Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia

Weina Shi; Junwan Lu; Chunlin Feng; Mengdi Gao; Anqi Li; Shuang Liu; Lei Zhang; Xueya Zhang; Qiaoling Li; Hailong Lin; Xi Lin; Kewei Li; Hailin Zhang; Yunliang Hu; Guangli Wang; Qiyu Bao; Weiyan Jiang

doi:10.3389/fcimb.2022.1097561

Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia

Front Cell Infect Microbiol. 2023 Jan 9:12:1097561. doi: 10.3389/fcimb.2022.1097561. eCollection 2022.

Authors

Weina Shi^{1

2}, Junwan Lu³, Chunlin Feng², Mengdi Gao², Anqi Li², Shuang Liu², Lei Zhang², Xueya Zhang^{1

2}, Qiaoling Li^{1

2}, Hailong Lin^{1

2}, Xi Lin², Kewei Li², Hailin Zhang¹, Yunliang Hu¹, Guangli Wang⁴, Qiyu Bao^{1

2

3}, Weiyan Jiang^{1

2}

Affiliations

¹ The Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, China.
² Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education, China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, China.
³ Medical Molecular Biology Laboratory, School of Medicine, Jinhua Polytechnic, Jinhua, China.
⁴ School of Medicine and Health, Lishui University, Lishui, China.

Abstract

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.

Keywords: APH(9)-Ic; Stenotrophomonas maltophilia; aminoglycoside 9-phosphotransferase; aminoglycoside resistance; aminoglycoside-modifying enzyme.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aminoglycosides / pharmacology
Anti-Bacterial Agents / pharmacology
Drug Resistance, Bacterial / genetics
Kanamycin Kinase / genetics
Kinetics
Microbial Sensitivity Tests
Spectinomycin*
Stenotrophomonas maltophilia* / genetics

Substances

Kanamycin Kinase
Spectinomycin
Anti-Bacterial Agents
Aminoglycosides

Grants and funding

This study was supported by the Science & Technology Project of Wenzhou City, China (Y2020112, N20210001, N20210100), Zhejiang Provincial Natural Science Foundation of China (LY19C060002, LQ17H190001) and Natural Science Foundation of China (81973382).