Elucidating the mechanisms of antibiotic tolerance in Staphylococcus aureus biofilms

摘要 金黄色葡萄球菌是美国细菌感染的主要原因之一。侵袭性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 即使在使用适当的抗生素治疗时，发病率和死亡率也很高。形成生物膜的能力， 微生物衍生的群落，细胞附着在表面或以细菌聚集体的形式生长 被复杂的细胞外基质包围，与持续的金黄色葡萄球菌感染有关。一个 包括金黄色葡萄球菌在内的细菌生物膜的主要特征是对抗生素具有高度的耐受性。尽管 生物被膜感染中抗生素耐受性的临床意义及其机制 发生的原因还知之甚少。抗生素已被证明能穿透金黄色葡萄球菌的生物膜，这表明 除了物理遮挡之外，耐受还涉及其他机制吗？一种可能的解释是 抗生素耐受性的提高是耐受氧化应激的能力增强，这在生物膜的生长中可以看到。 鉴于越来越多的文献表明氧化应激在抗生素介导的杀戮中起到了作用，我们 假设促进细菌氧化应激反应的蛋白质产生的变化导致 生物膜介导的金黄色葡萄球菌感染中的抗生素耐药表型。此外，鉴于 过渡金属锰和铁在金黄色葡萄球菌毒力和氧化应激中的重要作用 我们预计，调节这些金属水平及其在金黄色葡萄球菌中分布的能力 生物膜是金黄色葡萄球菌生物膜对抗生素耐药表型不可或缺的一部分。为了检验这一假设， 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。 荧光显微镜和基质辅助激光相结合的金黄色葡萄球菌生物膜 解吸/电离成像质谱仪(MALDI-IMS)与激光烧蚀电感耦合等离子体 质谱仪(LA-ICPMS)，(2)鉴定葡萄球菌耐药相关基因 利用转座子测序(TnSeq)和蛋白质组学实验进行生物膜生长，以及(3)证明了 利用慢性生物膜介导的金黄色葡萄球菌骨髓炎动物模型研究这些发现的活体意义 感染。这项工作的总结不仅将导致对元素是什么的更好的理解 负责金黄色葡萄球菌感染的抗生素耐受性，但它也将提供有用的新见解 在开发治疗方法和治疗方法方面，旨在减少因S。 金星。

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.