The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.

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.

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.