Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin
Abstract
:1. Introduction
2. Results
2.1. CHD and CDCHD Were Effective against Clinically Relevant Uropathogens and Displayed Resistance-Breaking Properties In Vitro
2.2. CHD Resistance Development in Target Pathogens Revealed Mutation of ramR in K. pneumoniae
2.3. Absence of Cross-Resistance to CDCHD
2.4. Resistance to CHD in K. pneumoniae Was Based on Efflux
2.5. ChdR Was Found to be the Primary Self-Resistance Factor in Natural CHD Producer A. sulphurea
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Antimicrobial Screening
4.3. In Vitro Selection of CHD-Resistant Klebsiella Mutants
4.4. Construction of a K. pneumoniae ramR Deletion Mutant
4.5. Construction of A. sulphurea Double Mutant and Complementation Vectors
4.6. In Vitro Selection of CHD-Resistant Amycolatopsis Mutants
4.7. Comparative Whole-Genome Sequencing
4.8. Quantification of Gene Expression by qPCR
4.9. Gene Expression Analysis by RNA-Seq
4.10. Preparation of an A. sulphurea Genomic Cosmid Library and Its Expression in A. mediterranei
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Isolate (n = Number of Strains) | MIC (µg/mL) | MBC (µg/mL) | ||
---|---|---|---|---|
CHD | CDCHD | CHD | CDCHD | |
Escherichia coli * | ||||
Sensitive (15) | 2 | 4 | 16 | 4 |
TEM β-lactamase (8) | 2–4 | 8 | 16 | 8 |
ESBL (7) | 4 | 4 | 32 | 8 |
Colistin-resistant (5) | 8 | 8 | 32 | 16 |
Enterobacter spp. * (8) | 4 | 4 | 16 | 8–16 |
Klebsiella spp. * | ||||
Sensitive (7) | 4 | 4 | 16 | 8 |
ESBL (2) | 4 | 4–8 | 32 | 8–16 |
Carbapenem-resistant (2) | 4 | 4–8 | 32–64 | 8 |
Enterococcus faecalis (19) | 8 | 16 | >64 | 16 |
Enterococcus faecium (6) | 4 | 8 | 32 | 16 |
Proteus spp. | ||||
Sensitive (7) | 4 | 8 | 8–16 | 64 |
ESBL (2) | 4 | 8–32 | 8–16 | 16–32 |
Pseudomonas aeruginosa (10) | >64 | 32–64 | >64 | >64 |
Test Condition | Isolate (n=) | MIC (µg/mL) | MBC (µg/mL) | ||
---|---|---|---|---|---|
CHD | CDCHD | CHD | CDCHD | ||
Artificial urine | Escherichia coli (7) | 1 | 1 | 8 | 2 |
Klebsiella pneumoniae (3) | 1 | 1 | 8 | 4 | |
CAMHB | Escherichia coli (7) | 4 | 4 | 32 | 8 |
Klebsiella pneumoniae (3) | 4 | 4 | 32 | 8 |
TET-Resistant Strain | Resistance Gene | Resistance Mechanism | MIC (µg/mL) | |
---|---|---|---|---|
CDCHD | TET | |||
E. coli | tetB | Efflux | 4 | >64 |
E. coli | tetM | Ribosomal protection | 0.5 | 64 |
E. coli | tetW | Ribosomal protection | 1 | >64 |
E. coli 49 | tetB | Efflux | 4 | >64 |
E. coli 74 | tetB | Efflux | 8 | >64 |
Serratia liquefaciens | tetB; tet34 | Efflux; enzymatic inactivation | 2 | >64 |
Pseudomonas pseudoalcaligenes | tetB; tet34 | Efflux; enzymatic inactivation | 2 | >64 |
K. pneumoniae 3 | tetA | Efflux | 8 | >64 |
K. pneumoniae 8 | tetA | Efflux | 32 | >64 |
K. pneumoniae 24 | tetA | Efflux | 8 | >64 |
Gene | Mt8.1 | Mt8.2 | Mt8.3 | Mt8.4 | Mt8.5 | Mt8.6 | Mt8.7 | Mt8.8 |
---|---|---|---|---|---|---|---|---|
Efflux | ||||||||
acrA | 2.65 | 1.72 | 1.97 | 2.16 | 2.03 | 2.33 | 1.98 | 2.88 |
acrB | 3.27 | 2.82 | 2.01 | 2.70 | 2.88 | 2.48 | 3.03 | 2.31 |
tolC | 2.97 | 2.00 | 2.25 | 2.15 | 2.10 | 3.39 | 2.12 | 3.96 |
Regulators | ||||||||
ramR | 5.80 | 4.44 | 4.35 | 6.78 | 4.89 | 5.59 | 4.98 | 3.01 |
ramA | 16.57 | 11.79 | 10.47 | 13.40 | 9.89 | 13.73 | 10.91 | 6.11 |
soxR | 1.02 | 1.03 | 3.22 | 0.70 | 0.93 | 3.83 | 0.81 | 0.73 |
soxS | 1.13 | 1.09 | 18.09 | 0.81 | 1.12 | 16.80 | 0.86 | 0.91 |
marR | 5.22 | 1.49 | 17.79 | 4.16 | 1.13 | 20.16 | 1.11 | 2.93 |
marA | 2.58 | 1.14 | 11.69 | 2.65 | 0.83 | 11.66 | 0.75 | 0.78 |
marB | 2.84 | 1.71 | 17.47 | 3.86 | 1.23 | 13.69 | 1.30 | 1.30 |
acrR | 0.63 | 1.09 | 1.01 | 0.67 | 0.95 | 0.76 | 1.10 | 0.41 |
Protease | ||||||||
lon | 0.78 | 0.86 | 0.28 | 0.76 | 0.87 | 0.35 | 0.84 | 0.92 |
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Hennessen, F.; Miethke, M.; Zaburannyi, N.; Loose, M.; Lukežič, T.; Bernecker, S.; Hüttel, S.; Jansen, R.; Schmiedel, J.; Fritzenwanker, M.; et al. Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin. Antibiotics 2020, 9, 619. https://doi.org/10.3390/antibiotics9090619
Hennessen F, Miethke M, Zaburannyi N, Loose M, Lukežič T, Bernecker S, Hüttel S, Jansen R, Schmiedel J, Fritzenwanker M, et al. Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin. Antibiotics. 2020; 9(9):619. https://doi.org/10.3390/antibiotics9090619
Chicago/Turabian StyleHennessen, Fabienne, Marcus Miethke, Nestor Zaburannyi, Maria Loose, Tadeja Lukežič, Steffen Bernecker, Stephan Hüttel, Rolf Jansen, Judith Schmiedel, Moritz Fritzenwanker, and et al. 2020. "Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin" Antibiotics 9, no. 9: 619. https://doi.org/10.3390/antibiotics9090619