Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics.

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Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics

Global and Tropical Health Division, Menzies School of Health Research, Darwin, Northern Territory, Australiaa; Department of Infectious Diseases and Northern Territory Medical Program, Royal Darwin Hospital, Darwin, Northern Territory, Australiab; Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, USAc

Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia.

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he Gram-negative soil-dwelling bacterium Burkholderia pseudomallei is the etiologic agent of melioidosis, an often deadly tropical disease that can be difficult to diagnose, particularly in nonendemic or resource-poor regions where cases are not expected and appropriate microbiological diagnostic tools are not readily available (1). Diabetics are particularly susceptible to melioidosis. B. pseudomallei infection can be acquired from contaminated soil or water by percutaneous inoculation, inhalation, aspiration, or ingestion, and no vaccine targeting this organism is available (2). In 2012, B. pseudomallei was reclassified by U.S. federal agencies as a tier 1 select agent, the highest risk category for a biological entity, due to concerns that this bacterium would pose a severe threat to humans and animals in the event of its deliberate misuse (3). The B. pseudomallei genome exhibits high homologous recombination rates. On a per-allele basis, recombination is estimated to occur between 18 and 30 times more frequently than mutation (4). This extensive lateral gene transfer can confound population analyses, particularly those that are based on studying limited geographic regions (e.g., the Northern Territory, Australia [5]) due to high rates of homoplasy observed among genetic variants. In contrast, genomic analyses of B. pseudomallei populations on a continental scale have revealed a clear separation of B. pseudomallei isolates between Asia and Australia (4, 6, 7). Bayesian analysis of B. pseudomallei genome variation points to an ancient separation, with migration out of Australia into Asia occurring tens of thousands of years ago during the Pleistocene (4). The rarity of pathogen movement is due largely to one factor: new melioidosis cases

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almost always result from bacterial infection acquired from the local environment, with human-to-human and zoonotic transmission of this pathogen being exceedingly rare (8). In support of the rarity of B. pseudomallei movement across major biogeographic boundaries, the definitive transmission of B. pseudomallei from Asia into Australia has not yet been observed. Nevertheless, melioidosis cases imported into nonendemic locations via travelers are being increasingly reported, as is recognition of locations that are endemic for melioidosis outside the classical regions of Southeast Asia and Australia (9). With modern global travel and

Received 15 September 2015 Accepted 13 November 2015 Accepted manuscript posted online 25 November 2015 Citation Price EP, Sarovich DS, Smith EJ, MacHunter B, Harrington G, Theobald V, Hall CM, Hornstra HM, McRobb E, Podin Y, Mayo M, Sahl JW, Wagner DM, Keim P, Kaestli M, Currie BJ. 2016. Unprecedented melioidosis cases in northern Australia caused by an Asian Burkholderia pseudomallei strain identified by using large-scale comparative genomics. Appl Environ Microbiol 82:954 –963. doi:10.1128/AEM.03013-15. Editor: H. Goodrich-Blair Address correspondence to Erin P. Price, [email protected]. * Present address: Yuwana Podin, Institute of Health and Community Medicine, Universiti Malaysia Sarawak, Sarawak, Malaysia. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.03013-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Applied and Environmental Microbiology

February 2016 Volume 82 Number 3

Downloaded from http://aem.asm.org/ on January 26, 2016 by Washington University in St. Louis

Erin P. Price,a Derek S. Sarovich,a Emma J. Smith,a,b Barbara MacHunter,a Glenda Harrington,a Vanessa Theobald,a Carina M. Hall,c Heidie M. Hornstra,c Evan McRobb,a Yuwana Podin,a* Mark Mayo,a Jason W. Sahl,c David M. Wagner,c Paul Keim,c Mirjam Kaestli,a Bart J. Curriea,b

Asian Origin for Australian Melioidosis Cases

MATERIALS AND METHODS Ethics statement. Ethical approval for the DPMS was obtained through the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research, approval no. HREC 02/38 (“Clinical and epidemiological features of melioidosis”). All patient data were deidentified prior to analysis. Culturing, DNA extraction, and WGS. Human and animal B. pseudomallei isolates were initially subcultured from clinical specimens (excluding blood samples) using a combination of Ashdown’s broth and subsequent plating onto Ashdown’s agar (Oxoid; Thebarton, South Australia, Australia). Blood samples were incubated in blood culture bottles until microbial growth was detected, and cultures were subsequently incubated onto chocolate and horse blood agar under aerobic and anaerobic conditions; B. pseudomallei was confirmed using the highly specific type three secretion system I assay (21). Environmental samples from soil, water, and air were cultured according to methods described elsewhere (22, 23), with soil samples in the current study collected at multiple depths (10 cm, 15 cm, or 20 cm). For genomic DNA extraction, isolates were subcultured once for purity on chocolate agar (Oxoid). The isolates were grown under aerobic conditions at 37°C for 24 h prior to extraction. DNA for PCR only was extracted using 5% Chelex 100, heat treated at 95°C for 20 min, and diluted 1:10 in molecular-grade H2O prior to PCR. DNA for WGS was extracted as previously described (24), quality checked, and quantified using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Scoresby, Victoria, Australia). Genomic data were generated from paired-end Illumina reads using the HiSeq 2000 platform (Illumina, Inc., San Diego, CA, USA). WGS was performed at Macrogen, Inc. (n ⫽ 184; Geumcheon-gu, Seoul, Republic of Korea) or the Center for Microbial Genetics and Genomics and Translational Genomics Research Institute (n ⫽ 132; Flagstaff, AZ, USA).


