Transfer of copper-resistance genes in the soil microbiome and relationship to horizontal gene transfer of antimicrobial resistance genes

Programme website: http://inspire-dtp.ac.uk

Project Rationale:
Our current NERC study investigates antimicrobial resistance horizontal gene transfer, principally via plasmid conjugation transfer of third generation extended spectrum beta lactamase (ESBL) and fourth generation carbapenemase from E. coli and Klebsiella pneumoniae donor strains to agricultural soil microbiomes, and vice versa.
Some authors have speculated that antimicrobial resistance genes on plasmids are co-selected with heavy metal resistance genes, in particular due to feeding copper as a supplement to animal feed to boost health and productivity; recently highlighted in an EFSA Report (2016). Studies on copper addition to animals or ingress into soil ecosystems are limited and inconclusive, necessitating more rigorous scientific study to investigate whether copper resistance/tolerance genes can be co-selected with antibiotic resistance genes.
The goal of this studentship proposal is to determine the effect of copper exposure on the transfer of antibiotic resistance genes among and between soil microbiota and pathogens.
Soil microbiomes will be screened for copper resistance/tolerance and antibiotic resistance by isolation on copper selective agar media containing various classes of antibiotics.
Common copper resistance genes will also be screened using PCR, including multicopper oxidase cueO which protects periplasmic enzymes from copper-mediated damage, cus which confers copper and silver resistance, and the copA and pcoABCD operon efflux mechanisms responsible for pumping excess copper out of the cytoplasm.

Methodology:
1. Screen three different soil types for natural copper resistant species by selecting on agar containing 0.1mM, 1mM and 5mM copper sulphate. Pick off copper resistant strains and screen for resistance to antibiotics using disc diffusion assay.
2. Add copper sulphate to one or more of the soils at previously determined copper concentrations. Incubate for 7 days, and screen soil again for copper resistant species. Pick off copper resistant strains and screen for resistance to antibiotics using disc diffusion assay.
3. Add azide resistant, copper sensitive, antibiotic sensitive E. coli to the soils. Incubate for 7 days and recover by plating on azide plates. Then screen the E. coli to determine whether it has gained copper resistance and antibiotic resistance.
4. Repeat (3) but with ESBL and carbapenemase resistant pathogens and assess whether copper and or antibiotic resistance gene transfer accelerates between these and the microbiome.
5. Sequence the genomes of selected bacterial isolates
6. Characterise the ability of the selected bacterial isolates to spread antibiotic resistance or other pathogenicity determinants.

Training:
The INSPIRE DTP programme provides comprehensive personal and professional development training alongside extensive opportunities for students to expand their multi-disciplinary outlook through interactions with a wide network of academic, research and industrial/policy partners. The student will be registered at the University of Southampton and hosted at School of Biological Sciences. Specific training will
include:
The cultivation of soil bacteria; performing horizontal gene transfer assays; quantitative PCR; high-throughput sequencing of amplicons; whole genome sequencing and (meta)genomic analysis