Molecular Phylogeny of Burkholderia pseudomallei from a Remote Region of Papua New Guinea

Background The island of New Guinea is located midway between the world's two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales. Methodology/Principal Findings We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability. Conclusions/Significance Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.


Introduction
Melioidosis is a severe disease caused by Burkholderia pseudomallei, a saprophytic Gram-negative b-Proteobacteria frequently isolated from soil and water in tropical and subtropical regions worldwide [1]. A feature of the disease is spatial clustering of clinical incidence linked to environmental prevalence of the organism. Whilst it is well established that infection follows environmental exposure, factors contributing to the environmental persistence and dispersal of B. pseudomallei remain to be elucidated. Molecular typing techniques have allowed detailed phylogenetic studies. Multi-locus sequence typing (MLST) has been used to examine the diversity of B. pseudomallei from various geographical regions [2,3,4,5] and revealed patterns of geographical partitioning between Australian and Southeast Asian isolates. Also, it has been determined that Australian isolates are more likely to carry an ancestral B. thailandensis-like flagellum and chemotaxis gene cluster (BTFC), whilst isolates from Asia almost exclusively carry a Yersinia-like fimbrial gene cluster (YLF) [6]. This suggests that these populations are genetically distinct due to broad scale biogeographical factors associated with establishment and persistence of the organism. More recently, whole genome sequencing has resolved that Asian isolates of B. pseudomallei share an Australian ancestral root [7].
Recent studies from Papua New Guinea (PNG) have identified a focus of melioidosis endemicity in a rural community in the Western province. A feature of these isolates is their narrow genetic diversity over spatial and temporal scales [8]. The island of New Guinea is positioned midway between the world's two major melioidosis endemic regions; Northern Australia and Southeast Asia. Detailed phylogenetic study of isolates from this region will advance understanding of the biogeography of melioidosis. This will provide insight into the ecology of B. pseudomallei through elucidation of the mechanisms involved in evolution, speciation and dispersal, and assist predictive mapping of disease prevalence. This study employed molecular typing techniques in an attempt to elucidate the origins of the PNG isolates and therefore the role of New Guinea in the dispersal of B. pseudomallei.

Ethics Statement
Approval and ethical clearance for this study was granted by the Medical Research Advisory Committee (MRAC) of Papua New Guinea under MRAC No 10.03. All clinical isolates collected originate from diagnostic specimens, and as such patients did not provide written informed. MRAC is the appropriate body in PNG to grant approval for the later use of clinical samples in research, as was the case for this study.

Bacterial isolates and DNA extraction
This study analyzed 13 clinical (from eight patients) and 26 environmental isolates of B. pseudomallei (Table 1), which were retrieved from the Balimo region of PNG ( Figure 1) as previously described [8,9]. Bacteria were stored in the James Cook University culture collection at 280uC in double strength Luria-Bertani broth supplemented with an equal volume of glycerol. Isolates were plated onto Ashdown's agar [10] and cultivated at 37uC for 48 hours prior to DNA extraction. A single colony from each plate was removed for DNA extraction using the RBC Genomic DNA extraction kit (RBC Bioscience, Chung Ho City, Taiwan) as per the manufacturer's directions.

Genotyping using MLST and MLVA
PCRs for MLST were performed using standard reagents from RBC and contained: 50 ng template DNA, 1 6 buffer (containing 2 mM MgCl and BSA), 0.8 mM mixed primers, 0.2 mM dNTPs, 0.25 U Taq polymerase, and molecular biology grade H 2 O (to 30 ml). Primers for MLST were as described [3] with the recommended amendments listed on the B. pseudomallei MLST website (http://bpseudomallei.mlst.net). Cycling conditions consisted of an initial denaturation period of 3 min at 95uC, followed by 40 cycles of 95uC for 30 sec, 62uC for 30 sec and 72uC for 30 sec and a final elongation of 72uC for 10 min. Sequencing products were analyzed by electrophoresis using a 1.5% agarose gel to ascertain correct fragment size, concentration and purity. Sequencing reactions generating insufficient or multiple products were discarded and repeated. Reactions were purified and sequenced by Macrogen (Seoul, South Korea), using the ABI PRISM3700 automated sequencing instrumentation (Applied Biosystems, Foster City, CA). New alleles and sequence types (STs) were submitted to the Burkholderia MLST website curator.
Phylogenetic reconstructions were performed using the maximum likelihood algorithm provided by PhyML [11] with the Tamura-Nei nucleotide substitution model against the entire MLST database (http://bpseudomallei.mlst.net), which as of September 2010 encompasses almost 700 distinct STs from more than 1,900 isolates and at least three other closely related species. Bootstrapping was performed 50 times, and trees were optimized for topology, rather than branch lengths and rate parameters. Trees were visualized and modeled using FigTree 1.1.2 software (http://tree.bio.ed.ac.uk).
Assignment of STs into either the Australian or Southeast Asian population was achieved using Structure 2.2 [12]. Structure uses allelic frequency data to identify population structure and assign individuals to populations. We used MLST allelic data downloaded from the B. pseudomallei MLST database for Structure analyses. Briefly, 100,000 iterations with a burn-in period of 30,000 iterations were used to determine population assignments of STs using the ''admixture'' model and assuming two populations as previously established [7]. Here, we report the percentage of iterations in which each PNG ST was placed into the Australian population rather than the Southeast Asian population.
Multiple-locus variable number of tandem repeat (MLVA) characterization was carried out as previously described [13]. Amplicons from 23 VNTR loci were used to characterise the 29 isolates. Amplicon sizes for all isolates at all loci were determined by two independent scorers to reduce bias. The Neighbor-Joining algorithm [14] in PAUP 4.0b [15] was used to illustrate patterns of relatedness among samples.

