Co-evolutionary Signals Identify Burkholderia pseudomallei Survival Strategies in a Hostile Environment.

The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of "co-selection"-that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions, highlighting the conditions as one of the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed a BPSL1661 role under nutrient deprivation, and explored the functional basis of co-selection gene network surrounding BPSL1661 in facilitating the bacterial survival under nutrient depletion. Our findings suggest that nutrient-limited conditions have been the common selection pressure acting on this species, and allelic variation of BPSL1661 may have promoted B. pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.

Burkholderia pseudomallei-absent soil bacterial community results in secondary metabolites that kill this pathogen.

Burkholderia pseudomallei is a Gram-negative bacterium found in soil and the causative agent of a severe disease in humans and animals known as melioidosis. It is intrinsically resistant to many antibiotics and has been reported resistant to the drugs of choice; ceftazidime. Microbial communities in soil in the presence and absence of B. pseudomallei were investigated using metagenomics approach. The variation in bacterial species diversity was significantly higher in soil samples without B. pseudomallei. Abundances of phyla Actinobacteria and Firmicutes were found significantly higher in B. pseudomallei-negative soils. Bacillus amyloliquefaciens KKU1 in phylum Firmicutes was discovered from negative soil and its secondary metabolites could inhibit clinical, environmental and drug resistant isolates of B. pseudomallei, together with some pathogenic Gram-negative but not Gram-positive bacteria. The antimicrobial activity from KKU 1 against B. pseudomallei was abolished when treated with proteinase K, stable in a wide range of pH and remained active after heating at 100 °C for 15 min. Precipitated proteins from KKU1 were demonstrated to cause lysis and corrugated surfaces of B. pseudomallei. The minimum inhibitory concentrations and minimum bactericidal concentrations of the precipitated proteins from KKU1 against B. pseudomallei were 0.97 µg/ml and 3.9 µg/ml. Interestingly, Native SDS-PAGE showed small active compounds of less than 6 kDa, along with other information collectively suggesting the properties of antimicrobial peptides. For the first time, culture-independent information in melioidosis endemic area could lead to a suspected source of metabolites that may help defense against B. pseudomallei and other pathogenic Gram-negative bacteria.

Secondary metabolites from Bacillus amyloliquefaciens isolated from soil can kill Burkholderia pseudomallei.

Bacillus species are Gram-positive bacteria found in abundance in nature and their secondary metabolites were found to possess various potential activities, notably antimicrobial. In this study, Bacillus amyloliquefaciens N2-4 and N3-8 were isolated from soil and their metabolites could kill Burkholderia pseudomallei, a Gram-negative pathogenic bacterium also found in soil in its endemic areas. Moreover, the metabolites were able to kill drug resistant isolates of B. pseudomallei and also inhibit other pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii but not the non-pathogenic Burkholderia thailandensis, which is closely related to B. pseudomallei. Since the antimicrobial activity of N3-8 was not partially decreased or abolished when treated with proteolytic enzymes or autoclaved, but N2-4 was, these two strains should have produced different compounds. The N3-8 metabolites with antimicrobial activity consisted of both protein and non-protein compounds. The inhibition spectrum of the precipitated proteins compared to the culture supernatant indicated a possible synergistic effect of the non-protein and peptide compounds of N3-8 isolates against other pathogens. When either N2-4 or N3-8 isolates was co-cultured with B. pseudomallei the numbers of the bacteria decreased by 5 log10 within 72 h. Further purification and characterization of the metabolites is required for future use of the bacteria or their metabolites as biological controls of B. pseudomallei in the environment or for development as new drugs for problematic pathogenic bacteria.

THE CONTRIBUTION OF SOIL PHYSICOCHEMICAL PROPERTIES TO THE PRESENCE AND GENETIC DIVERSITY OF BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei (Bp), the causative agent of melioidosis, is unevenly distributed in the complex soil environment. Physicochemical factors in the soil have been reported to affect microbial communities in the soil. The effect of physicochemical factors on the number and diversity of organisms in the soil has not been reported. Twenty-five each B. pseudomallei-positive and -negative soil samples were collected from a melioidosis-endemic area. The amount of Bp in each soil sample was measured by culture and quantitative PCR (qPCR). The following physicochemical properties from each soil sample were measured: pH, total organic carbon (TOC), total nitrogen (TN), carbon to nitrogen ratio (C:N ratio), exchangeable calcium (EC) and extractable iron (EI). All the physico- chemical properties measured were significantly different between the Bp-positive and -negative soil samples. The Bp-positive soil samples had lower C:N ratios and lower EC and a higher EI (p < 0.05) than the Bp-negative samples. The average pH was lower (3.7-5.0) in the Bp-negative samples. Among the Bp-positive soil samples, the EC was negatively correlated with the PCR copy number. The amount of bacteria detected with the qPCR method was higher than with the culture method, suggesting the presence of unculturable forms of bacteria that might re-grow when the environmental conditions was suitable. A total of 117 Bp isolates obtained from the soil samples were classified into 25 groups using BOX-PCR. The genetic diversity of Bp, did not correlate with the physicochemical factors investigated. A suitable pH range and C:N ratio may be important for the presence of Bp. The EI supports the needs and EC probably alters the growth of Bp. The genetic diversity of the bacteria was not influenced by the soil factors investigated in this study. This information shows the environment conducive to the growth of Bp. This gives us information about how to potentially control or decrease Bp in the soil in the future.

Response of free-living nitrogen-fixing microorganisms to land use change in the Amazon rainforest.

The Amazon rainforest, the largest equatorial forest in the world, is being cleared for pasture and agricultural use at alarming rates. Tropical deforestation is known to cause alterations in microbial communities at taxonomic and phylogenetic levels, but it is unclear whether microbial functional groups are altered. We asked whether free-living nitrogen-fixing microorganisms (diazotrophs) respond to deforestation in the Amazon rainforest, using analysis of the marker gene nifH. Clone libraries were generated from soil samples collected from a primary forest, a 5-year-old pasture originally converted from primary forest, and a secondary forest established after pasture abandonment. Although diazotroph richness did not significantly change among the three plots, diazotroph community composition was altered with forest-to-pasture conversion, and phylogenetic similarity was higher among pasture communities than among those in forests. There was also 10-fold increase in nifH gene abundance following conversion from primary forest to pasture. Three environmental factors were associated with the observed changes: soil acidity, total N concentration, and C/N ratio. Our results suggest a partial restoration to initial levels of abundance and community structure of diazotrophs following pasture abandonment, with primary and secondary forests sharing similar communities. We postulate that the response of diazotrophs to land use change is a direct consequence of changes in plant communities, particularly the higher N demand of pasture plant communities for supporting aboveground plant growth.