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The wide dissemination of New Delhi metallo-β-lactamase genes ( bla  NDM ) has resulted in the treatment failure of most available β-lactam antibiotics, with IncX3-type bla  NDM-5 -carrying plasmids recognised as having spread worldwide. In China, bacteria carrying these plasmids are increasingly being detected from diverse samples, including hospitals, communities, livestock and poultry, and the environment, suggesting that IncX3 plasmids are becoming a vital vehicle for bla  NDM dissemination. To elucidate the fitness cost of these plasmids on the bacterial host, we collected bla  NDM -negative strains from different sources and tested their ability to acquire the bla  NDM-5 -harboring p3R-IncX3 plasmid. We then measured changes in antimicrobial susceptibility, growth kinetics, and biofilm formation following plasmid acquisition. Overall, 70.7% (29/41) of our Enterobacteriaceae recipients successfully acquired the bla  NDM-5 -harboring p3R-IncX3 plasmid. Contrary to previous plasmid burden theory, 75.9% (22/29) of the transconjugates showed little fitness cost as a result of plasmid acquisition, with 6.9% (2/29) of strains exhibiting enhanced growth compared with their respective wild-type strains. Following plasmid acquisition, all transconjugates demonstrated resistance to most β-lactams, while several strains showed enhanced biofilm formation, further complicating treatment and prevention measures. Moreover, the highly virulent Escherichia coli sequence type 131 strain that already harbored mcr - 1 also demonstrated the ability to acquire the bla  NDM-5 -carrying p3R-IncX3 plasmid, resulting in further limited therapeutic options. This low fitness cost may partly explain the rapid global dissemination of bla  NDM-5 -harboring IncX3 plasmids. Our study highlights the growing threat of IncX3 plasmids in spreading bla  NDM-5 .

Fitness Cost of blaNDM-5-Carrying p3R-IncX3 Plasmids in Wild-Type NDM-Free Enterobacteriaceae