Investigating phenotypic changes in wound biofilms in response to antimicrobial treatment using Raman Spectroscopy.

伤口感染，如糖尿病足溃疡(DFU)，在临床管理方面可能是非常有问题的。这种感染带来了巨大的社会经济负担，在2017/18年度，NHS估计花费了83亿GB。DFU的死亡率很高，5年死亡率与癌症相当。然而，令人担忧的是，与癌症研究相比，这并没有反映在慢性伤口的资金水平上。临床医生在治疗慢性伤口时面临的主要困难是伤口部位有感染的风险，这是由于创面床上积累的微生物聚集体引起的。感染伤口是由复杂的生物膜形成的，这些生物膜在很大程度上对抗生素治疗具有抵抗力：其中大多数本质上是多菌的。这进一步使治疗制度复杂化，以减轻混合社区内的特定病原微生物。鉴于创面微生物群的日益复杂以及抗菌素耐药性的日益威胁，临床医生面临着靶向局部治疗或全身抗生素的挑战性前景。这可能是极其困难的，因为伤口护理中的护理点技术在微生物检测方面是有限的，临床医生依赖基础培养或分子方法学，可能需要几天的时间才能取得结果。因此，仍然需要快速、灵敏、非侵入性的床边微生物鉴定方法，以帮助伤口处理期间的临床治疗选择。拉曼是一种信息量丰富的振动技术，可以在亚细胞水平提供无标记的生化信息，但单细胞水平的高分辨率成像是一个相对较慢的过程，这使得它对活细胞成像具有挑战性。受激拉曼散射(SRS)是一种更敏感、更快速的技术，与普通拉曼相比，它可以提供关于更大细胞群体的高分辨率信息。SRS提供亚细胞分辨率的详细生化信息，包括蛋白质、脂肪和核酸位置的测定。由于它的速度和以无标记方式成像更大区域的能力，它在成像细菌生物膜方面具有巨大的潜力，既可以提供生物膜形成的生化信息，也可以提供生物膜形成的结构信息，以及抗菌素处理后生物膜的变化。到目前为止，有证据表明，不同的细菌物种表现出独特的光谱特征，这取决于它们在细胞水平上的化学结构。现有的研究在很大程度上局限于单细胞，很少有使用SRS来研究生物膜动力学的研究。本提案的总体目标是测试使用SRS监测生物膜形成的可行性，并评估与伤口感染相关的不同细菌和真菌物种的独特的拉曼光谱曲线或特征。1使用SRS监测单一物种伤口生物膜模型的生长动力学--比较不同临床相关微生物物种(革兰氏+菌、革兰氏细菌和真菌生物)在生物膜附着、成熟和扩散过程中的光谱曲线。2使用各种微生物、分子、显微和光谱技术--SRS用于评估生物膜中对细菌(或真菌)静态或杀伤性抗生素和其他新化合物的化学特征。3使用SRS评估多物种生物膜模型中生物膜形成的变化。可视化包含不同微生物物种的生物膜化学结构的表型变化。4用于描述真实世界生物膜的SRS(将通过现有的EPSRC赠款EP/V005839/1生成的伤口感染的临床样本生成的SRS)。利用已知成分的简单生物膜模型通过拉曼光谱分析确定的化学结构的特征变化。

Wound infections such as diabetic foot ulcers (DFUs) can be highly problematic in terms of clinical management. Such infections carry a significant socioeconomic burden, costing the NHS an estimated £8.3b in 2017/18. DFUs have significant mortality rates, with 5-year mortality rates comparable with cancer. However, worryingly this is not reflected in the level of funding for chronic wound compared to cancer research.The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site, arising from the accumulation of microbial aggregates in the wound bed. Infected wounds arise from the formation of complex biofilms that can be largely resistant to antibiotic therapy: most of which are polymicrobial in nature . This further complicates treatment regimes to alleviate specific pathogenic microorganisms within the mixed community. Given the increased complexity of the wound microbiome as well as the increasing threat of antimicrobial resistance, clinicians are faced with the challenging prospect of targeted topical therapy or systemic antibiotics. This can be extremely difficult as point-of-care technologies in wound care are limited with regards to microbial detection, with clinicians relying on basic culture or molecular methodologies that can take days for results. Thus there remains a requirement for rapid, sensitive, yet non-invasive methodologies for microbial identification at the bedside, to aid clinical therapy choices during wound management.Raman is an information rich vibration technique that gives label-free biochemical information at the sub-cellular level, however high-resolution imaging at a single cell level is a relatively slow process making it challenging for live cell imaging. Stimulated Raman scattering (SRS) is a more sensitive and much faster technique which can provide high resolution information on larger cell populations compared with normal Raman. SRS provides detailed biochemical information with sub-cellular resolution including determination of protein, lipids and nucleic acid location. Due to its speed and ability to image larger areas in a label free manner, it has great potential for imaging bacterial biofilms giving both biochemical information as well as structural information on biofilm formation, as well as changes to biofilm in response to antimicrobial treatments. To date there is evidence that different bacterial species exhibit unique spectral profiles depending on their chemical structures at a cellular level. Existing research is largely limited to single cells, with few studies investigating biofilm kinetics using SRS.The overall aims of the proposal are to test the feasibility of using SRS to monitor biofilm formation and assess unique Raman spectral profiles or signatures of different bacterial and fungal species related to wound infections.1 Monitoring growth dynamics of mono-species wound biofilm models using SRS - comparing spectral profiles in different clinically-relevant microbial species (gram+, gram- bacterial and fungal organisms) during biofilm attachment, maturation and dispersal.2 Investigating mono-species biofilm response to antibiotic therapy using a variety of microbiological, molecular, microscopic and spectroscopic techniques - SRS to be used to assess chemical signatures in the biofilms in response to bacterio- (or fungi-) static or -cidal antibiotics and other novel compounds.3 Assessing changes in biofilm formation in a multi-species biofilm model using SRS. Visualizing phenotypic changes in biofilm chemical structures containing a consortia of different microbial species.4 SRS to be used to profile real-world biofilms (those generated from clinical samples from wound infections which will be generated through an existing EPSRC grant EP/V005839/1). Characteristic changes in chemical structures identified by Raman Spectroscopy using simple biofilm models with known composition.