Clinical isolates. For the 25 years from October 1989 to August 2014, 888 melioidosis cases were enrolled in the DPMS. Of these, epidemiological and clinical case history data have identified 473 (53.2%) cases that were acquired in the Darwin urban region, the most densely populated region and the capital city of the Northern Territory. This region encompasses approximately 300 km2 and extends east to the greater Palmerston area, north to Buffalo Creek, and south to the Channel Island region. A viable B. pseudomallei culture was not able to be obtained for 18 (3.8%) of these clinical cases. All other isolates (n ⫽ 455) are available and were therefore examined in this study. Environmental isolates. Since 2001, we have conducted environmental sampling of B. pseudomallei in the Darwin urban region as part of ongoing surveillance and public health monitoring of this pathogen. These sampling efforts have yielded 223 unique B. pseudomallei-positive sites. All isolates were obtained from soil, with the exception of one isolate from an air sample (23) and six from water (see Table S3 in the supplemental material). Animal isolates. We also examined 17 B. pseudomallei isolates from 10 melioidosis-afflicted animals (sheep, cattle, camel, and parrot) that presented with melioidosis between 1993 and 2014. These animals were housed in an area where ST-562 was identified in the environment. MLST analyses. The B. pseudomallei MLST database (http: //bpseudomallei.mlst.net/) currently harbors the greatest number of B. pseudomallei strain genotypes from around the globe, making it a useful tool for geographic attribution. As of 18 May 2015, this database contained 1,092 unique STs among 3,649 submitted B. pseudomallei isolates. Of these isolates, 3,283 isolates (90%) originate from either Asia or Australia, constituting 1,020 STs. These 1,020 STs were analyzed by eBURST (25) to determine the probable geographic origin of ST-562. A total of 521 (51%) STs were from Australia, 495 (49%) STs were from Asia, and 4 STs (ST-89, ST-105, ST-562, and ST-849) were found in both regions. All isolates from Bangladesh, Cambodia, China, Hong Kong, India, Indonesia, Laos, Malaysia, Pakistan, the Philippines, Singapore, Sri Lanka, Taiwan, Thailand, Vietnam, and Yap were included in the Asian group. To simplify the analysis, only STs sharing 6 loci within group 1 (founder, ST-48) were examined by eBURST. MLST data have previously been generated for 398/455 clinical and 88/223 environmental isolates (see Table S3 in the supplemental material) from the Darwin urban region. Due to the high cost and labor-intensive nature of MLST, isolates lacking MLST data for this study were genotyped using novel ST-specific single-nucleotide polymorphism (SNP) real-time PCR assays targeting the four most common Darwin region STs (ST-36, ST-109, ST-132, and ST-562), using the methods described below. Genomic analyses. To identify ST-specific SNPs for ST-36, ST-109, ST-132, and ST-562 and to identify the probable geographic origin of ST-562, we performed comparative genomic analysis across a large B. pseudomallei isolate data set. We used WGS data for 455 predominantly Australian B. pseudomallei isolates comprising 217 STs (see Table S2 in the supplemental material). Additional autochthonous isolates from Brazil, China, Malaysia, Pakistan, Papua New Guinea, Singapore, Thailand, and Vietnam and an allochthonous isolate from a melioidosis case diagnosed in Arizona, USA, were included in our analysis. WGS data were either obtained from public databases (n ⫽ 139) or generated in-house (n ⫽ 316). For publicly available genomes lacking Illumina data, synthetic paired-end reads were generated with ART (version VanillaIceCream) (26) using quality shift values of 10. The comparative genomics pipeline SPANDx version 2.7 (27) was used to identify orthologous core-genome biallelic SNPs among the 455 B. pseudomallei genomes using default parameters. The B. pseudomallei Thai strain K96243 (28) (GenBank accession numbers NC_006351 and NC_006351 for chromosomes 1 and 2, respectively) was used as the reference genome for read mapping alignment. SNPs specific for ST-36, ST-109, ST-132, and ST-562 were identified initially from the SPANDx-generated SNP matrix (comprising 223,719 SNPs) using PLINK version 1.07 (29). The SNPs with the largest distance separating them from other SNPs were preferentially chosen for


