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| Status |
Public on Jan 01, 2017 |
| Title |
LLQR E. coli ATCC 25922 vs wild-type E. coli ATCC 25922 |
| Platform organisms |
Escherichia coli O157:H7 str. EDL933; Escherichia coli CFT073; Escherichia coli O157:H7 str. Sakai; Escherichia coli str. K-12 substr. MG1655 |
| Sample organism |
Escherichia coli ATCC 25922 |
| Experiment type |
Expression profiling by array
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| Summary |
Global response to ciprofloxacin in low level quinolone resistant Escherichia coli: a shorter path to survival. Background: Bactericidal activity of quinolones in bacteria has been related to a combination of DNA fragmentation, ROS production and programmed dead cell systems. Subjacent molecular systems responsible for reduction of bactericidal effect in low-level quinolone resistance (LLQR) phenotypes remain to be clarified. To answer this question and to define new possible antimicrobial targets, the transcriptomic profile in isogenic Escherichia coli harbouring quinolone resistance mechanisms in the presence of ciprofloxacin was evaluated.
Materials and methods: E. coli 25922 was used as background strain. Four LLQR isogenic strains were tested for transcriptomic assays: ATCC 25922 (wild-type), EC14 (coding for QnrS1), EC19 (marR deletion and coding for QnrS1) and EC24 (Ser83Leu substitution in GyrA and coding for QnrS1). Cells in exponential phase (DO600=0.4) were exposed to 1 mg/L of ciprofloxacin (breakpoint for reduced susceptibility according to CLSI) during 1 hour and, further, RNA was purified. Gene expression analysis was performed using AGILENT technology. Data obtained for each strain were always normalized to the wild-type E. coli ATCC 25922. Specific ROS modulation targets were validated by genetic and biochemical approach.
Results: A radical differential response to ciprofloxacin in LLQR strains, either up or downregulation, was observed (proportional to the MIC values). Multiple genes implicated in ROS production (related to TCA cycle, aerobic respiration or detoxification systems) were upregulated (sdhC up to 63.5-folds) in LLQR mutants. SOS system components were downregulated (recA up to 30.7-folds). yihE, coding for a protective kinase of programmed cell death, was also upregulated (up to 5.2-folds). SdhC inhibition sensitized LLQR phenotypes (up to Log=2.3 after 24 hours).
Conclusions: At clinical relevant concentration of ciprofloxacin, the pattern of genes expression of critical systems for bacterial survival and mutant development were significantly modified in LLQR phenotypes. This approach allowed validating ROS modulation as an interesting target in terms of bacterial sensitization.
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| Overall design |
Two-condition experiment, LLQR E. coli vs wild-type E. coli. Biological replicates: 2 control replicates, 2 replicates for each LLQR strain. E. coli ATCC 25922 (wild-type) and EC14, EC19, EC24 (LLQR) isogenic strains were tested to evaluate the global response to relevant fix concentration of ciprofloxacin (1 mg/L, breakpoint for reduced susceptibility according to CLSI). All of them were susceptible to quinolones according to CLSI breakpoints. Cultures were started from single colonies and grown overnight in 25 ml of LB. These cell were diluted 1:100 and growth to cell concentration of 4x108 cells/ml (OD600 nm=0.4, exponential phase) for treatment. Three biological replicates per genotype were incubated at 1 mg/L of ciprofloxacin during 60 minutes (that means 250xMIC for E. coli ATCC 25922, 8xMIC for EC14, 2xMIC for EC19 and 1xMIC for EC24). Approximately 109 cells (2 ml) were taken for RNA isolation. The pellet was immediately processed. Pellet was pre-incubated in RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Contaminating DNA was removed from RNA samples with TURBO DNA-free (Ambion, USA). The amount of RNA was measured on the NanoDrop ND-1000 (Thermo Scientific). The integrity of the RNA samples was analyzed with the BioAnalyzer 2100 (Agilent Technologies) using the RNAnano 6000 kit (Agilent Technologies). Labeling, microarray hybridization, scanning, and data processing were performed at the Genomic Unit of the Centro Nacional de Biotecnología (http://wwwuser.cnb.csic.es/~genomica/servicos.htm). Data obtained for each LLQR mutant were always compared to the E. coli ATCC 25922 wild-type strain. FIESTA program was used for analysis (http://bioinfogp.cnb.csic.es) and p-value was determined according to FDR algorithm (p<0.05 was considered significant).
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| Contributor(s) |
MACHUCA J, RECACHA E, BRIALES A, DÍAZ-DE-ALBA P, Blazquez J, PASCUAL A, RODRÍGUEZ-MARTÍNEZ J |
| Citation(s) |
28769919 |
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| Submission date |
Sep 01, 2016 |
| Last update date |
Jun 27, 2019 |
| Contact name |
Jose Manuel Rodriguez Martinez |
| E-mail(s) |
jmrodriguez@us.es
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| Phone |
+34 954552863
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| Organization name |
University of Seville
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| Street address |
Snchez Pizjuan SN
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| City |
Seville |
| ZIP/Postal code |
41009 |
| Country |
Spain |
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| Platforms (1) |
| GPL13360 |
Agilent-020097 E. coli Gene Expression Microarray (Feature Number version) |
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| Samples (8)
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| GSM2300525 |
Wild-type E. coli ATCC 25922 replicate 1 |
| GSM2300526 |
Wild-type E. coli ATCC 25922 replicate 2 |
| GSM2300527 |
E. coli ATCC 25922 QnrS replicate 1 |
| GSM2300528 |
E. coli ATCC 25922 QnrS replicate 2 |
| GSM2300529 |
E. coli ATCC 25922 marR deletion+QnrS replicate 1 |
| GSM2300530 |
E. coli ATCC 25922 marR deletion+QnrS replicate 2 |
| GSM2300531 |
E. coli ATCC 25922 Ser83Leu+QnrS replicate 1 |
| GSM2300532 |
E. coli ATCC 25922 Ser83Leu+QnrS replicate 2 |
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| Relations |
| BioProject |
PRJNA341481 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE86341_RAW.tar |
19.8 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE86341_fold_change.xlsx |
178.3 Kb |
(ftp)(http) |
XLSX |
| Processed data included within Sample table |
| Processed data are available on Series record |
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