Progression of ampC amplification during de novo amoxicillin resistance development in E. coli
ABSTRACT Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in Escherichia coli. However, the time c...
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| Language: | English |
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American Society for Microbiology
2025-02-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.02982-24 |
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| author | Luyuan Nong Martijs Jonker Wim de Leeuw Meike T. Wortel Benno ter Kuile |
| author_facet | Luyuan Nong Martijs Jonker Wim de Leeuw Meike T. Wortel Benno ter Kuile |
| author_sort | Luyuan Nong |
| collection | DOAJ |
| description | ABSTRACT Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in Escherichia coli. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of ampC amplification and ampC promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. AmpC promoter mutations occurred by day 2, while the approximately eight-fold amplification occurred after more than 6 days of amoxicillin exposure. The combination of the amplification and the promoter mutations increased the ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the ampC gene complementation strain (CompA), but not in ∆ampC, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and ampC amplification, the DNA mutations acquired during resistance evolution by the WT, ∆ampC, and CompA were analyzed. Compared to evolved ∆ampC, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆ampC did not show amplification of the fragment around the original ampC position but exhibited a large duplication or triplication at another position, suggesting the essential role of the duplicated genes in resistance development.IMPORTANCEAmoxicillin is the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These ampC-containing fragments have been identified in extended-spectrum beta-lactamase plasmids, which are considered the main cause of beta-lactam resistance. In this study, we document the time course of two important factors for ampC transcription enhancement, ampC amplification and ampC promoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification ampC region, that the sigma factor 70 regulates ampC overexpression, and that these combined form the backbone of a putative mechanism for ampC amplification. |
| format | Article |
| id | doaj-art-7ac2589ad1f6492991753c6add58312a |
| institution | Kabale University |
| issn | 2150-7511 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | American Society for Microbiology |
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| series | mBio |
| spelling | doaj-art-7ac2589ad1f6492991753c6add58312a2025-02-05T14:00:47ZengAmerican Society for MicrobiologymBio2150-75112025-02-0116210.1128/mbio.02982-24Progression of ampC amplification during de novo amoxicillin resistance development in E. coliLuyuan Nong0Martijs Jonker1Wim de Leeuw2Meike T. Wortel3Benno ter Kuile4Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the NetherlandsRNA Biology & Applied Bioinformatics, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the NetherlandsRNA Biology & Applied Bioinformatics, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the NetherlandsBiology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the NetherlandsBiology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the NetherlandsABSTRACT Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in Escherichia coli. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of ampC amplification and ampC promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. AmpC promoter mutations occurred by day 2, while the approximately eight-fold amplification occurred after more than 6 days of amoxicillin exposure. The combination of the amplification and the promoter mutations increased the ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the ampC gene complementation strain (CompA), but not in ∆ampC, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and ampC amplification, the DNA mutations acquired during resistance evolution by the WT, ∆ampC, and CompA were analyzed. Compared to evolved ∆ampC, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆ampC did not show amplification of the fragment around the original ampC position but exhibited a large duplication or triplication at another position, suggesting the essential role of the duplicated genes in resistance development.IMPORTANCEAmoxicillin is the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These ampC-containing fragments have been identified in extended-spectrum beta-lactamase plasmids, which are considered the main cause of beta-lactam resistance. In this study, we document the time course of two important factors for ampC transcription enhancement, ampC amplification and ampC promoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification ampC region, that the sigma factor 70 regulates ampC overexpression, and that these combined form the backbone of a putative mechanism for ampC amplification.https://journals.asm.org/doi/10.1128/mbio.02982-24AmpCbeta-lactamasegene amplificationresistance evolutionantimicrobialIS1 element |
| spellingShingle | Luyuan Nong Martijs Jonker Wim de Leeuw Meike T. Wortel Benno ter Kuile Progression of ampC amplification during de novo amoxicillin resistance development in E. coli mBio AmpC beta-lactamase gene amplification resistance evolution antimicrobial IS1 element |
| title | Progression of ampC amplification during de novo amoxicillin resistance development in E. coli |
| title_full | Progression of ampC amplification during de novo amoxicillin resistance development in E. coli |
| title_fullStr | Progression of ampC amplification during de novo amoxicillin resistance development in E. coli |
| title_full_unstemmed | Progression of ampC amplification during de novo amoxicillin resistance development in E. coli |
| title_short | Progression of ampC amplification during de novo amoxicillin resistance development in E. coli |
| title_sort | progression of ampc amplification during de novo amoxicillin resistance development in e coli |
| topic | AmpC beta-lactamase gene amplification resistance evolution antimicrobial IS1 element |
| url | https://journals.asm.org/doi/10.1128/mbio.02982-24 |
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