Fimbrial gene cluster analysis
Detection of BTFC and YLF gene clusters was performed using a multiplex real-time PCR melt procedure as previously described [6]. Briefly, 15 ml PCRs were comprised of RBC reagents and contained: 5 ng template DNA, 1 6 buffer (containing 2 mM MgCl and BSA), 0.3 mM of each primer, 0.2 mM dNTPs, 0.25 U Taq polymerase, 5 mM SYTO 9 (Invitrogen, Mulgrave, Australia)

Antimicrobial Sensitivity Testing
Comparative antimicrobial sensitivity testing was performed using the disk diffusion conditions for Burkholderia cepacia as per CLSI antimicrobial testing standards (January 2009). Resistance/ susceptibility was defined on the basis of the absence or presence of a visible zone of inhibition (ZOI) around the antibiotic disk. The assay was controlled using Escherichia coli ATCC 25922. Antibiotics tested were; chloramphenicol (30 mg), meropenem (10 mg), gentamicin (10 mg), amoxycillin/clavulanic acid (30 mg) and tetracycline (30 mg).

Results
MLST of the 39 isolates resolved three unique STs. A single ST comprised 32 of the isolates from this area (ST267). The remaining seven Balimo isolates comprised two STs (ST667; n = 6 and ST668; n = 1), differing from each other by a single nucleotide polymorphism in the ace gene, but by four and five locus variations from ST267, respectively. Phylogenetic analysis of the entire B. pseudomallei MLST database revealed distinct clustering of Asian isolates (including Malaysian, Singaporean, Philippine and Indonesian STs) separate from those of Australian origin (data not shown). All three STs from Balimo were located in clades heavily dominated by Australian isolates (Figure 2). Population genetic analysis using Structure 2.2 also demonstrated that all three STs from Balimo were more closely associated with the Australian population than the Southeast Asian population. ST267 and ST668 were both assigned to the Australian population in 100% of iterations and ST667 was assigned to the Australian population in 99.9% of iterations. Real-time PCR for the fimbrial gene cluster however, determined that all PCR products had distinct melt peaks at 88uC, which is consistent with the YLF type gene cluster found predominantly among isolates of Southeast Asian origin.
To obtain further resolution of the PNG STs, we employed the high-resolution 23-locus MLVA technique to these isolates. MLVA resolved 24 genotypes into three distinct clades, which corresponded with the three different STs (Figure 3). Clinical isolates from the same patient were identical by MLVA on two occasions (C5 & C6 and C11 & C13), but were divergent by one to three loci in an ancestral topography during all other cases (C2 &  C3, C7 & C8 and C9 & C10), consistent with in vivo mutation as previously reported [16].
All isolates demonstrated susceptibility to meropenem, amoxycillin/clavulanic acid and tetracycline. Resistance to chloramphenicol however, was confined to a single MLVA clade (Figure 3; box A).

