Anti-MRSA activity of a Bacillus pumilus natural isolate

描述(申请人提供)：传染病是全球主要的死亡原因之一。抗生素耐药细菌的出现引起了严重的担忧，甚至对曾经可以用抗生素或其他抗菌剂治疗的感染也是如此。一个明显的例子是最近爆发的耐甲氧西林金黄色葡萄球菌(MRSA)感染。金黄色葡萄球菌感染是医院内的，主要局限于易患金黄色葡萄球菌的人。最初，MRSA菌株只在卫生保健环境中发现(HA-MRSA)；然而，最近，MRSA菌株出现在社区(CA-MRSA)中，这些个体没有感染的危险因素。此外，报告的耐甲氧西林金黄色葡萄球菌严重侵袭性感染病例越来越多，2005年期间在美国已造成18650人死亡，估计有18650人死亡。虽然万古霉素仍然是治疗MRSA感染的最后手段，但在HA和CA-MRSA分离株中已经开始出现对万古霉素耐药的菌株，因此有必要开发新的策略来对抗MRSA感染。从短小芽孢杆菌天然分离物C9中鉴定出抗菌物质C9ASA。C9ASA对一些革兰氏阳性菌有效，对金黄色葡萄球菌，包括HA-和CA-MRSA，以及A组链球菌化脓性链球菌显示出最高的活性。金黄色葡萄球菌和A组链球菌都在NIAID重新出现的病原体名单中。C9ASA不是由其他7个短小芽孢杆菌菌株产生的，与以前从短小芽孢杆菌中分离和报道的抗菌物质不同，表明它是一个新的化合物。这项研究的长期目标是开发一种治疗MRSA和A组链球菌感染的新药，并以C9ASA和金黄色葡萄球菌为模型，阐明革兰氏阳性病原菌对某些抗菌药产生耐药性的机制。在特定目标1中，部分纯化的C9ASA将用高效液相色谱进一步纯化，并用串联质谱仪测定其相对分子质量。利用质子核磁共振和多维核磁共振技术对C9ASA的结构进行解析。在具体目标2中，C9ASA的作用模式将通过分离和鉴定对该物质具有抗药性的金黄色葡萄球菌突变体来确定。此外，还将进行转录组分析，以确定C9ASA诱导的金黄色葡萄球菌中的基因，从而可能发现潜在的C9ASA靶标。在特定目标3中，为了便于在特定目标4中提出的实验，将建立一个研究短小芽孢杆菌的遗传系统。基于短小芽孢杆菌由于缺乏枯草杆菌能力所必需的coms基因而缺乏自然能力的假设，将coms基因导入短小芽孢杆菌C9中，通过最近开发的接合系统赋予C9菌株天然能力。在特定目标4中，将使用Mini-Tn10突变来识别C9ASA生产所需的基因。这些基因可能包括生物合成基因和参与C9ASA生产的调节基因。公共卫生相关性：侵袭性MRSA感染是对全球公共卫生的严重威胁。这项建议描述了旨在表征什么可能是一种有希望的抗MRSA物质来对抗这种重新出现的传染病威胁的研究。

DESCRIPTION (provided by applicant): Infectious diseases are one of the leading causes of death worldwide. Emergence of antibiotic resistant bacteria causes serious concerns even for infections that once were treatable with antibiotics or other antimicrobial agents. A notable example is a recent outbreak of infections with methicillin resistant Staphylococcus aureus (MRSA). S. aureus infections were nosocomial and mostly confined to predisposed individuals. Originally, MRSA strains were found only in health care settings (HA-MRSA); however, more recently MRSA strains appear in the community (CA-MRSA) among individuals who bear no risk factor for the infections. In addition, increasing numbers of severe invasive infections with MRSA have been reported, which have resulted in and estimated 18,650 deaths during 2005 in the United States. Although vancomycin remains the last resort for treating MRSA infections, vancomycin resistant strains have begun to emerge among HA- and CA-MRSA isolates, necessitating the development of new strategies to combat MRSA infections. The antimicrobial substance C9ASA was identified from a natural isolate C9 of Bacillus pumilus. C9ASA is effective against some Gram-positive bacteria and exhibits the highest activity against S. aureus, including HA- and CA-MRSA, as well as Streptococcus pyogenes, a group A Streptococcus. Both S. aureus and group A Streptococcus are among the NIAID list of re-emerging pathogens. C9ASA is not produced by seven other B. pumilus strains thus far tested and is different from antibacterial substances that were previously isolated and reported from B. pumilus, suggesting that it is a novel compound. The long-term objectives of the proposed research are to develop a new drug to treat infections caused by MRSA and group A Streptococcus, and by using C9ASA and S. aureus as a model, to elucidate the mechanism by which Gram-positive pathogenic bacteria become resistant to certain antimicrobial agents. In Specific Aim 1, partially purified C9ASA will be further purified using HPLC and the molecular weight will be determined using tandem mass spectrometry. Proton NMR and multidimensional NMR will be applied to solve the structure of C9ASA. In Specific Aim 2, the mode-of-action of C9ASA will be determined by isolating and characterizing S. aureus mutants that are resistant to the substance. In addition, transcriptome analysis will be carried out to identify genes in S. aureus induced by C9ASA, which might uncover potential C9ASA targets. In Specific Aim 3, a genetic system for study of B. pumilus will be developed in order to facilitate experiments proposed in Specific Aim 4. Based on the hypothesis that B. pumilus lacks natural competence due to the absence of comS, a gene essential for B. subtilis competence, the comS gene will be introduced into B. pumilus C9 using a recently developed conjugative system to endow the C9 strain with natural competence. In Specific Aim 4, genes required for C9ASA production will be identified using mini-Tn10 mutagenesis. These genes likely include biosynthesis genes and regulatory genes involved in C9ASA production. PUBLIC HEALTH RELEVANCE: Invasive MRSA infections are a serious threat to public health worldwide. This proposal describes studies aimed at characterizing what might be a promising anti-MRSA substance for combating this re-emerging infectious threat.