First Detection of Tet(X4)-positive Enterobacterales in Retail Vegetables and Clonal Spread of Escherichia Coli ST195 Producing Tet(X4) in Animals, Foods, and Humans Across China and Thailand

The mobile tigecycline-resistant gene tet(X4), which confers resistance to all tetracyclines, has been identified in bacterial isolates from various sources. However, there are no reports on the occurrence of tet(X4) in bacterial isolates of ready-to-eat fresh vegetables. In this study, 113 vegetable samples from farmers' markets were screened for tigecycline-resistant strains. Ten Escherichia coli (two ST195, two ST48, and one ST10, ST58, ST88, ST394, ST641, and ST101) and one Klebsiella pneumoniae (ST327) recovered from nine vegetable samples (7.96 %) were identified as carrying tet(X4). The core genome sequences of the two E. coli ST195 isolates showed a close relationship (14-41 single-nucleotide polymorphisms) with 31 tet(X4)-bearing E. coli ST195 isolates from humans, pigs, pork, and bird in China and Thailand, and the 33 E. coli ST195 isolates producing Tet(X4) shared similar resistance genes and plasmid replicons. Nanopore sequencing and conjugation experiments confirmed that the tet(X4) genes were located on the hybrid plasmids IncFIA-HI1A-HI1B (n = 6), IncX1 (n = 3), and IncFII2 (n = 1) in E. coli, and IncFII plasmid in K. pneumoniae. IncFIA-HI1A-HI1B and IncX1 plasmids shared highly similar structures with plasmids from various sources in the GenBank database. This is the first study to report the observation of tet(X4)-positive bacteria in retail vegetables. The epidemic clones and plasmids contribute to tet(X4) dissemination in vegetables. The clonal spread of Tet(X4)-producing E. coli ST195 across multiple niches and countries could pose a potential threat to public health.