Elucidating the mechanisms of antibiotic tolerance in Staphylococcus aureus biofilms

总结 金黄色葡萄球菌是美国细菌感染的主要原因之一。入侵S。 金黄色葡萄球菌感染（如骨髓炎、心内膜炎和血流感染）与相对 高发病率和死亡率，即使使用适当的抗生素治疗。形成生物膜的能力， 微生物衍生的群落，其中细胞生长附着于表面或作为细菌聚集体 被复杂的细胞外基质包围，与持续性S。金黄色葡萄球菌感染一 细菌生物膜的定义特征，包括S.金黄色葡萄球菌生物膜，对抗生素具有高耐受性。尽管 在生物膜介导的感染中观察到的抗生素耐受性的临床意义， 发生的事情知之甚少。抗生素已被证明可以穿透S。金黄色葡萄球菌生物膜，这表明， 除了生理性咬合外，还有其他机制参与耐受。一个可能的解释 抗生素耐受性增加是耐受氧化应激的能力增强，这在生物膜生长中可见。 鉴于越来越多的文献表明氧化应激在肿瘤介导的杀伤中的作用，我们 假设促进细菌氧化应激反应的蛋白质产生变化导致 在生物膜介导的S.金黄色葡萄球菌感染此外，鉴于 过渡金属锰和铁在硫中的重要作用。金黄色葡萄球菌毒力与氧化应激 响应，我们预计，调节这些金属的水平和它们在S.金黄色 生物膜是在S.金黄色葡萄球菌生物膜为了验证这一假设， 我们将（1）确定抗生素暴露对金属调节和氧化应激反应的影响， S.应用荧光显微术沿着与基质辅助激光的组合检测金黄色葡萄球菌生物膜 解吸/电离成像质谱（MALDI-IMS）和激光烧蚀电感耦合等离子体 质谱法（LA-ICP-MS），（2）鉴定葡萄球菌中负责抗生素耐受性的基因， 使用转座子测序（TnSeq）和蛋白质组学实验的生物膜生长，和（3）证明了在生物膜生长过程中， 这些发现的体内意义使用骨髓炎的动物模型，慢性生物膜介导的S。金黄色 感染这项工作的总和不仅会使我们更好地理解这些元素是什么， 负责S.金黄色葡萄球菌感染，但它也将提供新的见解， 在开发治疗和治疗，旨在降低发病率和死亡率的感染，由于S。 金黄色。

SUMMARY Staphylococcus aureus is one of the leading causes of bacterial infections in the United States. Invasive S. aureus infections (such as osteomyelitis, endocarditis, and bloodstream infections) are associated with relatively high rates of morbidity and mortality even when treated with appropriate antibiotics. The ability to form biofilms, microbially derived communities where cells grow attached to a surface or as a bacterial conglomerate surrounded by a complex extracellular matrix, has been associated with persistent S. aureus infections. A defining feature of bacterial biofilms, including S. aureus biofilms, is a high tolerance to antibiotics. Despite the clinical significance of the antibiotic tolerance seen in biofilm-mediated infections, the mechanism by which this occurs is poorly understood. Antibiotics have been shown to penetrate S. aureus biofilms, suggesting that there are other mechanisms besides physical occlusion involved in tolerance. One possible explanation for the increase in antibiotic tolerance is an enhanced ability to tolerate oxidative stress, which is seen in biofilm growth. Given the growing body of literature suggesting a role for oxidative stress in antibiotic-mediated killing, we hypothesize that changes in protein production that promote the bacterial oxidative stress response lead to the antibiotic tolerance phenotype seen during biofilm-mediated S. aureus infections. Furthermore, given the important role of the transition metals manganese and iron in S. aureus virulence and the oxidative stress response, we anticipate that the ability to regulate the levels of these metals and their distribution within S. aureus biofilms is integral to the antibiotic tolerance phenotype seen in S. aureus biofilms. In order to test this hypothesis, we will (1) determine the impact of antibiotic exposure on metal regulation and the oxidative stress response in S. aureus biofilms using a combination of fluorescence microscopy along with matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), (2) identify the staphylococcal genes responsible for antibiotic tolerance in biofilm growth using transposon sequencing (TnSeq) and proteomic experiments, and (3) demonstrate the in vivo significance of these findings using an animal model of osteomyelitis, a chronic biofilm-mediated S. aureus infection. The sum of this work will result in not only a better understanding of what the elements are that are responsible for antibiotic tolerance in S. aureus infections, but it will also provide new insight that will be useful in developing therapeutics and treatments aimed at reducing the morbidity and mortality of infections due to S. aureus.