How do biocides interact with bacterial membranes to disinfect?

过去几年，越来越多的医院获得性感染以及由受污染的固体表面传播的Covid感染对我们有效对抗有害微生物的能力提出了挑战。阳离子表面活性剂（季氮化合物或简称quats）是应用最广泛的消毒剂之一。尽管它们被广泛使用，但对于阳离子表面活性剂如何与微生物膜相互作用以及如何在其他添加剂（如硬水离子、非离子表面活性剂）存在的情况下杀死病原体，人们知之甚少。这种情况限制了我们设计和配制有效消毒剂的能力。弱或无效的消毒剂，当用于硬表面清洁（手术设备，床和通风系统，甚至食品和乳制品加工设施）时，可能导致疫情爆发，可能造成数百万美元的损失，在某些情况下，还会造成人员伤亡。配制的阳离子quat消毒剂清洁产品通常含有非离子表面活性剂以提供清洁效果。尽管经过几十年的研究和发展，人们对非离子在消毒中的作用知之甚少。这种理解的缺乏在新产品开发时造成了真空。如果没有这类混合物的界面杀生物作用的重要信息，就很难平衡产品配方中的quats水平。杀菌剂分子可以通过强大的静电吸引与微生物膜结合，并通过结构破坏和膜泄漏杀死病原体。从扫描电子显微镜和共聚焦显微镜等成像研究中观察到的细菌和真菌崎岖或破碎的表面很好地支持了这种作用模式。通过膜渗透探针、荧光检测、zeta电位测量和动态光散射等脂膜模型，如扩散脂质单层、支撑脂质双层和小单层囊泡（suv），也监测了膜的破坏。与实际微生物测量的高一致性验证了模型膜方法。然而，目前的技术对膜内通常在1-5纳米范围内的结构变化没有灵敏度或分辨率。在生物杀灭剂结合过程中缺乏跟踪结构变化的能力使得区分一种生物杀灭剂和另一种生物杀灭剂或探索不同膜的影响变得困难。中子反射（NR）和散射（SANS）是仅有的两种技术，可以在有或没有非离子表面活性剂的情况下，深入了解脂质膜在四离子结合时的结构变化。在这个探索性项目中，中子实验的成功演示取决于(a)在中子实验的运行、氘化表面活性剂的合成以及ISIS团队的数据分析和解释方面的中子专业知识的投入，(b)曼彻斯特团队的抗菌工作和模型脂质膜的选择方面的专业知识，以及(c) Arxada积极参与将模型界面研究与实际quat配方联系起来。项目团队共同制定了具有挑战性的工作计划，该计划将由一位非常能干的PDRA廖明瑞博士负责，他在目前的PDRA工作中已经掌握了四价杀菌剂的先验知识，并在他的抗菌肽博士研究中进行了中子实验。该项目的成功成果将为合作团队向BBSRC和EU发送联合资助申请，以寻求更系统的中子实验，参与这一新的研究领域奠定坚实的基础。研究小组还将在JACS和Nat common等国际领先期刊上发表他们的研究结果。

The growing Hospital Acquired Infections as well as Covid infections transmitted from contaminated solid surfaces over the past few years have challenged our ability to fight harmful microorganisms effectively. Cationic surfactants (quaternary nitrogen compound or abbreviated as quats) are one of the most widely used disinfectants. Despite their intensive use, little is actually known about how cationic surfactants interact with microbial membranes and kill the pathogens in the presence of other additives, e.g. hard water ions, nonionic surfactants. This situation limits our ability to design and formulate effective disinfectants. Weak or ineffective disinfectants, when used in hard surface cleaning (surgical devices, beds and airing systems and even food and diary processing facilities), could lead to outbreaks that could cost millions and, in some cases, have casualties. A formulated cationic quat disinfectant cleaning product often contains nonionic surfactants to provide cleaning efficacy. Despite decades of research and development, little is known about the role of the nonionics in disinfection. This lack of understanding creates a vacuum when new products are developed. Without this vital information about the interfacial biocidal action of such blends it is difficult to balance the levels of quats in product formulation.Biocidal quat molecules can bind (and usually do) to microbial membranes via strong electrostatic attraction and kill pathogens by structural disruption and membrane leakage. This mode of action is well supported by the rugged or disrupted surfaces of bacteria and fungi viewed from imaging studies such as scanning electron microscopy and confocal microscopy. Membrane disruptions have also been monitored by membrane permeation probes and fluorescence detection, zeta potential measurements and dynamic light scattering using lipid membrane models, such as spread lipid monolayer, supported lipid bilayer and small unilamellar vesicles (SUVs). High consistency to real microbial measurements validates the model membrane approaches. However, current techniques do not have the sensitivity or resolution to structural changes within the membrane which is typically in the region 1-5 nm. Lack of capability to follow structural changes during a biocide binding makes it difficult to distinguish one biocide from another or explore the impacts of different membranes. Neutron reflection (NR) and scattering (SANS) are about the only techniques that offer the insights into the structural changes across lipid membranes upon quat binding, with and without nonionic surfactant. Successful demonstration of the neutron experiments in this exploratory project replies on (a) input of neutron expertise in the running of the neutron experiments, synthesis of the deuterated surfactants and data analysis and interpretation from the ISIS team, (b) the expertise of antimicrobial work and selection of model lipid membranes from the Manchester team and (c) the active participation of Arxada in relating model interfacial studies to real quat formulations. The project teams have worked together to devise the challenging workplan that will be delivered by a highly able PDRA, Dr Mingrui Liao, who has already had prior knowledge of the quat biocides in his current PDRA work and neutron experiments from his PhD research on antimicrobial peptides. Successful outcomes from this project will form a strong basis for the collaborating teams to send joint grant applications to BBSRC and EU to engage in this new area of research by seeking more systematic neutron experiments. The teams will also publish their results in leading international journals such as JACS and Nat Commun.