Analysis of fine-scale phylogeny of Burkholderia pseudomallei in relation to regional geography and drug susceptibility in Thailand.

Melioidosis caused by Burkholderia pseudomallei (Bp) is a public health threat. Genomic-epidemiology research on this deadly disease is scarce. We investigated whole-genome sequences of Bp isolates in relation to environmental source and drug susceptibility. In total, 563 Bp isolates were collected from 11 Northeast Thai provinces during the period 2004-2021. Patients (n = 530 isolates), infected animals (n = 8), and environmental sources (n = 25) provided samples. Phylogenetic analysis revealed genetic diversity among the Bp isolates, including numerous well-supported clusters of varying sizes. Through in-depth analysis of 38 monophyletic clades (MCs), we found eleven associated with province of origin (p-value < 0.001). Closely related clusters (CRCs) within MCs resembled MLST-identified "sequence types" (STs). We found 102 known and 52 novel STs. ST-70 was the most prevalent in this area (n = 78; 13.85%). Sample type (human/environmental) and sampling time intervals were not correlated with genetic distance among clonal Bp isolates. Some members of 12 CRCs had acquired resistance to co-trimoxazole and one against amoxicillin-clavulanic acid. Within Northeast Thailand, there is an association between Bp genotype and geographical origin.

MALDI-TOF MS analysis of Burkholderia pseudomallei and closely related species isolated from soils and water in Khon Kaen, Thailand.

Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to the presence of closely related species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacterial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and "other bacterial species"). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp.

Chitosan biological molecule improves bactericidal competence of ceftazidime against Burkholderia pseudomallei biofilms.

Biofilm-associated Burkholderia pseudomallei infections (melioidosis) are problematic because of reduced sensitivity to antibiotics and high frequency of relapse. Biofilm dispersal agents are essential to liberate the biofilm-encased cells, which then become planktonic and are more susceptible to antibiotics. This study aimed to evaluate the ability of deacetylated chitosan (dCS), an antimicrobial and antibiofilm biological macromolecule, to disrupt established biofilms, thus enabling ceftazidime (CAZ) to kill biofilm-embedded B. pseudomallei. We combined dCS with CAZ using a mechanical stirring method to generate dCS/CAZ. In combination, 1.25-2.5 mg ml-1 dCS/1-2 µg ml-1 CAZ acted synergistically to kill cells more effectively than did either dCS or CAZ alone. Notably, a combination of 5-10 mg ml-1 dCS with 256-512 µg ml-1 CAZ, prepared either by mechanical stirring (dCS/CAZ) or mixing (dCS + CAZ), drastically improved bactericidal activities against biofilm cells leading to a 3-6 log CFU reduction. Confocal laser-scanning microscope (CLSM) images revealed that 10 mg ml-1 dCS/512 µg ml-1 CAZ is by far the best formulation to diminish B. pseudomallei biofilm biomass and produces the lowest live/dead cell ratios of B. pseudomallei in biofilm matrix. Collectively, these findings emphasize the potential of novel therapeutic antibacterial and antibiofilm agents to fight against antibiotic-tolerant B. pseudomallei biofilm-associated infections.

Growth and biosorption of Purple guinea and Ruzi grasses in arsenic contaminated soils.

Increasing mining and industrial discharge of untreated wastewater, as well as excessive use of fertilizers for agricultural purposes, and heavy metal contamination in soil have become one of the serious environmental problems worldwide. In the present study, pot experiments were conducted to evaluate the influence of arsenic contamination and other factors on the growth and development of local forage grasses like Purple guinea and Ruzi grasses under controlled conditions. Influence of arsenic concentration, soil properties, and fertilizers on biosorption and withstanding potential of grasses was studied using model soil and real-time arsenic-contaminated mine soil. High arsenic contents in soil significantly affected the growth as well as biomass production of grasses and declined the overall biomass production concerning exposure durations. Purple guinea and Ruzi grasses showed growth tolerance in arsenic-contaminated soils with concentrations of 100 and 150 mg/kg respectively. Grass species, soil compositions, and properties, fertilizers, growth duration, etc. potentially influenced arsenic accumulation in grasses. Both local forage grasses showed <1 bio-accumulation factor (BAF) and bio-concentration factor (BCF) after 45 days that indicates the minimum harvesting time of 45 days, and biosorption rate was found significant to the exposure duration. Maximum translocation factor (TF) values observed in Purple guinea and Ruzi grasses were 0.65 and 0.95, respectively which are < 1, therefore, these local forage grasses could be labeled as arsenic-metallophytes and ability to tolerate high levels of heavy metals without much biosorption. The results confirmed that local forage grasses have much growth tolerance potential against arsenic in real-time mine soil with desired fertilizers and these species could be used for sustainable management of ecological health of the Thung Kum gold mine area in Thailand.

Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm.

Antibacterial activity of chitosan against Burkholderia pseudomallei.

The ability of Burkholderia pseudomallei to persist and survive in the environment is a health problem worldwide. Therefore, the antibacterial activities of chitosan against four environmental isolates of B. pseudomallei from soil in Khon Kaen, Thailand, were investigated. Antibacterial activities were assessed by a plate count technique after treatment with 0.2, 0.5, 1, 2 or 5 mg ml-1 chitosan for 0, 24 and 48 hr. Chitosan at 5 mg ml-1 completely killed all four B. pseudomallei isolates within 24 hr, whilst 2 mg ml-1 chitosan lowered the viability of B. pseudomallei by 20% within the same time span. Chitosan may act by disruption of the cell membrane, releasing intracellular components that can be detected spectrophotometrically at 260 and 280 nm. Transmission electron microscopy inspection of chitosan-treated B. pseudomallei revealed damage to the bacterial membranes. This study demonstrated the effective antibacterial activity by chitosan against B. pseudomallei. Chitosan causes disruption of the bacterial cell membrane, release of intracellular constituents and cell death. This study revealed the inhibitory potential of chitosan for mitigating B. pseudomallei occurrences.

Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei.

Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0-6 h using the plate count technique on Ashdown's agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist.

Morphological Alteration and Survival of Burkholderia pseudomallei in Soil Microcosms.

The resilience of Burkholderia pseudomallei, the causative agent of melioidosis, was evaluated in control soil microcosms and in soil microcosms containing NaCl or FeSO4 at 30°C. Iron (Fe(II)) promoted the growth of B. pseudomallei during the 30-day observation, contrary to the presence of 1.5% and 3% NaCl. Scanning electron micrographs of B. pseudomallei in soil revealed their morphological alteration from rod to coccoid and the formation of microcolonies. The smallest B. pseudomallei cells were found in soil with 100 µM FeSO4 compared with in the control soil or soil with 0.6% NaCl (P < 0.05). The colony count on Ashdown's agar and bacterial viability assay using the LIVE/DEAD(®) BacLight(™) stain combined with flow cytometry showed that B. pseudomallei remained culturable and viable in the control soil microcosms for at least 120 days. In contrast, soil with 1.5% NaCl affected their culturability at day 90 and their viability at day 120. Our results suggested that a low salinity and iron may influence the survival of B. pseudomallei and its ability to change from a rod-like to coccoid form. The morphological changes of B. pseudomallei cells may be advantageous for their persistence in the environment and may increase the risk of their transmission to humans.

Physicochemical factors affecting the growth of Burkholderia pseudomallei in soil microcosm.

Burkholderia pseudomallei causes melioidosis, the third most common cause of death from infectious diseases in northeast Thailand. Four physicochemical factors were set so that their values covered the range of the northeast, which is an endemic area. The soil pH was set at pH 4-10, soil salinity was 0.0-5.0% NaCl, total iron was 50-150 mg/kg soil, and carbon to nitrogen ratio (C/N) was 10:1 to 40:1. The experiments were carried out at 37°C, and soil moisture was maintained for 7 days. The number of viable bacterial cells was counted daily. Soil pH, salinity, Fe, and C/N ratio affected the bacterial growth. The bacterial colony was significantly (P < 0.05) reduced at soil pH > 8, soil salinity > 1% NaCl, and C/N ratio > 40:1. However, the growth of B. pseudomallei was enhanced by increasing the concentrations of iron significantly (P < 0.05). We propose using these findings to control B. pseudomallei in situ.

Environmental factors affecting Burkholderia pseudomallei biofilm formation.

Melioidosis is highly prevalent in Northeast Thailand which is associated with high incidence of Burkholderia pseudomallei present in the soil of this region. B. pseudomallei when present in biofilm becomes resistant to numerous environmental factors and also to certain antibiotics. In this study, we examined the effects of several environmentally relevant factors (salinity, iron, manganese and temperature) on biofilm formation of four clinical ribotypes of B. pseudomallei commonly found in Northeast Thailand. The results showed that biofilm formation increased when B. pseudomallei were grown in modified Vogel and Bonner's medium containing 0.85-1.7 M NaCl or 100-500 microM iron (FeSO4). Low temperature (20 degrees C) also induced more biofilm formation than 30 degrees C or 37 degrees C. On the other hand, protease production and bacterial motility were adversely affected but not in the case of low temperature. Results from this study should be useful in the development of prevention measures or controlling B. pseudomallei biofilm formation in the environment.