Purpose: 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 bla _AFM-1, bla _OXA-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 bla _AFM-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 bla _AFM-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 bla _AFM-1 gene.
Conclusion: Our investigation showed that plasmid-borne resistance genes can contribute to antibiotic resistance in A. hydrophila  SS332. A novel bla _NDM-like gene, bla _AFM-1, was verified to be functional and associated with novel ISCR19 -like elements. This fact indicated the risk of spread of bla _AFM-1 genes and ISCR19 -like elements.
Keywords: Aeromonas hydrophila , whole-genome sequencing, plasmid-borne resistance genes, bla _AFM-1, ISCR19 -like elements