Polymicrobial dynamics in transfer of vancomycin resistance to MRSA

描述（由申请人提供）：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，主要用万古霉素治疗。自2002年以来，vana型万古霉素耐药已通过质粒从万古霉素耐药肠球菌（VRE）转移到MRSA，形成VRSA。这种情况在感染过程中至少独立发生过12次，主要发生在糖尿病患者的四肢病变。所有感染万古霉素耐药转座子的金黄色葡萄球菌均属于单一金黄色葡萄球菌分支CC5。为了发生水平传播，VRE和MRSA必须在相同的环境中有足够的数量和距离才能发生转移。我假设CC5 MRSA和VRE与其他MRSA谱系相比表现出独特的相互作用，包括促进协同作用的合作代谢能力，与宿主衍生因子的共同聚集，以及物种内和物种间细胞信号的调节。初步数据确定血清依赖性共聚集是CC5-MRSA和VRE对所特有的。为了确定影响多物种结合的因素，将纯化的血清成分添加到共培养物中，并通过显微镜和光密度检测共聚集。任何水平传播的增强将通过与含有抗性质粒的肠球菌共培养后的MRSA受体的选择性镀板来量化。我在VRE /MRSA共培养的转录组反应中发现了VRE基因信息素信号和fsr毒力操纵子的下调。细胞信号调节的影响将通过转录组学和靶向诱变的推测信息素信号显示我们的微阵列数据来研究。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。由于只有万古霉素存在时才会诱导VanA型耐药，我推测金黄色葡萄球菌产生dLac可能对VRE与金黄色葡萄球菌共培养有优势。这将通过测量万古霉素暴露后两种物种在共培养和单培养中的存活率以及万古霉素治疗前用dLac孵育的单培养的VRE来进行测试。这些结果将最终揭示ST5-MRSA和VRE特异性多微生物相互作用的性质，以及它们如何导致万古霉素耐药性转移。

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.