Inhibitory effects of Trichoderma asperellum culture filtrates on pathogenic bacteria, Burkholderia pseudomallei.

Abstract

Background: Burkholderia pseudomallei is a soil- and water-dwelling bacterium that causes the life-threatening infection melioidosis. Patients typically acquire this infection through environmental exposure, so reducing B. pseudomallei levels in the environment could mitigate the risk of infection. Trichoderma asperellum is a biological control agent that synthesizes a diverse range of antimicrobial substances targeting other microorganisms. This study therefore examined the antibacterial and anti-biofilm activities of T. asperellum culture filtrate against B. pseudomallei.

Methods: The antibacterial activities of T. asperellum culture filtrates, collected at various time intervals, were assessed against B. pseudomallei using the agar well diffusion method. Subsequently, the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and anti-biofilm activities of the culture filtrate exhibiting the highest inhibitory effect were determined. Bactericidal efficacy was further evaluated via a time-kill assay. The mechanisms underlying inhibition were then investigated using scanning electron microscopy and crystal violet uptake assays.

Results: Filtrate collected from 7-day old cultures of T. asperellum (TD7) exhibited the strongest inhibitory effect on B. pseudomallei, with an inhibition zone of 30.33 ± 0.19 mm. The MIC of TD7 against B. pseudomallei was 7.81 ± 0.00 mg/mL and the MBC ranged from 7.81 ± 0.00 to 11.72 ± 1.75 mg/mL. Time-kill studies with TD7 confirmed its bactericidal activity, with complete elimination of B. pseudomallei occurring within 30 min treatment at 62.48 mg/mL (8xMIC) and 24 h treatment at 7.81 mg/mL (1xMIC). At a concentration of  7.81 mg/mL, TD7 also significantly reduced B. pseudomallei biofilm formation. Scanning electron microscopy revealed surface roughening and cell shrinkage of TD7-treated B. pseudomallei. TD7-treated bacteria were also found to absorb more crystal violet dye than untreated cells, indicating that TD7 might inhibit and kill B. pseudomallei by disrupting cell membrane permeability.

Conclusions: Our findings demonstrate that T. asperellum culture filtrates possess bactericidal activity and effectively disrupt biofilm formation by B. pseudomallei. This suggests that T. asperellum could potentially be used to reduce the presence of B. pseudomallei in the environment and, consequently, lower the incidence of melioidosis.