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

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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.