Disappearance of rifampin resistance in MRSA foreign body osteomyelitis

项目摘要 由于人口老龄化和医疗技术的进步， 频率.人工关节感染（PJI）影响高达2%的人工关节。葡萄球菌是 PJI的常见原因，金黄色葡萄球菌是最常见的病原体， 表皮葡萄球菌是第二常见的葡萄球菌。治疗 由于大多数抗微生物剂对微生物生物膜的活性有限，PJI是有问题的。利福平， 然而，具有抗生物膜活性，并且是控制葡萄球菌PJI的基础药物， 通过清创术和种植体保留（DAIR）进行管理。不幸的是，利福平耐药，介导的 靶基因中的单碱基对突变，很容易被选择，并且预计会损害 使用DAIR策略管理葡萄球菌PJI。然而，我们的初步数据令人惊讶地表明， 选择性利福平耐药可能不一定会对将来使用利福平为基础的治疗产生负面影响， 疗法具体来说，我们已经表明，正如预期的那样，利福平耐药选择后，利福平 在耐甲氧西林S.金黄色异物性骨髓炎（FBO）。但是， 令人惊讶的是，利福平耐药性在停止治疗两周后“消失”， 敏感S.金黄色葡萄球菌是可检测的。我们已经证明，利福平耐药不会降低 当细菌在体外或体内单独生长时， 利福平敏感细菌在体外和体内共同生长时。当用两种药物治疗FBO时， 利福平单药治疗第二轮后，耐药率低于第一轮 一轮利福平暴露这些观察结果表明，利福平可能是生物膜中的一种有用的药物- 先前已选择利福平耐药的相关感染。 因此，我们提出的研究的中心假设是，在实验性FBO中，利福平治疗 即使先前已经选择了利福平耐药性，也可以成功。我们的目标是发展一个 实验FBO模型，该模型允许从同一动物中真实的时间采样细菌，并使用 模型来定义利福平耐药性的选择和消失的动力学。而在传统 在骨髓炎模型中，在单个时间点分析微生物（即，当处死动物时）， 所提出的模型将允许随着时间的推移分析来自同一动物的细菌。我们将执行整个 对在一轮和两轮化疗期间和之后的多个时间点回收的细菌进行基因组测序， 利福平治疗这将允许定义利福平抗性和适应性相关突变， 出现在初始利福平治疗期间和之后，随后再次暴露于利福平。这种方法 将查明可能发生在利福平敏感细菌中的突变， 在他们的选择性优势超过他们的利福平耐药同行，即使挑战与 利福平最后，我们将比较用利福平单药治疗FBO， 利福平联合治疗或单独的利福平联合治疗。这将使我们能够确定 含利福平的方案可能是治疗利福平耐药的PJI的一种选择， 以前选的。拟议研究的结果将确定利福平的体内选择动力学 耐药性，并确定在利福平耐药细菌的竞争中发挥作用的突变， 对利福平敏感此外，我们的研究将为以下情况下的临床治疗方案提供信息： 选择利福平耐药性，并为未来的实验奠定基础，确定最佳治疗， 利福平耐药性的选择。

Project Summary Due to an aging population and advances in medical technology, prosthetic joint placement is increasing in frequency. Prosthetic joint infection (PJI) affects up to 2% of prosthetic joints. Staphylococci are the most common causes of PJI, with Staphylococcus aureus being the most common pathogen overall and Staphylococcus epidermidis being the second most common staphylococcal species involved. Treatment of PJI is problematic due to the limited activity of most antimicrobial agents against microbial biofilms. Rifampin, however, has anti-biofilm activity, and is a cornerstone drug in the management of staphylococcal PJI, when managed with débridement and implant retention (DAIR). Unfortunately, resistance to rifampin, mediated by single base pair mutations in the target gene, is readily selected and would be expected to compromise management of staphylococcal PJI using a DAIR strategy. Our preliminary data surprisingly suggest, however, that selected rifampin resistance may not necessarily negatively impact future treatment using rifampin-based therapy. Specifically, we have shown that, as expected, rifampin resistance is selected following rifampin monotherapy in a rat model of methicillin-resistant S. aureus foreign body osteomyelitis (FBO). But, surprisingly, rifampin resistance “disappears” two weeks following cessation of treatment, with only rifampin susceptible S. aureus being detectable. We have shown that rifampin resistance does not confer decreased fitness when bacteria are grown alone in vitro or in vivo; however, rifampin-resistant bacteria are out-competed by rifampin-susceptible bacteria when grown together in vitro and in vivo. And, when treating FBO with two rounds of rifampin monotherapy, resistance is observed at a lower frequency following the second than the first round of rifampin exposure. These observations suggest that rifampin may be a usable agent in biofilm- associated infections in which rifampin resistance has been previously selected. The central hypothesis of our proposed studies is therefore that in experimental FBO, treatment with rifampin can be successful even if rifampin resistance has been previously selected. Our objectives are to develop an experimental FBO model that allows sampling of bacteria in real time from the same animal and to use the model to define the kinetics of selection and disappearance of rifampin resistance. Whereas in traditional osteomyelitis models, microorganisms are analyzed at single time-points (i.e., when animals are sacrificed), the proposed model will allow analysis of bacteria from the same animal over time. We will perform whole genome sequencing on bacteria recovered at multiple time-points during and following one and two rounds of rifampin treatment. This will allow definition of rifampin resistance- and fitness-associated mutations that emerge during and following initial rifampin treatment, and subsequent re-exposure to rifampin. This approach will pinpoint mutations that may occur in rifampin-susceptible bacteria allowing their persistence or conferring upon them a selective advantage over their rifampin-resistant counterparts, even when challenged with rifampin. Finally, we will compare outcomes of FBO treated with either rifampin monotherapy followed by rifampin combination therapy or rifampin combination therapy alone. This will allow us to determine whether rifampin-containing regimens may be an option in the treatment of PJI in which rifampin resistance has been previously selected. Results of the proposed studies will define the kinetics of in vivo selection of rifampin resistance and identify mutations that play a role in out-competition of rifampin-resistant bacteria by those that are rifampin-susceptible. Additionally, our studies will inform clinical treatment options in the scenario of selected rifampin resistance and lay the groundwork for future experiments defining optimal therapy following selection of rifampin resistance.