OneAMRdx: real-time, sequencing-based diagnostics for the detection and prevention of antimicrobial resistance (AMR)

抗菌素耐药性（AMR）是对公众健康的严重威胁，最近的一项研究发现，2019年全球有130万人死于AMR。按照目前的轨迹，到2050年死亡人数将增加到1000万。抗生素耐药性是一个全球性问题，抗生素的过度使用加剧了这一问题，如果不加以控制，我们将没有任何有效的药物来治疗感染，即使是小手术也会使患者面临感染的风险。肺炎克雷伯菌（Klebsiella pneumoniae）是一种经常对所有抗生素具有耐药性的细菌，是医院获得性感染（如败血症和肺炎）的主要原因。与同一物种的非耐药菌株相比，耐药感染与住院时间更长、治疗费用增加以及患者预后更差有关。为了治疗感染，医生需要知道它是由什么微生物引起的，以及什么样的抗生素对它有效。抗微生物药物敏感性测试的金标准包括培养或培养微生物，以找到能阻止其生长的最低抗生素浓度，即MIC值。这一过程在大型医院实验室中通常是自动化的，但仍然需要两天到几周的时间才能完成。医生会开一种广谱抗生素，然后观察治疗是否有效。这意味着一定比例的患者服用的抗生素是无效的，这实际上增加了耐药性。如果有技术可以快速识别耐药性，它将提高早期治疗的有效性，从而获得更好的结果，减少住院时间，减少临床环境中的耐药性。其中一项技术是纳米孔测序，当单个DNA分子通过一个微小的蛋白质孔时，它们就会被读取。序列读数可用于确定引起感染的原因以及治疗感染的最佳抗生素。这个过程被称为临床宏基因组测序，这意味着测序不需要分离或培养。宏基因组学在发现方面很强大，但有时一种有针对性的方法，如寻找特定的东西，更敏感，因为筛选的背景噪音更少，而且成本更低。另一项技术是单细胞测序，这是一种将单细胞捕获在微小液滴中的方法。这是通过一种微流体装置来完成的，这是一小块带有通道的塑料，其中悬浮在水和油中的细胞通过通道遇到连接点。因为油和水不能混合，结果是一种含有数百万个细胞的小液滴的乳液，这些细胞可以单独分析。在这个项目中，我将开发利用纳米孔测序和单细胞方法检测抗生素耐药性的靶向方法。这些液滴就像一个袋子，确保细胞中没有任何东西，比如质粒，它是一种小的DNA环，通常含有抗性基因，从细胞的其他内容物中分离出来，这对准确的抗性预测很重要。使用纳米孔测序可以在几分钟内完成读取，而不是像现有的培养方法那样在几天内完成。这个项目正在开发技术，以便将来可以进行快速、廉价的测试，这些测试可以在全科医生诊所或医院进行，让医生做出自信的处方决定，而不会导致耐药性。更好的处方加上新开发的药物和非药物干预措施将保护抗生素的有效性，并避免抗生素的末日，即人们死于无法治疗的感染，甚至无法进行常规手术。抗菌素耐药性合作者。2019年全球细菌抗微生物药物耐药性负担：系统分析。柳叶刀。2022年2月12日；399(10325):629-655。

Antimicrobial resistance (AMR) is a serious threat to public health with a recent study finding that 1.3M deaths were attributable to AMR globally in 2019[1]. This would increase to 10M deaths in 2050 on the current trajectory. AMR is a global problem that has been accelerated by the overuse of antibiotics, if not brought under control it will leave us without any effective drugs to treat infections and putting patients at risk of infection even with minor procedures. One bacterial species, Klebsiella pneumoniae, is frequently resistant to all antibiotics and is a major cause of hospital-acquired infections such as sepsis and pneumonia. Resistant infections are associated with longer hospital stays, increased costs of treatment and worse outcomes for patients when compared with non-resistant strains of the same species. In order to treat an infection a doctor needs to know what organism it is caused by and what antibiotics will be effective against it. The gold standard antimicrobial susceptibility testing involves growing or culturing the organism to find the lowest concentration of antibiotic that will stop it growing, a value known as the MIC. This process is often automated in large hospital laboratories but can still take between two days and several weeks to complete. Doctors will prescribe a broad-spectrum antibiotic and wait to see if the treatment is effective. This means a certain percentage of patients receive antibiotics that are ineffective which actually increases resistance. If technology was available that could rapidly identify resistance it would improve the effectiveness of the early therapy leading to better outcomes, reduced hospital stays and less resistance in the clinical environment.One such technology is nanopore sequencing in which individual DNA molecules are read as they pass through a tiny protein pore. The sequence read-off can be used to determine what is causing an infection and which antibiotics would be the best to treat it. This process is known as clinical metagenomic sequencing which means sequencing without the need for isolation or culture. Metagenomics is powerful for discovery but sometimes a targeted approach i.e. looking for something specific, is more sensitive as there is less background noise to sift through and it is cheaper. Another technology is single-cell sequencing which is a way to trap single-cells inside tiny droplets. This is done using a microfluidics device, a small piece of plastic with channels running through it, in which cells suspended in water and oil travelling through the channels meet junctions. Because oil and water don't mix the result is an emulsion with millions of tiny droplets containing individual cells which can be analysed separately.In this project I will develop targeted methods for detecting antibiotic resistance using nanopore sequencing and single-cell methods. The droplets act as a bag to make sure nothing from the cell e.g. plasmids which are small rings of DNA often containing resistance genes, gets separated from the rest of the cells' contents which is important for accurate resistance prediction. The use of nanopore sequencing allows the read-out to be performed in minutes rather than days like for existing culture methods. This project is developing the technology so that rapid, cheap tests will be available in future, tests that can be performed in a GP surgery or hospital allowing the doctor to make confident prescribing decisions that don't lead to resistance. Better prescribing coupled with newly developed drugs and non-pharmaceutical interventions will protect the effectiveness of antibiotics and avoid an antibiotic apocalypse where people die of untreatable infections and even routine operations are impossible to perform.Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022 Feb 12;399(10325):629-655.