The evolutionary dynamics of multiazole resistance in pathogenic Aspergillus fungi

真菌烟曲霉在全球范围内普遍存在于环境中，存在于腐烂的植被和土壤中，在养分循环中发挥着宝贵的作用。这种真菌对健康人的健康威胁最小。然而，患有囊性纤维症、癌症或接受过器官移植并正在接受皮质类固醇治疗的患者，有患侵袭性曲霉病的风险。目前的估计表明，整个欧洲每年有超过6.3万名患者患上这种真菌疾病。控制感染的主要方法是使用唑类抗真菌药物。然而，我们和其他人已经显示出烟曲霉菌对一线唑类抗真菌药物的抗药性急剧增加，这些高危患者组的死亡率高得令人无法接受。使烟曲霉对这些药物产生抗药性的突变似乎是在环境中进化的，而不是在患者身上。唑类化合物也被用作杀菌剂来控制作物病害。这导致了一种假设，即在农作物中广泛使用唑类抗真菌喷雾剂会导致对烟曲霉的耐药性进行环境选择，从而导致感染后患者存活率下降。我们的项目旨在通过使用含有抗真菌药物的生长介质对环境人口进行采样来确定英国对唑类抗药性和对唑类敏感的烟曲霉的相对比例，从而验证这一假设。这种环境暴露评估方法将针对作物抗真菌药物的使用量从高到低的环境，并使我们能够从统计上检查环境中大量使用这些以唑为基础的化合物与抗药性烟曲霉菌的发生之间是否存在联系。然后，我们将使用强大的技术对数百种对唑类抗真菌药物敏感或耐药的烟曲霉的基因组进行测序。我们已经通过一个广泛的项目合作伙伴网络从世界各地预先收集了大量分离株，我们现在知道，广泛发生的两种主要的唑类耐药性突变。我们的计划是使用我们的基因组序列和尖端的统计遗传学方法来确定这些突变起源于全球的时间和地点，使用我们新分离的样本来测试它们是否发生在英国的环境和患者群体中，它们是否正在传播到入侵这里和其他地方的新环境，以及是否存在新的未描述的抗性突变。烟曲霉菌能够有性繁殖和无性繁殖。在这种情况下，新进化的抗性突变整合到新的遗传背景中的速度取决于烟曲霉种群的繁殖力。因此，为了直接测量烟曲霉菌种群的“性感”，我们将使用测序分离株进行有性杂交，测序分离株不仅代表我们遇到的遗传多样性的范围，而且代表对唑类抗药性突变的范围。通过测量后代的数量和适合度，我们将能够确定耐药突变重组到新的遗传背景的速度，并发现未知的耐药机制。通过解决这些问题，我们将直接测量使用抗真菌化合物对非靶标真菌物种进化耐药性的风险，并回答有关这些空气传播的真菌能够传播和共享基因的距离的重要问题。我们的发现不仅对卫生保健专业人员具有很高的相关性，直接为重症监护环境中的诊断方案和疾病管理提供信息，而且还将为目前关于在环境中广泛使用抗菌化合物的成本的辩论提供信息。这些目标都直接为NERC提供了新的战略方向--环境业务。

The fungus Aspergillus fumigatus is globally ubiquitous in the environment, being present on decaying vegetation and in soils, where it performs a valuable role in nutrient recycling. The fungus is a minimal health threat to healthy individuals. However, patients that suffer from cystic fibrosis, cancer or have received organ transplants and are undergoing corticosteroid therapy, are at risk from 'invasive aspergillosis'. Current estimates indicate that over 63,000 patients develop this fungal disease annually across Europe. The primary method for controlling infections is by administering azole antifungal drugs. However, we and others have shown a sharp increase in the resistance of A. fumigatus to frontline azole antifungals, with unacceptably high mortality rates in these at-risk patient groups. The mutations that confer resistance of A. fumigatus to these drugs appear to have evolved in the environment, rather than in the patient. Azole compounds are also used as fungicides to control crop diseases. This has led to the hypothesis that the widespread use in agricultural crops of azole antifungal sprays is leading to the environmental selection for resistance in A. fumigatus, which is then resulting in decreased patient survival following infection.Our project aims to examine this hypothesis by determining the relative proportions of azole-resistant and azole-sensitive A. fumigatus in the UK by sampling environmental populations using growth media containing antifungal drugs. This environmental exposure assessment approach will target environments that have had high to low applications of crop-antifungals and will enable us to statistically examine whether there are links between the intensive use of these azole-based compounds in the environment and the occurrence of drug-resistant A. fumigatus. We will then use powerful technologies to sequence the genomes of many hundreds of A. fumigatus that are sensitive, or resistant, to azole antifungals. We already have numerous isolates pre-collected from around the world though a broad network of project partners, and we now know that there are two main azole-resistance mutations that widely occur. Our plan is to use our genome sequences and cutting-edge statistical genetic methods in order to determine when and where these mutations originated globally, use our newly isolated samples to test whether they occur within the UK environment and patient populations, whether they are spreading to invade new environments here and elsewhere, and whether novel undescribed resistance mutations exist.A. fumigatus is capable of sexual, as well as asexual, reproduction. In this case, the rate at which a newly-evolved resistance mutation can be integrated into new genetic backgrounds depends on the fertility of the A. fumigatus populations. In order to directly measure the 'sexiness' of the A. fumigatus populations, we will therefore perform sexual crosses using sequenced isolates that represent not only the range of genetic diversity that we encounter, but also the range of azole-resistance mutations. By measuring the number and fitness of progeny, we will be able to determine the rate at which resistance mutations can recombine into new genetic backgrounds, and also discover unknown drug-resistance mechanisms.By addressing these questions, we will directly measure the risk that the use of antifungal compounds has on evolving resistance in non-target fungal species, and also answer important questions on the distance that these airborne fungi are able to spread and share genes with one another. Our findings will not only be of high relevance to health care professionals, directly informing diagnostic protocols and disease management in intensive-care settings, but will also inform current debates on the costs of widespread use of antimicrobial compounds in the environment. These goals all directly feed into NERCs new strategic direction 'The Business of the Environment'.