Polymicrobial dynamics in transfer of vancomycin resistance to MRSA

描述(申请人提供)：耐甲氧西林金黄色葡萄球菌(MRSA)是抗生素耐药感染的主要原因，主要使用万古霉素治疗。自2002年以来，VANA型万古霉素耐药从耐万古霉素肠球菌(VRE)转移到MRSA，形成VRSA。在感染过程中，这种情况至少独立发生了12次，主要发生在糖尿病患者的肢体皮损中。所有接受万古霉素耐药转座子的金黄色葡萄球菌都是一株金黄色葡萄球菌，CC5。要进行水平传播，VRE和MRSA必须以足够的数量和距离占据相同的环境才能进行传输。我推测，与其他MRSA谱系相比，CC5 MRSA和VRE表现出独特的相互作用，包括促进协同作用的协同代谢能力，与宿主衍生因子的共同聚集，以及对种内和种间细胞信号的调节。初步数据证实，血清依赖的共聚集是CC5-MRSA和VRE对所特有的。为了确定是什么影响多物种结合，纯化的血清成分将被添加到共培养中，并通过显微镜和光密度分析共聚集。任何水平传播的增强都将通过与含有耐药质粒的肠球菌共培养后对MRSA接受者进行选择性培养来量化。我发现在VRE/MRSA共培养的转录组反应中，VRE基因对信息素信号和FSR毒力操纵子的下调。细胞信号调节的影响将通过转录和我们的微阵列数据揭示的信息素信号的定向突变来研究。D-乳酸(DLac)通常由金黄色葡萄球菌在发酵过程中产生。DLac对VANA型万古霉素耐药也是必需的。由于VANA型耐药性只有在万古霉素存在的情况下才被诱导，我推测金黄色葡萄球菌dLac的产生在与金黄色葡萄球菌共培养时可能为VRE提供优势。这将通过测量万古霉素暴露后两个物种在共培养和单一培养中的存活率以及在万古霉素治疗之前与dLac孵育的单一培养中的VRE来进行测试。这些结果将最终揭示ST5-MRSA和VRE所特有的多菌相互作用的性质以及它们如何导致万古霉素耐药性转移。 公共卫生相关性：在感染过程中，万古霉素耐药至少12次从对万古霉素耐药的肠球菌(VRE)转移到甲氧西林耐药金黄色葡萄球菌(MRSA)，导致VRSA。金黄色葡萄球菌对万古霉素耐药性的出现，危及了一种治疗侵袭性细菌感染的常见原因的关键的最后一线杀菌药物。我将确定和描述导致万古霉素耐药性传播到金黄色葡萄球菌的因素，目标是开发新的策略来遏制它。在MRSA中，万古霉素耐药性尚未牢固确立，所产生的结果可能导致保留一种重要的最后一线药物的效用的策略。

DESCRIPTION (provided by applicant): Methicillin resistant S. aureus (MRSA) are leading causes of antibiotic resistant infection, treated mainly with vancomycin. Since 2002, VanA-type vancomycin resistance has moved on plasmids from vancomycin resistant enterococci (VRE) to MRSA, forming VRSA. This has happened at least 12 times independently during the course of infection, mainly in lesions on the extremities of diabetics. All S. aureus recipients of the vancomycin resistance transposon have been of a single S. aureus clade, CC5. For horizontal transmission to occur, VRE and MRSA must occupy the same environment in sufficient number and proximity for transfer to occur. I hypothesize that CC5 MRSA and VRE exhibit unique interactions compared to other MRSA lineages, which include cooperative metabolic abilities promoting synergy, co-aggregation with host-derived factors, and modulation of intra- and interspecies cell signaling. Preliminary data identified serum-dependent co-aggregation as being unique to CC5-MRSA and VRE pairs. To determine what affects multi-species binding, purified serum components will be added to co-cultures and co-aggregation will be assayed by microscopy and optical density. Any enhancement of horizontal transmission will be quantified by selective plating of MRSA recipients after co-culture with enterococci containing a resistance plasmid. I have found down-regulation of VRE genes for pheromone signaling and the fsr virulence operon in the transcriptome responses of VRE /MRSA in co-culture. The impact of cell signaling modulation will be studied by transcriptomics and targeted mutagenesis of the putative pheromone signal revealed by our microarray data. D-lactate (dLac) is commonly produced by S. aureus during fermentation. dLac is also necessary for VanA-type vancomycin resistance. As VanA type resistance is induced only when vancomycin is present, I hypothesize that S. aureus dLac production may provide an advantage to VRE in co-culture with S. aureus. This will be tested by measuring survival of both species in co- and mono-culture after vancomycin exposure as well as VRE in mono-culture incubated with dLac prior to vancomycin treatment. These results will ultimately reveal the nature of polymicrobial interactions specific to ST5-MRSA and VRE and how they lead to vancomycin resistance transfer. PUBLIC HEALTH RELEVANCE: Vancomycin resistance has transferred de novo from vancomycin resistant enterococci (VRE) into methicillin resistant S. aureus (MRSA) at least 12 times during the course of infection, resulting in VRSA. The emergence of vancomycin resistance in S. aureus compromises a key last line bactericidal drug for treating a very common cause of invasive bacterial infection. I will identify and characterize factors that contribute to he spread of vancomycin resistance into S. aureus, with the goal of developing new strategies for its containment. Vancomycin resistance is not yet firmly established in MRSA, and the results generated may lead to strategies to preserve the utility of an important last line drug.