Abstract
Biofilms are structured microbial communities that are the leading cause of numerous chronic infections which are difficult to eradicate. Within the lungs of individuals with cystic fibrosis (CF), Pseudomonas aeruginosa causes persistent biofilm infection that is commonly treated with aminoglycoside antibiotics such as tobramycin. However, sublethal concentrations of this aminoglycoside were previously shown to increase biofilm formation by P. aeruginosa, but the underlying adaptive mechanisms still remain elusive. Herein, we combined confocal laser scanning microscope analyses, proteomics profiling, gene expression assays and phenotypic studies to unravel P. aeruginosa potential adaptive mechanisms in response to tobramycin exposure during biofilm growth. Under this condition, we show that the modified biofilm architecture is related at least in part to increased extracellular DNA (eDNA) release, most likely as a result of biofilm cell death. Furthermore, the activity of quorum sensing (QS) systems was increased, leading to higher production of QS signaling molecules. We also demonstrate upon tobramycin exposure an increase in expression of the PrrF small regulatory RNAs, as well as expression of iron uptake systems. Remarkably, biofilm biovolumes and eDNA relative abundances in pqs and prrF mutant strains decrease in the presence of tobramycin. Overall, our findings offer experimental evidences for a potential adaptive mechanism linking PrrF sRNAs, QS signaling, biofilm cell death, eDNA release, and tobramycin-enhanced biofilm formation in P. aeruginosa. These specific adaptive mechanisms should be considered to improve treatment strategies against P. aeruginosa biofilm establishment in CF patients’ lungs.
Highlights
Bacterial biofilm forms a highly structured community of cells that are attached to each other and/or a surface and are enclosed in a complex matrix of extracellular polymeric substances (EPS).[1,2] Biofilms enable bacteria to colonize different environments and are prevalent in natural, industrial and medical environments
They observed lower Previous studies showed enhanced P. aeruginosa biofilm formaabundances of proteins associated with nucleotide metabolism, tricarboxylic acid (TCA), carbon metabolism and energy derivation, tion upon exposure to tobramycin and other aminoglycosides by using colorimetric assays based on crystal violet staining.[19,20,21]
Through Confocal laser scanning microscopy (CLSM) observations, whole biofilm proteome analysis, gene expression Reverse transcription-quantitative PCR analysis (RT-qPCR) assays, and phenotypic approaches, we present further insights into the adaptation mechanisms leading to increased biofilm formation in P. aeruginosa in response to sub-MIC of tobramycin
Summary
Bacterial biofilm forms a highly structured community of cells that are attached to each other and/or a surface and are enclosed in a complex matrix of extracellular polymeric substances (EPS).[1,2] Biofilms enable bacteria to colonize different environments and are prevalent in natural, industrial and medical environments. P. aeruginosa is a problematic Gram-negative pathogen representing a serious threat to individuals and public health This opportunistic pathogen causes both acute and chronic infections that are strongly related to its planktonic and biofilm lifestyles, respectively. Within the lungs of cystic fibrosis (CF) individuals, biofilms are gradually formed by P. aeruginosa cells surrounded by a self-produced matrix of EPS such as polysaccharides, proteins, extracellular DNA (eDNA), metabolites, and siderophores.[2,13,14,15] As a result of their ability to form biofilms and their high tolerance levels towards a broad spectrum of antimicrobials, P. aeruginosa chronic lung infections are almost impossible to eradicate.[13,16,17] Tobramycin, an aminoglycoside antibiotic, is used in the treatment of P. aeruginosa infections.[18] exposure to sub-MIC of this aminoglycoside[19,20,21,22] and of. Published in partnership with Nanyang Technological University other antibiotics such as quinolones[23] and tetracycline[20,21] In this context, we sought to elucidate adaptive mechanisms enhances P. aeruginosa biofilm formation.
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