The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification

Nicoloff, Hervé; Hjort, Karin; Levin, Bruce R.; Andersson, Dan I.

doi:10.1038/s41564-018-0342-0

Cited by 312 publications

(388 citation statements)

References 54 publications

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“…Gene amplification has been linked to the cause of temporary antibiotic resistance seen in a sub-population of bacteria and is known as heteroresistance. Heteroresistance is often lost after multiple generations in the absence of antibiotic selective pressure, due to the fitness cost imposed by the production of extra proteins as a result of amplification [19, 20]. While the mechanism of amplification of the bla TEM is not well known, recent studies have found that the amplified bla TEM has been co-located on a segment of DNA containing other antibiotic resistance genes, such as aadA and sulI , termed a genomic resistance module [15].…”

Section: Introductionmentioning

confidence: 99%

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofbla_TEM-1B

Hubbard

Mason

Parry

et al. 2020

Preprint

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A novel phenotype of Escherichia coli and Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3rd generation cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the β-lactamase blaTEM, however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a β-lactamase but lacked mutations within β-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS26-flanked composite transposon containing several antibiotic resistance genes, including blaTEM-1B, which was designated Tn6762. These resistance genes are also found to be present on a translocatable unit which had excised from Tn6762 in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn6762 following exposure to TZP and capture the TU in a plasmid containing a copy of IS26. Subsequent amplification of the TU, and by extension blaTEM-1B, leads to β-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value = <0.0001), we found that the TZP-resistant isolate was as fit as the susceptible ancestor. This mechanism of gene amplification, and the subsequent hyperproduction, of blaTEM-1B is an important consideration when using genomic data to predict resistance/susceptibility to TZP.

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Section: Introductionmentioning

confidence: 99%

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofbla_TEM-1B

Hubbard

Mason

Parry

et al. 2020

Preprint

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“…However, such AMR mechanisms are increasingly being recognized as commonly occurring in clinical isolates, including in the serious infections described in our cohort (37, 62–64). This would suggest that organisms with the capability to amplify AMR genes are widespread and capable of causing significant human infections especially under antibiotic selective pressure (7, 9, 10, 65). Moreover, the most recent systematic data on CRE in the U.S. found no difference in outcomes between patients with CRE and non-CP-CRE suggesting that circulating non-CP-CRE organisms may not have significant fitness defects (4).…”

Section: Discussionmentioning

confidence: 99%

“…In addition to possibly imposing a fitness cost on the organism, AMR gene amplifications have also been associated with the presence of antimicrobial heteroresistance (65, 67). The interplay between AMR gene amplification, heteroresistance and fitness is likely reflected in the genotype changes of patient 10 isolates given the development and subsequent reversion of porin mutations observed in those strains (Table 1).…”

Section: Discussionmentioning

confidence: 99%

Concurrence of Porin Loss and Modular Amplification of β-Lactamase Encoding Genes Drives Carbapenem Resistance in a Cohort of Recurrent Enterobacterales Bacteremia

