A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli
- Inserm UMR-S 1250 P3Cell, SFR CAP-Santé, Université de Reims-Champagne-Ardenne, France
- Assistance Publique-Hôpitaux de Paris, Department of Clinical Microbiology, Necker-Enfants Malades University Hospital, Université de Paris, 75015 Paris, France. INSERM U1151-Equipe 1, Institut Necker-Enfants Malades, Université de Paris, 75015 Paris, France. Division of Infectious Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA., United States
- Inserm UMR-S 1250 P3Cell, SFR CAP-Santé, Université de Reims-Champagne-Ardenne, Reims, France. Laboratoire de Bactériologie-Virologie-Hygiène Hospitalière-Parasitologie- Mycologie, CHU Reims, Hôpital Robert Debré, France
- Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, United States
- Institut Pasteur, Unité Plasticité du Génome Bactérien, CNRS UMR3525, France
- Université de Limoges, Inserm, CHU Limoges, RESINFIT, UMR 1092, France
- Chimie ParisTech, PSL Research University, CNRS, Institute of Chemistry for Life and Health Sciences, France
- Laboratoire d’Hématologie, CH de Troyes, France
Figures
Figure 1
qnrD genes are carried by small plasmids.
(A) The two qnrD-plasmid archetypes: p2007057 and pDIJ09-518a. (B) Distribution of the qnrD-plasmids among the 53 qnrD fully sequenced plasmids available in GenBank.
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Figure 1—source data 1
qnrD genes in small plasmids.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig1-data1-v2.xlsx
Figure 2 with 2 supplements
qnrD regulation is SOS-mediated and aminoglycosides induce the SOS in E. coli because of the qnrD-plasmid backbone.
(A) The qnrD SOS-box conservation by visualization of the consensus sequence logo generated from the 53 fully qnrD-plasmid sequences. The consensus sequence for E. coli is indicated below. (B) …
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Figure 2—source data 1
Relative expression of qnrD in E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-data1-v2.xlsx
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Figure 2—source data 2
Relative expression of sulA in E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-data2-v2.xlsx
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Figure 2—source data 3
Relative expression of sulA in E. coli/pDIJ09-518a.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-data3-v2.xlsx
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Figure 2—source data 4
GFP fluorescence in a E. coli MG1655 WT.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-data4-v2.xlsx
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Figure 2—source data 5
Relative expression of qnrD in E. coli MG1656 and isogenic strains with chormosomal complementation.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-data5-v2.xlsx
Figure 2—figure supplement 1
The viability of E. coli.
E. coli MG1656 carrying the qnrD-plasmid exposed to sub-minimum inhibitory concentration (MIC) of tobramycin is not impaired and the plasmid is stable in an antibiotic-free medium. (A) The curves …
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Figure 2—figure supplement 1—source data 1
OD600 measured for E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 2
qnrD-plasmid carriage does not promote the SOS response induction by tobramycin in Providencia rettgeri.
(A) Relative expression of sulA in P. rettgeri, carrying the pDIJ09-518a strains exposed to sub-minimum inhibitory concentrations (MICs) of tobramycin or mitomycin C, in comparison to expression in …
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Figure 2—figure supplement 2—source data 1
Relative expression of sulA in P. rettgeri/pDIJ09-518a.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig2-figsupp2-data1-v2.xlsx
Figure 3 with 1 supplement
Small qnrD-plasmid promotes nitrosative stress in E.coli.
(A) Reactive oxygen species (ROS) formation for E. coli MG1656 (WT) and its derivative carrying pDIJ09-518a cultured in lysogeny broth (LB) or exposed to tobramycin. Production of ROS was calculated …
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Figure 3—source data 1
DHR-123 fluorescence for ROS formation in E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig3-data1-v2.xlsx
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Figure 3—source data 2
Relative expression of katG in E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig3-data2-v2.xlsx
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Figure 3—source data 3
DAF-2A fluorescence for NOS formation in E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig3-data3-v2.xlsx
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Figure 3—source data 4
DAF-2A fluorescence obtained using a FACS-based approach, in E. coli WT and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig3-data4-v2.xlsx
Figure 3—figure supplement 1
Schematic approach for fluorometric detection of free intercellular reactive oxygen species (ROS) and nitric oxide (NO).
<bold>Figure 4.</bold> with 1 supplement
Aminoglycosides induce SOS in E. coli/pDIJ09-518a due to overwhelmed GO-repair pathway associated with inactivated Hmp.
