Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Objective: To characterize biofilm production by clinical (n=21) and environmental (n=11) isolates of Burkholderia pseudomallei and evaluate the production of proteases, hemolysins and siderophores. Methods: Initially, the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1% glucose (MH-1% glucose) and BHI broth-1% glucose, using the crystal violet staining technique. Subsequently, growing (48 h) and mature (72 h) biofilms were evaluated by confocal microscopy. Finally, the production of proteases, hemolysins and siderophores by planktonic aggregates, growing biofilms and mature biofilms was evaluated. Results: All isolates produced biofilms, but clinical isolates had significantly higher biomass in both MH-1% glucose (P<0.001) and BHI-glucose 1% (P=0.005). The structural analyses by confocal microscopy showed thick biofilms, composed of multiple layers of cells, homogeneously arranged, with mature biofilms of clinical isolates presenting higher biomass (P=0.019) and thickness of the entire area (P=0.029), and lower roughness coefficient (P=0.007) than those of environmental isolates. Protease production by growing biofilms was significantly greater than that of planktonic (P<0.001) and mature biofilms (P<0.001). Hemolysin release by planktonic aggregates was higher than that of biofilms (P<0.001). Regarding siderophores, mature biofilms presented higher production than growing biofilms (P<0.001) and planktonic aggregates (P<0.001). Conclusions: Clinical isolates have higher production of biofilms than their environmental counterparts; protease and siderophores seem important for growth and maintenance of Burkholderia pseudomallei biofilms.