SVants – A long-read based method for structural variation detection in bacterial genomes

Hanson, Blake; Johnson, Jethro; Leopold, Shana R.; Sodergren, Erica; Weinstock, George M.

doi:10.1101/822312

Cited by 4 publications

(4 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Plasmid copy number contributions were based on normalized coverage depths of full-length plasmid harboring β-lactamase = 1.5×, which indicates approximately 50% of the population has 2 copies of the β-lactamase-positive plasmid. *,MGE + PCN context resolved in part through extraction of individual long reads using SVants ( 74 ). e Incomplete and/or short-read assemblies only preclude an estimate of genomic context of β-lactamase gene amplification.…”

Section: Resultsmentioning

confidence: 99%

Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections

et al. 2022

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 99%

Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections

et al. 2022

View full text Add to dashboard Cite

show abstract

“…CARD, PlasmidFinder, ISFinder 32 and BLAST webtools were utilized during manual inspection of the assemblies. SVAnts v0.1 33 b-lactamase gene copy number, transcript level and impact of b-lactamase expression on susceptibility analysis Quantitative PCR (qPCR) and qRT-PCR were used to assess DNA copy number and RNA transcript levels, respectively. qPCR and qRT-PCR primers and probe sequences are provided in Table S2.…”

Section: Genome Annotation Amr Gene and Mobile Genetic Element (Mge) ...mentioning

confidence: 99%

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort

Shropshire

Aitken

Pifer

et al. 2020

Journal of Antimicrobial Chemotherapy

View full text Add to dashboard Cite

Background Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms. Objectives To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population. Methods This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT–PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively. Results Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure. Conclusions These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

show abstract

“…Plasmid Copy Number contributions were based on normalized coverage depths of full-length plasmid harboring β-lactamase = 1.5X, which indicates approximately 50% of the population has 2 copies of the β-lactamase positive plasmid. *MGE + PCN context resolved in part through extraction of individual long reads using SVAnts(70)…”

mentioning

confidence: 99%

Systematic Analysis of Mobile Genetic Elements Mediating β-lactamase Gene Amplification in Non-Carbapenemase-Producing Carbapenem Resistant Enterobacterales Bloodstream Infections

Shropshire

Konovalova

McDaneld

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Non-carbapenemase-producing carbapenem resistant Enterobacterales (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem resistant Enterobacterales (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole genome sequencing (WGS) was used to elucidate carbapenem non-susceptibility determinants in Enterobacterales bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem non-susceptible Enterobacterales (CNSE) mechanisms through a combination of phylogenetic analysis, antimicrobial resistant (AMR) gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%) whereas 25.3% (20/79) were carbapenem intermediate Enterobacterales (CIE) and 22.8% (18/79) were carbapenemase producing Enterobacterales (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum β-lactamase (ESBL) genes (Wilcoxon Test; p-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6X; n= 17) and K. pneumoniae (median CNV = 3.2X, n = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., ompC/ompF or ompK36/ompK35). Completely resolved CNSE genomes revealed that IS26 and ISEcp1 structures harboring blaCTX-M variants along with other AMR elements were the primary drivers of gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE mediated β-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS26 translocatable units, or segmental duplication, typically due to ISEcp1 transposition units. Non-CP-CRE strains were the most prevalent cause of CRE bacteremia with carbapenem non-susceptibility driven by concurrent porin loss and MGE-mediated amplification of blaCTX-M genes. IMPORTANCE: Carbapenem resistant Enterobacterales (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase producing Enterobacterales (CPE) even though non-carbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating β-lactamase gene amplification. By generating complete genomes of 65 carbapenem non-susceptible Enterobacterales (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.

show abstract

SVants – A long-read based method for structural variation detection in bacterial genomes

Cited by 4 publications

References 17 publications

Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections

Systematic Analysis of Mobile Genetic Elements Mediating β-Lactamase Gene Amplification in Noncarbapenemase-Producing Carbapenem-Resistant Enterobacterales Bloodstream Infections

IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort

Systematic Analysis of Mobile Genetic Elements Mediating β-lactamase Gene Amplification in Non-Carbapenemase-Producing Carbapenem Resistant Enterobacterales Bloodstream Infections

Contact Info

Product

Resources

About