|
| Status |
Public on Jun 06, 2017 |
| Title |
4_ESBL019 Reverted Repl 1 |
| Sample type |
RNA |
| |
|
| Source name |
ESBL019 Reverted
|
| Organism |
Escherichia coli |
| Characteristics |
strain: beta-lactamase (ESBL)-producing UPEC (ESBL019)
morphology: Reverted (reverted back from a Filamented shape into a coli shape)
|
| Treatment protocol |
Four morphologic states of ESBL019 were used during the experiments. A first ESBL019 morphological state was prepared by resuspension of ESBL019 in cell culture medium (CCM) without ceftibuten supplementation prior to its inoculation of primary human bladder epithelial cells (HBEP) (designated; ESBL019 Coliform). A second ESBL019 morphological state was prepared by resuspension in CCM and then used to inoculate HBEP cells with ceftibuten supplementation (480ng/mL) (designated; ESBL019 Transition). This transition from coliform into filamented occurs in the presence of HBEP cells. A third ESBL019 morphological state was prepared by resuspension and pre-incubation for 3 hours in CCM with ceftibuten in a tube, in order to induce complete filamentation of all bacteria. The third pre-filamented ESBL019 fraction was used to inoculate HBEP cells in the presence of ceftibuten to keep the bacteria filamented (designated; ESBL019 Filamented). The CCM of the pre-filamented ESBL019 fraction was replaced with CCM without ceftibuten and the filamented ESBL019 were allowed to revert completely back to its coliform during 1 hour in a tube prior to the inoculation of HBEP cells without ceftibuten (designated; ESBL019 Reverted). Prior to inoculation the bacterial concentrations of all fractions were adjusted in order to inoculate HBEP cells with a multiplicity of infection (MOI) of 10. All inoculated cells were incubated for 4 hours in 5% CO2 at 37oC after which supernatants were collected and centrifuged 5 min at 5000 x g to collect the bacteria.
|
| Growth protocol |
The E. coli isolate ESBL019 was originally isolated from a patient at Örebro University hospital, Sweden and was maintained on tryptic soy agar (TSA) (Becton Dickinson, Le Pont Claix, France). ESBL019 was grown in Luria broth (Difco Laboratories, Detroit, MI, USA) overnight on shake at 200 rpm 37 °C prior to experiments. The bacteria were resuspended in sterile phosphate buffered saline prior to inoculation of CnT-21 cell culture medium (CCM) with or without ceftibuten (480 ng/mL).
|
| 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 > 9 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 |
ESBL019 Reverted Repl 1
|
| Data processing |
75th percentile shift normalisation and baseline adjusted
|
| |
|
| Submission date |
Jun 05, 2017 |
| Last update date |
Jan 23, 2018 |
| Contact name |
Robert Kruse |
| E-mail(s) |
robert.kruse@oru.se
|
| Organization name |
Örebro University
|
| Department |
Medical Sciences
|
| Street address |
S grevrosengatan
|
| City |
Örebro |
| ZIP/Postal code |
703 62 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL13359 |
| Series (1) |
| GSE99661 |
Transcriptional alterations of virulence-associated genes in extended spectrum beta-lactamase (ESBL)-producing uropathogenic Escherichia coli during morphologic transitions induced by ineffective antibiotics |
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