Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm
BackgroundAminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminogl...
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1224464/full |
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| author | Yuning Sha Yuning Sha Naru Lin Guozhi Zhang Yuan Zhang Jingxuan Zhao Junwan Lu Tingting Zhu Xueya Zhang Qiaoling Li Hailin Zhang Xi Lin Kewei Li Qiyu Bao Qiyu Bao Qiyu Bao Dong Li Dong Li |
| author_facet | Yuning Sha Yuning Sha Naru Lin Guozhi Zhang Yuan Zhang Jingxuan Zhao Junwan Lu Tingting Zhu Xueya Zhang Qiaoling Li Hailin Zhang Xi Lin Kewei Li Qiyu Bao Qiyu Bao Qiyu Bao Dong Li Dong Li |
| author_sort | Yuning Sha |
| collection | DOAJ |
| description | BackgroundAminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention.MethodsThe bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3′)-Id gene and its phylogenetic relationship with other AMEs.ResultsA novel aminoglycoside 3′-O-phosphotransferase gene designated aph(3′)-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3′-O-phosphotransferase APH(3′)-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3′)-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3′)-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M−1/s−1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M−1/s−1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M−1/s−1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M−1/s−1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3′)-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3′)-Id gene.ConclusionIn this work, a novel aminoglycoside 3’-O-phosphotransferase gene designated aph(3′)-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene. |
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| spelling | doaj.art-41cf7ec4dbb64cafb648df0744965d8f2023-08-28T12:28:42ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-08-011410.3389/fmicb.2023.12244641224464Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farmYuning Sha0Yuning Sha1Naru Lin2Guozhi Zhang3Yuan Zhang4Jingxuan Zhao5Junwan Lu6Tingting Zhu7Xueya Zhang8Qiaoling Li9Hailin Zhang10Xi Lin11Kewei Li12Qiyu Bao13Qiyu Bao14Qiyu Bao15Dong Li16Dong Li17The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaKey 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, ChinaMedical Molecular Biology Laboratory, School of Medicine, Jinhua Polytechnic, Jinhua, ChinaThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaKey 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, ChinaKey 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, ChinaThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaKey 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, ChinaMedical Molecular Biology Laboratory, School of Medicine, Jinhua Polytechnic, Jinhua, ChinaThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, ChinaKey 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, ChinaBackgroundAminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention.MethodsThe bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3′)-Id gene and its phylogenetic relationship with other AMEs.ResultsA novel aminoglycoside 3′-O-phosphotransferase gene designated aph(3′)-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3′-O-phosphotransferase APH(3′)-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3′)-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3′)-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M−1/s−1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M−1/s−1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M−1/s−1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M−1/s−1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3′)-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3′)-Id gene.ConclusionIn this work, a novel aminoglycoside 3’-O-phosphotransferase gene designated aph(3′)-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1224464/fullKluyvera intermediaresistance geneAPH(3′)-Idaminoglycoside 3’-O-phosphotransferasekinetic parameter |
| spellingShingle | Yuning Sha Yuning Sha Naru Lin Guozhi Zhang Yuan Zhang Jingxuan Zhao Junwan Lu Tingting Zhu Xueya Zhang Qiaoling Li Hailin Zhang Xi Lin Kewei Li Qiyu Bao Qiyu Bao Qiyu Bao Dong Li Dong Li Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm Frontiers in Microbiology Kluyvera intermedia resistance gene APH(3′)-Id aminoglycoside 3’-O-phosphotransferase kinetic parameter |
| title | Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm |
| title_full | Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm |
| title_fullStr | Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm |
| title_full_unstemmed | Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm |
| title_short | Identification and characterization of a novel chromosomal aminoglycoside 3’-O-phosphotransferase, APH(3′)-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm |
| title_sort | identification and characterization of a novel chromosomal aminoglycoside 3 o phosphotransferase aph 3 id from kluyvera intermedia dw18 isolated from the sewage of an animal farm |
| topic | Kluyvera intermedia resistance gene APH(3′)-Id aminoglycoside 3’-O-phosphotransferase kinetic parameter |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1224464/full |
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