We report 16 Burkholderia pseudomallei genomes, including 5 new multilocus sequence types, isolated from rivers in Laos. The environmental bacterium B. pseudomallei causes melioidosis, a serious infectious disease in tropical and subtropical regions. The isolates are geographically clustered in one clade from around Vientiane, Laos, and one clade from further south.
ABSTRACT
We report 16 Burkholderia pseudomallei genomes, including 5 new multilocus sequence types, isolated from rivers in Laos. The environmental bacterium B. pseudomallei causes melioidosis, a serious infectious disease in tropical and subtropical regions. The isolates are geographically clustered in one clade from around Vientiane, Laos, and one clade from further south.
ANNOUNCEMENT
Burkholderia pseudomallei causes the human infectious disease melioidosis and is found in tropical and subtropical soils and freshwater (1). Survival and replication in various ecological niches and within hosts is possibly enabled by the large and highly variable accessory genome of B. pseudomallei (2, 3). Genome descriptions of B. pseudomallei isolates contribute to research on links between environment-associated and disease-associated genes of B. pseudomallei and their functions (3, 4).
We sequenced the genomes of 16 B. pseudomallei isolates from 14 filtered water samples and two sediment samples from rivers in Laos, cultured and confirmed as previously described (5). After storage at −80°C and pure culture on nutrient agar in air at 37°C for 24 h, genomic DNA was extracted using the Qiagen DNeasy blood and tissue kit and submitted to Microsynth AG (Balgach, Switzerland) for Nextera XT library preparation and sequencing using an Illumina NextSeq 500 instrument (paired-end [PE], 150-bp reads). Reads were quality trimmed using Trimmomatic 0.36 (slidingwindow:4: 8, minlen:127) (6) and assembled using SPAdes 3.11.1 (-careful, -mismatch-correction, -k 21, 33, 55, 77, 99, 127 bp) (7). Pilon 1.22 (8) was applied to improve the quality of the draft assemblies. Scaffolds of <200 bp or with low coverage were removed. Finally, contaminants were removed manually using a BLAST search against the NCBI nucleotide database. The quality and completeness of the de novo-assembled genomes were accessed using BUSCO 3.0.1 (lineage, Betaprotebacteria odb9) (9), and basic assembly statistics were compared using QUAST 4.6.3 (10). The genomes were annotated automatically using the NCBI Prokaryotic Annotation Pipeline 4.11 (11). Default settings were used for all software unless otherwise specified. A summary of the assembly results is provided in Table 1. The 16 isolates were found to belong to 6 different sequence types using the multilocus sequence typing pipeline (12, 13), 5 of which were new. Sequence type 54 (ST54) (two isolates) was previously described and is common in neighboring Thailand (14). To unravel the phylogenetic relationship of the isolates, we first constructed a core single nucleotide polymorphism genome alignment using Snippy 4.4.3 with B. pseudomallei MSHR4503 (15) as the reference. Then, we built a maximum likelihood tree using RAxML 8.2.11 (16) with a general time-reversible nucleotide substitution model including 1,000 bootstraps (Fig. 1). The six main branches of the tree correspond to the sequence types and are geographically clustered in two different clades. One clade includes isolates from or around Vientiane, Laos (city and province), whereas the other consists of isolates from further south. The sediment isolate from Xe Bangnouan, Laos, is more closely related to the sediment isolate from the Mekong River than to the corresponding water isolate (Fig. 1). However, with relatively few samples taken at one point in time from rivers with large catchment areas, the interpretation of these clusters remains speculative. It is hoped that sequencing more isolates of B. pseudomallei from Laos will improve our understanding of the phylogeography of the organism within the country and enable comparisons to be made between clinical and environmental isolates.
TABLE 1.
