Use of a variable amplicon typing scheme reveals considerable variation in the accessory genomes of isolates of Burkholderia pseudomallei

Abstract

Melioidosis, a disease caused by the bacterium Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We used suppression subtractive hybridization (SSH) to identify sequences that varied between two B. pseudomallei isolates from Australia and determined the distribution of 45 SSH-derived sequences among a panel of B. pseudomallei and B. thailandensis isolates. Sequences exhibiting variable prevalence were included in a variable amplicon typing (VAT) scheme designed to score the presence or absence of 14 PCR amplicons. VAT analysis was carried out with 48 isolates from Thailand, which were typed by multilocus sequence typing (MLST), and 44 isolates from Australia of known MLST type. The VAT scheme could be used to divide the 48 isolates from Thailand into 23 VAT types and the 44 isolates from Australia into 36 VAT types. Some of the sequences included in the VAT scheme were more commonly PCR positive among isolates from Australia than among isolates from Thailand, and vice versa. No isolate from Australia was PCR positive for genomic island 11 or a putative transposase sequence, whereas four SSH-derived sequences were far more prevalent among the Australian isolates. Analysis based on the VAT scheme indicated that the isolates clustered into groups, some of which were mainly or exclusively from one geographical origin. One cluster included Australian isolates that were mostly associated with severe disease, including rare neurological melioidosis, suggesting that the content of the accessory genome may play an important role in determining the clinical manifestation of the disease.

FIG. 1.

PCR-based distribution analysis of SSH sequences. Filled boxes indicate PCR-positive results.

FIG. 2.

M-PCR analysis of B. pseudomallei isolates. The figure shows agarose gels of example PCR amplicons derived from Thai isolates (indicated above individual lanes) by M-PCR1 (a), M-PCR2 (b), M-PCR3 (c), and M-PCR4 (d). The first lane on each gel contains a 1 kb-plus DNA ladder (Invitrogen). +ve, positive control.

FIG. 3.

Cluster analysis of VAT profiles. The VAT profiles were clustered by using a multivariate analysis for clustering of observations in the MINITAB software package. A dendrogram was constructed by using average linkage and Pearson distance. One isolate of B. thailandensis (isolate E52) was included in the analysis as an outlier. B. pseudomallei isolates P1 to P50 were from Thailand. All other isolates with the exception of isolates 139 to 141 and 314 were from Australia. The cluster that includes the neurotropic isolates is bracketed.