Bacterial motility and chemotaxis as drivers of antimicrobial resistance in biofilms

抗菌素耐药性(AMR)的演变和传播现在被认为是对全球健康、粮食安全和社会经济发展的主要威胁之一，据预测，每年将导致1000万人死亡，到2050年累计造成100万亿美元的损失。因此，AMR成为一个主要的研究主题并成为BBSRC基金等研究资金的关键战略优先事项也就不足为奇了。AMR研究中的一个重要问题是理解AMR最初是如何出现的，大量文献对这一主题做出了贡献。从这些研究中，我们现在知道，例如，亚致死浓度的抗生素和其他抗菌剂促进了抗菌素耐药性的演变，但我们仍然对到底是什么推动了这种影响知之甚少。重要的是，绝大多数抗生素研究都集中在同质培养上，通常是在摇瓶中，这与细菌在自然环境中的经历形成了鲜明对比。细菌通常附着在表面被称为生物膜的群落中，这种群落对抗菌压力特别有弹性。在生物膜中，细菌经历了陡峭而稳定的营养和抗生素等有毒化合物的梯度，但令人惊讶的是，我们对它们如何对这种梯度做出反应知之甚少。特别是，我们不知道生物膜细菌如何对抗生素梯度做出反应，以及这种反应如何影响抗生素耐药性的出现。我在这里的研究将解决这个重要但很少被探索的话题。更具体地说，我将阐明生物膜细胞如何在抗生素的梯度中控制它们的运动性，以及这种行为如何影响抗生素耐药性的演变。虽然我们对自由漂浮的细菌如何游泳和改变它们的运动有详细的了解，但我们对生物膜细胞的运动性控制的了解非常有限。直到最近，我们甚至不知道表面附着的细菌是否可以跟踪化学梯度(趋化性)。我解决了这个问题，开发了基于微流控梯度和大规模并行自动跟踪的新型分析方法，以研究人类病原体铜绿假单胞菌的趋化行为，并发现单个生物膜细菌可以有效地跟踪化学梯度。特别是，我已经证明了生物膜细菌控制着名为IV型菌毛的抓握状钩子，以爬上营养和其他典型的化学诱导剂的梯度。生物膜细胞并不像人们经常描绘的那样行动迟缓，而是以亚微米级的精度在群落中定位。这种新的生物被膜行为如何有助于它们对抗微生物压力的内在抵抗力？为了回答这个问题，我最近一直在使用同样的分析方法来了解生物膜细胞如何在稳定和明确的抗生素梯度中控制它们的运动，并发现生物膜细菌确实在这种梯度中偏向它们的运动。出人意料的是，单个细胞积极地转向增加抗生素的浓度，达到极高的浓度，在同质条件下很容易杀死它们，进一步的研究表明，这种非凡的能力依赖于表型耐药性。这种行为的遗传基础是什么？它为什么会演变？重要的是，运动控制是否有助于出现对抗生素的遗传耐药性？这些都是我将在这个研究项目中解决的问题。我将进行基因筛查，以澄清哪些细菌基因对微流控梯度中的抗生素产生偏向运动至关重要。此外，我将阐明生物膜细胞如何控制它们的运动性，以应对在附近生长的产生抗生素的物种，这将有助于我们了解这种行为是如何在自然环境中进化的。此外，我将结合微生物基因组学和数学模型来精确量化细菌的运动性控制如何有助于抗生素耐药性的进化。

The evolution and spread of antimicrobial resistance (AMR) is now considered one of the major threats to global health, food security, and socio-economic development, being predicted to cause 10 million human deaths annually, and a cumulative cost of $100 trillion by 2050. It is thus not surprising that AMR became a major research topic, and a key strategic priority for research funding such as BBSRC funding.An important question in AMR research is understanding how AMR emerges in first place, and a wealth of literature has contributed to this topic. From these studies we now know for example that sublethal concentrations of antibiotics and other antimicrobials facilitate the evolution of antimicrobial resistance, but we still know very little about what exactly drives this effect. Importantly, the vast majority of antibiotic research focuses on homogenous cultures, often in shaking flasks, which is in sharp contrast to what bacteria experience in natural settings. Bacteria often live attached to surface in communities called biofilms, which are particularly resilient to antimicrobial stress. In biofilms, bacteria experience steep and stable gradients of nutrients and toxic compounds such as antibiotics, but we know surprisingly little about how they respond to such gradients. In particular we do not know how biofilm bacteria respond to antibiotic gradients and how this respond affects the emergence of antibiotic resistance. My research here will address this important but poorly explored topic. More specifically, I will clarify how biofilm cells control their motility in gradients of antibiotics, and how this behaviour affects the evolution of antibiotic resistance.While we have detailed understanding of how free-floating bacteria swim and bias their motion, our understanding of motility control in biofilm cells is very limited. Until recently we did not even know if surface-attached bacteria could track chemical gradients (chemotaxis). I addressed this problem by developing novel assays based on microfluidic gradients and massively-parallel automated tracking to study the chemotactic behaviour of the human pathogen Pseudomonas aeruginosa, and found that individual biofilm bacteria can effectively track chemical gradients. In particular, I have showed that biofilm bacteria control grappling-like hooks called type IV pili to climb gradients of nutrients and other canonical chemoattractants. Rather than being sluggish as often pictured, biofilm cells position themselves within the community with submicron precision. How does this novel biofilm behaviour contribute to their intrinsic resistance to antimicrobial stress? To answer this question, I have recently been using the same assays to understand how biofilm cells control their motility in stable and well-defined gradients of antibiotics and found that biofilm bacteria indeed biased their motion in such gradients. Unexpectedly, individual cells actively move towards increasing concentrations of antibiotics, reaching extremely high concentrations that would readily kill them in homogenous conditions, and further studies showed that this remarkable ability relies on phenotypic resistance. What is the genetic basis of this behaviour? And why did it evolve? Importantly, does motility control contribute the emergence of genetic resistance to antibiotics? These are the questions I will address in this research project.I will conduct a genetic screen to clarify which bacterial genes are critical for biased motility towards antibiotics in microfluidic gradients. Moreover, I will clarify how biofilm cells control their motility as a response to antibiotic-producing species growing in the neighbourhood, which will help us to understand how the behaviour evolved in natural settings. Additionally, I will combine microbial genomics and mathematical modelling to precisely quantify how motility control in bacteria contributes to the evolution of antibiotic resistance.