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commerce, the potential for long-range transmission and ecological establishment of B. pseudomallei is increasing. Lower-resolution genotyping methods, such as multilocus sequence typing (MLST) (10–13) and 16S internal transcribed spacer typing (14), support the continental separation of B. pseudomallei observed on the genomic level (see, e.g., references 5 and 15), and these molecular tools have proven useful for source tracing B. pseudomallei in regions that are nonendemic for the organism (14, 15). However, these genotyping methods have their limitations, as evidenced by a recent study in which two multilocus sequence types (STs) shared between Australia and Cambodia were found to be genetically unrelated on the whole-genome level (7). Thus, whole-genome sequencing (WGS) is essential for confirming the true geographic origin of B. pseudomallei strains. Darwin, the capital city of the Northern Territory, Australia, has an exceptionally high incidence of melioidosis (16), with published rates paralleled only in northeastern Thailand (17). Since October 1989, the Darwin Prospective Melioidosis Study (DPMS) has documented all known melioidosis cases in Darwin and the greater Top End region of the Northern Territory (18, 19). In 2001, we began undertaking environmental sampling in Darwin to detect, characterize, and better understand the distribution of B. pseudomallei in this region. In this study, we detail the unprecedented occurrence and rise in prevalence of an Asian B. pseudomallei clone, ST-562 (20), in the Darwin region. Prior to 2005, this ST was not observed in Australia, with other STs (ST-36, ST-109, and ST-132) being dominant in clinical, animal, and environmental isolates from this region. Surprisingly, ST-562 is now the most common ST affecting melioidosis patients in the Darwin region, having surpassed the prevalence of ST-36, ST-109, and ST-132 in the 2013-2014 northern Australian wet season.

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TABLE 1 Effect of genetic recombination on B. pseudomallei diversity in ST-36, ST-109, ST-132, and ST-562a No. of SNPs after default filter (ⱖ3 SNPs within 10 bp)

No. of SNPs after heavy filter (ⱖ2 SNPs within 2 kb)

% SNPs removed by heavy filter

ST-36 ST-109 ST-132 ST-562

28 36 21 31

379 9,536 615 56

297 331 99 56

22 97 84 0

a SNP, single-nucleotide polymorphism. The default SNP density filter in SPANDx (27) was compared with a stringent SNP density filter, which removes the majority of recombinogenic SNPs.

assay design due to a decreased likelihood of recombination in these regions and for increased probability of primer binding specificity. The locus specificity for each ST SNP assay was confirmed with BLAST. The Yersinia-like fimbrial (YLF) and Burkholderia thailandensis-like flagellum and chemotaxis (BTFC) gene clusters were identified in silico using the BEDCov presence/absence matrix generated by SPANDx. In silico MLST was performed on all genome-sequenced isolates to confirm ST assignments, and new isolate and ST data were uploaded to the public B. pseudomallei MLST database. To assess within-ST genomic diversity, each ST-36, ST-109, ST-132, and ST-562 strain was analyzed against a reference genome of the same ST. The publicly available genomes for B. pseudomallei MSHR0305 (GenBank accession numbers NC_021884 and NC_021877) (30), MSHR4462 (GenBank accession no. JPQM00000000) (31), MSHR465J (GenBank accession no. JPZW00000000) (31), and MSHR5858 (GenBank accession numbers CP008892 and CP008891) (32) were used as alignment references for the ST-36, ST-109, ST-132, and ST-562 Illumina data, respectively. In addition to the default SPANDx SNP filtering parameters (i.e., exclusion of SNPs in which ⱖ3 are found within 10 bp of each other), phylogenetic reconstruction was also carried out using a heavy SNP recombination density filter (i.e., exclusion of SNPs for which ⱖ2 are found within 2 kb of each other) to mitigate the effects of recombination on observed strain diversity (Table 1; see also Fig. S2 in the supplemental material) (7, 33). The ST genome phylogenies used both SNP and insertion-deletion (indel) variants to improve the epidemiological associations, as we described elsewhere (5). Recombinogenic SNPs were also identified using the default parameters in Gubbins (see Fig. S3 in the supplemental material) (34). ST SNP typing. Isolates lacking MLST data in our isolate data set were screened with an inexpensive PCR method to determine their ST. Candidate SNPs specific for the four STs were converted to SYBR green-based mismatch amplification mutation assays (SYBR MAMAs) (see Fig. S1 in the supplemental material). We chose the SYBR MAMA format (also known as allele-specific real-time PCR) due to its speed, simplicity, and low cost (35, 36). The ST-specific primers are as follows, in 5= to 3= format (lowercase bases indicate deliberate mismatches [36], and underlined bases indicate the SNP position): ST36-only-T (CCGACGAGCCGACGt GT), ST36-others-C (CGACGAGCCGACGtGC), and ST36-For (TCGT GCGTGACGATGATGAC); ST109-only-T (GGTCCGCGAGCATCGa CT), ST109-others-A (GTCCGCGAGCATCGaCA), and ST109-For (GG CTTCTCCGTGCCGAAC); ST132-only-T (CGCAGGCCGCGATGtAT), ST132-others-C (CGCAGGCCGCGATGtAC), and ST132-Rev (CGAGA GCATCGTCTGGGACAC); and ST562-others-T (AACGAATATGTCGT GACCGTcAT), ST562-only-C (AACGAATATGTCGTGACCGTcAC), and ST562-Rev (GTGCCCTTGTCGAACAGYTAG). To assess the accuracy of the four SYBR MAMAs, 139 B. pseudomallei isolates with WGS data were screened across the ST SNP assays; all four assays performed with 100% accuracy. The ST SNP assays were subsequently screened across all Darwin region isolates lacking MLST data. Real-time PCRs were conducted in a 5-␮l total reaction volume in 384well optical PCR plates using 1⫻ SYBR green PCR master mix (Applied

