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

人类使用的许多类型的抗生素(AB)(例如，氯霉素及其衍生物)也用于农场(例如，甲磺酸基氯霉素的衍生物)，用于治疗或预防疾病。它们具有相似的抗菌谱，可能会显着增加临床病原体对人类药物产生交叉耐药性的可能性。肠道微生物区系是抗生素耐药(AR)产生的震中来源，但对AB的选择性压力的反应尚未得到充分研究。绝大多数细菌不能在实验室条件下培养，这限制了我们对这些物种可能拥有的潜在AR决定因素的了解，并以群落依赖的方式表达。元基因组学用于识别细菌种群中存在的编码代谢途径，揭示了微生物群落中许多新的、可能处于休眠状态的基因，以及预测和检测到的抗性基因之间的巨大差异。虽然元基因组学主要专注于将DNA作为基因组信息的来源进行分析，但RNA也可以作为遗传物质。最近对细菌系统的高通量RNA测序(RNA-Seq)分析有两个关键发现。首先，细菌基因组的表达受到非编码(NC)RNA的广泛调控，包括反义(AS)RNAs(一类编码在目标基因相反链上的ncRNA)。AsRNAs通过RNA-RNA相互作用调节基因表达，从而调节mRNA的翻译和稳定性。AsRNAs最早发现于用于AR基因在不同细菌之间水平转移的可移动元件(噬菌体、质粒或转座子)上。然而，这些asRNA调节AR相关基因的表达以及可能的获取和传播的机制尚不清楚。其次，微生物转录组包含双链(DS)RNA序列，来自与相应DNA不匹配的未鉴定噬菌体。有趣的是，这些dsRNA具有编码大量未知功能的新蛋白的潜力。AB在医学和农业中的使用在细菌群落中触发了许多适应反应。微生物群对AB反应的这种功能变化背后的动力学和机制仍然难以捉摸。这可能涉及到一些与抗性相关的基因(如转座酶、蛋白水解酶或外排泵基因)、羟色胺的表达被激活，或者基因和非编码DNA/RNA元件在可移动结构上的动员。因此，关键问题是：代谢综合征对抗生素治疗有何反应？动物肠道微生物区系中是否含有与它们的DNA元基因组不对应的dsRNA？如果是，那么这些dsRNA在这个生态系统中扮演什么角色？因此，我们的目标是对从动物粪便样本中提取的dsRNAs进行RNA序列分析，以确定dsRNAs对抗生素治疗的反应。这些结果将导致识别一系列尚未探索的新遗传信息，包括非编码调控元件和与AR机制相关的新的开放阅读框架。这反过来将改变我们对新dsRNA在细菌群落中AR的发育和调节中所起作用的看法。这些结果还将导致检测受dsRNA调控的新的或休眠的途径，以产生对抗性敏感性较低的次生代谢物，并发现与AR的发育、传递和调节有关的新的遗传信息。这一开创性的研究项目有可能为理解环境来源的AR的传播和调节提供一个范式转变，并指导未来新型抗菌药的战略开发。

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.