Carbapenemase-Encoding Gene Copy Number Estimator (CCNE): a Tool for Carbapenemase Gene Copy Number Estimation

Jiang, Jianping; Chen, Liang; Chen, Xin; Li, Pei; Xu, Xiaogang; Fowler, Vance G.; Duin, David van; Wang, Minggui

doi:10.1128/spectrum.01000-22

Cited by 6 publications

(3 citation statements)

References 35 publications

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“…The importance of improving phenotypic-based AST, biochemical assays and utilising predictions from genomic data is highlighted by the TZP-R/3GC-S phenotype presented in this study, and the need to account for gene copy number and predicted expression levels when predicting resistance to BL/BLI combinations. Scores for BL/BLI resistance prediction from genomic data have been developed (72) in addition to β-lactamase copy number calculators (73) and tools like these will be vital to support decisions for use of new BL/BLI combinations and ensure their future efficacy is safeguarded. Alternative diagnostic methods, based on protein detection, such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technology, may provide an alternative strategy to rapidly detect β-lactamase hyperproduction (74).…”

Section: Discussionmentioning

confidence: 99%

A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam

Fraser,

Ball,

Cantillon

et al. 2024

Preprint

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Resistance to the β-lactam/β-lactamase inhibitor (BL/BLI) combination antibiotic piperacillin/tazobactam (TZP) predominantly occurs via β-lactamase enzymes also leading to resistance to third-generation cephalosporins (3GCs). However, if β-lactamases inactive against 3GCs and inhibited by tazobactam are expressed at high levels leading to enzyme hyperproduction, the surplus enzyme escapes inhibition by tazobactam and inactivates the antibiotic piperacillin. Understanding this mechanism is clinically relevant as enzyme hyperproduction can emerge upon antibiotic administration, resulting in treatment failure despite initial resistance profiles supporting TZP use. We report the identification of an Escherichia coli isolate that developed resistance to TZP during patient treatment. Our whole genome sequencing (WGS) analyses show that TZP resistance evolved via IS26-mediated duplication of a blaTEM-1 containing gene cassette on a plasmid, resulting in hyperproduction of TEM-1 β-lactamase. We demonstrate that ten copies of blaTEM-1 induce resistance greater than 32-times the MIC and exposure to TZP further increases amplification of blaTEM-1. Furthermore, in the absence of TZP, gene copy number of IS26 and blaTEM-1 remains stable over five days, despite a 48,205 bp genome size increase compared to the pre-amplification isolate. We additionally detect phenotypic changes that might indicate host adaptation potentially linked to the additional genes in the amplified cassette. Our analysis advances the understanding of infections caused by isolates evolving β-lactamase hyperproduction, which represent a complex problem in both detection and treatment. As 40% of antibiotics active against WHO priority pathogens in the pre-clinical pipeline are BL/BLI combinations further investigations are of urgent concern.

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Section: Discussionmentioning

confidence: 99%

A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam

Fraser,

Ball,

Cantillon

et al. 2024

Preprint

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“…For the verification of duplicated ARGs on the plasmids, BLASTN was performed on all filtered long-read reads against a reference containing the tandem repeat units and relative flanking region, and the results were processed by an in-house R script ( 25 ). The short-read sequence data of 237 ST11 KPC-2-CRKP isolates were used to verify the frequency of evolution events in pKPC-2 plasmids, and the copy numbers of ARGs were estimated by CCNE ( 42 ).…”

Section: Methodsmentioning

confidence: 99%

Genome sequencing unveils bla _KPC-2 -harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae

Han,

Zhou,

et al. 2024

mSystems

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The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by bla KPC-2 -harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn 3 -mediated plasmid homologous recombination, IS 26- mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy bla KPC-2 , bla SHV-12 , and bla CTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum β-lactamase tended to develop multicopy bla KPC-2 . Notably, the presence of multicopy bla SHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of bla SHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments. IMPORTANCE This study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 ( bla KPC-2 -harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel β-lactam/β-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

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“…The bla KPC gene copy number was estimated with the ccne tool (v1.1.0) [ 29 ]. The presence of a mixed population of bla KPC variants was investigated by aligning raw reads to the bla KPC-31 reference sequence (CARD accession ARO:3005362) with the bwa-mem algorithm (v0.7.17) and by identifying the single-nucleotide polymorphisms (SNPs) with SAMtools (v1.4) and freebayes (v1.3.2) [ 30 ].…”

Section: Methodsmentioning

confidence: 99%

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy

Del Rio,

Fox,

Muresu

et al. 2023

Microorganisms

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Background: Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. Methods: Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020–2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. Results: All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including β-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel β-lactam/β-lactamase inhibitors (BL/BLI). Conclusion: WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

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Carbapenemase-Encoding Gene Copy Number Estimator (CCNE): a Tool for Carbapenemase Gene Copy Number Estimation

Abstract: Globally disseminated carbapenem-resistant Enterobacterales is an urgent threat to public health. The most common carbapenem resistance mechanism is the production of carbapenemases.

Cited by 6 publications

References 35 publications

A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam

A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam

Genome sequencing unveils bla _KPC-2 -harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy

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Carbapenemase-Encoding Gene Copy Number Estimator (CCNE): a Tool for Carbapenemase Gene Copy Number Estimation

Abstract: Globally disseminated carbapenem-resistant Enterobacterales is an urgent threat to public health. The most common carbapenem resistance mechanism is the production of carbapenemases.

Cited by 6 publications

References 35 publications

A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam

A high-resolution genomic and phenotypic analysis of resistance evolution of anEscherichia colistrain from a critical care patient treated with piperacillin/tazobactam

Genome sequencing unveils bla KPC-2 -harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy

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Genome sequencing unveils bla _KPC-2 -harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae