Global transcriptome analysis of Staphylococcus aureus biofilms in response to innate immune cells
ABSTRACT: S. aureus biofilms are associated with the organism's ability to cause disease. Biofilm associated bacteria must cope with the host's innate immune system. We used commercially available Affymetrix S. aureus GeneChips to compare the gene expression properties of 4 and 6 day established biofilms following short (1 hr)- and long (24 hr)- term exposure to macrophages and neutrophils. S. aureus strain USA300 LAC biofilms where formed for 4 or 6 days. Established biofilms were then exposed to macrophages for 1 or 24 hr. Alternatively, biofilms were exposed to neutrophils for 1 or 4 hr. Total bacterial RNA was isolated and subjected to GeneChip hybridization and analysis. We sought to determine the regulatory effects of Macrophages and Neutrophils on established S. aureus biofilms.
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:Interactions between human keratinocytes and secreted factors from Staphylococcus aureus biofilm were investigated using microarray analysis. Relative to control cells, biofilm-secreted factors upregulated cytokine and chemokine genes in keratinocytes. Genes associated with DNA damage and oxidative stress were also induced in keratinocytes treated with secreted factors from S. aureus. Here we show that secreted factors from S. aureus biofilm cultures differentially impact several aspects of wound healing processes. Human keratinocytes were grown in co-culture with mature S. aureus biofilms for 24 hours. Keratinocytes exposed to S. aureus biofilm were analyzed in quadruplicate. Control cells were also analyzed in quadruplicate. Dye-swaps were performed.
Project description:Novel anti-infective agents targeting Staphylococcus aureus and capable of increasing S. aureus susceptibility towards antibiotics are needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. QS is a process by which bacteria produce and detect signal molecules and thereby coordinate their behaviour, virulence and biofilm formation in a cell-density-dependent manner. Hamamelitannin (HAM) was previously suggested to target the S. aureus QS system, thereby increasing the susceptibility of S. aureus biofilms towards vancomycin. However, mechanistic insights are still lacking. For this reason, we evaluated the effect of Hamamelitannin, vancomycin and combination treatment of Hamamelitannin and vancomycin on gene expression in S. aureus Mu50 biofilms.
Project description:RNA sequencing (RNA-Seq) was used in our study to elucidate the mechanism of Tea tree oil (TTO) as a potential antibacterial agent to evaluate differentially expressed genes and functional network analysis in S. aureus ATCC 29213 biofilms. Staphylococcus aureus biofilm cells were exposed for 60 minutes to TTO at concentration of 1/2×MBIC (1 mg/ml).2 samples including 2 control samples are analyzed.
Project description:Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we study protein distributions in bacterial biofilms using shotgun proteomics. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections.
Project description:S. anginosus, S. aureus LMG 10147 and P. aeruginosa DK2 are often co-isolated in sputum samples from cystic fibrosis patients. We found that S. anginosus LMG 14502 becomes less suceptible towards treatment with several antibiotics when it's grown together with S. aureus LMG 10147 and P. aeruginosa DK2, compared to when it's grown alone. In order to elucidate the molecular mechanisms responsible, we performed RNA-seq of an S. anginosus monospecies biofilm and of a multispecies bioiflm of S. anginosus, S. aureus and P. aeruginosa. First, biofilms of S. anginosus alone or in combination with S. aureus and P. aeruginosa were grown. Next, RNA was isolated. Subsequently, a Truseq stranded RNA library preparation kit (Illumina) was used to create strand specific libraries. After a quality and concentration control, the libraries were equimolarly pooled and sequenced using an Illumina NextSeq 500, generating 75bp unpaired reads.
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:Staphylococcus aureus (S. aureus) is one of the most important pathogens in humans and animals. The formation of S. aureus biofilm is considered an important mechanism of antimicrobial resistance. Therefore, finding effective drugs against the biofilm produced by S. aureus has been a high priority. Licochalcone A, a natural plant product, was reported to have antibacterial activities and showed good activity against all 21 tested strains of S. aureus biofilm and planktonic cells. To detect the possible molecular mechanism of Licochalcone A against S. aureus biofillm or planktonic cells, Affymetrix GeneChips were used to determine the global comparative transcription of S. aureus biofilm and planktonic cells triggered by treatment with sub-bactericidal and sub-inhibitory concentrations of Licochalcone A, respectively. Staphylococcus aureus planktonic cells and biofilm were exposed for 60 minutes to Licochalcone A at concentration of 2 μg/ml (1/2× MIC) and 64 μg/ml (4× MIBC), respectively. 4 samples including 4 control samples are analyzed.
Project description:The physiological and transcriptional response of Nitrosomonas europaea biofilms to phenol and toluene was examined and compared to suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors under continuous flow conditions of growth medium containing ammonia as growth substrate. The responses of N. europaea biofilms to the aromatic hydrocarbons phenol and toluene were determined during short-term (3 h) additions of each compound to the biofilms. Ammonia oxidation in the biofilms was inhibited 50% by 60 uM phenol and 100 uM toluene. These concentrations were chosen for microarray analysis of phenol- and toluene-exposed N. europaea biofilms. Liquid batch cultures of exponentially growing N. europaea cells were harvested alongside the biofilms to determine differential gene expression between attached and suspended growth of N. europaea. Four sample groups of N. europaea cells were used in this study, with biological triplicates of each group. Groups were: Control (untreated) biofilms, phenol-exposed biofilms, toluene-exposed biofilms, and exponentially growing suspended cells. Biofilms were grown in Drip Flow Biofilm Reactors containing 4 independent growth channels and subject to 2 hour inhibition tests. During each experiment, 2 biofilm channels served as control with no inhibitor present and the other 2 biofilm channels were exposed to either 60 uM phenol or 100 uM toluene. Nitrite production was monitored throughout the experiment, and the given concentrations of phenol and toluene resulted in 50% inhibition of ammonia oxidation by the biofilms. Suspended cells were grown in batch reactors. Three 4-plex NimbleGen microarray chips were used, and each chip contained one sample from each experimental group. QC of samples was determined by spectrophotometric methods and using Agilent bioanalyzer traces to determine purity and integrity of RNA and cDNA. A sample tracking report was used to verify the correct hybridization of each sample to the intended array.
Project description:Microtoming Coupled with Microarray Analysis to Evaluate Potential Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in Anode Biofilms Differences in the Metabolic Status of Geobacter sulfurreducens at Different Depths in A Current Producing Biofilm Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. In order to further compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, mature anode biofilms were treated to stop turnover over of mRNA and then encased in resin which was sectioned into 100 nm shavings with a diamond knife and pooled into inner (0-20 µm from anode surface) and outer (30-60 µm) fractions. Whole genome DNA microarray analysis of RNA extracted from the shavings revealed that, at a 2-fold lower threshold, there were 146 genes that had significant (p<0.05), differences in transcript abundance between the inner and outer portions of the biofilm. Only 1 gene, GSU0093, a hypothetical ABC transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting that cells in the outer biofilm had lower metabolic rates. However, the differences in transcript abundance were relatively low (<3-fold) and the outer biofilm did not have significantly lower expression of the genes for TCA cycle enzymes which previous studies have demonstrated are sensitive indicators of changes in rates of metabolism in G. sulfurreducens. There also was no significant difference in the transcript levels for outer-surface cell components thought to be important in electron transfer in anode biofilms. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of the metabolic staining and gene expression studies suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms. Three biological replicates were hybridized in triplicate on a coustom affimetrix tilling array using prokaryotic protocol (p69Affy, p75 Adobe) for labeling, hybridization and scanning.