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)是这两种疾病侵袭性感染的主要原因 社区获得性环境和医院相关环境。根据标准，耐甲氧西林金黄色葡萄球菌菌株具有内在的耐药性。 β-内酰胺类抗生素体外药敏试验。相比之下，甲氧西林敏感的金黄色葡萄球菌(MSSA) 菌株对许多标准的β-内酰胺类药物(如苯唑西林、奈福西林、头孢唑林)仍然高度敏感。 不推荐将β-内酰胺类药物用于治疗mrsa感染：i)mrsaβ-内酰胺类药物 2)它们与青霉素结合蛋白(PBP)2a结合相对较差(主要 负责细胞壁合成和分裂的耐甲氧西林金黄色葡萄球菌中的PBP)；iii)饱和所需的β-内酰胺水平 PBP 2a超过标准临床剂量方案所达到的人体血清水平；以及iv)治疗 β-内酰胺类抗生素的实验性耐甲氧西林金黄色葡萄球菌感染(如心内膜炎)一般无效。 最近几个实验室表明，补充标准MIC测试介质的碳酸氢盐可以 体外使部分(但不是全部)耐甲氧西林金黄色葡萄球菌菌株对β-内酰胺类抗生素和宿主防御肽(例如，来自 中性粒细胞；皮肤)。此外，MRSA菌株在体外表现出对碳酸氢盐敏感的表型(即， 在标准介质中对β-内酰胺耐药，但在含碳酸氢盐介质中敏感)有效 在选定的β-内酰胺类药物的小鼠菌血症模型中被根除。我们用四个例子放大了这些观察结果 MRSA原型菌株(LAC-USA-300；COL[USA 100]；MW-2[USA 400]；BMC1001[USA 300] 证明了以下关键结果：i)所有菌株在体外标准(MHB)中对这两种病毒都具有耐药性 苯唑西林(OX)和头孢唑林(CFZ)；两株菌株在小苏打中表现出对小苏打的反应表型。 补充MHb，变得对两种β-内酰胺高度敏感，而两种不敏感；ii)两种碳酸氢盐- 在群体分析中，响应菌株是异型的，而另外两个菌株是同型的(AUC 两株对碳酸氢盐反应的菌株都能有效地从所有靶器官中清除。 CFZ用于实验性心内膜炎(IE)，而两株碳酸氢盐不敏感菌株对治疗无效； (4)小苏打对mecA-PBP2a和SarA-SIGB遗传途径均有影响。 目前的建议将调查：i)重碳酸盐反应表型在体外的范围 β-内酰胺类抗生素在较大的临床耐甲氧西林金黄色葡萄球菌菌株集合中；ii)这些 侵袭性耐甲氧西林金黄色葡萄球菌感染(IE)相关体内模型的体外指标；以及iii)潜在的机制(S) 碳酸氢盐在耐甲氧西林金黄色葡萄球菌中的反应性。这一提议可能为关键的临床试验奠定基础(S) 利用碳酸氢盐对β-内酰胺类抗生素改良的MRSA体外检测的可预测性评价 标准媒体的补充。这项研究具有从根本上改变 目前耐甲氧西林金黄色葡萄球菌对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.