Burkholderia pseudomallei, the causative agent of melioidosis, is rare but ecologically established and widely dispersed in the environment in Puerto Rico

Abstract

Background: Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated.

Methodology/principal findings: We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp.

Conclusions/significance: B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America.

Fig 1. Locations in Puerto Rico where 1) environmental samples were collected in this study, 2) previous human melioidosis cases occurred, and 3) one previous B. pseudomallei-positive soil sample was collected.

Site numbers are located next to the shapes indicating the 60 sampling locations from this study; soil samples were collected at sites 1–50 and water samples were collected at sites 51–60. The locations of past human melioidosis cases and the one previous B. pseudomallei positive soil location [20] are indicated. This map was created using ArcGIS software by Esri.

Fig 2. Burkholderia pseudomallei global whole genome phylogeny.

Maximum-likelihood phylogeny of 414 globally-diverse B. pseudomallei isolates rooted with a B. thailandensis isolate; bootstrap values are reported on nodes. Two of the 55 B. pseudomallei isolates obtained from site 23 in this study are included (Bp9039 and Bp9110). (A) All 414 B. pseudomallei genomes with nodes collapsed. (B) Expanded nodes within the monophyletic group containing all included isolates (n = 44) from Africa, Central and South America, Mexico, and the Caribbean.

Fig 3. Genomic islands present in B. pseudomallei isolates from a single location in Puerto Rico.

(A) Reveals the structure of the four novel B. pseudomallei genomic islands (GI1-4) that were discovered in this study; contig names are listed on the far right. The red arrows reflect B. pseudomallei genes found within the genomic islands and the blue arrows reflect conserved flanking regions commonly found in other B. pseudomallei strains. (B) Circular phylogeny with genomic islands mapped on the outside of the phylogeny. This phylogeny was constructed using the same 414 B. pseudomallei isolates used to generate Fig 2 plus the 53 additional B. pseudomallei isolates from site 23 that were not included in Fig 2.

Fig 4. Burkholderia spp. whole genome phylogeny.

Core genome maximum-likelihood phylogeny of 781 Burkholderia spp. isolates rooted with Cupriavidus. The 781 isolates include 129 isolates obtained from Puerto Rico in this study (indicated with red lines; S1 Table) and 651 publicly available Burkholderia spp. isolates (S5 Table). Bootstrap values are reported on nodes.