Tn3-derived inverted-repeat miniature elements that mobilize antibiotic resistance genes.

Miniature inverted-repeat transposable elements (MITEs) are non-autonomous mobile genetic elements (MGEs) that can be mobilized by transposases provided by the relevant autonomous MGEs. MITEs originating from Tn3-family transposons were previously termed Tn3-derived inverted-repeat miniature elements (TIMEs). Composite transposon-like structures bounded by two copies of TIME, called TIME-COMPs, were shown to mobilize the intervening sequences. However, their association with antibiotic resistance genes (ARGs) has not yet been systematically studied. This study thus aimed to identify new TIME-COMP-like structures containing ARGs in the genomic sequences of the clinically important bacterial family Enterobacteriaceae in public databases. TIME-COMP-like structures were first searched for in the plasmid database PLSDB, focusing on small plasmids, using a self-against-self blastn approach to identify repeated elements. Then, newly and previously identified MITEs (including TIMEs) were searched for in the NCBI core nucleotide database to identify TIME-COMP-like structures located on other replicons. Bioinformatic analysis identified multiple previously unreported TIME-COMPs containing ARGs, which are bounded by directly or inversely oriented TIMEs, namely IS101, MITESen1 and a novel 244 bp TIME termed TIME244. TIME244 contains a putative resolution site related to that of Tn21. These TIMEs were predominantly detected in plasmids and very rarely in chromosomes. The ARGs embedded in newly identified TIME-COMPs were bla KPC-2, floR, qnrS1 and tet(A). Notably, the bla KPC-2 carbapenemase gene was found in TIME-COMPs bounded by TIME244 and a TIME-COMP bounded by IS101. These findings highlight a potential role for TIMEs in the spread of diverse ARGs.