Project description:Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, using genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1, we identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections. This dataset compares the expression of SAH108, a strain with enhanced antibiotic tolerance in the biofilm state, to expression in wild-type strains.
Project description:Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, using genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1, we identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections. This dataset compares the expression of SAH108, a strain with enhanced antibiotic tolerance in the biofilm state, to expression in wild-type strains. We compared the expression of two biological replicates from strain SAH108 to samples from three wild-type, reference strains. All samples were collected from exponentially-growing planktonic cultures.
Project description:ndvB is a gene expressed preferrentialy in biofilms of Pseudomonas aeruginosa and has been implicated in antibiotic resistance. This gene also has a role in signaling in some plant pathogens. A knockout ndvB strain was used to determine if it controlled any other gene expression related to antibiotic resistance We used microarrays of wildtype and ndvB knockout P. aeruginosa grown in biofilms to identify the role of ndvB in gene expression
Project description:Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip-flow reactors on a medium composed to mimic the exudate from a chronic wound (CWE). After 72 hours, the biofilms were treated with CWE (control biofilms) or CWE containing ciprofloxacin (treated biofilms) for an additional 24 hours. Planktonic samples were cultivated to early logarithmic phase in CWE. The biofilm specific growth rate was estimated via elemental balances to be approximately 0.37 h-1, or one-third of the planktonic maximum specific growth rate. Global analysis of gene expression indicated decreased anabolic activity in biofilms compared to planktonic cells. A focused transcriptomic analysis revealed the induction of multiple stress responses in biofilm cells, including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing.
Project description:ndvB is a gene expressed preferrentialy in biofilms of Pseudomonas aeruginosa and has been implicated in antibiotic resistance. This gene also has a role in signaling in some plant pathogens. A knockout ndvB strain was used to determine if it controlled any other gene expression related to antibiotic resistance We used microarrays of wildtype and ndvB knockout P. aeruginosa grown in biofilms to identify the role of ndvB in gene expression Wildtype and ndvB knockout PA14 biofilms were grown for 48 hours followed by RNA extraction. RNA was pooled from 2 wells for each condition to obtain a single biological replicate. Affymetrix microarray processing was performed on PA01 genechips and data analysis was carried out using R statistical framework with bio-conductor packages using RMA and MAS5.0 normalization procedures
Project description:We grew Pseudomonas aeruginosa biofilms on CFBE41o- human airway cells in culture, and we treated these biofilms with tobramycin. Microarray analysis was performed to gain an understanding of the global transcriptional changes that occur during antibiotic treatment. Keywords: Antibiotic Response
Project description:Transcriptome analysis was applied to characterize the physiological activities of Psuedomonas aeruginosa cells grown for three days in drip flow biofilm reactors when compared to the activities of P. aeruginosa grown planktonically to exponential phase in the same media. Here, rather than examining the effect of an individual gene on biofilm antibiotic tolerance, we used a transcriptomics approach to identify regulons and groups of related genes that are induced during biofilm growth of Pseudomonas aeruginosa. We then tested for statistically significant overlap between the biofilm-induced genes and independently compiled gene lists corresponding to stress responses and other putative antibiotic protective mechanisms. This data was evaluated and used to select strains that carry transposon mutations in genes that might play a role in antibiotic tolerance of biofilms. The strains were evaluated for defects in biofilm tolerance.
Project description:Pseudomonas aeruginosa is known to tolerate antibiotic therapy during infection. This prevents clearance of infection and negatively impacts patient outcomes. Here, we report the transcriptome sequence of antibiotic-treated and untreated P. aeruginosa cultures and the differential gene expression observed when treated cells are compared to untreated cells.
Project description:Transcriptome analysis was applied to characterize the physiological activities of Psuedomonas aeruginosa cells grown for three days in drip flow biofilm reactors when compared to the activities of P. aeruginosa grown planktonically to exponential phase in the same media. Here, rather than examining the effect of an individual gene on biofilm antibiotic tolerance, we used a transcriptomics approach to identify regulons and groups of related genes that are induced during biofilm growth of Pseudomonas aeruginosa. We then tested for statistically significant overlap between the biofilm-induced genes and independently compiled gene lists corresponding to stress responses and other putative antibiotic protective mechanisms. This data was evaluated and used to select strains that carry transposon mutations in genes that might play a role in antibiotic tolerance of biofilms. The strains were evaluated for defects in biofilm tolerance. One planktonic condition with four biological replicates; One drip flow biofilm condition grown for 72 hours with three biological replicates; One drip flow biofilm condition grown for 84 hours with three biological replicates.
Project description:Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The superior capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states, and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residues R152, K270, or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG sensing mechanism by RccR.