Shropshire

Aitken

Pifer³

et al. 2019

Preprint

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30 Background 31 Carbapenem resistant Enterobacterales (CRE) remain urgent antimicrobial resistance threats. 32 Approximately half of CRE clinical isolates lack carbapenem hydrolyzing enzymes and develop 33 carbapenem resistance through alternative mechanisms. The purpose of this study was to 34 elucidate the development of carbapenem resistance mechanisms from clonal, recurrent 35 extended-spectrum b-lactamase positive Enterobacterales (ESBL-E) bacteremia isolates in a 36 vulnerable patient population. 37 Methods 38 This study investigated a historical, retrospective cohort of ESBL-E bacteremia cases in the 39 University of Texas MD Anderson Cancer Center (MDACC) from January 2015 to July 2016. 40 Phylogenetic and comparative genomic analyses were performed to identify clonal, recurrent 41 ESBL-E isolates developing carbapenem resistance. Oxford Nanopore Technology (ONT) long-42 read and Illumina short-read sequencing data were used to generate consensus assemblies and to 43 identify signatures of mobile genetic element mediated amplification and transposition of 44 antimicrobial resistance genes. Serial passaging experiments were performed on a set of clinical 45 ST131 ESBL-E isolates to recapitulate in vivo observations. qPCR and qRT-PCR were used to 46 determine respective copy number and transcript levels of b-lactamase genes. 47 Results 48 116 ESBL-E bacteremia cases were identified, 16 of which had documented recurrent infections. 49 Four serial, recurrent isolates displayed a carbapenem resistant phenotype, three without the 50 acquisition of a known carbapenemase. These three isolates had non-carbapenemase-producing 51 CRE (non-CP-CRE) mechanisms driven by IS26-and ISEcp1-mediated amplification of 52 4 respective translocatable units (TU) and transposition units (TPU) harboring both blaOXA-1 and 53 blaCTX-M variants with concomitant outer membrane porin disruption. The TU and TPU 54 structures inserted into the open reading frames of outer membrane porin genes in a subset of 55 non-CP-CRE isolates. Serial passage of an index ST131 ESBL-E isolate under selective 56 carbapenem exposure resulted in chromosomal amplification of modular, TUs harboring β-57 lactamase genes with concomitant porin inactivation, recapitulating the in vivo carbapenem 58 resistance progression. Long-read sequencing of two additional MDACC bacteremia strains 59 identified similar non-CP-CRE mechanisms observed in the serial isolates. 60 Conclusions 61 Non-CP-CRE de novo mechanisms were the primary driver of CRE development in recurrent 62 bacteremia cases within this vulnerable patient population. The incorporation of long-read ONT 63 data into AMR surveillance platforms is critical to identify high-risk CRE isolates that are 64 difficult to identify with low-resolution phenotypic and molecular characterization methods. 65 66 Keywords: 67 Non-carbapenemase-producing carbapenem resistant Enterobacterales, Oxford Nanopore 68 Technologies MinION Sequencing, Mobile Genetic Element Amplifications, Mobile Genetic 69 Element Transpositions...

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“…The subsequent increase in the resistant subpopulation may eventually lead to the failure of an antibiotic treatment (6). Hypotheses for the underlying molecular mechanisms involving the stochasticity of genetic mutation, gene expression, and protein regulation (7-9), however, remain hard to test in dynamically changing cell subpopulations, partly due to the absence of appropriate single cell experimental technique (10). The need to better understand cell heterogeneity motivates the development of new techniques that link the single-cell phenotype with its in situ molecular information.…”

Section: Introductionmentioning

confidence: 99%

A hydrogel beads based platform for single-cell phenotypic analysis and digital molecular detection

Zhu

Lin

et al. 2019

Preprint

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Microfluidic platforms integrating phenotyping and genotyping approaches have the 19 potential to advance the understanding of single cell genotype-to-phenotype correlations. These 20 correlations can play a key role in tackling antibiotic heteroresistance, cancer cell heterogeneity, 21 and other related fundamental problems. Herein, we report a novel platform that enables both high-22throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-23 sized biocompatible polyethylene glycol hydrogel beads produced by a convenient and disposable 24 centrifugal droplet generation device. The hydrogel beads have been demonstrated enhanced 25 thermal stability, and achieved uncompromised efficiencies in digital polymerase chain reaction, 26 digital loop-mediated isothermal amplification, and single cell phenotyping. The crosslinked 27 hydrogel network highlights the prospective linkage of various subsequent molecular analyses to 28 address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. 29Our platform shows great potential for applications in clinical practice and medical research, and 30 promises new perspectives in mechanism elucidation of environment-evolution interaction and 31 other basic research areas. 32

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The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification

Cited by 312 publications

References 54 publications

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofbla_TEM-1B

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofbla_TEM-1B

Concurrence of Porin Loss and Modular Amplification of β-Lactamase Encoding Genes Drives Carbapenem Resistance in a Cohort of Recurrent Enterobacterales Bacteremia

A hydrogel beads based platform for single-cell phenotypic analysis and digital molecular detection

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The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification

Cited by 312 publications

References 54 publications

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofblaTEM-1B

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofblaTEM-1B

Concurrence of Porin Loss and Modular Amplification of β-Lactamase Encoding Genes Drives Carbapenem Resistance in a Cohort of Recurrent Enterobacterales Bacteremia

A hydrogel beads based platform for single-cell phenotypic analysis and digital molecular detection

Contact Info

Product

Resources

About

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofbla_TEM-1B

Within-patient evolution to piperacillin/tazobactam resistance in a clinical isolate ofEscherichia colidue to IS26-mediated amplification ofbla_TEM-1B