Next generation sequencing reveals limitation of qPCR methods in quantifying emerging antibiotic resistance genes (ARGs) in the environment

Background and aims: Only a few antimicrobials are effective against H. pylori, and antibiotic resistance is an increasing problem for eradication therapies. In 2017, the World Health Organization categorized clarithromycin resistant H. pylori as a “high-priority” bacterium. Standard antimicrobial susceptibility testing can be used to prescribe appropriate therapies but is currently recommended only after the second therapeutic failure. H. pylori is, in fact, a “fastidious” microorganism; culture methods are time-consuming and technically challenging. The advent of molecular biology techniques has enabled the identification of molecular mechanisms underlying the observed phenotypic resistance to antibiotics in H. pylori. The aim of this literature review is to summarize the results of original articles published in the last ten years, regarding the use of Next Generation Sequencing, in particular of the whole genome, to predict the antibiotic resistance in H. pylori.Methods: a literature research was made on PubMed. The research was focused on II and III generation sequencing of the whole H. pylori genome. Results: Next Generation Sequencing enabled the detection of novel, rare and complex resistance mechanisms. The prediction of resistance to clarithromycin, levofloxacin and amoxicillin is accurate; for other antimicrobials, such as metronidazole, rifabutin and tetracycline, potential genetic determinants of the resistant status need further investigation.

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Next-Generation Sequencing for Typing and Detection of Resistance Genes: Performance of a New Commercial Method during an Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli

Journal of Clinical Microbiology ◽

10.1128/jcm.00313-14 ◽

2014 ◽

Vol 52 (7) ◽

pp. 2454-2460 ◽

Cited By ~ 24

Author(s):

J. Veenemans ◽

I. T. Overdevest ◽

E. Snelders ◽

I. Willemsen ◽

Y. Hendriks ◽

...

Keyword(s):

Escherichia Coli ◽

Next Generation Sequencing ◽

Resistance Genes ◽

Beta Lactamase ◽

Next Generation ◽

Extended Spectrum Beta Lactamase ◽

Extended Spectrum ◽

Commercial Method ◽

Generation Sequencing

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Identification and characterization of candidateRlm4blackleg resistance genes inBrassica napususing next-generation sequencing

Plant Biotechnology Journal ◽

10.1111/j.1467-7652.2012.00716.x ◽

2012 ◽

Vol 10 (6) ◽

pp. 709-715 ◽

Cited By ~ 28

Author(s):

Reece Tollenaere ◽

Alice Hayward ◽

Jessica Dalton-Morgan ◽

Emma Campbell ◽

Joanne R.M. Lee ◽

...

Keyword(s):

Next Generation Sequencing ◽

Resistance Genes ◽

Next Generation ◽

Identification And Characterization ◽

Generation Sequencing

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Next-Generation Sequencing and Comparative Analysis of Sequential Outbreaks Caused by Multidrug-Resistant Acinetobacter baumannii at a Large Academic Burn Center

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02014-15 ◽

2015 ◽

Vol 60 (3) ◽

pp. 1249-1257 ◽

Cited By ~ 27

Author(s):

Hajime Kanamori ◽

Christian M. Parobek ◽

David J. Weber ◽

David van Duin ◽

William A. Rutala ◽

...

Keyword(s):

Next Generation Sequencing ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Reference Genome ◽

Index Case ◽

Multidrug Resistant ◽

Next Generation ◽

The Third ◽

Burn Center ◽

Generation Sequencing

Next-generation sequencing (NGS) analysis has emerged as a promising molecular epidemiological method for investigating health care-associated outbreaks. Here, we used NGS to investigate a 3-year outbreak of multidrug-resistantAcinetobacter baumannii(MDRAB) at a large academic burn center. A reference genome from the index case was generated usingde novoassembly of PacBio reads. Forty-six MDRAB isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and sequenced using an Illumina platform. After mapping to the index case reference genome, four samples were excluded due to low coverage, leaving 42 samples for further analysis. Multilocus sequence types (MLST) and the presence of acquired resistance genes were also determined from the sequencing data. A transmission network was inferred from genomic and epidemiological data using a Bayesian framework. Based on single-nucleotide variant (SNV) differences, this MDRAB outbreak represented three sequential outbreaks caused by distinct clones. The first and second outbreaks were caused by sequence type 2 (ST2), while the third outbreak was caused by ST79. For the second outbreak, the MLST and PFGE results were discordant. However, NGS-based SNV typing detected a recombination event and consequently enabled a more accurate phylogenetic analysis. The distribution of resistance genes varied among the three outbreaks. The first- and second-outbreak strains possessed ablaOXA-23-likegroup, while the third-outbreak strains harbored ablaOXA-40-likegroup. NGS-based analysis demonstrated the superior resolution of outbreak transmission networks for MDRAB and provided insight into the mechanisms of strain diversification between sequential outbreaks through recombination.

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Appearance of synthetic vector-associated antibiotic resistance genes in next-generation sequences

10.1101/392225 ◽

2018 ◽

Author(s):

George Taiaroa ◽

Gregory M. Cook ◽

Deborah A Williamson

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Sequence Data ◽

Antibiotic Resistance Genes ◽

Data Sets ◽

Next Generation ◽

Beta Lactam ◽

Sequence Contigs ◽

Beta Lactam Antibiotics ◽

Genome Shotgun Sequence

SynopsisBackgroundNext-generation sequencing methods have broad application in addressing increasing antibiotic resistance, with identification of antibiotic resistance genes (ARGs) having direct clinical relevance.ObjectivesHere, we describe the appearance of synthetic vector-associated ARGs in major public next-generation sequence data sets and assemblies, including in environmental samples and high priority pathogenic microorganisms.MethodsA search of selected databases – the National Centre for Biotechnology Information (NCBI) nucleotide collection, NCBI whole genome shotgun sequence contigs and literature-associated European Nucleotide Archive (ENA) datasets, was carried out using sequences characteristic of pUC-family synthetic vectors as a query in BLASTn. Identified hits were confirmed as being of synthetic origin, and further explored through alignment and comparison to primary read sets.ResultsSynthetic vectors are attributed to a range of organisms in each of the NCBI databases searched, including examples belonging to each Kingdom of life. These synthetic vectors are associated with various ARGs, primarily those encoding resistance to beta-lactam antibiotics and aminoglycosides. Synthetic vector associated ARGs are also observed in multiple environmental meta-transcriptome datasets, as shown through analysis of associated ENA primary reads, and are proposed to have led to incorrect statements being made in the literature on the abundance of ARGs.ConclusionsAppearance of synthetic vector-associated ARGs can confound the study of antimicrobial resistance in varied settings, and may have clinical implications in the nearfuture.

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