Molecular Mechanisms Of Daptomycin Resistance In Enterococci

描述（由申请人提供）：抗生素耐药细菌的出现是21世纪影响人类的最具挑战性的公共卫生问题之一。在这些细菌中，万古霉素耐药肠球菌（VRE）是美国各地医院最难治疗的细菌之一。目前只有两种抗菌化合物被fda批准用于治疗VRE感染；即利奈唑胺和奎奴普司汀-达福普司汀（Q/D）。然而，由于严重感染的治疗效果不理想，副作用的频繁发生以及利奈唑胺和Q/ d耐药VRE分离株的出现和广泛传播，这两种药物用于VRE的使用受到阻碍。达托霉素（DAP）是一种脂肽抗生素，其杀伤机制涉及与细菌细胞膜（CM）以钙依赖的方式相互作用。DAP是目前唯一具有抗VRE活性的杀菌抗生素。尽管DAP没有fda批准的用于治疗VRE感染的适应症，但由于缺乏更好的替代方案来治疗通常病情严重且免疫系统严重受损的VRE感染患者，临床医生经常被迫使用DAP。在VRE治疗期间，超说明书使用DAP已导致多例DAP耐药（DAP- r），从而使临床情况进一步恶化。我们的长期目标是了解在VRE治疗期间导致DAP-R发展的分子事件，以便能够i)设计改进的治疗策略以防止DAP-R的出现，ii)确定未来抗菌开发的新潜在靶点，目的是保护DAP对VRE的疗效。通过对dap易感和耐药的粪肠球菌（VREfs）和粪肠球菌（VREfm） VRE临床菌株对的全基因组比较、CM和细胞包膜超微结构分析，我们确定了两个极有可能参与DAP-R发展的基因：i)在VREfs和VREfm中编码心磷脂合成酶的基因（cls），参与细胞膜稳态；ii) VREfs与liaF基因同源，它是一个三组分基因系统的一部分，参与细菌对抗菌药物和抗菌肽的细胞包膜反应。因此，我们的目标是a)研究上述基因突变（VREfs和VREfm中的cls以及VREfs中的liaF）对DAP耐药的贡献，b)通过测试DAP剂量的增加以及DAP与氨苄西林（VREfs）和替加环素或利福平（VREfm）联合治疗在小鼠感染性心内膜炎模型中预防DAP-r出现的效果，评估优化DAP用于VRE的策略。我们预计这些研究将有助于更深入地了解CM磷脂稳态和细胞包膜调节在抗生素耐药性和抗菌肽作用发展中的作用，并肯定有助于在未来保留DAP作为治疗VRE感染的有用抗生素。

DESCRIPTION (provided by applicant): The emergence of antibiotic resistant bacteria is one of the most challenging public health problems affecting humankind in the 21st century. Among these bacteria, vancomycin-resistant enterococci (VRE) are one of the most difficult organisms to treat in hospitals across the US. Only two antimicrobial compounds are currently FDA-approved for the treatment of VRE infections; namely, linezolid and quinupristin-dalfopristin (Q/D). However, the use of these two agents against VRE has been hampered by suboptimal therapeutic outcomes in severe infections, frequent occurrence of side effects and the emergence and widespread dissemination of linezolid- and Q/D-resistant VRE isolates. Daptomycin (DAP) is a lipopeptide antibiotic whose mechanism of killing involves the interaction with the bacterial cell membrane (CM) in a calcium-dependent manner. DAP is the only bactericidal antibiotic currently available with activity against VRE. Although DAP does not have an FDA-approved indication for the treatment of VRE infections, clinicians are often pushed to use DAP due to the lack of better alternatives to treat patients infected with VRE who are often severely ill and with important compromise of the immune system. The off-label use of DAP during VRE therapy has led in several instances to the development of DAP resistance (DAP-R), thus, worsening the clinical scenario even further. Our long- term goal for this grant application is to understand the molecular events that lead to the development of DAP- R during VRE therapy to be able to i) design improved therapeutic strategies to prevent the emergence of DAP-R, and ii) identify new potential targets for antimicrobial development in the future with the aim of protecting the efficacy of DAP against VRE. Based on the information gathered from the comparative whole- genome, CM and cell envelope ultrastructural analysis of VRE clinical strain pairs of DAP-susceptible and DAP-resistant Enterococcus faecalis (VREfs) and E. faecium (VREfm), we have identified two genes that are highly likely to be involved in the development of DAP-R: i) a gene (cls) encoding a cardiolipin synthase enzyme in both VREfs and VREfm, involved in cell membrane homeostasis and ii) a VREfs homolog of the liaF gene, which is part of a three-component gene system involved in the bacterial cell envelope response to antimicrobials and antimicrobial peptides. Thus, we aim to a) investigate the contribution of mutations in the above genes (cls in both VREfs and VREfm and liaF in VREfs) to DAP-resistance, and b) evaluate strategies to optimize the use of DAP for VRE by testing the effect of escalating doses of DAP and combination therapies of DAP with i) ampicillin (for VREfs), and ii) with tigecycline or rifampin (for VREfm), in preventing emergence of DAP-R using a murine model of infective endocarditis. We anticipate that these studies will contribute to a deeper understanding of the role of CM phospholipid homeostasis and cell envelope regulation in the development of antibiotic resistance and antimicrobial peptide action and will certainly facilitate the preservation of DAP as a useful antibiotic to treat VRE infections in the future. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the molecular strategies used by vancomycin-resistant enterococci (VRE, a common hospital-associated pathogen) to develop resistance to the antibiotic daptomycin; a compound of last resource to treat VRE infections. We aim to design strategies to prevent the emergence of daptomycin resistance in VRE and potentially characterize novel targets for the development of antimicrobial agents with activity against enterococci and other multidrug resistant organisms.