Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of genes important for response of Pseudomonas aeruginosa biofilms to ciprofloxacin exposure using TraDIS-Xpress

ABSTRACT: Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotics challenge remain unexplored. In this study we employed a high-density TraDIS-Xpress library of P. aeruginosa PAO1 to assay the impact of gene disruptions or altered gene expression on biofilm formation at different concentrations of ciprofloxacin. Gene fitness was analysed by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Gene determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin were identified.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Pseudomonas aeruginosa

SUBMITTER: Meng Wang

PROVIDER: E-MTAB-16928 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 M-bM-^HM-^Frmf 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 M-BM-5m 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.

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Identification of genes important for response of Pseudomonas aeruginosa biofilms to ciprofloxacin exposure using TraDIS-Xpress

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