Decoding the microbial bioburden of diabetic foot ulcers: A metagenomic approach

 描述（由申请方提供）：定植和感染病原体（伤口生物负载）对糖尿病足溃疡（DFU）结局的作用尚不清楚。我们的研究小组已经表明，使用高通量16 S核糖体RNA（rRNA）基因测序测量的DFU中微生物总数与溃疡持续时间、溃疡深度和血糖控制不良直接相关。然而，生物负载随时间的变化仅提供了对这些变化对DFU结局的作用的有限见解。尽管如此，生物负载可能会影响DFU结果，但使用16 S rRNA分析可能无法识别重要的具体维度。这种分子技术的主要局限性包括它没有揭示微生物群的功能潜力，并且通常不可能解析到物种或菌株水平。为了规避这些限制，我们将采用全基因组鸟枪（WGS）宏基因组测序和分析，一个强大的透镜破译微生物群落的功能潜力。我们的总体假设是，动态DFU微生物组的特定维度可以预测结果。此外，我们假设不同的治疗方案对DFU微生物组有不同的影响。我们已经组建了一个多学科的专家团队来执行这项提案的目标，他们拥有伤口微生物组和宏基因组学方面的基础科学专业知识，以及DFU和感染方面的临床和研究专业知识。为了检验我们的假设，我们将完成以下目标：目标1：确定DFU宏基因组的致病物种/菌株和总体微生物多样性是否与特定DFU结果相关。我们将描述从我们先前的研究中招募的107名DFU受试者的宏基因组随时间的变化，从出现到最终结果，每2周收集一次样本。我们将确定1）病原体的特定物种或菌株（例如β-溶血性链球菌）的存在;和2）低微生物多样性是否与较低的愈合率或感染相关并发症（即，溃疡恶化、骨髓炎或截肢）。目的2：确定DFU宏基因组中的致病基因是否与特定DFU结果相关。我们将检查赋予1）生物膜形成潜力，2）抗生素抗性和3）毒力因子的基因的存在和相对丰度，以确定它们是否相关 治愈率和感染相关并发症的发生率较低。目的3：确定积极的锐性清创术和抗生素治疗对DFU宏基因组的影响。这是第一次使用WGS宏基因组测序对DFU微生物组对DFU结果的作用进行纵向研究，也是第一次研究两种常用的抗菌干预对DFU宏基因组的影响。

 DESCRIPTION (provided by applicant): The role of colonizing and infecting pathogens (wound bioburden) on diabetic foot ulcer (DFU) outcomes remains unclear. Our group has shown that the totality of microorganisms in DFUs measured using high throughput 16S ribosomal RNA (rRNA) gene sequencing, is directly related to: ulcer duration, ulcer depth, and poor glycemic control. However, changes in bioburden over time provided only limited insights into the role of these changes on DFU outcomes. Nonetheless, bioburden may be influencing DFU outcomes, but the specific dimensions of importance may not be discernable using 16S rRNA profiling. Major limitations of this molecular technique include the fact that it does not disclose functional potential of the microbiota and resolution to species or strain level is often impossible. To circumvent these limitations, we will employ whole genome shotgun (WGS) metagenomic sequencing and analysis, a powerful lens for deciphering the functional potential of microbial communities. Our overarching hypothesis is that specific dimensions of the dynamic DFU microbiome are predictive of outcome. Additionally, we hypothesize that different treatment regimens have different effects on the DFU microbiome. We have assembled a multi-disciplinary team of experts to execute the aims of this proposal, with basic science expertise in wound microbiome and metagenomics and clinical and research expertise in DFUs and infection. To test our hypothesis, we will complete the following aims: Aim 1: Determine if pathogenic species/strains and overall microbial diversity of the DFU metagenome are associated with specific DFU outcomes. We will characterize the changes in metagenomes over time for 107 subjects with DFUs recruited from our previous studies, from whom specimens were collected every 2 weeks from presentation to final outcome. We will determine if 1) the presence of specific species or strains of pathogens (e.g. beta-hemolytic Streptococcus); and 2) low microbial diversity are directly associated with lower rates of healing or infection-related complications (i.e., ulcer deterioration, osteomyelitis or amputation). Aim 2: Determine if pathogenicity genes in the DFU metagenome are associated with specific DFU outcomes. We will examine the presence and relative abundance of genes conferring 1) biofilm-formation potential, 2) antibiotic resistance and, 3) virulence factors, to determine if they are association with lower rates of healing and infection-related complications. Aim 3: Determine the effects of aggressive sharp debridement and antibiotic treatment on the DFU metagenome. This is the first longitudinal study of the role of the DFU microbiome on DFU outcomes using WGS metagenomic sequencing as well as the first to examine the impact of two commonly employed antimicrobial interventions on the DFU metagenome.