Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes

Guitor, Allison K; Raphenya, Amogelang R.; Klunk, Jennifer; Kuch, Melanie; Alcock, Brian; Surette, Michael G.; McArthur, Andrew G.; Poinar, Hendrik N.; Wright, Gerard D.

doi:10.1128/aac.01324-19

Cited by 80 publications

(103 citation statements)

References 58 publications

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“…Targeted methods are limited by their inability to identify novel AMR determinants, whereas a functional SMS approach might provide more detail 52 . Using an in-solution probe-and-capture strategy 53 , researchers suggested that designed probes should target sequences with up to 15% nucleotide sequence divergence from a reference sequence, which would widen their applicability and target capacity toward newly characterized members of AMR gene families, which often differ from other members by only a few nucleotides 28 .…”

Section: Discussionmentioning

confidence: 99%

“…For targeted methods, new (larger) panels are constantly being released, e.g. 37,826 probes targeting over 2,000 nucleotide sequences associated with AMR using a probe-and-capture strategy 28 , or 78,600 non-redundant genes (including 47,806 putative ARGs) using targeted metagenomics 51 .…”

Section: Discussionmentioning

confidence: 99%

“…However, because reads corresponding to ARGs can be low (e.g. less than 2% of the total sequencing data in 28 ), targeted approaches (including an initial amplification of a panel of ARGs) with increased sensitivity have been developed. As new methods and new commercial panels become available, it is currently unclear what method is best to study resistomes when no pre-existing information is available, e.g.…”

Section: Introductionmentioning

confidence: 99%

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A preliminary investigation of major bacterial antibiotic resistance genes present in Australian fecal samples using shotgun metagenomics and targeted sequencing

Guernier

Chamings

Collier

et al. 2020

Preprint

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24The gut microbiota is an immense reservoir of antimicrobial resistance genes (ARGs); however, in 25 Australia the profile of the gut 'resistome', or ensemble of ARGs, has not been investigated. This 26 study provides a first preliminary mapping of the major bacterial ARGs present in human, domestic 27 dog and wild duck fecal samples collected from south-eastern Victoria, Australia; and evaluates 28 the use of shotgun metagenomics sequencing (SMS) and targeted amplification of ARGs. We 29 analysed SMS data using an in-house method and web-based bioinformatics tools: ResFinder and 30 KmerResistance. We examined targeted sequences using One Codex or the PanBacterialAnalysis 31 Torrent Suite plugin. All methods detected ARGs in all samples, with resistance to up to 13 32 classes of antibiotics detected overall. ARGs were more abundant in the human and dog samples 33 than the duck samples. They mostly conferred resistance to three classes of antibiotics that are 34 the most frequently prescribed in Australia: tetracycline, b-lactams and MLSB (macrolide, 35 lincosamide, streptogramin B). Targeted sequencing significantly improved sensitivity for detection 36 of ARGs included in the panel; however, SMS provided quantitative information and allowed 37 tentative identification of the host bacteria. For SMS, web-based and in-house methods gave 38 comparable results, with discrepancies mostly due to different reference databases. The in-house 39 method allowed manually checking results and potential errors, while web-based methods were 40 user-friendlier and less time-consuming. More samples need to be investigated to fully describe 41 the resistome in humans and animals in Australia. 43Antimicrobial resistance (AMR) occurs naturally in bacteria and other microorganisms, but 48 increases when antimicrobials (antibiotics, antivirals, antifungals) are used 1 . According to WHO, 49AMR accounts for an estimated 700,000 deaths per year and is one of the ten threats to global 50 health in 2019 2 . Reservoirs of multi drug-resistant (MDR) bacteria are ubiquitous, and the concept 51 of an "antibiotic resistome" was first coined to describe the large collection of bacterial 52 antimicrobial resistance genes (ARGs) found in a specific environment 3 . Although AMR is most 53 clinically relevant in pathogenic bacteria, ARGs exist in both pathogenic and non-pathogenic 54 bacteria, and large reservoirs of these genes exist in all ecosystems, including in commensals at 55 all sites of the human or animal body 4 . 57The adult human gastrointestinal tract harbours up to 1,000 molecular species or phylotypes often 58 referred to as operational taxonomic units (OTUs) in 16S rRNA metagenomics 5,6 . This vast array 59 of resident bacteria responds to environmental conditions inside the host, including altered dietary 60 intake and antibiotic-induced disturbances. That can result in modifications in the microbial 61 community structure and genetics, including the enrichment of ARGs within the gut microbiota. 62Recent studies have suggested pot...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A preliminary investigation of major bacterial antibiotic resistance genes present in Australian fecal samples using shotgun metagenomics and targeted sequencing