Characteristics and accession numbers of the genomes of 16 B. pseudomallei isolates from rivers in Laos
| Isolatea | Rivera | Latitudea | Longitudea | Raw read SRA no. | GenBank assembly accession no. | Sequence type | No. of contigs | Assembly length (Mbp) | GC content (%) | N50 (kbp) | No. of paired reads (million) | Genome coverage (×) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 40S_S61 | Mekong | 15.11316 | 105.80506 | SRR11097786 | GCA_014713055.1 | ST1787 | 186 | 7.15 | 68.19 | 119.6 | 4.8 | 203 |
| 43W_S62 | Xe Bangnouan | 16.00286 | 105.47903 | SRR11097785 | GCA_014713065.1 | ST1801 | 205 | 7.17 | 68.14 | 107.1 | 4.7 | 183 |
| 43S_S63 | Xe Bangnouan | 16.00286 | 105.47903 | SRR11097778 | GCA_014713085.1 | ST1787 | 176 | 7.15 | 68.19 | 128.2 | 4.4 | 195 |
| 44W_S64 | Mekong | 16.00421 | 105.42515 | SRR11097777 | GCA_014713025.1 | ST1801 | 191 | 7.17 | 68.14 | 123.3 | 5.5 | 231 |
| 45W_S65 | Xe Banghieng | 16.09798 | 105.37699 | SRR11097776 | GCA_014713015.1 | ST1815 | 184 | 7.15 | 68.15 | 126.5 | 4.6 | 194 |
| 46W_S66 | Mekong | 17.39898 | 104.80098 | SRR11097774 | GCA_014712945.1 | ST1815 | 188 | 7.15 | 68.14 | 132.9 | 5.3 | 219 |
| 47W_S67 | Xe Bangfai | 17.07787 | 104.98503 | SRR11097775 | GCA_014712955.1 | ST1801 | 209 | 7.17 | 68.15 | 119.7 | 5.3 | 221 |
| 49W_S68 | Nam Kading | 18.32559 | 104.00002 | SRR11097773 | GCA_014712965.1 | ST1801 | 193 | 7.17 | 68.14 | 127.8 | 5.3 | 223 |
| 50W_S69 | Nam Xan | 18.39103 | 103.65572 | SRR11097772 | GCA_014712935.1 | ST1852 | 192 | 7.24 | 68.1 | 138.5 | 5.0 | 208 |
| 51W_S70 | Nam Gniep | 18.41756 | 103.60212 | SRR11097771 | GCA_014712915.1 | ST1852 | 222 | 7.25 | 68.26 | 110.8 | 4.9 | 201 |
| 52W_S71 | Nam Mang | 18.37017 | 103.19838 | SRR11097784 | GCA_014712835.1 | ST54 | 168 | 7.04 | 68.09 | 134.4 | 5 | 213 |
| 53W_S72 | Nam Ngum | 18.17874 | 103.05594 | SRR11097783 | GCA_014712895.1 | ST54 | 182 | 7.04 | 68.27 | 126.7 | 4.4 | 188 |
| 58W_S73 | Nam Ngum | 18.20194 | 102.58669 | SRR11097782 | GCA_014712875.1 | ST1852 | 184 | 7.25 | 68.1 | 120.9 | 4.7 | 195 |
| 60W_S74 | Nam Sang | 18.22297 | 102.14228 | SRR11097781 | GCA_014712825.1 | ST1852 | 186 | 7.24 | 68.11 | 123 | 4.6 | 189 |
| 64W_S75 | Mekong | 17.97309 | 102.50404 | SRR11097780 | GCA_014712815.1 | ST1869 | 212 | 7.3 | 68.1 | 97.6 | 4.8 | 197 |
| 91W_S76 | Nam Ngum | 18.3555 | 102.57198 | SRR11097779 | GCA_014712775.1 | ST1869 | 194 | 7.21 | 68.11 | 87.9 | 4.1 | 171 |
Data from reference 5.
FIG 1.
Maximum likelihood phylogeny and geographic locations of 16 environmental B. pseudomallei isolates. Clusters of isolates are displayed in different colors and correspond to sequence types (ST); circles represent water isolates, and squares represent sediment isolates. B. pseudomallei strain MSHR4503 from northern Australia (15) was used as the reference. The tree scale indicates changes per nucleotide; bootstrap values were calculated from 1,000 bootstrap replicates and are reported as percentages. The phylogenetic tree was visualized using Microreact (17), and the map was adapted from reference 5.
Data availability.
Illumina raw reads and genome assemblies were deposited at the NCBI and DDBJ/ENA/GenBank, respectively. The accession numbers are listed in Table 1. The isolates are linked to the respective sequence types on the PubMLST database.
ACKNOWLEDGMENTS
We are very grateful to the director and staff of the Microbiology Laboratory, Mahosot Hospital, to Bounthaphany Bounxouei, past director of Mahosot Hospital, to Bounnack Saysanasongkham, past director of the Department of Health Care, Ministry of Health, and to H. E. Bounkong Syhavong, Minister of Health, Lao PDR.
The project was funded by the U.S. Defense Threat Reduction Agency Cooperative Biological Engagement Program (contract HDTRA-16-C-0017).
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Associated Data
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Data Availability Statement
Illumina raw reads and genome assemblies were deposited at the NCBI and DDBJ/ENA/GenBank, respectively. The accession numbers are listed in Table 1. The isolates are linked to the respective sequence types on the PubMLST database.