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RESULTS

MLST analyses suggest that ST-562 is Asian in origin. To investigate the likely origin of ST-562, we first performed a basic interrogation of the B. pseudomallei MLST database. Of the 46 ST-562 isolates deposited in the MLST database to date, 45 have been epidemiologically linked to the Darwin region or have been retrieved from the Darwin environment, with one isolate from a melioidosis patient in Hainan, southern China (see Table S1 in the supplemental material). A search for ST-562 single-locus variants (SLVs) (i.e., 6/7 MLST loci shared with ST-562) identified several STs: ST-99, ST-167, ST-303, ST-388, ST-704, ST-1257, and ST1349. All isolates harboring these STs are Asian in origin, being represented by clinical or environmental isolates from Cambodia, China, Malaysia, the Philippines, and Thailand. Using lessstringent criteria to identify ST-562 double-locus variants (DLVs) (i.e., 5/7 loci shared with ST-562), we again failed to find any Australian STs, although additional isolates from Bangladesh, Kenya, Laos, Pakistan, Papua New Guinea, and Vietnam were found. We next performed eBURST (25) analysis of the MLST data from all available Asian and Australian isolates (n ⫽ 3,283 isolates; 1,020 STs) to determine the likely origin of the ST-562 clone. eBURST is a popular tool for assessing and reconstructing the evolutionary relatedness of bacterial strains with associated MLST data (25), although its accuracy is diminished in highly recombinogenic species such as B. pseudomallei (7, 38). Nevertheless, this tool can provide useful, albeit not definitive, phylogeographic information for this species. Although not conclusive, our eBURST analysis demonstrated that ST-562 probably resides within an Asian clade, as it grouped with other Asian STs and is well separated from all Australian STs (Fig. 1). Comparative genomic analysis confirms an Asian origin for Australian ST-562. To conclusively identify the geographic origin of the ST-562 clone found in northern Australia, we performed additional WGS on 316 isolates comprising primarily Australian B. pseudomallei (n ⫽ 305) isolates. These data were combined with 129 publicly available B. pseudomallei genomes representing Asian, Australasian, and South American isolates, for a total of 455




ST

No. of genomes

Biosystems, Foster City, CA, USA), primers at a concentration of 250 nM, and molecular-grade water. Each allele-specific primer was paired with the common primer and run as two separate reactions per ST assay. Appropriate controls (target ST, nontarget ST, and no-template controls [NTCs]) were included for each run to ensure accuracy. Thermocycling was carried out using default parameters on the ABI Prism 7900HT instrument (Applied Biosystems); a dissociation curve was included at the end of each run to ensure specificity. All samples were run in duplicate using 1 ␮l of DNA template. Phylogenetic analysis. Maximum parsimony trees were generated from relevant SPANDx .nex outputs using PAUP* 4.0b10 (37). For within-ST genomic comparisons, SNP and small indel variants were combined for increased resolution and improved epidemiological signal (5). For the 455-genome phylogeny, bootstrapping using 100 replicates was performed to determine the robustness of the relevant branches. Maximumlikelihood trees were generated from the Gubbins analyses as part of the Gubbins software (34). Nucleotide sequence accession numbers. Illumina sequence data for the 107 ST-36, ST-109, ST-132, and ST-562 B. pseudomallei genomes described in this study have been deposited into the Sequence Read Archive database under accession no. SRP065595. Other non-publicly available genomes described in this study are available from the authors upon request.


isolates, and purple shading indicates STs that overlap between Australia and Asia. All other colors were generated by the eBURST program and are explained elsewhere (25). One overlapping ST, ST-89, was not included in this diagram, as explained in Materials and Methods. This analysis hints that ST-562 might be Asian in origin.

genomes (see Table S2 in the supplemental material). In addition to the publicly available ST-562 genome (B. pseudomallei MSHR5858), we included a further 31 ST-562 isolates from the Darwin region. We also sequenced several ST-36 (n ⫽ 28), ST-109 (n ⫽ 36), and ST-132 (n ⫽ 21) isolates to enable a comparison of ST-562 with these other common STs from the Darwin region. Despite high levels of recombination in B. pseudomallei, phylogenetic reconstruction of the 223,719 SNPs identified robust clustering of STs into distinct phylogeographic clades and confirmed that ST-562 definitively falls within the Asian clade (Fig. 2). All other Australian genomes fell into the Australian clade. The genomic data were also used to determine the distribution of the mutually exclusive BTFC/YLF gene clusters (39) in the 455 B. pseudomallei strains in silico. The BTFC cluster is common in Australian strains (between 79 and 88% in other published studies [39, 40] and 65% in our data set), whereas Asian strains almost exclusively carry YLF, as evidenced by 30/31 (97%) of the Asian genomes in our data set containing YLF (see Table S2 in the supplemental material). The ST-562 strains all harbored YLF, adding further weight to the hypothesis of an Asian origin for these strains. ST-562 emergence in Darwin melioidosis cases. ST profiles for our isolates were first determined using either MLST or, where MLST data were lacking, SYBR MAMA PCR assays, which were developed as part of the current study to aid in the rapid and inexpensive detection of ST-36, ST-109, ST-132, and ST-562 (see Fig. S1 in the supplemental material). Of the 455 clinical isolates collected from melioidosis patients in the Darwin region since 1989, 40% have been attributed to four common STs: 49 cases (11%), 71 cases (16%), 40 cases (9%), and 28 cases (4%) were attributed to ST-36, ST-109, ST-132, and ST-562, respectively. These STs are less common outside the Darwin region (5), and ST-562 has not been identified in other Australian locales. The first clinical ST-562 isolate, B. pseudomallei MSHR1967, was observed in a single case during the 2004-2005 northern Australian wet season, with the next case observed in the 2006-2007 wet season. All subsequent cases occurred during or after the 2008-2009


