Comment on “the IS6 family, a clinically important group of insertion sequences including IS26” by Varani and co-authors

The insertion sequence IS26 has long been known to play a major role in the recruitment of antibiotic resistance genes into the mobile resistance gene pool of Gram-negative bacteria and IS26 also plays a major role in their subsequent broad dissemination. Related IS, IS431/257 and IS1216 are important in the same roles in Gram positive bacteria. However, until recently the properties of IS26 movement that could potentially explain this ability had not been explored. A much needed insight has come from our recent demonstration that IS26 uses a novel targeted mechanism that is conservative. The targeted conservative mechanism is much more efficient than the known replicative mechanism, which is now more accurately called copy-in. A recent review “The IS6 family, a clinically important group of insertion sequences including IS26” by Varani, He, Siguier, Ross and Chandler published in Mobile DNA has substantially misrepresented the recent studies on the targeted conservative mechanism and at the same time incorrectly implied that any mechanism established for IS26 can be assumed to apply to a range of IS that are at best very distantly related. A few of the most important issues are examined in this comment. Readers are advised to consult the original literature to check facts before drawing firm conclusions.

Response from Varani et al. to “Comment on ‘the IS6 family, a clinically important group of insertion sequences including IS26’ by Ruth M. Hall”

The IS6 family of insertion sequences is a large but coherent group which was originally named to avoid confusion between a number of identical or nearly identical IS that were identified at about the same time and given different names (IS15D, IS26, IS46, IS140, IS160, IS176). The underlying common mechanistic feature of all IS6 family members which have been investigated is that they appear to transpose by replicative transposition and form pseudo compound transposons with the flanking IS in direct repeat and in which associated genes are simply transferred to the target replicon and lost from the donor.In the accompanying letter Hall raises a number of very serious and wide-ranging criticisms of our recent review article concerning the IS6 family of insertion sequences. She clearly feels that we have undervalued her work and that we question or ignore certain of her in vivo results. This impression is almost certainly the result of the standard of proof we generally apply to mechanistic aspects of transposition where we think it important to identify transposition intermediates including the types of synaptic, strand cleavage and strand transfer complexes involved.

Genetic Characterization and Passage Instability of a Hybrid Plasmid Co-Harboring bla IMP-4 and bla NDM-1 Reveal the Contribution of Insertion Sequences During Plasmid Formation and Evolution

In this study, we characterized a novel plasmid from a carbapenem-resistant K. michiganensis (CRKM) isolate, which harbors two metallo-β-lactamases (MBLs), IMP-4 and NDM-1, is capable of transconjugation and contains three replicons. Our results first expand the diversity of plasmids co-harboring carbapenemase genes in Enterobacterales , which exhibits epidemic importance in bacterial resistance.

Alterations in chromosomal genes nfsA, nfsB, and ribE are associated with nitrofurantoin resistance in Escherichia coli from the United Kingdom

Antimicrobial resistance in enteric or urinary Escherichia coli is a risk factor for invasive E. coli infections. Due to widespread trimethoprim resistance amongst urinary E. coli and increased bacteraemia incidence, a national recommendation to prescribe nitrofurantoin for uncomplicated urinary tract infection was made in 2014. Nitrofurantoin resistance is reported in <6% urinary E. coli isolates in the UK, however, mechanisms underpinning nitrofurantoin resistance in these isolates remain unknown. This study aimed to identify the genetic basis of nitrofurantoin resistance in urinary E. coli isolates collected from north west London and then elucidate resistance-associated genetic alterations in available UK E. coli genomes. As a result, an algorithm was developed to predict nitrofurantoin susceptibility. Deleterious mutations and gene-inactivating insertion sequences in chromosomal nitroreductase genes nfsA and/or nfsB were identified in genomes of nine confirmed nitrofurantoin-resistant urinary E. coli isolates and additional 11 E. coli isolates that were highlighted by the prediction algorithm and subsequently validated to be nitrofurantoin-resistant. Eight categories of allelic changes in nfsA, nfsB, and the associated gene ribE were detected in 12412 E. coli genomes from the UK. Evolutionary analysis of these three genes revealed homoplasic mutations and explained the previously reported order of stepwise mutations. The mobile gene complex oqxAB, which is associated with reduced nitrofurantoin susceptibility, was identified in only one of the 12412 genomes. In conclusion, mutations and insertion sequences in nfsA and nfsB were leading causes of nitrofurantoin resistance in UK E. coli . As nitrofurantoin exposure increases in human populations, the prevalence of nitrofurantoin resistance in carriage E. coli isolates and those from urinary and bloodstream infections should be monitored.

Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures

Background We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation “short-read” and third-generation “long-read” sequencing methods. Results We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a “long-read first” approach. Conclusions Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Operon formation by insertion sequence IS3 in Escherichia coli

Operons are a hallmark of the genomic and regulatory architecture of prokaryotes. However, the mechanism by which two genes placed far apart gradually come close and form operons remains to be elucidated. Here, we propose a new model of the origin of operons: Mobile genetic elements called insertion sequences can facilitate the formation of operons by consecutive insertion-deletion-excision reactions. This mechanism barely leaves traces of insertion sequences and is difficult to detect in evolution in nature. We performed, to the best of our knowledge, the first experimental demonstration of operon formation, as a proof of concept. The insertion sequence IS3 and the insertion sequence excision enhancer are genes found in a broad range of bacterial species. We introduced these genes into insertion sequence-less Escherichia coli and found that, supporting our hypothesis, the activity of the two genes altered the expression of genes surrounding IS3, closed a 2.7 kilobase pair gap between a pair of genes, and formed new operons. This study shows how insertion sequences can facilitate the rapid formation of operons through locally increasing the structural mutation rates and highlights how coevolution with mobile elements may shape the organization of prokaryotic genomes and gene regulation.

Insertion sequences associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteremia.

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in dissemination and modification of antibiotic resistance phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over four days from an inpatient with prolonged bacteremia. Isolates from both species harbored similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events, and IS-medicated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the IS in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium offering a new paradigm for the study of daptomycin-resistance and novel insights into the route of daptomycin resistance dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.