Mechanisms and Circumvention of Daptomycin Resistance in Streptococcus mitis

摘要 绿色群链球菌(VGS)，尤其是米氏链球菌，是引起多种肺炎的重要病原菌。 侵袭性血管内感染，包括：1)中性粒细胞减少的“突破性菌血症”和“中毒性休克” 2)感染性心内膜炎(IE)。鉴于青霉素耐药性和其他疾病的全球趋势 β-内酰胺类最低抑菌浓度在米蒂斯链球菌中“爬行”，严重感染的比例相对或完全 β-内酰胺耐药(R)菌株令人不安。此外，在这种情况下，使用替代方案的临床结果 治疗方案(如万古霉素)一直令人失望，可能与高患病率有关 对万古霉素“耐受”这类菌株。这促使了新型杀菌剂的使用，如达托霉素 (DAP)用于对β内酰胺类耐药菌株的严重米非司氏菌综合征。令人震惊的是，最近人们认识到 DAP疗法诱导的快速、持久和高水平的DAP-R显著降低了人们对此的热情 接近了。此外，S.mitis组的DAP MIC值是所有其他VGS组的2-10倍。这个 出现DAP-R的侵袭性米氏葡萄球菌感染的临床报告病例数量有限，原因是 迄今为止，DAP在这类感染中的使用相对较少。然而，米氏链球菌β-内酰胺-R的渐进性上升 此外，VANC治疗此类综合征的结果不一致实际上确保了DAP使用量的增加， 导致DAP-R S.mitis感染。此外，DAP使用率较高的医疗中心最近证实 大量的最低抑菌浓度在肠球菌中“爬行”。对DAP-R发生机制的理解(S) 因此，规避其进化的策略具有重要的临床意义。 我们的初步数据显示，DAP-R在S.mitis中的结果可能是两个因素的多因素 表型水平和基因水平。最有趣的是，我们现在已经展示了两种形式的令人信服的证据 “DAP超积累”：链球菌链中的单个细胞可以超捕获DAP和 要么死亡(“利他主义自杀”)，要么抵抗DAP杀戮，以保护剩余的细胞群体免受 DAP暴露和致命性。这显然是革兰氏阳性菌中DAP-R的一种独特机制 病原体。 在这份提案中，我们将使用战略荧光显微镜、流式细胞术和多维 生理讯问和单细胞分选+基因分型以揭示DAP-R的机制 米氏假单胞菌能抵抗DAP暴露。最后，我们将使用两个具有良好特征的血管内模型 体外感染(小室模型)和体内感染(实验性兔IE)，以确定DAP方案对两者 避免DAP-R的出现，加强对米氏葡萄球菌的清除。总而言之，这些研究将泄露 预防DAP-R在米氏葡萄球菌和其他革兰氏阳性病原体中出现的临床策略。

ABSTRACT Viridans group streptococci (VGS), especially Streptococcus mitis, are pivotal pathogens in a variety of invasive endovascular infections,including: i) “breakthrough bacteremias” and “toxic shock” in neutropenic cancer patients; and ii) infective endocarditis (IE). Given world-wide trends in penicillin-resistance and other β-lactam MIC “creeps” amongst S. mitis strains, the proportion of serious infections caused by relatively or fully β-lactam-resistant-(R) strains is disturbing. Moreover, clinical outcomes in such cases utilizing alternate regimens (e.g., vancomycin) have been disappointing, presumably related to the high prevalence of vancomycin “tolerance” in such strains. This has prompted use of newer bactericidal agents, like daptomycin (DAP) for severe S. mitis syndromes in strains with β-lactam-resistance. Alarmingly, recent recognition of rapid, durable and high-level DAP-R induced by DAP therapy has significantly reduced enthusiasm for such approaches. In addition, DAP MICs in the S. mitis group are 2-10-fold higher than all other VGS groups. The number of reported clinical cases of invasive S. mitis infections in which DAP-R has emerged is limited, due to the relatively infrequent use of DAP in such infections to-date. However, progressive rise in S. mitis β-lactam-R plus the inconsistent outcomes of VANC therapy in such syndromes virtually assures increased DAP use, leading to DAP-R S. mitis infections. Moreover, medical centers with high DAP usage have recently confirmed substantial MIC “creeps” amongst enterococci. Understanding mechanism(s) of emergence of DAP-R in S. mitis, plus strategies to circumvent its evolution are, thus, of great clinical significance. Our Preliminary Data showed that DAP-R outcomes in S. mitis are likely to be multifactorial on both phenotypic and genotypic levels. Most interestingly, we have now shown compelling evidence of two forms of “DAP hyperaccumulation” in which individual cells in a given streptococcal chain can hyper-capture DAP and either die (“altruistic suicide”) or resist DAP killing, in order to protect the remainder of the cell population from DAP exposures and lethality. This is an apparently unique mechanism of DAP-R amongst gram-positive pathogens. In this proposal, we will use strategic fluorescence microscopy, flow cytometry with multidimensional physiologic interrogations, and single cell sorting plus genotyping to divulge mechanisms by which DAP-R S. mitis can resist DAP exposures. Finally, we will use two well-characterized models of endovascular infections, ex vivo (chamber model) and in vivo (experimental rabbit IE), to define DAP regimens to both circumvent emergence of DAP-R and enhance clearance of S. mitis. In summary, these studies will divulge clinical strategies to forestall emergence of DAP-R in S. mitis and perhaps other Gram-positive pathogens.