wet season. The number of melioidosis cases attributable to infection with the ST-562 clone has risen dramatically since the 20082009 season (Fig. 3), and in the 2013-2014 wet season, ST-562 was identified in 9/34 (26%) Darwin clinical cases, the highest percentage seen to date. In contrast, the numbers of cases caused by ST-36, ST-109, and ST-132 during that period were only 4 (12%), 4 (12%), and 2 (6%), respectively. Environmental ST-562 in Darwin is geographically restricted. Since 2001, we have conducted environmental sampling of B. pseudomallei in the Darwin urban region as part of the ongoing surveillance and public health monitoring of this pathogen. We were therefore able to investigate the environmental distribution of this ST in the Darwin region over time. Of 223 B. pseudomallei-positive environmental samples, just two (soil sample MCAPS118B and air sample ASDA11A) harbored ST-562. Both samples were obtained during a squally rain storm from the same site on the same day in 2011 (23). Five other B. pseudomalleipositive environmental samples from this suburb, which were collected in earlier wet seasons, were ST-109. No additional environmental samples have been collected in this suburb since 2011. In contrast, we have identified 37 ST-36-positive samples, 62 ST109-positive samples, and 13 ST-132-positive samples in the Darwin region since the 2001-2002 wet season (see Table S3 in the supplemental material). Emergence of ST-562 in Darwin animal melioidosis cases. We examined 17 B. pseudomallei isolates from 10 melioidosisafflicted animals (sheep, cattle, camel, and parrot) that presented with melioidosis between 1993 and 2014. These animals were all housed in the suburb where ST-562 was identified in the environment. The two most recently afflicted animals, which were diagnosed with melioidosis in 2009 and 2014, were both infected with ST-562. In contrast, the other eight animals were diagnosed in 2005 or earlier, and none were infected by ST-562. Genomic comparison of ST-562 with other common Darwin STs. Genomic analysis of ST-36, ST-109, ST-132, and ST-562 isolates was used to provide a measure of comparative genetic


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FIG 1 eBURST analysis of Australian and Asian B. pseudomallei sequence types (STs). Red highlighting indicates Australian isolates, no shading indicates Asian

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diversity among the Australian ST-562 isolates. Using default SNP density filtering parameters in SPANDx (27), ST-109 was found to be the most diverse ST, with 9,536 SNPs and 211 kb (⬃3%) of the 7.3-Mbp MSHR4462 reference genome being variably present among the 36 genomes (Table 1). ST-36 and ST-132 were less diverse, with 379 and 615 SNPs identified in 28 and 21 genomes, respectively, and 119 kb and 62 kb of the genome being variably present among these ST genomes, respectively. In comparison, we identified only 56 SNPs among the 31 ST-562 isolates. However, 127 kb of the genome was variably present among the ST-562 isolates, approximately double the amount of locus variation seen in ST-132 (see Fig.

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S2 in the supplemental material). One ST-562 strain, B. pseudomallei MSHR8014, had an ⬃89-kb deletion, and another strain, B. pseudomallei MSHR8055, had a separate ⬃10-kb deletion, which accounted for most of the locus variation in the Australian ST-562 isolates. Given that recombination may lead to artificially high diversity measurements, we explored the role of recombination on ST-36, ST-109, ST-132, and ST-562 SNP diversity. Using heavy SNP density filtering to remove recombined regions, the proportion of SNPs in ST-36, ST-109, and ST-132 decreased by between 22% and 97%, whereas that of ST-562 did not change (Table 1; see also Fig. S2 in the supplemental material). ST-132 strains still had ap-




FIG 2 Pinpointing the geographic origin of ST-562 using phylogenetic analysis of 455 B. pseudomallei genomes. Maximum parsimony phylogenetic reconstruction using 223,719 core-genome orthologous SNPs demonstrates that B. pseudomallei possesses a strong phylogeographic signal on the whole-genome level, despite very high levels of recombination. ST-562 isolates retrieved from melioidosis cases and the environment in Darwin, Australia, cluster with Asian isolates. No other Australian STs (n ⫽ 191) cluster with Asian strains. This analysis also confirms that identical STs cluster together (gray shading) with high confidence. SNPs specific for the four target STs (ST-36, ST-109, ST-132, and ST-562) were identified from the core-genome orthologous SNP output and were used for ST-specific assay design. The overall tree consistency index (CI) was 0.15, and the ST-562 subclade CI was 0.93 (see Fig. S2 in the supplemental material). Relevant bootstrap values are shown.