Discussion
Structure and MLST analyses suggest that isolates from the Balimo region are more closely related to those from Australia than Southeast Asia. Although MLST data from B. pseudomallei does not provide good phylogenetic resolution due to extremely high rates of lateral gene transfer in relation to mutation, phylogenetic analyses from this and other studies show a tendency for groups of isolates from Australia to cluster independently of Asian isolates. Approaches that compare allele frequencies across populations in order to determine population assignments have been successfully used with MLST data from B. pseudomallei and other highly recombinogenic species. Here too, our analyses showed that the three STs identified in Balimo are more closely related to the Australian population rather than the Asian population. Conversely, all of the isolates in this study contained the YLF gene cluster, which is much more common among Asian isolates (98%) than those from Australia (12%) [6]. Given the lower frequency of this gene cluster in members of the Australian population and the close proximity of New Guinea to both Australia and Indonesia, it is possible that B. pseudomallei from PNG represent unique genotypes that share characteristics with both major populations.
It has been suggested that geographical ties between PNG and Australia have aided dispersal of the organism during the Last Glacial Maximum (LGM) [7]. The LGM was during the Pleistocene period (approximately 21,500 years ago) at which time sea levels were lower than present, resulting in a continuous land mass from Australia to New Guinea forming the Sahul continent [17,18]. Similarly, mainland Asia was united with the Indonesian and Philippine islands forming the continent of Sunda, separated from Sahul by the Java and Philippine oceanic trenches [19] (Figure 1a). Concurrently, global temperatures were substantially lower than present, most likely compressing the endemic boundaries of ancient B. pseudomallei populations toward the equator. As such, it is likely that human and/or animal movements through New Guinea [20] played a pivotal role in the dissemination of B. pseudomallei, however, analysis of a broader range of isolates from the island are required for confirmation.
To elucidate small-scale partitioning of B. pseudomallei isolates from Balimo, we employed higher resolution genotyping in the form of MLVA. Studies employing this technique have recently determined that analysis of multiple clinical isolates is necessary due to the high rate of in vivo mutation [16,21]. However, the concept of measuring in vivo evolutionary patterns using molecular techniques such as MLVA was in its infancy at the time of sample collection. Despite this shortcoming, the genotyping of this dataset retains its value because the samples typed probably represent the highest frequency in vivo genotype. Moreover, the high levels of genetic homogeneity observed among environmental isolates collected over several years (Figure 3; box A) compared to clinical isolates from a single patient collected suggests that both environmental and in vitro mutation rates may be limited in comparison to those in vivo.
While MLST data suggest low genetic diversity among these isolates, MLVA indicates that genetic diversity exists within the three STs. As only six of the 24 genotypes are represented by more than one isolate, it is likely that our sampling does not reflect the full extent of MLVA diversity. In many cases, branch lengths between isolates are relatively long, reflecting considerable evolutionary divergence. One exception to this is the short branches connecting isolates in box A (Figure 3) suggesting that members of this group (almost half of all the isolates) are closely related to each other. Antimicrobial sensitivity testing bolsters the MLVA in determining that only these isolates are resistant to chloramphenicol; an antibiotic which, due to the limited availability of due to the limited availability of ceftazidime, has been extensively used for the treatment of melioidosis in the region [8]. Given that highly resistant chloramphenicol mutants emerge in vivo among 7.1% of patients [22], that chloramphenicol is nonbactericidal [22], and that the excretion of viable B. pseudomallei in feces occurs in approximately one quarter of human melioidosis cases [23], it is possible that excretion and cycling of antibiotic resistant genotypes in this subsistent village based community is responsible for the low diversity demonstrated in box A (Figure 3).
The relatively long branches within the other two groups suggest that these lineages of B. pseudomallei have persisted in this area for a long period; however, growth rates in the environment and the effects of mutational saturation on branch lengths remains to be determined. Despite this, it is likely, given the eleven clonal isolates in box A (Figure 3), which were collected over six years and from various geographical locations, that this clade represents a stable population of B. pseudomallei congruent with our hypothesis of long term stability in the environment, as opposed to recent importation. As only a single isolate from ST668 was collected, it is possible that this ST has not been prevalent in this region for as long as the other STs. Alternatively, as this ST differs from ST667 by just one base substitution at a single MLST locus, it is possible that further sampling will lead to the recovery of more isolates from ST668.
The stability and fragmentation of B. pseudomallei populations around Balimo is at least partially impacted by anthropogenic influences. Previously, the introduction of B. pseudomallei into nonendemic regions has been linked to the importation of infected animal carriers [24,25], and anthropogenic influences are highly likely to have been responsible for the dissemination of B. pseudomallei from Australia into Asia. Recent studies have estimated that the introduction on B. pseudomallei into Asia has occurred within a time-frame congruent with the arrival of the first humans into Austronesia [7]. PNG is highly diverse in terms of culture, language and human genetics due to limited human mobility in the region [26]. If human movement has facilitated dispersal of B. pseudomallei between regions of endemicity in other parts of the world, it stands to reason that in regions where human movement is limited, so should the distribution and diversity of B. pseudomallei isolates. In conclusion, the role of PNG in the global distribution of B. pseudomallei remains to be fully elucidated. Closer analysis of the PNG isolates and phylogenetic reconstructions using a broader range of isolates from the island and adjacent regions such as the Torres Strait will undoubtedly provide a deeper insight into the biogeography of melioidosis.