Anti-MRSA activity of a Bacillus pumilus natural isolate

描述（由申请人提供）：传染病是世界范围内导致死亡的主要原因之一。抗生素耐药细菌的出现甚至对曾经可以用抗生素或其他抗菌剂治疗的感染也引起了严重的担忧。一个显著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院内感染，大多局限于易感个体。最初，MRSA菌株仅在医疗机构中发现（HA-MRSA）；然而，最近MRSA菌株出现在社区（CA-MRSA）中没有感染风险因素的个体中。此外，据报道，耐甲氧西林金黄色葡萄球菌严重侵袭性感染的数量不断增加，2005年在美国估计有18650人死亡。尽管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万古霉素耐药菌株，这就需要开发新的策略来对抗MRSA感染。从矮芽孢杆菌天然分离物C9中鉴定出抑菌物质C9ASA。C9ASA对一些革兰氏阳性细菌有效，对金黄色葡萄球菌（包括HA-和CA-MRSA）以及化脓性链球菌（a组链球菌）具有最高的活性。金黄色葡萄球菌和A群链球菌都在NIAID重新出现的病原体名单中。C9ASA是目前已检测的另外7株矮螺旋体菌株所不能产生的，与此前从矮螺旋体中分离和报道的抗菌物质不同，提示它是一种新化合物。本研究的长期目标是开发一种新的药物来治疗MRSA和a群链球菌引起的感染，并以C9ASA和金黄色葡萄球菌为模型，阐明革兰氏阳性致病菌对某些抗菌药物产生耐药性的机制。在Specific Aim 1中，部分纯化的C9ASA将使用HPLC进一步纯化，并使用串联质谱法测定分子量。质子核磁共振和多维核磁共振将用于解析C9ASA的结构。在Specific Aim 2中，C9ASA的作用模式将通过分离和表征对该物质具有抗性的金黄色葡萄球菌突变体来确定。此外，将进行转录组分析，以鉴定C9ASA诱导的金黄色葡萄球菌中的基因，这可能会发现潜在的C9ASA靶点。在Specific Aim 3中，将建立一个研究小圆菇的遗传系统，以促进在Specific Aim 4中提出的实验。基于矮螺旋藻由于缺乏comS基因而缺乏自然能力的假设，本文将comS基因引入到矮螺旋藻C9菌株中，利用新开发的接合系统使C9菌株具有自然能力。在Specific Aim 4中，将使用mini-Tn10诱变技术鉴定产生C9ASA所需的基因。这些基因可能包括参与C9ASA产生的生物合成基因和调控基因。公共卫生相关性：侵袭性MRSA感染是全球公共卫生的严重威胁。本提案描述了旨在描述可能是一种有前途的抗mrsa物质的研究，以对抗这种重新出现的传染性威胁。

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.