in this region relative to the three other common STs since the first case was identified in 2005. In the 2013-2014 season, ST-562 was, for the first time, the most common ST isolated from melioidosis cases in the Darwin region.

proximately twice the number of SNPs as ST-562, and ST-36 and ST-109 had approximately five and six times more SNPs than ST-562, respectively (Table 1). Gubbins analysis also identified recombinogenic SNPs among the ST-36, ST-109, and ST-132 genomes, but none were found in ST-562 (see Fig. S3 in the supplemental material). DISCUSSION

In this study, we confirmed the unprecedented transmission of the pathogenic bacterium B. pseudomallei from one region endemic for the pathogen to another, in this case, from Asia to Australia. Unlike most other human pathogens, B. pseudomallei almost always causes infection through contact with contaminated soil or water, with only a small number of zoonotic, nosocomial, or human-to-human cases reported in the literature (e.g., reference 41). Therefore, the use of high-resolution molecular tools in combination with epidemiological data provides a robust and powerful way to identify the geographic origin of a given B. pseudomallei infection. Here, we used MLST-, WGS-, and PCR-based methods to identify the presence of an Asian B. pseudomallei clone, ST-562, in several clinical, animal, and environmental isolates from northern Australia. ST-562 was not detected in Australia prior to 2005, pointing to an emerging clone with recent transmission into northern Australia from Southeast Asia. Although very infrequent, the transmission of B. pseudomallei across major geographic barriers has been a necessary occurrence for the global dispersal of this bacterium. In 2009, Pearson and colleagues (4) used WGS of 43 Burkholderia spp. to show that B. pseudomallei likely originated in Australia, followed by one of more rare dissemination events into Southeast Asia across the Wallace Line sometime between 16,000 and 225,000 years ago. The Wallace Line is a major biogeographic barrier between Asia and Australia that has led to restricted genetic flow among the flora and fauna between these two regions. Other studies have since affirmed the ancestral nature of Australian B. pseudomallei and the rare historical transmission of B. pseudomallei from Australia to Asia (6, 15). The ancient separation of these two populations has enabled a strong phylogeographic signal between Asian and Australian clades to evolve despite high levels of recombination across the B. pseudomallei genome (4, 6). The clarity of this geographic signal is a consequence of both localized evolution and the rarity of transmission, and thus gene flow, between B. pseudomallei strains from these regions.


The generation of substantially more B. pseudomallei genomic data in recent years has enabled us to increase the resolution of the phylogeny first presented by Pearson et al. (4) by ⬎10-fold. In the current study, we reconstructed a midpointrooted maximum parsimony phylogeny using 223,719 core-genome orthologous SNPs identified across 455 global B. pseudomallei genomes from Australia, Brazil, Papua New Guinea, Southeast Asia, and South Asia (Fig. 2). This tree represents the most extensive and in-depth global phylogeny of B. pseudomallei constructed to date. Like Pearson and colleagues, we noted a strong geographic signal whereby Australian strains were phylogenetically distinct from Asian and Brazilian strains, with the exception of ST-562, an emerging clone in northern Australia that unequivocally grouped with Asian strains. MLST analysis of 3,283 Australian and Asian isolates supported our WGS findings (Fig. 1), with all SLVs and DLVs of ST-562 being Asian in origin. In further support of an Asian origin for ST-562, this ST was recently documented in clinical isolates from Hainan, southern China (20), and in clinical and environmental isolates from the Er Ren Basin region of southern Taiwan (42). Three of 110 melioidosis cases diagnosed between 2008 and 2012 in Hainan, the southernmost province of China, were identified as ST-562 (20). ST-562 has also been reported in an unpublished clinical case from Hainan (BPC003; identification [ID] 3875; http: //bpseudomallei.mlst.net). In the Er Ren Basin, 15/59 (25%) B. pseudomallei-positive soil samples collected between 2005 and 2007 and 2/194 (1%) clinical cases from this region diagnosed between 2004 and 2010 were identified as ST-562 (42). Interestingly, two different pulsed-field gel electrophoresis (PFGE) types were identified among environmental and clinical Taiwanese ST562 isolates (42), pointing to ST-562 genetic diversity in this region and thus a possible ancestral origin for ST-562 in Taiwan. We initially used MLST to determine the probable origin of ST-562, as the MLST database has the greatest abundance of data for global B. pseudomallei strains. In addition to ST-562, the global MLST data set contains three other instances of B. pseudomallei ST overlap between Asia and Australia: ST-89, ST-105, and ST-849 (Fig. 1). It was recently demonstrated that ST-105 and ST-849, which have each been observed in both Australian and Cambodian B. pseudomallei isolates, are in fact phylogeographically distinct on a whole-genome level. The isolates from Cambodia with these shared STs are not genetically related to the isolates


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FIG 3 Incidence of melioidosis cases in the Darwin region attributed to common STs since 1990. The Asian clone, ST-562, has increased in prevalence

