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

Abstract

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...

Evaluation of metagenomic, 16S rRNA gene and ultra-plexed PCR-based sequencing approaches for profiling antimicrobial resistance gene and bacterial taxonomic composition of polymicrobial samples

“…A commonly used single target for profiling bacterial communities is the 16S rRNA gene which possesses both highly conserved regions facilitating the use of universal primers, and hypervariable regions which can be used to discriminate between bacterial taxa 11,12 . Increasingly, targeted ultra-highly multiplexed PCR panels have been developed to enable amplicon-based evaluation of the presence/absence and diversity of specific features of a metagenome associated with a key phenotype, for example AMR gene diversity [13][14][15] , or of a subset of organisms associated with disease, for example respiratory viruses. One example is Illumina AmpliSeq TM which can be used with both Illumina-and community-curated panels (primer pools).…”