Abstract
The high tolerance of biofilm-grown Burkholderia cepacia complex bacteria against antimicrobial agents presents considerable problems for the treatment of infected cystic fibrosis patients and the implementation of infection control guidelines. In the present study, we analyzed the tolerance of planktonic and sessile Burkholderia cenocepacia J2315 cultures and examined the transcriptional response of sessile cells to treatment with chlorhexidine. At low (0.0005%) and high (0.05%) concentrations, chlorhexidine had a similar effect on both populations, but at intermediate concentrations (0.015%) the antimicrobial activity was more pronounced in planktonic cultures. The exposure of sessile cells to chlorhexidine resulted in an upregulation of the transcription of 469 (6.56%) and the downregulation of 257 (3.59%) protein-coding genes. A major group of upregulated genes in the treated biofilms encoded membrane-related and regulatory proteins. In addition, several genes coding for drug resistance determinants also were upregulated. The phenotypic analysis of RND (resistance-nodulation-division) efflux pump mutants suggests the presence of lifestyle-specific chlorhexidine tolerance mechanisms; efflux system RND-4 (BCAL2820-BCAL2822) was more responsible for chlorhexidine tolerance in planktonic cells, while other systems (RND-3 [BCAL1672-BCAL1676] and RND-9 [BCAM1945-BCAM1947]) were linked to resistance in sessile cells. After sessile cell exposure, multiple genes encoding chemotaxis and motility-related proteins were upregulated in concert with the downregulation of an adhesin-encoding gene (BCAM2143), suggesting that sessile cells tried to escape the biofilm. We also observed the differential expression of 19 genes carrying putative small RNA molecules, indicating a novel role for these regulatory elements in chlorhexidine tolerance.