Transcription profiling of Pseudomonas aeruginosa reveals biofilms and type III secretion are not mutually exclusive
ABSTRACT: Biofilm formation and type III secretion have been shown to be reciprocally regulated in P. aeruginosa, and it has been suggested that factors related to acute infection may be incompatible; with biofilm formation. We investigated how growth conditions influence the production of virulence factors by studying the inter-relationships between colonies, biofilms and planktonic cells. We found that biofilms in our growth conditions express the type III secretion and these lifestyles are therefore not mutually exclusive in P. aeruginosa. Experiment Overall Design: Pseudomonas aeruginosa cells grown in five different conditions were analysed with three biological replicates for each sample. The five different conditions were planktonic cells in exponential phase, planktonic cells in stationary phase, colonies on agar plates incubated for 15 or 40 hours and biofilms in a continuous flow system after three days of growth.
Project description:Biofilm formation and type III secretion have been shown to be reciprocally regulated in P. aeruginosa, and it has been suggested that factors related to acute infection may be incompatible with biofilm formation. We investigated how growth conditions influence the production of virulence factors by studying the inter-relationships between colonies, biofilms and planktonic cells. We found that biofilms in our growth conditions express the type III secretion and these lifestyles are therefore not mutually exclusive in P. aeruginosa. Keywords: Comparison of different growth modes and growth phases of Pseudomonas aeruginosa Overall design: Pseudomonas aeruginosa cells grown in five different conditions were analysed with three biological replicates for each sample. The five different conditions were planktonic cells in exponential phase, planktonic cells in stationary phase, colonies on agar plates incubated for 15 or 40 hours and biofilms in a continuous flow system after three days of growth.
Project description:Transcription profile of Escherichia coli cells in biofilms under static batch culture was compared to that of E. coli cells in planktonic cultures. Both E. coli biofilm and planktonic cultures were cultivated for 18 h in 10% Luria-Bertani broth at room temperature (20 degree Celsius). Biofilms were grown in static batch culture in petri dishes. Both planktonic culture and biofilms were homogenized and run through a separated protocol. Two condition experiments: E. coli biofilm vs E. coli planktonic cultures. Two biological replicates with independently grown and harvested biofilms or planktonic cultures. Each biological replicate has two technical replicates of hybridization on microarray slides. Each slide has three built-in replicates for each probe.
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. Overall design: Three biological replicates were prepared and analyzed for the following three growth conditions: (1)Pseudomonas aeruginosa was grown planktonically to early log phase. (2) Pseudomonas aeruginosa was grown in drip flow biofilm reactors on hydroxyapetite-coated glass slides for four days. (3) Pseudomonas aeruginosa was grown in drip flow biofilm reactors on hydroxyapetite-coated glass slides for three days and then treated with 1 mg/ml ciprofloxacin for an additional day.
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:Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptional responses of Pseudomonas aeruginosa growing in biofilms by using microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNAs for these housekeeping genes were low in cells at the bottom of the biofilms. Selective GFP labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNAs levels for genes regulated by the hypoxia induced regulator, Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-termanoxia. Transcripts for ribosomal proteins were primarily associated with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (rmf and PA4463) at the bottom of the biofilms. A P. aeruginosa ∆rmf strain had increased uptake of the membrane integrity stain, propidium iodide. Using selective GFP labeling and cell sorting, we showed that the dividing cells were more susceptible to tobramycin and ciprofloxacin than the dormant subpopulation. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. 52-hour Pseudomonas aeruginosa TSA colony biofilms were cryoembedded, thin sectioned, and laser dissected (LCM) to obtain samples from the top and bottom 50 µm of the biofilms. 9 sections per biofilm were pooled. RNA was extracted with the RNeasy Micro kit, Turbo DNase treated, poly(A) tailed, and amplified using the Quantitect WTA kit. After clean up, the resulting product was fragmented and end labeled before hybridization.
