Quantifying Antibiotic Resistance Evolution in Clinically-Relevant Microbes

当我们用通常花费巨大成本发现或设计的药物治疗受感染患者时，经常观察到耐药性，这些药物的明确目的是治愈人们的传染病。例如，这种情况发生在艾滋病患者、疟疾患者身上，或者当大肠杆菌等致病微生物进入某人的血液时。癌症很快就会对我们投给它们的化疗药物产生抗药性，这一切都是因为进化。耐药性的进化过程可能需要时间。在引入一种新药之后，我们可能需要数年甚至数十年的时间才能看到对它的临床耐药性的证实，而十年的时间尺度被认为是许多抗生素的典型。不幸的是，这阻止了制药公司寻找新的抗生素分子。毕竟，他们为什么要花费10年的时间，花费巨大的成本，用一种在市场上只能再盈利10年的药物来治疗疾病呢？有趣的是，肿瘤的耐药性在病人身上出现的时间要短得多，有时是在化疗开始的几个月内，这取决于使用的药物、肿瘤类型和病人的个人情况。那么，为什么我们不应该观察到抗生素的类似现象呢？事实上，我们确实如此，而且我们现在看到数据集的出现，表明细菌病原体可以在个体患者体内进化出耐药性，因为细菌的DNA在短短几周，甚至几天内发生了变化；&它可能是致命的。该提案引用了2015年的一项研究（Blair等人，PNAS），该研究追踪了血液传播沙门氏菌感染对抗生素治疗的耐药性，一周一周，持续了20周，随后患者死亡。这项全基因组测序研究使用了一系列旨在实时跟踪进化的计算机和物理建模技术，非常精确地显示了沙门氏菌群体中多种蛋白质的表达水平和结构的改变如何迅速改变了感染的抗性谱。在一周内，这个群体产生的外排蛋白量增加了一倍，而且，它现在产生的外排蛋白甚至比原来的更好，感染了沙门氏菌。外排蛋白被用来将抗生素从沙门氏菌细胞中抽出来，以阻止抗生素击中目标，因此它们停止工作，但这只是已确定的多种机制之一，这些机制与治疗期间发生的耐药性变化有关。重要的是要提到“质粒”，即由不同微生物物种传播到地球各地的DNA环，这些微生物物种对一系列抗生素具有耐药性，鉴于这些，如果不采取措施减缓这种快速进化，我们未来应对微生物感染的能力似乎处于非常危险的状态。但我们能做些什么呢？重要的是，2015年的研究暗示了这种可能性。它表明，细菌对某些抗生素变得敏感，因为它们对其他抗生素的抵抗力增强了；换句话说，在治疗过程中会出现交叉或侧络敏感性。所以，有时候，一个人可以用一种抗生素，然后用另一种抗生素。这并不奇怪，这是一个已经在幽门螺杆菌感染的临床试验中的想法，但除此之外，我们现在需要找到新的交叉敏感性。我们还需要将抗生素组合成新型鸡尾酒的新方法，这里也提出了其中一些方法。我认为，通过采用现代的数学建模和数据分析工具，对实验室中使用抗生素的微生物进行分析，通过观察它们的反应，我们可以找到它们防御的薄弱环节，这将有助于临床医生设计新的治疗方法，并为制药公司提供新的分析方法。事实上，这种情况现在正在发生，我正在寻求资金来继续我的小组在这项任务中的努力。

Drug resistance is often observed when we treat infected patients with drugs that were discovered,or designed, usually at great cost, with the express purpose of curing people of their infectious disease.This happens, for example, to HIV patients, malaria sufferers or when a pathogenic microbe, like E. coli, finds its way into someone's bloodstream. Cancers can soon become resistant to the chemotherapeutic agents we throwat them too, & all because of evolution.The evolutionary march towards drug resistance can take time. It can be years, ordecades, after the introduction of a new drug before we see confirmation of clinical resistance to it anda ten-year timescale is thought typical of many antibiotics. Unfortunately, this stops pharmaceutical companies fromseeking new antibiotic molecules. After all, why should they spend 10 years, at great cost, seeking to curea disease with a pill that is profitable in the marketplace for only 10 more years?Intriguingly, drug resistance in tumours is seen in patients on a much shorter timescale,sometimes within months of the start of chemotherapy, depending on the drug used, the tumourtype, and on the individual patient. So why should we not observe a similar phenomenon for antibiotics?In fact, we do, & we are now seeing the emergence of datasets showing that bacterial pathogenscan evolve resistance within individual patients because of changes to the DNA of that bacteriumin a matter of mere weeks, even days; & it can be lethal.This proposal cites a 2015 study (Blair et al, PNAS) whereby resistance to antibiotic treatment in ablood-borne Salmonella infection was traced, week-by-week, over a 20-week period, whereupon the patient died.That whole-genome sequencing study, using a range of computer and physical modelling techniquesdesigned to track evolution in real time, showed very precisely how the resistance profile of the infection quicklychanged by altering expression levels and structures of a variety of proteins within the Salmonella population.Within a week the population had doubled the amount of efflux protein it was making, moreover, it was now making even better efflux proteins than the original, infecting Salmonella. The efflux proteins are used to pump the antibiotics from inside Salmonella cells to prevent the antibiotic from hitting its target, so they stop working, but this was just one of a variety of mechanisms identified that were shown to correlate with the changes in drug resistance that took place during treatment.It is important to mention 'plasmids', loops of DNA that are disseminated across the planet by differentmicrobial species that provide resistance to a range of antibiotics, given these, it seems our future ability to deal with microbial infection sits in a terribly parlous state if something is not done to mitigate such rapid evolution. But what can be done?Importantly, the 2015 study hints at possibilities. It shows that bacteria become susceptible to someantibiotics as they increase resistance to others; in other words there are cross- or collateral-sensitivities that emergeduring treatment. So, sometimes, one could use one, and then another antibiotic. This is not outlandish, it is anidea that has been trialled in the clinic for Helicobacter pylori infections, but little else, so we now need to findnovel cross sensitivities. We also need new ways of combining antibiotics into novel cocktails, & some of those areproposed here too.I claim that by bringing to bear modern tools of mathematical modelling and data analysis on microbes thatare subjected to antibiotics in the laboratory, by observing how they respond, we can find weak spotsin their defences that will help clinicians design new therapies & give pharma companies newmethodologies to use within their analysis pipelines. Indeed, this is happening now & I am seeking funding to continue the efforts of my group in this task.