ABSTRACTSeveral variants of the plasmid-carried tigecycline resistance gene cluster,tmexCD-toprJ, have been identified. This study characterized another novel variant,tmexC6D6-toprJ1b, located on the chromosome of environmental-originPseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity oftmexC6D6-toprJ1band negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression oftmexC6D6-toprJ1b.tmexC6D6-toprJ1bwas found in the plasmids or chromosomes of differentPseudomonasspecies from six countries. Two genetic backgrounds, class 1 integrons andint-carrying integrase units, were found adjacent to thetmexC6D6-toprJ1bgene cluster and might mediate the transfer of this novel efflux pump gene cluster inPseudomonas. Further phylogenetic analysis revealedPseudomonasas the major reservoir oftmexCD-toprJvariants, warranting closer monitoring in the future.IMPORTANCETigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster,tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another noveltmexCD-toprJvariant,tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. AlthoughtmexC6D6-toprJ1bwas found only inPseudomonasspecies,tmexC6D6-toprJ1bmight spread toEnterobacteriaceaehosts via mobile genetic elements resembling those of othertmexCD-toprJvariants, compromising the therapeutic strategies. Meanwhile, novel transferabletmexCD-toprJvariants are constantly emerging and mostly exist inPseudomonasspp., indicatingPseudomonasas the important hidden reservoir and origin oftmexCD-toprJvariants. Continuous monitoring and investigations oftmexCD-toprJare urgent to control its spread.