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Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae
OBJECTIVE: In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression. METHODS: We created the a...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Dove
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10461740/ https://www.ncbi.nlm.nih.gov/pubmed/37645559 http://dx.doi.org/10.2147/IDR.S421598 |
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author | Li, Guiling Wang, Li Zhang, Heguang Luan, Ying Sun, Qi Duo, Libo |
author_facet | Li, Guiling Wang, Li Zhang, Heguang Luan, Ying Sun, Qi Duo, Libo |
author_sort | Li, Guiling |
collection | PubMed |
description | OBJECTIVE: In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression. METHODS: We created the ampG gene deletion mutant strains Kp1-ΔampG and Kp NTUH-K2044-ΔampG with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-ΔampG-RC drug resistance model strains with plasmid pACYC184. We constructed the ampG knock-in strains by introducing the ampG genes of Kp1, Enterobacter cloacae 029M, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC25922, and Salmonella typhimurium LT2 into the ampG gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of ampC and ampG mRNAs. RESULTS: Compared with Kp1, the induction phenotype of the ampC of Kp1-ΔampG strain disappeared, the ampC expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 μg/mL to 1 μg/mL. Based on Kp1, five strain were successfully constructed to complement the ampG genes from five knock-in strain, and all of the above complemented strains showed inducible expression of ampC and restored the expression of ampG to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime (P < 0.05). The ampC and ampG genes were barely expressed in Kp NTUH-K2044-ΔampG-RC when compared with Kp NTUH-K2044-RC. The expressions of ampG and ampC in each knock-in strain were recovered, the induction phenotype of ampC was restored, and the MIC values of cefoxitin and ceftazidime were increased. (P < 0.05). CONCLUSION: In this study, we found that ampG was an essential regulator for the plasmid-mediated ampC-induced expression in K. pneumoniae. |
format | Online Article Text |
id | pubmed-10461740 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Dove |
record_format | MEDLINE/PubMed |
spelling | pubmed-104617402023-08-29 Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae Li, Guiling Wang, Li Zhang, Heguang Luan, Ying Sun, Qi Duo, Libo Infect Drug Resist Original Research OBJECTIVE: In this study, we constructed ampG knock-out and knock-in strains from a clinically isolated Kp1strain carrying ampR-ampC in its plasmid and compared them with the Kp NTUH-K2044 strain to investigate the relationship between ampG and ampR-ampC-induced expression. METHODS: We created the ampG gene deletion mutant strains Kp1-ΔampG and Kp NTUH-K2044-ΔampG with pKO3-km plasmid using homologous recombination technology. We constructed the Kp NTUH-K2044-RC and Kp NTUH-K2044-ΔampG-RC drug resistance model strains with plasmid pACYC184. We constructed the ampG knock-in strains by introducing the ampG genes of Kp1, Enterobacter cloacae 029M, Pseudomonas aeruginosa PAO1, Escherichia coli ATCC25922, and Salmonella typhimurium LT2 into the ampG gene-deleted strains with carrier pet-30a. Real-time polymerase chain reaction (real-time PCR) was used to detect the relative expressions of ampC and ampG mRNAs. RESULTS: Compared with Kp1, the induction phenotype of the ampC of Kp1-ΔampG strain disappeared, the ampC expression was reduced, and the minimal inhibitory concentration (MIC) values of cefoxitin and ceftazidime significant decrease from 128 μg/mL to 1 μg/mL. Based on Kp1, five strain were successfully constructed to complement the ampG genes from five knock-in strain, and all of the above complemented strains showed inducible expression of ampC and restored the expression of ampG to varying degrees, as well as restored resistance to the antimicrobial drugs cefoxitin and ceftazidime (P < 0.05). The ampC and ampG genes were barely expressed in Kp NTUH-K2044-ΔampG-RC when compared with Kp NTUH-K2044-RC. The expressions of ampG and ampC in each knock-in strain were recovered, the induction phenotype of ampC was restored, and the MIC values of cefoxitin and ceftazidime were increased. (P < 0.05). CONCLUSION: In this study, we found that ampG was an essential regulator for the plasmid-mediated ampC-induced expression in K. pneumoniae. Dove 2023-08-24 /pmc/articles/PMC10461740/ /pubmed/37645559 http://dx.doi.org/10.2147/IDR.S421598 Text en © 2023 Li et al. https://creativecommons.org/licenses/by-nc/3.0/This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/ (https://creativecommons.org/licenses/by-nc/3.0/) ). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). |
spellingShingle | Original Research Li, Guiling Wang, Li Zhang, Heguang Luan, Ying Sun, Qi Duo, Libo Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae |
title | Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae |
title_full | Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae |
title_fullStr | Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae |
title_full_unstemmed | Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae |
title_short | Study on the Role of ampG in the Regulation of Plasmid-Mediated ampC -Induced Expression in Klebsiella pneumoniae |
title_sort | study on the role of ampg in the regulation of plasmid-mediated ampc -induced expression in klebsiella pneumoniae |
topic | Original Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10461740/ https://www.ncbi.nlm.nih.gov/pubmed/37645559 http://dx.doi.org/10.2147/IDR.S421598 |
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