Burkholderia thailandensis Isolated from the Environment, United States

Burkholderia thailandensis, an opportunistic pathogen found in the environment, is a bacterium closely related to B. pseudomallei, the cause of melioidosis. Human B. thailandensis infections are uncommon. We isolated B. thailandensis from water in Texas and Puerto Rico and soil in Mississippi in the United States, demonstrating a potential public health risk.


Additional Methods
Environmental sampling was conducted in Puerto Rico and Texas using methods previously described (1,2) and were adapted from international consensus guidelines (3) with additional modifications developed by the Menzies School of Health and Research in Darwin, Australia (4). Permission was obtained from landowners to collect soil and/or water samples on their property and, when necessary, permits were received to collect from reserve lands. Briefly, at each site, we collected 10 to 100 soil samples at a depth of 30 cm from holes spaced 2.5 meters apart in one to two 10-hole transects or in a grid for each site as previously described (1).
Water samples (150 mL) were collected along a linear transect with 2.5 meters between each sampling location, when possible, using one of two sampling approaches: For approach A water samples were collected (20 samples per site in Puerto Rico and 10 samples per site in Texas) approximately 1 meter from the shoreline, avoiding flowing water, whereas for approach B water samples were collected (10 samples per site in Puerto Rico) at five sampling locations with the first sample collected directly on the water's edge and another sample collected 1 meter from the shoreline (5). All water samples were filtered the day of collection using a Sartorius water filtration manifold with 0.22 µm nitrocellulose filters, as previously described (1).
Environmental scrapes were only collected in Texas at one site from a partially empty residential 500-gallon water holding tank. Briefly, the bottom and sides of the tank were scraped, and the scrape contents were placed into a sterile 2 mL screw-cap tube (2). All collected samples were kept from direct UV exposure and shipped at ambient temperature to NAU for further processing. Upon arrival, samples were stored in the dark at ambient temperature except for the sampling approach B water filters, which were stored at 4°C.
To prepare the environmental scrapes for culturing, the 2 mL tubes containing the scrapes were first vortex at high speed for 1 minute and then sonicated for 5 minutes using a Branson sonicator bath set to 70W, 42kHz at room temperature.
All samples were processed for detection and isolation of Burkholderia spp. as previously described (1), with the following modifications. Each water sample was filtered onto one filter, which was cut in half and only one half was used for the inoculation of 30 mL of Ashdown's broth. The entire contents of the environmental scrape were transferred to the 30 mL of Ashdown's broth. The soil was processed in the same way as Hall et al., 2022 (2). Also, a B. Once a pure B. thailandensis isolate was obtained after at least three isolation streaks, long term glycerol stocks were created and high molecular weight gDNA was obtained using a QIAGEN DNeasy Blood & Tissue kit, following the Gram-Positive protocol in the manufacturer's instructions. Whole genome sequencing was performed as previously described (1).

Environmental sampling (soil, water, and plant material) was conducted in Mississippi in
July of 2022 using methods previously described (1) and adapted from international consensus guidelines (3). Samples were shipped to the CDC in Atlanta, GA for culturing. Culturing of the environmental samples occurred as previously described (1) thailandensis isolate was sent to the CDC in Atlanta, GA.
Both ST1785 and ST1772 have all the same alleles as ST101, which was assigned to the Arkansas, Louisiana, and Texas B. thailandensis clinical samples, except for the gltB and ace difference described above.
Coding region sequences (CDSs) were identified that had a BSR value ≥0.9 in Puerto Rican isolates an a BSR value <0.4 in all other B. thailandensis genomes.
The 113 genes unique to the B. thailandensis isolates from Puerto Rico are listed in S1