Guernier

Chamings

Collier

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Melting temperature (Tm) was predicted using the OligoArrayAux function melt.pl (settings, -n RNA -t 65 -C 1.89e -9 ) and used to remove probes with a Tm <55°C or >105°C (16). To prevent off-target hybridization between the probes and any non-viral DNA or RNA, the candidate set of probes was compared against GenBank's nucleotide database using high-throughput BLASTN (default settings) (13,17). Probes with high-scoring segment pairs (HSPs) >50 nt and high sequence similarity (>80%) to non-viral targets were discarded.…”

Section: Designmentioning

confidence: 99%

Rapid Design of a Bait Capture Platform for Culture- and Amplification-Free Next-Generation Sequencing of SARS-CoV-2

Nasir¹,

Speicher²,

Kozak³

et al. 2020

Preprint

Self Cite

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SARS-CoV-2 is a novel betacoronavirus and the aetiological agent of the current COVID-19 outbreak that originated in Hubei Province, China. While polymerase chain reaction is the front-line tool for SARS-CoV-2 surveillance, application of amplification-free and culture-free methods for isolation of SARS-CoV-2 RNA, partnered with next-generation sequencing, would provide a useful tool for both surveillance and research of SARS-CoV-2. We here release into the public domain a set of bait capture hybridization probe sequences for enrichment of SARS-CoV-2 RNA from complex biological samples. These probe sequences have been designed using rigorous bioinformatics methods to provide sensitivity, accuracy, and minimal off-target hybridization. Probe design was based on existing, validated approaches for detecting antimicrobial resistance genes in complex samples and it is our hope that this SARS-CoV-2 bait capture platform, once validated by those with samples in hand, will be of aid in combating the current outbreak.

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“…Indexed library samples will undergo hybridisation with the pathogen baits for between 2 and 12 hours (final sensitivity testing pending) at 55°C-65°C followed by magnetic purification using streptavidin-coated magnetic beads to enrich the level of pathogen versus human DNA in the sample. 30 Sequencing Enriched samples will undergo NGS using an Illumina HiSeq 1500flx sequencing platform in the Farncombe Family Digestive Health Research Institute, McMaster University. 6 31 32 We will use three biological replicates per sample group where possible, with a minimum of two technical repetitions.…”

Section: Pathogen-targeted Enrichmentmentioning

confidence: 99%

Fast I(n)dentification of Pathogens in Neonates (FINDPATH-N): protocol for a prospective pilot cohort study of next-generation sequencing for pathogen identification in neonates with suspected sepsis

Klowak

Helou

Pernica

et al. 2020

bmjpo

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IntroductionSepsis is a major source of morbidity and mortality in neonates; however, identification of the causative pathogens is challenging. Many neonates have negative blood cultures despite clinical evidence of sepsis. Next-generation sequencing (NGS) is a high-throughput, parallel sequencing technique for DNA. Pathogen-targeted enrichment followed by NGS has the potential to be more sensitive and faster than current gold-standard blood culture. In this pilot study, we will test the feasibility and pathogen detection patterns of pathogen-targeted NGS in neonates with suspected sepsis. Additionally, the distribution and diagnostic accuracy of biomarkers cell-free DNA and protein C levels at two time points will be explored.Methods and analysisWe will conduct a prospective, pilot observational study. Neonates over 1 kg with suspected sepsis from a single tertiary care children’s hospital will be recruited for the study. Recruitment will be censored at 200 events or 6 months’ duration. Two blood study samples will be taken: the first simultaneous to the blood culture (time=0 hour, for NGS and biomarkers) via an exception to consent (deferred consent) and another 24 hours later after prospective consent (biomarkers only). Neonates will be adjudicated into those with clinical sepsis, culture-proven sepsis and without sepsis based on clinical criteria. Feasibility parameters (eg, recruitment) and NGS process time will be reported.For analysis, NGS results will be described in aggregate, compared with the simultaneous blood culture (sensitivity and specificity) and reviewed via expert panel for plausibility. Pilot data for biomarker distribution and diagnostic accuracy (sensitivity and specificity) for distinguishing between septic and non-septic neonates will be reported.Ethics and disseminationEthics approval has been granted by the Hamilton Integrated Research Ethics Board. We will seek publication of study results in peer-reviewed journals.

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Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes

Cited by 80 publications

References 58 publications

A preliminary investigation of major bacterial antibiotic resistance genes present in Australian fecal samples using shotgun metagenomics and targeted sequencing

A preliminary investigation of major bacterial antibiotic resistance genes present in Australian fecal samples using shotgun metagenomics and targeted sequencing

Rapid Design of a Bait Capture Platform for Culture- and Amplification-Free Next-Generation Sequencing of SARS-CoV-2

Fast I(n)dentification of Pathogens in Neonates (FINDPATH-N): protocol for a prospective pilot cohort study of next-generation sequencing for pathogen identification in neonates with suspected sepsis

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