(A, D) Relative expression of sulA in E. coli MG1656 (WT) isogenic strains carrying pDJJ09-518a, overexpressing the GO-repair system protein MutT and the hmp-deleted mutant, exposed to tobramycin, …
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Figure 4—source data 1
Relative expression of sulA in E. coli MG1656/ pDJJ09-518a overexpressing MutT.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig4-data1-v2.xlsx
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Figure 4—source data 2
GFP fluorescence in a E. coli MG1655 ΔrecB and ΔrecF.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig4-data2-v2.xlsx
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Figure 4—source data 3
Ratio of colony-forming units.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig4-data3-v2.xlsx
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Figure 4—source data 4
Relative expression of sulA in E. coli MG1656/pDJJ09-518a with deleted hmp.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig4-data4-v2.xlsx
Figure 4—figure supplement 1
hmp deletion and empty vector carriage do not promote the SOS response induction.
(A) Relative expression of sulA in hmp-deleted E. coli MG1656, E. coli MG1656 carrying the empty vector, in comparison to expression in wild-type (WT) E. coli MG1656 strain and E. coli co-carrying …
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Figure 4—figure supplement 1—source data 1
Relative expression of sulA in E. coli MG1656 Δhmp and E. coli MG1656 carrying empty vector.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Relative expression of sulA in hmp mutant and derivatives strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig4-figsupp1-data2-v2.xlsx
Figure 5 with 1 supplement
Deletion of ORF3 decreases the SOS response induction, after tobramycin treatment and ORF4 regulates the Hmp nitric oxide detoxification pathway.
(A) Relative expression of sulA in E. coli MG1656 (WT) derived isogenic strains carrying pDIJ09-518a with ORF3 and/or ORF4 deleted and complemented, exposed to mitomycin C (dark blue) or tobramycin …
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Figure 5—source data 1
Relative expression of sulA in E. coli MG1656 (WT) derived isogenic strains #1.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig5-data1-v2.xlsx
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Figure 5—source data 2
Relative expression of sulA in E. coli MG1656 (WT) derived isogenic strains #2.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig5-data2-v2.xlsx
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Figure 5—source data 3
Relative expression of sulA in E. coli MG1656 (WT) derived isogenic strains #3.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig5-data3-v2.xlsx
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Figure 5—source data 4
DAF-2A fluorescence in E. coli MG1656 (WT) and complemented strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig5-data4-v2.xlsx
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Figure 5—source data 5
Relative expression of hmp in E. coli MG1656 (WT) and isogenic strains.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig5-data5-v2.xlsx
Figure 5—figure supplement 1
Spontaneous mutation ratio after treatment with sub-minimum inhibitory concentration (MIC) of tobramycin.
Bacteria were grown overnight in lysogeny broth (LB) supplemented or not with sub-MIC of tobramycin (0.001 µg/ml). Appropriate dilutions were plated on LB plate, and 1 ml of culture was centrifuged …
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Figure 5—figure supplement 1—source data 1
CFU counting after treatment of sub-MIC of tobramycin.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig5-figsupp1-data1-v2.xlsx
Figure 6
Aminoglycosides potentiate the selection of higher fluoroquinolone resistance in E.coli harbouring the small qnrD-plasmid.
Mutant prevention concentrations (MPCs) of isogenic E. coli ATCC25922 strains with or without exposure to sub-MIC of tobramycin. E. coli ATCC25922 (red circle), E. coli ATCC25922/pDIJ09-518a (green …
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Figure 6—source data 1
CFU counting for MPC assay.
- https://cdn.elifesciences.org/articles/69511/elife-69511-fig6-data1-v2.xlsx
Figure 7
Model of SOS response induction by aminoglycosides in E. coli bearing the small qnrD-plasmid.
Schematic representation of the network leading to SOS induction in E. coli/pDIJ09-518a when not (A) or exposed (B) to sub-minimum inhibitory concentration (MIC) of aminoglycosides. NOS, nitric …
Author response image 1
Tables
Table 1
Minimal inhibitory concentration of quinolones.
| Strain* | MIC (μg/ml)† | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| NAL | LVX | OFX | CIP | ||||||||
| E. coli MG1656 | 3 | S | 0.023 | S | 0.006 | S | 0.004 | S | |||
| E. coli MG1656/pDIJ09-518a | >256 | R | 0.19 | S | 0.25 | S | 0.094 | S | |||
| E. coli MG1656 + CIP | 2 | S | 0.023 | S | 0.006 | S | 0.004 | S | |||
| E. coli MG1656/pDIJ09-518a + CIP | >256 | R | 0.25 | S | ×1.3 | 0.38 | I | x 1.5 | 0.19 | S | ×2 |
| E. coli MG1656 + TOB | 2 | S | 0.032 | S | 0.006 | S | 0.006 | S | |||
| E. coli MG1656/pDIJ09-518a + TOB | >256 | R | 0.38 | S | ×2 | 0.38 | I | x 1.5 | 0.25 | S | ×2.7 |
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*
+CIP and +TOB stand for strains exposed to sub-MIC of ciprofloxacin and tobramycin, respectively, prior to MIC assessment.
