A novel approach to reducing multiple-drug resistance in foodborne bacteria: application of CRISPR technology

The testable hypothesis is that the CRISPR-Cas systems may be exploited to reduce the burden of drug resistance in bacterial populations in animal production such that (a) the risk of resistance passing into the human food chain is dramatically reduced and (b) that targeted removal of specific resistance will permit use of the antibiotic, thus expanding the potential utility of all antibiotics. The system consists of a sequence-specific nuclease (Cas9) and aguide RNA (gRNA) that provides sequence specificity to its DNA cleavage activity. CRISPR-Cas9 is a versatile and highly specific next-generation antimicrobial that allows targeting of specific pathogenic bacteria while leaving the remaining microbiome intact.
We will devise a CRISPR-Cas system that effectively reduces the incidence of selected test antibiotic resistance genes for use in poultry in the first instance. Four objectives will be delivered. (1) In silico analysis of WGS data from commercial broiler poultry microbiomes will be undertaken to identify antibiotic resistance genes, their prevalence and probable vectors. Targets tested by BLAST searches for specificity to the target gene and to identify suitable gene positive/negative strains for in vitro and in vivo testing and information on the distribution of CRISPR-Cas systems within those strains. (2) Targe t sequences will be synthesised and assembled into Folium vectors constitutively expressing Cas9 or Type I CRISPR-Cas genes. The testing of the utility of the guide sequences is empirical using gene positive/negative recipients from [1] above. The constructs will be mobilised into test strains and loss of the targeted resistance monitored both phenotypically and genotypically. (3) In vitro gut poultry models will be exploited to determine the dynamics of delivery of the CRISPR-Cas system to the poultry gut microbial community and assess reduction of the target resistance gene and its associated vector. Mathematical modelling of rates of transfer and diversity of recipient microbes will provide information related to enhancing and/or specifying range of conjugation recipients. (4) The CRISPR-Cas probiotic strain developed in 1-3 above will then be used in commercial broiler chicks with a target strain using appropriate third-party chicken containment facilities. Molecular profiling will be undertaken to examine dissemination of the CRISPR-Cas system, the loss of the targeted AMR gene and any population shifts in the gut microbiome.