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，假体关节置放在 频率假体关节感染(PJI)影响多达2%的假体关节。葡萄球菌是最多的 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种最常见的葡萄球菌。治疗 PJI是有问题的，因为大多数抗菌剂对微生物生物膜的活性有限。利福平， 然而，具有抗生物被膜活性，是治疗葡萄球菌肺炎的基石药物，当 通过脱桥和种植固位(DAIR)进行管理。不幸的是，利福平的耐药性是由 靶基因中的单碱基对突变是很容易选择的，预计会造成损害 用DAIR策略管理葡萄球菌肺炎。然而，我们的初步数据出人意料地表明， 选定的利福平耐药性不一定会对未来使用利福平为基础的治疗产生负面影响 心理治疗。具体地说，我们已经证明，正如预期的那样，利福平耐药性是在利福平之后选择的 单一疗法治疗耐甲氧西林金黄色葡萄球菌异物骨髓炎(FBO)大鼠模型。但, 令人惊讶的是，停止治疗两周后，利福平耐药性就消失了，只有利福平 敏感的金黄色葡萄球菌可被检测到。我们已经证明，利福平耐药性不会降低。 细菌在体外或体内单独生长时的适合性；然而，对利福平具有抗药性的细菌在竞争中处于劣势 在体外和体内共同生长时，由利福平敏感细菌产生。而且，当用两种药物治疗FBO时 单轮利福平治疗后，观察到第二轮耐药的频率低于第一轮 一轮利福平暴露。这些观察表明，利福平可能是生物膜中的一种有用的试剂。 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 即使之前已经选择了利福平耐药，也可以成功。我们的目标是开发一种 实验FBO模型，允许对同一动物的细菌进行实时采样并使用 模型来定义利福平耐药的选择和消失的动力学。而在传统的 骨髓炎模型，在单个时间点(即，当动物被宰杀时)分析微生物， 拟议的模型将允许随着时间的推移对同一动物的细菌进行分析。我们将完整地表演 细菌基因组测序在一轮和两轮之后的多个时间点恢复 利福平治疗。这将允许定义与利福平耐药性和适应性相关的突变 在最初的利福平治疗期间和之后出现，随后再次暴露于利福平。这种方法 将精确定位利福平敏感细菌中可能发生的突变，从而使其持续存在或授予 在他们身上，即使在受到挑战时，他们也比耐利福平的同行具有选择性优势 利福平。最后，我们将比较单一利福平治疗FBO的效果。 利福平联合治疗或单独利福平联合治疗。这将使我们能够确定 含有利福平的方案可能是治疗PJI的一种选择，在PJI中利福平耐药 以前选择的。建议的研究结果将确定利福平的体内选择动力学 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，我们的研究将为临床治疗方案提供信息 选择利福平耐药性，并为未来确定最佳治疗方案的实验奠定基础 利福平耐药菌株的筛选。

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.