Identification of novel double-stranded RNA elements in developing antibiotic resistance in the agricultural environment

Many types of antibiotics (AB) which are used in humans (e.g. chloramphenicol and its derivatives) are also used in farms (e.g. thiamphenicol, a methyl-sulfonyl derivative of chloramphenicol) either to treat or prevent disease. They have a similar antibacterial spectrum and may significantly increase a possibility that clinical pathogens will develop cross-resistance to drugs used in human medicine. The intestinal microbiota is the epicentre but underexplored source for antibiotic resistance (AR) emergence in response to the selective pressure of AB. The vast majority of bacteria cannot be cultured in laboratory conditions and this limits our knowledge of the potential AR determinants these species may possess and express in a community-dependent manner.
Metagenomics, for identification of encoded metabolic pathways present in bacterial populations, has revealed many novel, possibly dormant genes in microbial communities as well as a large discrepancy between predicted and detected resistance genes. While metagenomics has primarily focused on analysis of DNA as the source of genomic information, RNA can also serve as genetic material. Recent high-throughput RNA-sequencing (RNA-Seq) analyses of bacterial systems have made two critical discoveries. Firstly, expression of the bacterial genome is extensively regulated by non-coding (nc) RNAs, including antisense (as) RNAs (a class of ncRNA that are encoded on the opposite strand of their target genes). asRNAs regulate gene expression via RNA-RNA interactions, thus leading to modulation of mRNA translation and stability. asRNAs were firstly found on mobile elements (phages (bacterial viruses), plasmids or transposons) which are used for horizontal transfer (HT) of AR genes between different bacteria. However, the mechanism by which these asRNAs regulate expression and possibly acquisition and spread of genes involved in AR is unknown. Secondly, the microbial metatranscriptome contains double stranded (ds) RNA sequences, derived from uncharacterised phages which do not match to the corresponding DNA. Intriguingly, these dsRNAs have coding potential for a large proportion of novel proteins of unknown function.
The use of AB in medicine and agriculture triggers a number of adaptation responses in bacterial communities. Dynamics and mechanisms underlying such functional changes in microbiomes in response to AB are still elusive. This may involve activation of expression of a number of genes relevant to resistance (e.g. transposases, proteases or efflux pump genes), HT or mobilisation of genes and non-coding DNA/RNA elements on mobile structures. Thus, key questions are: How do as-metatranscriptomes respond to antibiotic treatments? Does the animal gut microflora contain dsRNAs that do not correspond to their DNA metagenomes? If yes, then what roles do these dsRNAs play in this ecosystem? Does this uncharacterised genetic information play a role in adaptation responses, including AR?
Our aim therefore is to undertake RNA-Seq of dsRNAs extracted from animal faecal samples in order to identify dsRNAs metatranscriptome in response to antibiotic therapy. The results will lead to identification of an unexplored array of novel genetic information, including non-coding regulatory elements and new open reading frames relevant to AR mechanisms. This in turn, will transform our view on the role novel dsRNAs play in the development and regulation of AR in bacterial communities. The results will also lead to detecting novel or dormant pathways which are regulated by dsRNAs for the production of secondary metabolites with low susceptibility to resistance and discovery of novel genetic information involved in development, transmission and regulation of AR. This ground breaking research project has the potential to provide a paradigm shift in the understanding of transmission and regulation of AR originated from environment and direct the future strategic development of novel antimicrobials.

This work will lead to a fundamental shift in our view on the development, transmission and regulation of AR and bacterial pathogenicity in agricultural settings. This project will result in identification of new targets for novel and possibly existing antibacterials, thus paving the way for industry to re-stimulate research in antibiotic drug discovery. Results from this proposal will inform the UK government and other policymakers (via One Health framework) on the use of clinically relevant drugs in agricultural settings, such as farms in order to prevent potential appearance of clinically relevant AR determinants on mobile elements which are controlled by anti-sense RNAs.
Exploitation of our work has significant potential for Pharmaceutical industries in developing novel strategies and designing new generation therapies based on novel targets. Results will be shared via Antibiotic Action initiative groups across the globe.
The results of this proposal in the long term will be important for development of new combination therapies, as this research has a great potential to identify new regulatory elements (coding and non-coding), AR determinants associated with bacterial communication and mechanisms which can be regulated in response to antibiotic pressure (e.g. switching on adaptive mechanisms, including the excludon). This could lead to follow up studies to this project by screening new therapies based on the use of novel agents (asRNAs or novel RNA-based drugs) to silence mechanisms responsible for transmission of AR in conjunction with known antibacterial to treat the infection. Mutations in the targets (as a mechanism of developing AR) of asRNAs are unlikely to influence the efficiency of asRNA-mRNA binding and, therefore therapy. asRNAs are from tens to hundreds of nucleotides, so are generally long enough to maintain a strong interaction despite mutations in mRNA targets. Resulting new antibacterial with a reduced capacity for resistance long term would significantly benefit the NHS and the cost of care, increase efficiency of health care, life expectancy and reduce the risk of invasive surgery and treatments. This will result in saving millions of lives world-wide and save the National Health Service millions of pounds.
The results from this blue-sky research will set a solid foundation for future work in our lab to address current and potentially forthcoming challenges associated with AR. Identification of novel RNA-based regulatory elements will stimulate further efforts to characterise their roles in AR not just in our lab but also in the real world. This in turn will transform the research data into identification of novel drug targets for potential drug discovery.
The experimental work will also add to the collaborative momentum generated on AR. Understanding the role of ds-metatranscriptome in AR within our lab would directly stimulate future studies planned by the collaboration and also by other groups. The results will therefore be shared with other researchers within the Faculty and around the world (via The Joint Programming Initiative on Antimicrobial Resistance) and collaborators to get a better understanding of the role of novel regulatory RNAs in developing AR.
This and future research will provide opportunities for our Master/PhD students as well as for Warwickshire College students to gain first hand experience in cutting edge multidisciplinary research, deep-sequencing, molecular biology, biochemistry, microbiology and bioinformatics. This will significantly enhance our research-based teaching for both undergraduate and postgraduate programmes and therefore be beneficiary for their experience.
Given the global nature of this proposed study this research will benefit Britain also via (i) creation of high-quality bioscience research jobs, (ii) retention of high quality staff in the UK (iii) support the global reputation of the UK as location to support pioneering advanced science based research.