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

 描述(由申请人提供)：定植和感染病原体(伤口生物细菌)在糖尿病足溃疡(DFU)预后中的作用尚不清楚。我们的团队已经证明，使用高通量16S核糖体RNA(RRNA)基因测序测量DFUS中的微生物总数，与溃疡持续时间、溃疡深度和血糖控制不良直接相关。然而，随着时间的推移，生物负荷的变化只提供了对这些变化对DFU结果的作用的有限的见解。尽管如此，生物污染可能正在影响DFU的结果，但使用16S rRNA图谱可能无法区分重要性的特定维度。这种分子技术的主要局限性包括它没有揭示微生物区系的功能潜力，而且通常不可能分辨出物种或菌株水平。为了绕过这些限制，我们将使用全基因组鸟枪(WGS)元基因组测序和分析，这是破译微生物群落功能潜力的强大透镜。我们的主要假设是动态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微生物群有不同的影响。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，其中包括伤口微生物组和元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。为了验证我们的假设，我们将完成以下目标：目标1：确定DFU元基因组的致病物种/菌株和总体微生物多样性是否与特定的DFU结果相关。我们将描述107名从我们以前的研究中招募的患有DFU的受试者的元基因组随时间的变化，从提出到最终结果每两周收集一次样本。我们将确定1)特定种类或菌株的病原体(例如，β-溶血性链球菌)的存在；以及2)微生物多样性较低是否与较低的愈合率或感染相关的并发症(如溃疡恶化、骨髓炎或截肢)直接相关。目的2：确定DFU亚基因组中的致病基因是否与特定的DFU结局相关。我们将检查1)生物膜形成潜力、2)抗生素耐药性和3)毒力因子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较低。目的3：确定侵袭性清创和抗生素治疗对DFU元基因组的影响。这是使用WGS元基因组测序首次纵向研究DFU微生物组对DFU结果的影响，也是第一次检查两种常用的抗菌素干预对DFU元基因组的影响。

 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.