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

Abstract

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

Acknowledgments

The authors thank all the colleagues and the reviewers who helped with this work. This study was supported by the Natural Science Foundation of Zhejiang Province, China [LQ17H010003, LY19C060002 and LQ17H190001], the National Natural Science Foundation of China [81700011, 81973382 and 81960381], the Science & Technology Project of Wenzhou City, China [Y20170205 and 2019Y0358] and the Science & Technology Project of Inner Mongolia Autonomous Region, China (201802125).

Ethics Approval and Consent to Participate

Individual patient data were not involved, and only anonymous clinical residual samples were used in this study. This study followed the principles stated in the Declaration of Helsinki.

Disclosure

The authors report no conflicts of interest in this work.