Fig. 5: lasB mRNA is also present in eDNA fibres of human clinical sputum sample.

smiFISH performed on clinical sputum samples highly enriched with P. aeruginosa. a Confocal micrograph of 0.1 mg ml⁻¹ DNaseI pretreated human clinical sputum sample HP0005 highly enriched in P. aeruginosa showing eDNA fibres (TOTO−1 stain; green), lasB mRNA (lasB mRNA specific smiFISH probe; red streaks), merged image of lasB mRNA and eDNA fibres and colocalised region of eDNA fibres with lasB mRNA (yellow). A Mander’s coefficient of 56.72 % (n = 8 images) was determined for the clinical sample based on the overlap of the lasB mRNA signal with TOTO−1 stained (green) eDNA fibres (colocalised region panel; yellow). Scale bars represent 10 µm. b Fluorescence intensity quantification (n = 4) of extracellular DNA (green bars) and lasB mRNA (red bars) expression in human clinical sputum sample positive for P. aeruginosa biofilms treated with 0, 0.05, and 0.1 mg ml⁻¹ DNase I. c Clinical sputum sample treated with 0.4 mg ml⁻¹ of DNase-I at 37 °C for 1 h showing complete dispersal of sputum and increased solution turbidity d smiFISH confocal micrograph of 0.4 mg ml⁻¹ DNase-I treated human clinical sample at 37 °C for 1 h (n = 3), showing eDNA staining with TOTO−1 dye (green) after DNase treatment and lasB signal as visualised with lasB-specific oligoribonucleotide smFISH probes (red), and colocalisation of eDNA and lasB (yellow). e smiFISH confocal micrograph (n = 4) of clinical sputum sample negative for P. aeruginosa after 0.1 mg ml⁻¹ DNase1 pre-treatment showing dead cells (green) and no lasB oligoribonucleotide smFISH probes signal (red). f smiFISH confocal micrograph (n = 4) of clinical sputum sample positive for P. aeruginosa after 0.1 mg ml⁻¹ DNase1 pre-treatment following staining with scrambled lasB-specific oligoribonucleotide smFISH probe (red), and eDNA specific TOTO−1 stain (green) showing eDNA fibres. The scale bars represent 10 µm. The standard deviation bars indicated in 5b are generated based on the mean values calculated from biological triplicates. Relevant source data for Fig. 5b are provided as a source data file.