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.

Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.

Comparison of stress adaptation and survival rate between Burkholderia pseudomallei with mutant and wild type bfmR.

Burkholderia pseudomallei (Bp) is highly adaptable to a wide range of environmental changes for survival and pathogenesis. However, the underlying mechanisms of such adaptability are still unclear. Two-component system (TCS) is a common signal transduction used by bacteria in response to environmental changes. A gene designated as bfmR (locus tag of BPSL2024) has been proposed to encode a response regulator, a member of the TCS, and was studied by mutagenesis and comparison of its phenotypic changes compared with those of the wild type. The growth rates of the mutant Bp at temperatures of 37 degrees-39 degrees C and pH 5-8 were significantly lower than the wild type strain (p < 0.05), especially at 39 degrees C (p = 0.01) and pH 7 (p = 0.01). The survival rate of the mice infected with the mutant strain is not significantly different from mice infected with wild type strain. The defective phenotypes were recovered in the complemented strain. These results indicated that bfmR is involved in adaptation of Bp to thermal- and pH-induced stress conditions.

LYTIC CAPABILITY OF BACTERIOPHAGES (FAMILY MYOVIRIDAE) ON BURKHOLDERIA PSEUDOMALLEI.

Burkholderia pseudomallei, a gram-negative bacillus found in soil and water, is the causative agent of melioidosis. It can produce a biofilm, which increases resistance to antibacterial agents. Bacteriophages (phages) have been suggested as alternative antibacterial agents. In this study, the ability of six phages (family Myoviridae) to lyse B. pseudomallei isolates was examined using a microplate phage virulence assay. The six phages were more efficient in lysing soil than clinical B. pseudomallei isolates. Phage ST79 had the highest lytic capability, independent of inoculating phage quantity with a 4-log reduction of bacterial numbers after a 4 hour treatment. Three modified derivatives of ST79 were developed by multiple passages on phage-resistant B. pseudomallei isolates, leading to an increase in lytic capability from 62% to 80%. Phage ST79 at a multiplicity of infection (MOI) of 10 significantly reduces biofilm formation determined by a colorimetric method. The recovery of B. pseudomallei growth following phage treatment needs to be overcome if these lytic phages are to be used as biocontrol agents of B. pseudomallei in the environment.

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.

Tn5-OT182 should not be used to identify genes involved in biofilm formation in Burkholderia pseudomallei.

Burkholderia pseudomallei, a gram-negative bacterium, is the causative agent of melioidosis. One of the important virulence properties of this bacteria is its ability to form a biofilm. Genes involved in biofilm formation in B. pseudomallei have not been thoroughly studied. In this study, Tn5-OT182 mutagenesis was used to isolate of B. pseudomallei strain A2 mutants unable to produce biofilm. Ten biofilm-defective transposon mutants were isolated and analyzed. Flanking DNA from each transposon mutant were self-cloned and sequenced, then the sequences were analyzed with the BLAST program. To confirm these genes are involved in biofilm formation, we constructed three gene deletion mutants marked with a tetracycline resistance gene. The constructed tet(r)-marked deletion mutants were checked for correct structure and size by polymerase chain reaction. When subjected to biofilm assay, all tested tet(r)-marked deletion mutants were still able to produce biofilm, indicating the three genes are not involved in biofilm formation. These results suggest integration of Tn5-OT182 in genes not involved in biofilm production can render B. pseudomallei unable to produce biofilm by an unknown mechanism. This information demonstrates Tn5-OT182 is not a reliable tool for identifying genes involved in biofilm formation unless a confirmatory experiment is carried out in parallel.

Role of Burkholderia pseudomallei in the invasion, replication and induction of apoptosis in human epithelial cell lines.

Burkholderia pseudomallei is a bacterial pathogen causing melioidosis. In the present study, we demonstrated the effects of B. pseudomallei capsular polysaccharide on the invasion, intracellular replication, induction of cytotoxicity and apoptosis of human epithelial HeLa and A549 cells. The B. pseudomallei capsule mutant had a significantly greater ability to invade both cell lines (p < 0.05). The B. pseudomallei capsule mutant had a greater ability to induce apoptosis in A549 cells than wild type B. pseudomallei. These results indicate the capsular polysaccharide of B. pseudomallei plays an important role in inhibiting invasion by the bacteria but has no effect on intracellular multiplication, induction of cytotoxicity or apoptosis in epithelial cells.

Construction and characterization of an unmarked aroC deletion mutant of Burkholderia pseudomallei strain A2.

Using a sucrose counter-selection strategy, we have constructed an unmarked aroC deletion mutant of B. pseudomallei strain A2 in order to investigate the possibility of an effective live-attenuated vaccine. The aroC deletion in the resultant mutant, designated A2DeltaaroC, was confirmed by PCR, Southern hybridization and failure of the mutant to grow in a defined medium without aromatic compounds. Compared to the parental wild type strain, A2DeltaaroC was highly attenuated for virulence following intraperitoneal introduction into BALB/c and C57BL/6 mice. BALB/c mice immunized intraperitoneally with A2DeltaaroC were not protected against a challenge dose of 500 cfu (25 x LD50) of the parental strain A2, whereas C57BL/6 mice similarly immunized intraperitoneally with A2DeltaaroC were significantly protected against a challenge dose of 6,000 cfu (20 x LD50).