Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

Abstract

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Phage-layer interferometry: a companion diagnostic for phage therapy and a bacterial testing platform

“…T7 was used as the phage biosensor to prototype PLI because it has high therapeutic potential. It is active in vivo, can be engineered to expand host range to target an array of pathogens, and can be used as a phagemid delivery system 15 , 16 , 21 , 43 . To synthesize phage-functionalized BLI sensors, biotin-streptavidin bioconjugation was used.…”

A systematic review of antimicrobial therapy in children with tracheostomies

“…The long‐term effect of antimicrobial exposure on microbial communities and the presence of antimicrobial resistance genes within them (their “resistome”) is not fully understood and remains an active area of research 23,24 . Nevertheless, repeated cycles of broad‐spectrum antimicrobials increase the risk of drug‐resistant organisms 25 .…”

“…The long-term effect of antimicrobial exposure on microbial communities and the presence of antimicrobial resistance genes within them (their "resistome") is not fully understood and remains an active area of research. 23,24 Nevertheless, repeated cycles of broad-spectrum antimicrobials increase the risk of drugresistant organisms. 25 The increasing prevalence of antimicrobial resistance is a growing concern for the management of vulnerable patient groups, and healthcare in general; the World Health Organization even lists antimicrobial resistance as one of their top 10 threats to humanity.…”

Barcoded overexpression screens in gut Bacteroidales identify genes with new roles in carbon utilization and stress resistance

“…For example, dual barcoding of vectors (Dub-seq) has been used to construct libraries expressed in E. coli , but these are currently limited to ∼250,000 strains and are restricted to DNA sources with sequence assemblies, which are required for mapping. 24–27…”

“…For example, dual barcoding of vectors (Dub-seq) has been used to construct libraries expressed in E. coli, but these are currently limited to ~250,000 strains and are restricted to DNA sources with sequence assemblies, which are required for mapping. [24][25][26][27] Here, we exploit advances in long-read sequencing to develop a new workflow termed Bobaseq, or Barcoded Overexpression BActerial shotgun library sequencing, to perform large-scale gain-of-function screens using DNA barcoded vector libraries. We demonstrate this with overexpression in B. theta, although this approach is readily applicable to alternative expression strains and is agnostic to the source of DNA being assayed.…”