Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from
APH(9)-Ic
Stenotrophomonas maltophilia
aminoglycoside 9-phosphotransferase
aminoglycoside resistance
aminoglycoside-modifying enzyme
Journal
Frontiers in cellular and infection microbiology
ISSN: 2235-2988
Titre abrégé: Front Cell Infect Microbiol
Pays: Switzerland
ID NLM: 101585359
Informations de publication
Date de publication:
2022
2022
Historique:
received:
14
11
2022
accepted:
15
12
2022
entrez:
26
1
2023
pubmed:
27
1
2023
medline:
28
1
2023
Statut:
epublish
Résumé
The intrinsic resistance mechanism plays an essential role in the bacterial resistance to a variety of the antimicrobials. The aim of this study is to find the chromosome-encoded novel antimicrobial resistance gene in the clinical isolate. The function of the predicted resistance gene was verified by gene cloning and antibiotic susceptibility test. Recombinant protein expression and enzyme kinetic studies were performed to explore the in vivo activity of the enzyme. Expression of the resistance gene exposed to antimicrobial was determined by RT-qPCR. Whole genome sequencing and bioinformatic analysis were applied to analyze the genetic context of the resistance gene. The novel aminoglycoside (AG) resistance genes designated aph(9)-Ic and aph(9)-Ic1 confer resistance to spectinomycin, and a recombinant strain harboring aph(9)-Ic (pMD19-T-aph(9)-Ic/DH5α) showed a significantly increased minimum inhibitory concentration (MIC) level against spectinomycin compared with the control strains (DH5α and pMD19-T/DH5α). The result of the kinetic analysis of APH(9)-Ic was consistent with the MIC result for the recombinant pMD19-T-aph(9)-Ic/DH5α, showing the efficient catalytic activity for spectinomycin [kcat/Km ratio = (5.58 ± 0.31) × 104 M-1·s-1]. Whole-genome sequencing demonstrated that the aph(9)-Ic gene was located on the chromosome with a relatively conserved genetic environment, and no mobile genetic element was found in its surrounding region. Among all the function-characterized resistance genes, APH(9)-Ic shares the highest amino acid sequence identity of 33.75% with APH(9)-Ia. We characterized a novel AG resistance gene aph(9)-Ic and its variant aph(9)-Ic1 that mediated spectinomycin resistance from S. maltophilia. The identification of the novel AG resistance genes will assist us in elucidating the complexity of resistance mechanisms in microbial populations.
Sections du résumé
Background
The intrinsic resistance mechanism plays an essential role in the bacterial resistance to a variety of the antimicrobials. The aim of this study is to find the chromosome-encoded novel antimicrobial resistance gene in the clinical isolate.
Methods
The function of the predicted resistance gene was verified by gene cloning and antibiotic susceptibility test. Recombinant protein expression and enzyme kinetic studies were performed to explore the in vivo activity of the enzyme. Expression of the resistance gene exposed to antimicrobial was determined by RT-qPCR. Whole genome sequencing and bioinformatic analysis were applied to analyze the genetic context of the resistance gene.
Results
The novel aminoglycoside (AG) resistance genes designated aph(9)-Ic and aph(9)-Ic1 confer resistance to spectinomycin, and a recombinant strain harboring aph(9)-Ic (pMD19-T-aph(9)-Ic/DH5α) showed a significantly increased minimum inhibitory concentration (MIC) level against spectinomycin compared with the control strains (DH5α and pMD19-T/DH5α). The result of the kinetic analysis of APH(9)-Ic was consistent with the MIC result for the recombinant pMD19-T-aph(9)-Ic/DH5α, showing the efficient catalytic activity for spectinomycin [kcat/Km ratio = (5.58 ± 0.31) × 104 M-1·s-1]. Whole-genome sequencing demonstrated that the aph(9)-Ic gene was located on the chromosome with a relatively conserved genetic environment, and no mobile genetic element was found in its surrounding region. Among all the function-characterized resistance genes, APH(9)-Ic shares the highest amino acid sequence identity of 33.75% with APH(9)-Ia.
Conclusion
We characterized a novel AG resistance gene aph(9)-Ic and its variant aph(9)-Ic1 that mediated spectinomycin resistance from S. maltophilia. The identification of the novel AG resistance genes will assist us in elucidating the complexity of resistance mechanisms in microbial populations.
Identifiants
pubmed: 36699730
doi: 10.3389/fcimb.2022.1097561
pmc: PMC9868417
doi:
Substances chimiques
Kanamycin Kinase
EC 2.7.1.95
Spectinomycin
93AKI1U6QF
Anti-Bacterial Agents
0
Aminoglycosides
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1097561Informations de copyright
Copyright © 2023 Shi, Lu, Feng, Gao, Li, Liu, Zhang, Zhang, Li, Lin, Lin, Li, Zhang, Hu, Wang, Bao and Jiang.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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