Bicarbonate-Mediated Enhancement of Beta-Lactam-MRSA Killing: Mechanisms and Clinical Translatability

摘要 耐甲氧西林金黄色葡萄球菌（MRSA）是两种侵袭性感染的主要原因 社区获得的和医院相关的环境。根据标准，MRSA菌株具有内在耐药性 对β-内酰胺类抗生素的体外药敏试验。与此相反，甲氧西林敏感的S.金黄色葡萄球菌（MSSA） 菌株仍然对许多标准护理β-内酰胺（例如苯唑西林;萘夫西林;头孢唑啉）高度敏感。 不推荐β-内酰胺类药物治疗MRSA感染：i）MRSA β-内酰胺MIC高于 当前CLSI“折点”; ii）它们与青霉素结合蛋白（PBP）2a（主要 MRSA菌株中负责细胞壁合成和分裂的PBP）; iii）使细胞壁饱和所需的β-内酰胺水平 PBP 2a超过用标准临床剂量方案实现的人血清水平;和 实验性MRSA感染（例如，心内膜炎）与β-内酰胺类药物的联合使用通常无效。 几个实验室最近表明，标准MIC测试介质的重碳酸盐稀释可以 在体外使一些（但不是全部）MRSA菌株对β-内酰胺和宿主防御肽（例如，LL-37从 嗜中性粒细胞;皮肤）。此外，在体外表现出“碳酸氢盐响应性”表型的MRSA菌株（即， 在标准培养基中β-内酰胺耐药，但在含碳酸氢盐培养基中敏感）有效地 在鼠菌血症模型中用选定的β-内酰胺根除。我们使用四个放大这些观察结果 原型MRSA菌株（LAC-USA-300; COL [USA 100] ; MW-2 [USA 400]; BMC 1001 [USA 300]， 证明了以下关键结果：i）所有菌株在体外标准（MHB）中对以下两种都具有抗性： 苯唑西林（OX）和头孢唑啉（CFZ）;两种菌株在碳酸氢盐- 补充MHB，变得对两种β-内酰胺高度敏感，而两种没有; ii）两种碳酸氢盐- 在群体分析中，响应菌株为异型，而另外两个菌株为同型（AUC > 0.9）; iii）两种碳酸氢盐响应性菌株均被OX和 CFZ在实验性心内膜炎（IE），而两个碳酸氢盐无反应菌株是难治性; 和iv）碳酸氢盐影响mecA-pbp 2a和sarA-sigB遗传途径。 目前的建议将调查：i）碳酸氢盐反应表型的范围在体外， β-内酰胺类抗生素在临床MRSA菌株的较大集合中的应用; ii）此类抗生素的整体大规模可翻译性 侵袭性MRSA感染（IE）的相关体内模型的体外指标;和iii）潜在的机制 MRSA中的碳酸氢盐反应性。该提案可为关键临床试验奠定基础 利用碳酸氢盐评估改良MRSA对β-内酰胺类体外试验的可预测性 补充标准培养基。这项研究具有从根本上改变 目前MRSA对β-内酰胺类抗生素的体外药敏试验方法。

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of invasive infections in both community-acquired and hospital-associated contexts. MRSA strains are intrinsically resistant by standard in vitro susceptibility testing to β-lactam antibiotics. In contrast, methicillin-susceptible S. aureus (MSSA) strains remain highly susceptible to many standard-of-care β-lactams (e.g. oxacillin; nafcillin; cefazolin). β-lactams are not recommended for treating MRSA infections: i) MRSA β-lactam MICs are above current CLSI “breakpoints”; ii) they bind relatively poorly to penicillin-binding protein (PBP) 2a (predominant PBP in MRSA strains responsible for cell wall synthesis and division); iii) β-lactam levels required to saturate PBP 2a exceed human serum levels achieved with standard clinical dose-regimens; and iv) treatment of experimental MRSA infections (e.g., endocarditis) with β-lactams are generally ineffective. Several labs recently showed that bicarbonate supplemention of standard MIC testing media can “sensitize” some (but not all) MRSA strains in vitro to β-lactams and host defense peptides (e.g., LL-37 from neutrophils; skin). Further, MRSA strains exhibiting a “bicarbonate- responsive” phenotype in vitro (i.e., β-lactam-resistant in standard media, but susceptible in bicarbonate-containing media) were effectively eradicated in murine bacteremia models with selected β-lactams. We amplified these observations using four prototype MRSA strains (LAC-USA-300; COL [USA 100] ; MW-2 [USA 400]; BMC1001 [USA 300] which demonstrated the following key outcomes: i) all strains were resistant in vitro in standard (MHB) to both oxacillin (OX) and cefazolin (CFZ); two strains exhibited a bicarbonate-responsive phenotype in bicarbonate- supplemented MHB, becoming highly susceptible to both β-lactams, while two did not; ii) two bicarbonate- responsive strains were heterotypic on population analyses, while the other two strains were homotypic (AUCs > 0.9); iii) both bicarbonate-responsive strains were effectively cleared from all target organs by both OX and CFZ in experimental endocarditis (IE), while two bicarbonate-nonresponsive strains were refractory to therapy; and iv) bicarbonate impacted both the mecA-pbp2a and sarA-sigB genetic pathways. The current proposal will investigate: i) the scope of the bicarbonate-responsive phenotype in vitro to β-lactams among a larger collection of clinical MRSA strains; ii) the overall large-scale translatability of such in vitro metrics to a relevant in vivo model of invasive MRSA infection (IE); and iii) the mechanism(s) underlying bicarbonate-responsiveness in MRSA. This proposal could lay the foundation for pivotal clinical trial(s) assessing predictability of modified in vitro testing of MRSA to β-lactams, utilizing bicarbonate supplementation of standard media. This research has the overarching potential to fundamentally transform current MRSA in vitro susceptibility testing methods for β-lactams.