeoides, (28) D. acidovorans, (29) ultramicrobacterium, (30) R. gelatinosus, (31) B. parapertussis, (32) L. discophora, (33) B. pertussis, (34) B. denitrificans, (35) B. avium, (36) A. xylosoxidans, (37) A. indigens, (38) A. sinuosum, (39) A. faecalis, (40) S. maltophilia, (41) P. aerguinosa, (42) P. putida, (43) S. typhi, and (44) E. coli. The scale bar indicates a distance in substitutions per nucleotide.

FIG. 4

Phylogenetic analysis of the 16S rRNA genes of 16 members in the genus Burkholderia for which the complete or almost-complete 16S rRNA sequences were available. The scale bar indicates a distance in substitutions per nucleotide.

FIG. 5

Ethidium bromide-stained agarose gel electrophoresis of the amplified 16S rRNA genes of Ara⁻ and Ara⁺ B. pseudomallei isolates performed by using the multiplex primer set. M, 100-bp marker; lane 1, Ara⁻ B. pseudomallei isolate K94050; lane 2, Ara⁻ B. pseudomallei isolate K96243; lane 3, Ara⁺ B. pseudomallei isolate S95019; lane 4, Ara⁺ B. pseudomallei isolate TRF681; lane 5, negative control.