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in the environment in the Darwin region provides support for a locally acquired source of infection for all cases. The identification of animals housed in the Darwin region that were infected with ST-562 (see Table S1 in the supplemental material) further cements the autochthonous nature of ST-562 in Darwin. The limited geographic focus of ST-562 clinical cases is also strongly suggestive of a recent point-source introduction into the Darwin region. To investigate further, we performed comparative genomic analysis on 32 ST-562 isolates, three of which were environmental (including 2 colonies from the same positive soil sample) and the remainder clinical. This analysis revealed that there is some diversity among ST-562 isolates on the genome level, but this diversity is lower than that observed among ST-36, ST-109, and ST-132, even when SNPs within putative regions of recombination were omitted (see Fig. S2 and S3 in the supplemental material). Curiously, removal of the recombinogenic SNP loci decreased the number of SNPs identified among ST-36, ST-109, and ST-132 by between 22% and 97% but did not affect ST-562, for which no SNPs were removed by this filter (Table 1). This finding suggests that there has been insufficient time for recombination to shape the genome of Darwin ST-562 B. pseudomallei, and it supports our hypothesis of a recent introduction of ST-562 into northern Australia. However, it cannot be ruled out that this clone may undergo recombination events less frequently in the northern Australian environment than other Australian strains due to genetic distance between ST-562 and potential donor strains, a phenomenon recently reported by Nandi and coworkers (45). Alternatively, ST562 may exist in an ecological niche not shared by other B. pseudomallei strains, restricting opportunities for recombination. This hypothesis is supported by a lack of other STs being isolated from the two ST-562-positive environmental samples (see Table S3 in the supplemental material). However, the absence of other STs in environmental samples is not in itself uncommon (data not shown), and sampling and microbiological processing biases can be expected to greatly influence the cultivable B. pseudomallei population of any specimen. Nevertheless, based on the comparative genomic findings, a recent introduction of ST-562 remains the most plausible scenario. The precise route and date of the ST-562 introduction into northern Australia remain speculative, as there are several unknowns. What is known is that the ST-562 clone has been in Australia at least since 2005, when the first clinical case was identified, and the geographic distribution of ST-562 appears to be limited. Unfortunately, molecular clock modeling is unlikely to provide useful dating information of the Australian ST-562 clone for several reasons. First, putative restricted gene flow between ST-562 and Australian strains (45), coupled with high rates of recombination (4) in other strains, will dramatically influence the projected rates of genetic diversity. Second, there are no data on generation times or mutation rates in environmental B. pseudomallei strains, with evolution and doubling rates likely to vastly differ from those of in vitro- or mammalian-passaged strains. This issue of different generation times has recently been exemplified in an outbreak of Francisella tularensis (tularemia) in Sweden, where no correlations between spatial, temporal, and genetic distances were found (46). Third, there is insufficient resolution among the Australian ST-562 genomes, as evidenced by several polytomies in our ST-562 phylogenies (see




of the same ST from Australia but rather are the result of homoplasy (7). Less clear is the ST-89 overlap, which has been described in a B. pseudomallei isolate from a melioidosis patient from the Torres Strait Islands of Australia and in a Thai environmental isolate (43). Further review of the strains involved suggests that incorrect provenance likely accounts for this overlap, with the ST-89 strain listed as being from Thailand, while it is more likely to actually be an Australian strain (44). Unlike the ST-89 overlap, ST-562 has compelling genetic and genomic data linking the origin of this clone to Asia. Therefore, ST-562 is the only confirmed B. pseudomallei clone thus far that has been definitively documented in both Asia and Australia. The detection of ST-562 in northern Australian melioidosis cases is of concern for several reasons, not least of which is the higher melioidosis mortality rate in Asia (2), which may in part be attributable to heightened infectivity or virulence potential of Asian strains (40). Of the 455 melioidosis cases identified between October 1989 and August 2014 that have been epidemiologically linked to the Darwin region, 28 were caused by ST-562. We observed a dramatic spike in ST-562 incidence, particularly during the 2013-2014 northern Australian wet season (see Table S1 and Fig. 3 in the supplemental material), which, for the first time, surpassed the proportion of cases caused by other common Darwin STs (ST-36, ST-109, and ST132). The incidence of ST-562 began to rise in the 2008-2009 wet season and has steadily increased since then (Fig. 3). Although the reason for this increase is unclear, above-average rainfall in the Top End in the 2009-2010 wet season, a region which saw an unprecedented number of melioidosis cases (16), may have contributed to ST-562 dissemination due to higher water levels and erosion and thus greater exposure risk. Although we did not investigate this possibility in our study, ST-562 may possess an adaptive advantage that enables this clone to survive, persist, and even flourish in the northern Australian environment, thereby outcompeting other strains. The increase in ST-562 prevalence is alarming, as it suggests that cases caused by this emerging clone are on the rise in this area, with the possibility of elevated melioidosis mortality rates in future years should ST-562 be confirmed to be more virulent than the other common Darwin STs. Further data over the upcoming northern Australian wet seasons will be essential for investigating these possibilities. To confirm the in situ presence of ST-562 in the northern Australian environment and to rule out infection acquisition following travel to Asia, we conducted a complete survey of all B. pseudomallei-positive environmental samples collected in the Darwin region since 2001, the year our environmental sampling efforts began. An analysis of 223 unique samples identified just two ST-562-positive samples (one soil and one air), which were collected from the same urban site in 2011 (23). The very low ST-562 environmental detection rate is in contrast to the detection rate of the other common Darwin STs, which are readily isolated from the environment across the Darwin region (see Table S3 in the supplemental material). In combination with its lower genomic variation compared to that of other Darwin STs (Table 1; see also Fig. S2 and S3 in the supplemental material), our findings indicate that the Australian ST-562 clone is not yet widespread in the Darwin environment, although more detailed sampling efforts are needed to confirm this hypothesis. None of our patients had a history of travel to Asia, and the detection of ST-562


