mcr-1 and mcr-2 (mcr-6.1) variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015

AbuOun, Manal; Stubberfield, Emma; Duggett, Nick A; Kirchner, Miranda; Dormer, Luisa; Nuñéz-García, Javier; Randall, Luke; Lemma, Fabrizio; Crook, Derrick W.; Teale, Christopher; Smith, Richard P.; Anjum, Muna F.

doi:10.1093/jac/dky272

Cited by 108 publications

(101 citation statements)

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“…The pooled pig caeca had been collected as part of a separate study surveying the presence of AMR genes in E. coli in pigs from 56 farms across the UK [28]. In brief, caecal contents were sampled from 10 randomly selected healthy finishing pigs from each of the farms at 12 different abattoirs (March 2014-October 2015), and suspended in 22.5 mL of PBS (processing within 24 h of collection).…”

Section: Sample Types and Settingsmentioning

confidence: 99%

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Gweon

Shaw

Swann

et al. 2019

Environmental Microbiome

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on behalf of the REHAB consortium Abstract Background: Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short-and long-reads) to create highquality assemblies for comparison to the metagenomic data. To automate data processing, we developed an opensource software pipeline, 'ResPipe'. Results: Taxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve < 1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.Conclusions: Sequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/ hsgweon/ResPipe).

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Section: Sample Types and Settingsmentioning

confidence: 99%

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Gweon

Shaw

Swann

et al. 2019

Environmental Microbiome

Self Cite

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“…The pooled pig caeca had been collected as part of a separate study surveying the presence of AMR genes in E. coli in pigs from 56 farms across the UK (28). In brief, caecal contents were sampled from 10 randomly Table 4).…”

Section: Sample Types and Settingsmentioning

confidence: 99%

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Gweon

Shaw

Swann

et al. 2019

Preprint

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on behalf of the REHAB consortium. ABSTRACTBackground. Shotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short-and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an opensource software pipeline, 'ResPipe'. Results.Taxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve <1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially Gweon et al. 3 changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.Conclusions. Sequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

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“…Additionally, mcr‐1 and mcr‐2 homologues, mcr‐1.10 and mcr‐2.2 , respectively, in Moraxella isolated from pig farms in 2014–2015, have also been recently reported (AbuOun et al . ).…”

Section: Introductionmentioning

confidence: 97%

Longitudinal study on the occurrence in pigs of colistin‐resistant Escherichia coli carrying mcr‐1 following the cessation of use of colistin

et al. 2018

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mcr-1 and mcr-2 (mcr-6.1) variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015

Cited by 108 publications

References 1 publication

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

Longitudinal study on the occurrence in pigs of colistin‐resistant Escherichia coli carrying mcr‐1 following the cessation of use of colistin

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