Project description:In the present in vitro study, interactions between P. aeruginosa (sessile biofilms as well as planktonic cells) and PMNs were analyzed by means of DNA microarray based transcriptomics. We found that the P. aeruginosa wild type biofilms, in contrast to planktonic cultures and quorum sensing (QS) deficient strains, respond to PMN exposure in a rather aggressive manner. The response does not involve protective mechanisms such as those involved in oxidative stress. Rather it is dominated by QS controlled virulence determinants such as those encoded by pqs, phz, rhlAB, all of which are designed to cripple Eukaryotic cells including PMNs and macrophages. Our comparative analysis supports the view that QS plays a major role in mechanisms by which P. aeruginosa evades host defense systems. Keywords: Stress response The biofilms were allowed to grow and develop in the biofilm flow chambers for 3 days before challenge with PMNs. Fresh PMNs from human volunteers were isolated and resuspended in RPMI-1640 medium (BIOCHROM AG) as previously described by Bjarnsholt et al. 2005 (Bjarnsholt et al., 2005). Prior to PMN injection the flow was halted. Ten million PMNs were resuspended in 6 ml of RPMI media and injected into the flow chamber. The concentration of PMNs was found by microscopy to be approximately 1 PMN per 1000 bacterial cells. PMNs and biofilm were incubated for 2 hours. The flow chambers were rolled every 15 minutes to ensure that the entire biofilm was exposed to the PMNs. After incubation the fluid inside the chamber was discarded and the attached biofilm cells were loosened by rolling. The cells were then mechanically removed and collected in 6 ml of RNAlater®
Project description:Pseudomonas aeruginosa is one of the most frequent pathogen dominant in complicated urinary tract infections (UTI). To unravel the adaptation strategies of P. aeruginosa to the conditions in the urinary tract and to define the underlying regulatory network an artificial growth system mimicking the conditions in the urinary tract was established. Transcriptome analyses were used to investigate the physiological status of P. aeruginosa under this conditions. We performed comparisons to identify genes induced under artificial urinary tract conditions to unravel the adaptive strategies and the underlying regulatory network used by Pseudomonas aeruginosa during urinary tract infections using Affimetrix GeneChips. Pseudomonas aeruginosa wild type strain PAO1 was grown in an artificial in vitro growth system mimicking the conditions in the urinary tract. Therefore, biofilms were grown on the surface of membrane filters placed on agar plates at 37 °C up to the late logarithmic state under aerobic and anaerobic conditions (incubated in an anaerobic beanch). An artificial urine medium (AUM) simulating the averaged urine of an human adult was used as nutrient souce. 10-fold diluted Luria Bertani (LB)-medium was used as reference medium. For growth under oxygen depletion the media were supplemented with 50 mM KNO3 to sustain anaerobic respiration. The biofilms were harveted at this time points and resuspsended in 0.9% (w/v) NaCl. The OD578 of biofilm suspension was 0.8 for all tested conditions. First comparison: Identification of genes induced or repressed under aerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown aerobically for 18 h to the late logarithmic phase in biofilms on AUM with the transcriptome profile of the PAO1 strain, which was grown aerobically for 18 h to the late logarithmic phase in biofilms on 10-fold diluted LB. Second comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown anaerobically for 2 days up to the late logarithmic phase in biofilms on AUM supplemented with 50 mM nitrate with the transcriptome profile of the PAO1 strain, which was grown anaerobically for 2 days up to the late logarithmic phase in biofilms on 10-fold diluted LB supplemented with 50 mM nitrate.
Project description:In this work we have demonstrated increased mutability of Staphylococcus aureus and S. epidermidis in biofilms and have explored the mechanisms underlying the enhanced mutability. A novel static biofilm model, utilising cellulose filter disks, was developed to support the formation of mature biofilms with sufficiently high cell densities to permit determination of mutation frequencies. The mutability of biofilm cultures increased up to 60 fold and 4 fold for S. aureus and S. epidermidis, respectively, compared with planktonic cultures. Incorporation of antioxidants into S. aureus biofilms reduced mutation frequencies, indicating that increased oxidative stress underlies increased mutability in the biofilm. Transcriptional profiling revealed upregulation of the superoxide dismutase gene, sodA, in early biofilm cultures, also suggesting enhanced oxidative stress in these cultures. However, loss of the genes encoding superoxide dismutases or peroxidases did not specifically exacerabate biofilm mutability. In S. aureus SH1000, hydrogen peroxide was found to contribute to biofilm mutability. Three growth conditions (18 hr planktonic growth, 48 hr biofilm growth and 144 biofilm growth) of which there are three biological replicates of each
Project description:This SuperSeries is composed of the following subset Series:; GSE9989: Tobramycin Treatment of P. aeruginosa Biofilms Grown on CFBE41o- Cells; GSE9991: Tobramycin Treatment of Planktonic Pseudomonas aeruginosa Experiment Overall Design: Refer to individual Series
Project description:Microarray analysis was used to identify changes in the level of transcription of genes in P. aeruginosa drip flow biofilms in response to ciprofloxacin and tobramycin exposure. 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. Four drip flow biofilm conditions with three replicates each: (1) baseline controls at 72 hours, (2) tobramycin treated for 12 hours past baseline, (3) ciprofloxacin treated for 12 hrs past baseline, and (4) no treatment for 12 hrs past baseline.