Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp (Ctenopharyngodon idella) and evidence of blaNDM-5-bearing IncHI2 plasmid transfer between ducks and fish

Abstract

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou, China. A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes. Characterization of blaNDM-5 positive isolates and plasmids was determined by antimicrobial susceptibility testing, conjugation experiments, Illumina HiSeq, and Nanopore sequencing. One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as blaNDM-5 carriers (3.57%, 7/196). The blaNDM-5 genes were located on the IncX3 (n=5), IncHI2 (n=1), or IncHI2-IncF (n=1) plasmids. All blaNDM-5-bearing plasmids were transferred by conjugation at frequencies of ∼10-4- 10-6. Based on sequence analysis, the IncHI2 plasmid pHNBYF33-1 was similar to other blaNDM-5- carrying IncHI2 plasmids deposited in GenBank from Guangdong ducks. In all IncHI2 plasmids, blaNDM-5 was embedded in a novel transposon, Tn7 051 (IS3 000-ΔISAba125-IS5-ΔISAba125-blaNDM-5-bleMBL-trpF-tat- Δdct-IS26-ΔumuD-ΔISKox3-IS3 000), which was identical to the genetic structure surrounding blaNDM-5 found in some IncX3 plasmids. The IncHI2-IncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the blaNDM-5-carrying IncHI2 plasmid and a heavy-metal-resistant IncF plasmid through ΔTn1 721. To the best of our knowledge, this is the first report on the characterization of blaNDM-5-bearing plasmids in fish in China. The IncHI2 plasmid pHNBYF33-1 may be transmitted from ducks, considering the common duck-fish freshwater aquaculture system in Guangdong. Tn7 051 is likely responsible for the transfer of blaNDM-5 from IncX3 to IncHI2 plasmids in Enterobacteriaceae, resulting in the expansion of transmission vectors of blaNDM-5.