Molecular and Functional Characterization of a Novel Plasmid-Borne blaNDM-Like Gene, blaAFM-1, in a Clinical Strain of Aeromonas hydrophila

Abstract

Xi Lin,1,2 Junwan Lu,1,2 Changrui Qian,1,2 Hailong Lin,1– 3 Qiaoling Li,1– 3 Xueya Zhang,1– 3 Hongmao Liu,1– 3 Zhewei Sun,1,2 Danying Zhou,1,2 Wei Lu,1,2 Mei Zhu,4 Hailin Zhang,2,3 Teng Xu,5 Kewei Li,1,2 Qiyu Bao,1– 3 Li Lin2,3 1Key Laboratory of Medical Genetics of Zhejiang Province, Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, People’s Republic of China; 2Institute of Biomedical Informatics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, People’s Republic of China; 3The Second Affiliated Hospital and Children’s Hospital, Wenzhou Medical University, Wenzhou, 325027, People’s Republic of China; 4Department of Clinical Laboratory, Zhejiang Hospital, Hangzhou, Zhejiang, 310013, People’s Republic of China; 5Institute of Translational Medicine, Baotou Central Hospital, Baotou, 014040, People’s Republic of ChinaCorrespondence: : Li LinThe Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, 325027, People’s Republic of ChinaTel [phone]Email [email protected] BaoInstitute of Biomedical Informatics, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, 325035, People’s Republic of ChinaTel/Fax [phone]Email [email protected]: An increasing frequency of antibiotic resistance has been observed in both clinical and environmental Aeromonas hydrophila isolates in recent years. However, there are still very few in-depth studies regarding the role of plasmids in the antibiotic resistance of A. hydrophila. Hence, we investigated the molecular and functional characterization of a multidrug-resistant plasmid encoding an NDM-like metallo-β-lactamase, AFM-1, in the clinical A. hydrophila isolate SS332.Methods: The minimum inhibitory concentrations (MICs) of 24 antibiotics against A. hydrophila SS332 were measured by the agar dilution method. The genome of A. hydrophila SS332 was sequenced with PacBio and Illumina platforms. Six plasmid-borne antimicrobial resistance genes were chosen for cloning, including blaAFM-1, blaOXA-1, msr(E), mph(E), aac(6ʹ)-Ib10, and aph(3ʹ)-Ia. Phylogenetic analysis, amino acid sequence alignment, and comparative genomic analysis were performed to elucidate the active site requirements and genetic context of the blaAFM-1 gene.Results: A. hydrophila SS332 showed high levels of resistance to 15 antibiotics, especially those with MIC levels at or above 1024 μg/mL, including ampicillin, cefazolin, ceftriaxone, aztreonam, spectinomycin, and roxithromycin. Six plasmid-borne resistance genes from A. hydrophila were verified to be functional in E. coli DH5α. AFM-1 shared 86% amino acid identity with NDM-1 and showed resistance to ampicillin, cefazolin, cefoxitin, and ceftazidime. In addition, the blaAFM-1 gene was associated with three different novel ISCR19-like elements, designated ISCR19-1, ISCR19-2 and ∆ISCR19-3, which may be involved in the acquisition and mobilization of the blaAFM-1 gene.Conclusion: Our investigation showed that plasmid-borne resistance genes can contribute to antibiotic resistance in A. hydrophila SS332. A novel blaNDM-like gene, blaAFM-1, was verified to be functional and associated with novel ISCR19-like elements. This fact indicated the risk of spread of blaAFM-1 genes and ISCR19-like elements.Keywords: Aeromonas hydrophila, whole-genome sequencing, plasmid-borne resistance genes, blaAFM-1, ISCR19-like elements

Keywords

• aeromonas hydrophila
• whole-genome sequencing
• plasmid-encoded resistance genes
• blaafm-1
• novel iscr19-like elements