Silencing and reactivation of antimicrobial resistance genes in Enterobacteriaceae

越来越多的耐药性细菌感染威胁着全球公共卫生。细菌可以在移动的遗传元件（例如质粒）上获得抗微生物剂抗性基因（ARG），这使得细菌能够在单个获取事件中变得对多种抗微生物剂具有抗性。然而，当不存在抗生素时，质粒上存在的ARG通常会对细菌生长产生负面影响。一个原因是相对于细菌染色体，ARG可以含有比G + C更多的A + T碱基，并且A + T核苷酸的可用性在细菌中可能是有限的。为了避免这种情况，在没有抗菌剂的情况下，细菌可以使用一系列靶向富含A + T的DNA的机制来沉默质粒编码的ARG，包括组蛋白样蛋白H-NS。ARG沉默可导致表型和基因型之间的错配，其中细菌细胞含有ARG（基因型）但不表达它们，表现为表型易感（表型）。这种不匹配可能在感染治疗过程中引起问题，因为抗菌素耐药性（AMR）是使用表型抗菌药物敏感性测定来确定的，这有助于临床医生选择最合适的抗菌素来治疗感染。这是控制抗菌药物使用和限制治疗使用期间AMR发展的重要组成部分，称为抗菌药物管理。沉默ARG的重新激活可能导致抗菌治疗不再有效，这将对治疗和抗菌管理的结果产生负面影响。ARG沉默也使AMR预测和AMR感染数量的监测复杂化，这对于开发诊断抗菌剂耐药性感染和抗菌剂管理的测试至关重要。本项目的目的是了解肠杆菌科中质粒编码的ARGs的沉默和再激活，将包括三个主要部分：1。肠杆菌科临床分离株中的ARG沉默。ARG将以不同的A + T丰度合成，同时保持相同的氨基酸序列，并在质粒上引入肠杆菌科的临床分离株中。ARG沉默的频率将使用实验进化方法在营养丰富和营养缺乏的环境中确定，并使用qPCR和RT-PCR确认。这将提供关于影响ARG沉默的因素的重要信息，以及产生含有沉默ARG的临床分离株的文库。肠杆菌科临床分离株中的ARG再活化。使用实验进化方法，将选择重新激活的ARG，以增加ARG和其他不相关抗菌剂靶向的抗菌剂浓度。这将告诉我们抗菌剂浓度对ARG再激活的影响，以及使用无关的抗菌剂是否也可以再激活这些ARG。ARG再激活的分子机制。显示ARG再激活的分离株的全基因组和RNA测序将用于确定再激活的基本机制。该项目将填补关于ARG沉默在表型和基因型错配中的作用的重大知识空白，加强AMR的监测和预测。重要的是，这些数据将通过为治疗用途提供最佳抗菌剂的选择，改善治疗选择和治疗结果。参考文献：Enne，V. I.，等，2006年。大肠杆菌耐药基因沉默的证据。抗微生物剂和化疗。DOI：10.1128/AAC.00137 - 06方，F.C.和Rimsky，S. 2008.对H-NS转录调控的新见解。目前微生物学的观点。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.