Overnight cultures of ESBL7 from the original isolate, or isolates pre-exposed 20 times to CORM-2 or vehicle, were used to inoculate MS-medium, (OD620) of 0.1, followed by exposure to CORM-2 (250 µM) or vehicle for 30 min at 37 °C.
Growth protocol
Cultures were maintained on tryptic soy agar (TSA) (Becton Dickinson, Le Pont Claix, France). Overnight cultures were grown in Difco Luria-Bertani (LB) broth (Lennox; Franklin Lakes, NJ, USA) at 37 °C aerobically on a shaker at 200 rpm. Bacteria (picked from 5-10 colonies) were suspended in 1 ml of PBS, yielding a suspension corresponding to the turbidity of McFarland 0.5, and diluted 1:100 in minimal salt (MS)-medium (~106 CFU/ml). The suspension was exposed to CORM-2 (250 µM) or vehicle (2.5% DMSO) for 4 hours at 37 °C. A volume (10 µl) was spread onto TSA-agar plates and incubated at 37 °C overnight. This procedure was repeated 10 times (10x, ~45 generations) or 20 times (20x, ~90 generations).
Extracted molecule
total RNA
Extraction protocol
RNA isolation was performed using an RNeasy mini kit (Qiagen Technologies, Hilden, Germany), according to the manufacturer’s protocol. DNA decontamination treatment was performed using Turbo DNase (Qiagen) and the quantity and purity of the purified RNA samples were determined using a spectrophotometer Nanodrop-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA) by measuring the absorbance (A260, 230, 280) and calculating absorbance ratios (A260/A230 and A260/A280). All samples had A260/A230 and A260/A280 ratios above 1.9. The RNA integrity was analysed using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) in conjunction with RNA 6000 Nano LabChip kit (Agilent Technologies) according to the manufacturer’s protocol. RNA integrity number (RIN) values were > 8.5 for all samples.
Label
Cy3
Label protocol
The cDNA synthesis was performed by using WT Primer Mix and cDNA Master Mix (Agilent).
Hybridization protocol
Labelled samples were hybridised onto G4813A E. coli gene expression Microarray 8×15K glass slides (Agilent) containing 15 208 E. coli probes.
Scan protocol
Microarrays were scanned with a G2565 CA array laser scanner (Agilent) followed by image analysis and data extraction with Feature Extraction Software version 10.7.3.1 (Agilent).
Description
First exposure to sub-inhibitory levels of CORM-2
Data processing
75th percentile shift normalisation and baseline adjusted
Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli
Data table header descriptions
ID_REF
VALUE
75th percentile shift normalisation and baseline adjusted
