Burkholderia pseudomallei in Soil, US Virgin Islands, 2019

The distribution of Burkholderia pseudomallei in the Caribbean is poorly understood. We isolated B. pseudomallei from US Virgin Islands soil. The soil isolate was genetically similar to other isolates from the Caribbean, suggesting that B. pseudomallei might have been introduced to the islands multiple times through severe weather events.

The diagnosis of these cases showed the surveillance system of Senegal's capacity to quickly detect, isolate, and investigate those cases to take adequate control measures. Our findings indicate that the earliest cases in Senegal or sub-Saharan Africa were imported from Europe, implying that the particularly high volume of direct flights from Europe was a key factor in the spread of the virus in West Africa. However, we cannot exclude the possibility that a few COVID-19 cases were missed at that time in Senegal, including paucisymptomatic or asymptomatic cases (4,5). Our study emphasizes the imperative need for efficient epidemiologic investigations to identify the cases and characterize the transmission modes to prevent, control, and stop the spread of COVID-19.
B urkholderia pseudomallei is a gram-negative soildwelling bacterium and the causative agent of melioidosis (1). B. pseudomallei is endemic to tropical regions around the world (1), but its environmental distribution in the Caribbean remains poorly understood. Although it is rare but ecologically established in Puerto Rico (2,3), it has not been isolated from the environment in the neighboring US Virgin Islands (USVI). After the 2017 Caribbean hurricane season, melioidosis developed in 3 persons in the USVI (4), 2 in St. Thomas and 1 in St. John. We aimed to determine whether, as this cluster suggests, B. pseudomallei might be endemic to the USVI.
We collected 480 soil and 100 freshwater samples from 29 sites (24 terrestrial and 5 freshwater) on the 3 main USVI islands (i.e., St. Thomas, St. John, and St. Croix) during January 20-April 17, 2019. We selected study sites to maximize geographic distribution across the islands and epidemiologic connection to melioidosis cases in humans (Appendix Figure 1, https:// wwwnc.cdc.gov/EID/article/26/11/19-1577-App1. pdf). These efforts followed consensus guidelines for environmental sampling of B. pseudomallei (5) and The distribution of Burkholderia pseudomallei in the Caribbean is poorly understood. We isolated B. pseudomallei from US Virgin Islands soil. The soil isolate was genetically similar to other isolates from the Caribbean, suggesting that B. pseudomallei might have been introduced to the islands multiple times through severe weather events. methods previously reported (2) with 4 modifications: we collected 20 samples per site; we collected soil samples in 2 linear transects of 10 samples each; we collected 150 mL water per sample; and we used half of each sample for our analysis (the other half was archived). Although we strove for a sampling depth of 30 cm in soil, this was impossible at some sites because of rocks and debris (Appendix Table 1). We placed environmental samples in Ashdown's liquid media for Burkholderia spp. enrichment (2). After enrichment, we extracted DNA using QiaAmp kits (QIAGEN, https://www.qiagen.com) and screened it using a B. pseudomallei-specific TaqMan assay (Ther-moFisher Scientific, https://www.thermofisher.com) (6,7). We cultured samples to isolate pure B. pseudomallei and generate whole-genome sequences (WGSs). We conducted a phylogenetic analysis as previously described (2) and conducted genetic typing on these WGSs, WGSs from the 3 patients with melioidosis from USVI in 2017, and 43 additional B. pseudomallei WGSs available in GenBank from the Caribbean, the Americas, and Africa (Appendix Table 2).
We isolated B. pseudomallei from only 1 (≈4%) of 24 soil sites, a prevalence resembling that of nearby Puerto Rico (2), where another study isolated B. pseudomallei from 2 soil samples collected at only 1 (2%) of 50 sampled sites. We obtained the B. pseudomalleipositive sample from site 122 (Appendix Figure 1), Maximum-likelihood phylogeny of Burkholderia pseudomallei isolates from patients and the environment in the US Virgin Islands and reference isolates available in GenBank from other countries in the Americas, Africa, and the Caribbean.
which was adjacent to a paved roadway 76 meters above sea level on eastern St. John. We collected the soil sample, which was composed of dry gravelly loam and had a pH of 6.9, from a depth of 30 cm (8) (Appendix; Appendix Table 1, Figure 2).
Our phylogenetic analysis assigned the 4 isolates (3 from patients, 1 from the environment) from the USVI to a monophyletic clade with all other B. pseudomallei isolates from the Caribbean (except 1 from Aruba) (Figure). However, none of the 4 isolates from the USVI were close genomic matches. These isolates differed by 6,355-10,115 single-nucleotide polymorphisms (SNPs) in the core genome, exhibiting more genomic diversity than B. pseudomallei isolates within Puerto Rico and Martinique (Figure). The 2019 soil and 2017 human isolates from St. John were not closely related (differing by 10,115 core genome SNPs), suggesting multiple introductions of B. pseudomallei to this island. The closest genomic match to the St. John soil isolate (differing by 170 core genome SNPs) was a 2007 isolate from Road Town, Tortola, British Virgin Islands (9). Although the dispersal mechanism of B. pseudomallei to this region is unknown, a dispersal event between these 2 locations (≈11 km) might have been caused by aerosolization of B. pseudomallei during an extreme weather event, such as a hurricane (10). This mechanism of long-distance dispersal might also explain why the 2017 isolate from St. John is more closely related to isolates from Martinique than to the other isolates from USVI; this patient from the USVI was infected shortly after hurricane Maria (4). We placed the 2 isolates, despite differing by 6,355 core genome SNPs, from patients on St. Thomas in a single subclade; this pattern might suggest long-term endemicity on this island. However, these scenarios are based on an analysis of a relatively small number of B. pseudomallei WGSs from the Caribbean.
Our findings demonstrate that B. pseudomallei is rare in the environment in the USVI. The 2017 cases of melioidosis and the soil isolate from St. John indicate this bacterium might be ecologically established in the USVI. Additional environmental sampling will determine the environmental distribution of B. pseudomallei in the USVI, aiding the development of public health strategies to mitigate the risk for melioidosis.

Description of Burkholderia pseudomallei-Positive Soil Sample
The soil type and soil profile for this sample were very gravelly loam (Appendix Figure   2) and southgate-rock outcrop complex, respectively (Appendix Table 1). Southgate has a typical geologic profile of 0-5 inches: gravelly loam; 5-10 inches: very gravelly loam; 10-17 inches: