Project description:Purpose: The goals of this study are to compare RNA-Seq transcriptomic data of P. aeruginosa PAO1 in response to chronic exposure of clindamycin and rifampicin eluted from antibiotic-impregnated catheters in a continuous biofilm culture condition. Methods: Transcriptomic profiles of P. aeruginosa PAO1 grown with catheters without antibiotic coating or with clindamycin/rifampicin coating were generated by deep sequencing, in quadruplicates, using Illumina NextSeq 550 sequencer with SE-75. FastQC and Trimmomatic (v0.39) were used for sequencing quality assessment. Raw read counts of the annotated genes were calculated using HTSeq-count. The log2 fold change and false discovery rate (FDR) were measured by edgeR. Genes with |log2FC| > 1.0 and FDR < 0.05 were considered as differential expression genes. Transcripts per million (TPM) was measured by Kallisto, which is an alignment free expression estimation tool. Results: P. aeruginosa PAO1 RNA sequencing using Illumina NextSeq 550 sequencer generated 5,205,190 of average number of reads with average read length of 65 bp and a mean coverage of 54X. Differentially regulated proteins of 877 in the planktonic cells were identified, 55 of which displayed up-regulation and 822 displayed down-regulation. On the other hand, 178 proteins were differentially expressed in the biofilm cells containing 152 up-regulated and 26 down-regulated. Conclusions: Our study represents the first detailed analysis of transcriptomes generated by RNA-seq technology after P. aeruginosa PAO1 planktonic and biofilm cells were continuously exposed to clindamycin/rifampicin-impregnated catheters. Our results show that RNA sequencing offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content of strain PAO1 cells. We conclude that RNA-seq based transcriptome characterization would help the understanding for the mechanism of antimicrobial resistance and virulence in P. aeruginosa after exposure of antimicrobial-coated catheter.
Project description:Purpose : The goal of this study was to use RNA-seq to compare transcriptional profiles under biofilm conditions with planktonic growth and explore the correlation of gene expression of a collection of clinical P. aeruginosa isolates to various phenotypes, such as biofilm structure or virulence. Methods : mRNA profiles were generated for Pseudomonas aeruginosa clinical samples derived from various geographical locations by deep sequencing. The removal of ribosomal RNA was performed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2 Kit (Illumina). The samples were sequenced in single end mode on an Illumina HiSeq 2500 device or paired end mode on an Illumina Novaseq 6000. mRNA reads were trimmed and mapped to the NC_008463.1 (PA14) reference genome from NCBI using bowtie2 with default settings.
Project description:The aim of this study is to evaluate the evolutionary robustness of the quorum sensing inhibitor (QSI) furanone C-30 for the treatment of P. aeruginosa biofilm infections. We repeatedly exposed P. aeruginosa biofilms to furanone C-30 (with or without tobramycin) in the synthetic cystic fibrosis sputum medium (SCFM2) and characterized the genotype and phenotype of the evolved lineages. P. aeruginosa biofilms were grown in SCFM2 for 24 h after which the treatment in fresh SCFM2 was added to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. After each cycle the number of CFU was determined and an aliquot of the culture was stored at -80°C in Microbank vials to allow further tests on the evolved strains. A sample from the treated biofilm was used to prepare a new overnight culture, in order to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. Whole-genome sequencing was performed on the wild type P. aeruginosa PAO1 and on the exposed lineages after 5, 10 and 16 cycles.
Project description:P. aeruginosa PAO1 wild type and PA2663 mutant strains expression in biofilm cells relative to P. aeruginosa PAO1 wild type strain expression in biofilm cells. All samples cultured in LB with glass wool Keywords: Biofilm
Project description:P. aeruginosa was cultured in a MultiScreen-Mesh plate, which has a filter at the bottom of the wells. The plate was immersed in either in medium alone (control) or in medium inoculated with a mixture of five bacterial strains commonly found in cystic fibrosis sputum (\"microbiome\"). The filter prevented physical contact between P. aeruginosa and the other bacteria, yet soluble products could migrate through the filter into the P. aeruginosa biofilm. P. aeruginosa was then allowed to form biofilms in the wells for 72h, then the biofilm was harvested and a fraction of the harvested cells were used for re-inoculations. This was repeated for 18 cycles for a total of 54 days.
Project description:P. aeruginosa PAO1 PA2663-UW expression in biofilm cells relative to P. aeruginosa PAO1 WT-UW expression in biofilm cells. All samples cultured in LB with glass wool. Keywords: Mutation
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:In order to unearth the marker genes in the formation process of M. tuberculosis biofilm, Illumina sequencing technology was firstly used to catalog global gene expression profiling and functional network analysis of M. tuberculosis bioflim and planktonic populations in the world.
Project description:Nitrate-reducing iron(II)-oxidizing bacteria are widespread in the environment contribute to nitrate removal and influence the fate of the greenhouse gases nitrous oxide and carbon dioxide. The autotrophic growth of nitrate-reducing iron(II)-oxidizing bacteria is rarely investigated and poorly understood. The most prominent model system for this type of studies is enrichment culture KS, which originates from a freshwater sediment in Bremen, Germany. To gain insights in the metabolism of nitrate reduction coupled to iron(II) oxidation under in the absence of organic carbon and oxygen limited conditions, we performed metagenomic, metatranscriptomic and metaproteomic analyses of culture KS. Raw sequencing data of 16S rRNA amplicon sequencing, shotgun metagenomics (short reads: Illumina; long reads: Oxford Nanopore Technologies), metagenome assembly, raw sequencing data of shotgun metatranscriptomes (2 conditions, triplicates) can be found at SRA in https://www.ncbi.nlm.nih.gov/bioproject/PRJNA682552. This dataset contains proteomics data for 2 conditions (heterotrophic and autotrophic growth conditions) in triplicates.