Tn3-derived inverted-repeat miniature elements (TIMEs) that mobilize antibiotic resistance genes

Miniature inverted-repeat transposable elements (MITEs) are nonautonomous 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 blaKPC-2, floR, qnrS1, and tet(A). Notably, the blaKPC-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. IMPACT STATEMENTBacterial miniature inverted-repeat transposable elements (MITEs) are a group of short (50 bp-500 bp) nonautonomous transposable elements that are thought to have originated from insertion sequences or transposons. Although MITEs can theoretically mobilize antibiotic resistance genes (ARGs) in the presence of transposases, only a few studies have reported their association with ARGs, probably due to difficulties in identifying MITEs in genomic sequences. This study provides evidence, based on bioinformatic analysis of public Enterobacteriaceae genomes, that a subset of MITEs, called Tn3-derived inverted-repeat miniature elements (TIMEs), mobilizes ARGs by forming composite transposon-like structures. A novel 244-bp TIME, designated TIME244, was present in more than 100 Enterobacteriaceae plasmids in the current RefSeq database, suggesting its further transmission in bacterial populations through horizontal gene transfer. This study reveals that TIMEs were often overlooked when analyzing the genetic contexts of ARGs in previous studies. These findings highlight the importance of TIMEs in bacterial gene acquisition and underscore the need for new tools that can detect TIMEs in bacterial genomes for ARG surveillance. DATA SUMMARYAccession numbers of sequence data analyzed in this study are provided within the article or in supplementary data files.