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

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 bla_NDM-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 bla_NDM-5 carriers (3.57%, 7/196). The bla_NDM-5 genes were located on the IncX3 (n=5), IncHI2 (n=1), or IncHI2-IncF (n=1) plasmids. All bla_NDM-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 bla_NDM-5-carrying IncHI2 plasmids deposited in GenBank from Guangdong ducks. In all IncHI2 plasmids, bla_NDM-5 was embedded in a novel transposon, Tn7051 (IS3000-ΔISAba125-IS5-ΔISAba125-bla_NDM-5-ble_MBL-trpF-tat-∆dct-IS26-∆umuD-∆ISKox3-IS3000), which was identical to the genetic structure surrounding bla_NDM-5 found in some IncX3 plasmids. The IncHI2-IncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla_NDM-5-carrying IncHI2 plasmid and a heavy-metal-resistant IncF plasmid through ∆Tn1721. To the best of our knowledge, this is the first report on the characterization of bla_NDM-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. Tn7051 is likely responsible for the transfer of bla_NDM-5 from IncX3 to IncHI2 plasmids in Enterobacteriaceae, resulting in the expansion of transmission vectors of bla_NDM-5.