ACKNOWLEDGMENTS We thank Brian Spratt and Daniel Godoy (Imperial College, London, United Kingdom) for their extensive efforts with B. pseudomallei MLST development, data acquisition, and database curation over many years; Ian Harrington (Menzies School of Health Research) for his assistance with environmental sample collection in the Darwin region; and Apichai Tuanyok, Adina Doyle, Christopher Allender, Nicolette Janke, and Molly Bollig (Center for Microbial Genetics and Genomics, Northern Arizona University) for their assistance in generating the genome sequence data. We also thank the Rebecca L. Cooper Medical Research Foundation for the provision of the NanoDrop 2000 spectrophotometer.


FUNDING INFORMATION Defense Threat Reduction Agency provided funding to Dave M. Wagner and Paul Keim under grant number HDTRA1-12-C-0066. U.S. Department of Homeland Security (DHS) provided funding to Dave M. Wagner and Paul Keim under grant numbers HSHQDC-10-C-00139 and DHS2010-ST-108-000019. Department of Industry, Innovation, Science, Research and Tertiary Education, Australian Government | Australian Research Council (ARC) provided funding to Mirjam Kaestli and Bart J. Currie under grant number LP110100691. Department of Health | National Health and Medical Research Council (NHMRC) provided funding to Mirjam Kaestli, Bart J. Currie, Erin P. Price, Derek S. Sarovich, Mark Mayo, and Paul Keim under grant numbers 605820 and 1046812. The funders had no role in the study design, data collection and interpretation, or in the decision to submit the work for publication.

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Fig. S2 and S3 in the supplemental material), which may be a consequence of insufficient elapsed time since the introduction of ST-562 into Australia. WGS of Chinese and Taiwanese ST-562 strains will likely provide critical clues as to the timing of ST-562 introduction into Australia and may help resolve some of these existing molecular clock modeling issues. Based on previous B. pseudomallei studies, we hypothesize several scenarios of ST-562 transmission to Australia. The first involves importation via environmental seeding from B. pseudomallei-infected animals, similarly to the devastating Parisian zoo outbreak in the 1970s (47). Despite its strict quarantine legislation, Australia does have a history of importing melioidosissusceptible exotic animals from Asia (e.g., the introduction of Camelus spp. into Australia during the 1800s). As with humans (19), animals can be infected with B. pseudomallei but not display overt symptoms. Environmental seeding may have also occurred from B. pseudomallei being shed from a chronically infected human (e.g., via expectoration) returning or traveling from Asia at some stage in the past, or from the fecal matter of B. pseudomalleiinfected migratory or storm-driven birds flying south from Asia. B. pseudomallei carriage and infection have been documented in both native Australian and imported avian species and represent a possible route by which B. pseudomallei was introduced into New Caledonia and other Pacific islands from the Australian mainland (48). Coastal northern Australia is a stop-off point for some of the great migratory flyways, and shorebirds that have transited Asia on their southern migration from Siberia to Australia have been found to carry avian influenza viruses (49). The second scenario involves the inadvertent importation of B. pseudomallei-contaminated plant or soil material. B. pseudomallei can infect the rhizosphere, roots, and aerial parts of certain grasses, including exotic species (50). However, imported soil was ruled out as a source of B. pseudomallei infection in a highly unusual southern Arizonan melioidosis case (14); in addition, the high salinity of potting mix is not conducive to B. pseudomallei survival (51). A third scenario is that ST-562 has been in the northern Australian environment for several decades, or perhaps centuries, but has only recently been uncovered by anthropogenically driven environmental disturbances in the fast-growing Darwin region (51). A final possibility is importation via B. pseudomallei-contaminated medical products manufactured in Southeast Asia (19, 52); however, the environmental detection of ST-562 B. pseudomallei, the occurrence of animal cases, and epidemiological investigation of human cases render this scenario improbable. Ongoing investigation of ST-562 from both Australia and Asia will be essential for identifying the most probable transmission event/s into Australia.

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Growth, motility and resistance to oxidative stress of the melioidosis pathogen Burkholderia pseudomallei are enhanced by epinephrine.

Role of Burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis.

Melioidosis and end-stage renal disease in tropical northern Australia.

The concentrations of ambient Burkholderia pseudomallei during typhoon season in endemic area of melioidosis in Taiwan.

Involvement of L-selectin expression in Burkholderia pseudomallei-infected monocytes invading the brain during murine melioidosis.