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†
Susceptibility testing categories according to EUCAST clinical breakpoints. Nalidixic acid: R > 16 μg/ml. Levofloxacin: S ≤ 0.5 μg/m, R > 1 μg/ml. Ofloxacin: S ≤ 0.25 μg/ml, R > 0.5 μg/ml. Ciprofloxacin: S ≤ 0.25 μg/ml, R > 0.5 μg/ml. Fold-change increases of MIC are shown in comparison to the QnrD-producing WT strain.
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Similarly, the MICs increased for the qnrD-carrying E. coli exposed to sub-MIC of tobramycin as compared to growth in antibiotic-free medium (Table 1): 2-, 1.5-, and 2.7-fold higher for levofloxacin, ofloxacin and ciprofloxacin, respectively. These results showed that the aminoglycoside-induced SOS response increased quinolone (nalidixic acid and fluoroquinolones) MIC in line with the increased expression of qnrD in E. coli.
Table 2
Quinolone resistance-determining region (QRDR) mutations and minimal inhibitory concentration of quinolones for surviving mutants obtained in the mutant prevention concentration (MPC) assay.
| Strain* | Mutant | MPC(μg/ml) | QRDR mutations | MIC (μg/ml)† | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| GyrA | GyrB | ParC | ParE | NAL | CIP | OFX | LVX | |||||||
| E. coli ATCC25922/pDIJ09-518a | #1 | 1 | S83L | - | - | - | >256 | R | 0.125 | S | 0.38 | I | 0.19 | S |
| #2 | 1 | S83W | - | - | - | 24 | R | 0.25 | S | 0.5 | I | 0.38 | S | |
| #3 | 1 | S83W | - | - | - | >256 | R | 0.38 | I | 0.75 | R | 0.38 | S | |
| #4 | 1 | S83W | - | - | - | 24 | R | 0.19 | S | 0.5 | I | 0.25 | S | |
| #5 | 1 | S83W | - | - | - | >256 | R | 0.125 | S | 0.38 | I | 0.19 | S | |
| E. coli ATCC25922/pDIJ09-518a + TOB | #1 | 2 | S83W | - | - | - | >256 | R | 1 | R | 4 | R | 1 | I |
| #2 | 2 | S83W | - | G78D | - | >256 | R | 0.38 | I | 2 | R | 0.75 | I | |
| #3 | 2 | S83W | - | G78D | - | >256 | R | 1.5 | R | 4 | R | 0.75 | I | |
| #4 | 2 | S83W | - | G78D | - | >256 | R | 1 | R | 6 | R | 1 | I | |
| #5 | 2 | S83W | - | - | - | >256 | R | 1 | R | 4 | R | 1 | I | |
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*
+TOB stands for strains exposed to sub-MIC of tobramycin prior to MPC assay.
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†
Susceptibility testing categories according to EUCAST clinical breakpoints. Nalidixic acid: R > 16 μg/ml. Levofloxacin: S ≤ 0.5 μg/ml, R > 1 μg/ml. Ofloxacin: S ≤ 0.25 μg/ml, R > 0.5 μg/ml. Ciprofloxacin: S ≤ 0.25 μg/ml, R > 0.5 μg/ml.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69511/elife-69511-transrepform1-v2.docx
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Supplementary file 1
Genetic background of qnrD positive enterobacterial isolates.
- https://cdn.elifesciences.org/articles/69511/elife-69511-supp1-v2.docx
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Supplementary file 2
Minimum-inhibitory concentrations for antibiotics used as SOS-response inducers.
- https://cdn.elifesciences.org/articles/69511/elife-69511-supp2-v2.docx
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Supplementary file 3
Strains and plasmids.
- https://cdn.elifesciences.org/articles/69511/elife-69511-supp3-v2.docx
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Supplementary file 4
Primers used for this study.
- https://cdn.elifesciences.org/articles/69511/elife-69511-supp4-v2.docx
