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

A Burkholderia pseudomallei-like organism has recently been identified amon g some soil isolates of B. pseudomallei in an area with endemic melioidosis . This organism is almost identical to B. pseudomallei in terms of morpholo gical and biochemical profiles, except that it differs in ability to assimi late L-arabinose, These Ara(+) isolates are also less virulent than the Ara (-) isolates in animal models. In addition, clinical isolates of B. pseudom allei available to date are almost exclusively Ara(-). These features sugge sted that these two organisms may belong to distinctive species. In this st udy, the 165 rRNA-encoding genes from five clinical (four Ara(-) and one Ar a(+)) and nine soil isolates (five Ara(-) and four Ara(+)) of B, pseudomall ei were sequenced. The nucleotide sequences and phylogenetic analysis indic ated that the 165 rRNA-encoding gene of the Ara(+) biotype was similar to b ut distinctively different from that of the Ara(-) soil isolates, which wer e identical to the classical clinical isolates of B. pseudomallei. The nucl eotide sequence differences in the 16S rRNA-encoding gene appeared to be sp ecific for the Ara(+) or Ara(-) biotypes, The differences were, however, no t sufficient for classification into a new species within the genus Burkhol deria. A simple and rapid multiplex PCR procedure was developed to discrimi nate between Ara(-) and Ara(+) B. pseudomallei isolates. This new method co uld also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens.