Two tet(X4)-positive Enterobacter cloacae isolates TECL_1 and TECL_2 were isolated from pigs in China. S1-PFGE and Southern blotting showed that tet(X4) located on plasmids in the size of ∼290 kb and ∼190 kb in TECL_1 and TECL_2, respectively. Conjugation experiment demonstrated that the tet(X4)-harboring plasmid can transfer from the donor strain TECL_1 and TECL_2 to the recipient strain Escherichia coli J53, and the tigecycline resistance of transconjugants was increased by 128-fold and 64-fold compared with E. coli J53, respectively. We obtained the complete plasmid sequence of pTECL_2-190k-tetX4 (190,185 bp) from E. cloacae TECL_2 and found that the plasmid was a hybrid plasmid with replicon types of IncFIA, IncHI1A and IncHI1B. We further analyzed 85 tet(X4)-carrying plasmids in the public database and clarified that pTECL_2-190k-tetX4-like plasmid was widespread in multiple species of Enterobacteriaceae. IMPORTANCE We identified two tet(X4)-positive E. cloacae isolates, which has not been previously reported. We obtained the complete sequence of pTECL_2-190k-tetX4 and found that it was a hybrid plasmid with multiple replicon types, including IncFIA, IncHI1A and IncHI1B. By comparing all the known tet(X4)-carrying plasmids, we found that pTECL_2-190k-tetX4-like plasmid has been disseminated across various species in China. Our study expanded the identification of tet(X4)-positive species and emphasized that pTECL_2-190k-tetX4-like plasmid has spread widely in various species.
Keywords: Enterobacter cloacae; tet(X4); tigecycline resistance.