Molecular Mechanisms of Chlorhexidine Tolerance in Burkholderia cenocepacia Biofilms

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 adhesinencoding gene (BCAM2143), suggesting that sessile cells tried to escape the biofilm. We also observed the differential expression of 19 genes carying putative small RNA molecules, indicating a novel role for these regulatory elements in chlorhexidine tolerance.

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins

“…The primary features of the A. baumannii chlorhexidine resistance response, i.e., up-regulation of genes encoding RND and PCE family efflux systems, are conserved in other γ-proteobacteria (9), as well as β-proteobacteria (10). Given the phylogenetic distance of these organisms, this similarity of regulatory responses to a compound that has only been synthesized since the early 20th century is intriguing.…”

“…With the exception of the adeAB efflux transporter genes, only one gene was overexpressed by more than 10-fold in response to chlorhexidine exposure in A. baumannii, A1S_2063, which was annotated as encoding a hypothetical protein. Orthologs of A1S_2063 in P. aeruginosa and B. cenocepacia were also among the most overexpressed genes in response to chlorhexidine challenge (9,10). Therefore, these genes are a highly conserved component of a chlorhexidineresponsive regulatory circuit in several proteobacterial genera.…”

The Third Replicon of Members of the Burkholderia cepacia Complex, Plasmid pC3, Plays a Role in Stress Tolerance

“…Chlorhexidine is an antiseptic that acts via membrane disruption. It was previously shown to lead to the upregulation of a number of pC3 genes in J2315, including the aforementioned chaperonin-encoding genes BCAS0637 and BCAS0638 (8-and 22-fold, respectively) (29). B. cenocepacia H111 showed very significantly higher resistance to 5% chlorhexidine than H111⌬c3 (P ϭ 0.006618; the mean zone of inhibition tested by disc diffusion assay was 5.2 mm for H111 and 6.2 mm for H111⌬c3, with a standard error of 0.2 in both cases).…”

“…The second general factor that would be expected to limit the occurrence of pC3-null communities in the environment is the importance of this replicon to stress tolerance and environmental fitness. Orthologs of the B. cenocepacia J2315 chaperonin genes BCAS0637 and BCAS0638, which were shown to be upregulated under various stresses (28,29), were found in several strains of the Bcc species B. cenocepacia, B. multivorans, and B. ambifaria by BLAST analysis. As a result of these factors increasing the stability of pC3 within the Bcc, this nonessential replicon is highly prevalent throughout the complex and (A) Bacteria were grown overnight in LB medium and then subcultured into LB supplemented with additional NaCl to a total concentration of 2 M. Viability was measured before inoculation into high-salt LB broth and after 2.5, 5, and 24 h of incubation at 37°C with shaking in this medium.…”

MtvR Is a Global Small Noncoding Regulatory RNA in Burkholderia cenocepacia

“…A bioinformatics study performed by Coenye et al (12) identified 213 putative sRNAs on the genome of the highly epidemic clinical isolate B. cenocepacia J2315 (13), although the expression of only 4 sRNAs was confirmed (12). In another study on the transcriptional responses of planktonic and sessile cells of B. cenocepacia J2315 upon exposure to chlorhexidine, 19 intergenic sequences putatively encoding sRNAs were identified as differentially expressed, although their biological function remains unknown (14). More recently, 24 sRNAs from B. cenocepacia J2315 were identified and validated, based on the exploitation of the RNA binding ability of Hfq (15).…”