Silencing and reactivation of antimicrobial resistance genes in Enterobacteriaceae

越来越多的抗菌素耐药细菌感染威胁着全球公共健康。细菌可以在可移动的遗传元件上获得抗菌素耐药基因(ARG)，这使细菌能够在单个获得事件中对多种抗菌素产生抗药性。然而，当抗生素不存在时，质粒上的ARG通常会对细菌的生长产生负面影响。一个原因是相对于细菌染色体，ARG可以包含比G+C更多的A+T碱基，而A+T核苷酸在细菌中的可用性可能是有限的。为了绕过这一点，在缺乏抗菌剂的情况下，细菌可以使用一系列针对A+T丰富DNA的机制来沉默质粒编码的ARG，包括类似组蛋白的H-NS蛋白。Arg沉默可导致表型和基因之间的不匹配，细菌细胞含有ARG(基因)，但不表达它们，表现为表型敏感(表型)。这种不匹配可能会在感染的治疗过程中造成问题，因为抗菌素耐药性(AMR)是通过表型抗菌素敏感性分析来确定的，这有助于临床医生选择最合适的抗菌剂来治疗感染。这是控制抗菌药物使用和限制治疗期间AMR发展的重要部分，称为抗菌药物管理。沉默的ARGs的重新激活可能导致抗菌治疗不再起作用，这将对治疗结果和抗菌药物管理产生负面影响。Arg沉默还使AMR预测和AMR感染数量的监测复杂化，这对于开发诊断抗菌素耐药性感染和抗菌素管理的测试至关重要。本研究的目的是了解肠杆菌科细菌中编码Arg的质粒的沉默和再激活，包括三个主要组成部分：1.肠杆菌科临床分离株中的Arg沉默。在保持相同的氨基酸序列的同时，将合成具有不同A+T丰度的ARGs，并以质粒的形式引入肠杆菌科临床分离株。ARG沉默的频率将使用实验进化方法在营养丰富和营养贫乏的环境中确定，并使用qPCR和RT-PCR进行确认。这将提供关于影响ARG沉默的因素的重要信息，以及产生包含沉默的ARG的临床分离物的文库。肠杆菌科临床分离株的Arg再激活。使用实验进化方法，重新激活的ARG将被选择用于增加ARG和其他无关抗菌剂靶标的抗菌剂浓度。这将告诉我们抗菌素浓度对ARG重新激活的影响，以及使用无关的抗菌药是否也可以重新激活这些ARG。ARG再激活的分子机制。将使用显示ARG重新激活的分离株的全基因组和RNA测序来确定重新激活的基本机制。这将确定与ARG重新激活有关的任何基因表达的突变和/或变化。该项目将填补关于ARG沉默在表型和基因错配中所起作用的重要知识空白，加强对AMR的监测和预测。重要的是，这些数据将通过为治疗使用选择最佳抗菌剂来改善治疗选择和治疗结果。参考文献：enne，V.I.等人。2006年。大肠杆菌中抗生素耐药基因沉默的证据。抗菌剂和化疗。DOI：10.1128/AAC.00137-06 Fang，F.C.和Rimsky，S.2008。H-NS转录调控的新见解。微生物学的最新观点。DOI：10.1016/j.mib.2008.02.011.

The rising number of antimicrobial resistant bacterial infections threaten global public health. Bacteria can obtain antimicrobial resistant genes (ARG) on mobile genetic elements, such as plasmids, which enables bacteria to become resistant to multiple antimicrobials in a single acquisition event. However, ARGs present on plasmids often negatively affect bacterial growth when antibiotics are not present. One reason is that ARGs can contain more A+T bases than G+C relative to the bacterial chromosome and A+T nucleotide availability can be limited in bacteria. To circumvent this, in the absence of antimicrobials, bacteria can silence plasmid-encoded ARGs using a range of mechanisms that target A+T-rich DNA, including the histone-like protein H-NS. ARG silencing can cause mismatch between the phenotype and genotype, where the bacterial cell contains an ARGs (genotype) but does not express them, appearing phenotypically susceptible (phenotype). This mismatch can cause problems during the treatment of infections, as antimicrobial resistance (AMR) is determined using phenotypic antimicrobial susceptibility assays, which help clinicians choose the most appropriate antimicrobial to use to treat an infection. This is an important part of the control of antimicrobial usage and to limit the development of AMR during therapeutic use, termed antimicrobial stewardship. Reactivation of silenced ARGs can result in the antimicrobial treatment no longer working, which will negatively impact the outcomes of treatment and antimicrobial stewardship. ARG silencing also complicates AMR prediction and monitoring of the number of AMR infections, which are essential for the development of tests to diagnose antimicrobial resistant infections and antimicrobial stewardship. The aim of this project is to understand the silencing and reactivation of plasmid encoded ARGs in Enterobacteriaceae and will comprise of three main components:1. ARG silencing in clinical isolates of Enterobacteriaceae. ARGs will be synthesised with varying A+T-richness while maintaining an identical amino acid sequence and introduced into clinical isolates of Enterobacteriaceae on a plasmid. Frequency of ARG silencing will be determined using an experimental evolution approach in nutrient-rich and nutrient-poor environments and confirmed using qPCR and RT-PCR. This will provide important information on factors which effect ARG silencing, as well as producing a library of clinical isolates containing silenced ARGs.2. ARG reactivation in clinical isolates of Enterobacteriaceae. Using an experimental evolution approach, reactivated ARGs will be selected for in increasing concentrations of antimicrobials which are both targeted by the ARG and other unrelated antimicrobials. This will tell us the effect of antimicrobial concentration on ARG reactivation and whether the use of unrelated antimicrobials can also reactivate these ARGs.3. Molecular mechanisms of ARG reactivation. Whole genome and RNA sequencing of isolates displaying ARG reactivation will be used to identify the underlining mechanisms of reactivation. This will determine any mutations and/or changes in gene expression which are involved in ARG reactivation.This project will fill a significant knowledge gap about the role of ARG silencing in mismatch between phenotype and genotype, enhancing the monitoring and prediction of AMR. Importantly, this data will improve treatment options and treatment outcomes by informing the selection of the best antimicrobial for therapeutic use. References:Enne, V. I., et al. 2006. Evidence of Antibiotic Resistance Gene Silencing in Escherichia coli. Antimicrobial Agents and Chemotherapy. DOI: 10.1128/AAC.00137-06Fang, F.C. and Rimsky, S. 2008. New insights into transcriptional regulation by H-NS. Current Opinion in Microbiology. DOI: 10.1016/j.mib.2008.02.011.