Targeted IS-element sequencing uncovers transposition dynamics during selective pressure in enterococci

Abstract

Insertion sequences (IS) are simple transposons implicated in the genome evolution of diverse pathogenic bacterial species. Enterococci have emerged as important human intestinal pathogens with newly adapted virulence potential and antibiotic resistance. These genetic features arose in tandem with large-scale genome evolution mediated by mobile elements. Pathoadaptation in enterococci is thought to be mediated in part by the IS element IS256 through gene inactivation and recombination events. However, the regulation of IS256 and the mechanisms controlling its activation are not well understood. Here, we adapt an IS256-specfic deep sequencing method to describe how chronic lytic phage infection drives widespread diversification of IS256 in E. faecalis and how antibiotic exposure is associated with IS256 diversification in both E. faecalis and E. faecium during a clinical human infection. We show through comparative genomics that IS256 is primarily found in hospital-adapted enterococcal isolates. Analyses of IS256 transposase gene levels reveal that IS256 mobility is regulated at the transcriptional level by multiple mechanisms in E. faecalis, indicating tight control of IS256 activation in the absence of selective pressure. Our findings reveal that stressors such as phages and antibiotic exposure drives rapid genome-scale transposition in the enterococci. IS256 diversification can therefore explain how selective pressures mediate evolution of the enterococcal genome, ultimately leading to the emergence of dominant nosocomial lineages that threaten human health.

A metagenomics pipeline reveals insertion sequence-driven evolution of the microbiota

“…This is supported by the presence of ISOSDB45 (IS256 family) insertions that are associated with pathogenic bacteria (Fig. 2D) 19,42–44 .…”

“…These strains lack native copies of ISOSDB412 which allowed us to study this IS element in the context of a genetic arrangement that we found from our analysis of the microbiota. Following growth, we isolated their genomic DNA and performed IS-Seq which enriches for ISOSDB412 amplicons to identify their insertion locations 19 . Both Bt and Bf strains carrying the ISOSDB412 plasmid obtained numerous ISOSDB412 insertions throughout their genomes compared to controls that lacked the ISOSDB412 plasmid construct (Fig.…”

“…iORFs were common among MGE resistance and macrolide resistance ( ermF ) genes 76 . Both of these classes of genes have been reported to be targets of IS inactivation 19,33 . Since these are heterogenous populations that co-exist between genes carrying or lacking IS insertions, we hypothesize that IS insertions in these genes maintains a low level of population-wide MGE and antibiotic resistance and reduces the burden of expressing the resistance gene.…”

“…Genomic DNA was extracted from 1.5 mL cultures of Bacteroides strains using the Zymobionics DNA Miniprep kit (Zymo Research, Irvine, CA) and used as input for IS-Seq as described previously 19 . Briefly, NGS libraries were produced using the Illumina DNA Prep Kit (Illumina, San Diego, CA) and amplified using ISOSDB412 IS-Seq Step1 and p7 primers for 13 cycles using Q5 Master Mix (New England Biolabs, Ipswich, MA).…”

“…Additionally, IS elements can rapidly expand in bacterial genomes when commensal bacteria transition to become pathogens (15, 17, 18). Thus, IS elements have a profound impact on the evolution of bacterial genomes and the physiological traits of bacteria (19).…”