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<p>Diabetic foot infections (DFIs) are complex, polymicrobial conditions that delay wound healing, increase the risk of lower limb amputation, and contribute to higher mortality among patients with diabetes mellitus. Traditional culture-based diagnostic methods often fail to identify the full diversity of wound-associated microorganisms, particularly fastidious and anaerobic species. This study aimed to characterize the microbial composition of DFIs through 16S rRNA gene sequencing and to examine its relationship with disease severity and wound healing outcomes. A cross-sectional comparative study was conducted on 300 participants divided into three groups of equal numbers: healthy controls, patients with mild DFIs, and patients with severe DFIs. Superficial and deep wound swabs were collected under aseptic conditions. Bacterial genomic DNA was extracted, and the V3 to V4 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. Sequence processing and quality control were performed through the QIIME2 DADA2 pipeline, while taxonomic classification was assigned using the SILVA 138 database. Microbial diversity analyses included Shannon and Simpson indices for alpha diversity, however,&#xa0; Bray-Curtis and weighted UniFrac metrics for beta diversity. Significant differences in microbial community composition were observed among the study groups (PERMANOVA, <i>p</i> &lt; 0.001), although overall amplicon sequence variant richness showed no significant variation (<i>p</i> = 0.12). Alpha diversity was highest in mild DFIs and declined significantly in severe infections (<i>p</i> &lt; 0.001), indicating progressive microbial dysbiosis with increasing disease severity. <i>Proteobacteria</i> dominated all cohorts (&gt; 98.2%), with <i>Pseudomonas aeruginosa</i>, <i>Stenotrophomonas maltophilia</i>, and <i>Delftia acidovorans</i> among the most prevalent taxa. Beta diversity analyses demonstrated partial clustering according to clinical severity, suggesting subtle but biologically meaningful microbial restructuring. Reduced microbial diversity and increased abundance of opportunistic pathogens are associated with poor healing outcomes in DFIs. These findings highlight the clinical relevance of microbiome profiling to enhance understanding of disease progression and optimize therapeutic strategies. Severe DFIs are associated with reduced microbial diversity and enrichment of opportunistic Gram-negative pathogens, particularly within the order <i>Pseudomonadales</i>. These findings highlight the limitations of conventional culture methods and support the integration of microbiome-based diagnostics for improved risk stratification and targeted antimicrobial management in diabetic foot infections.</p>

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Characterization of microbiome diversity and its association with healing outcomes in diabetic foot ulcer patients

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月9日

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  • Muhammad Shakil Khan,
  • Aasia Kalsoom,
  • Awais Altaf,
  • Muhammad Sarwar,
  • Tahir Maqbool,
  • Ghulam Shabbir,
  • Aisha Tahir,
  • Hafiza Saba Safdar,
  • Muhammad Hassan,
  • Muhammad Amber Fareed

摘要

Diabetic foot infections (DFIs) are complex, polymicrobial conditions that delay wound healing, increase the risk of lower limb amputation, and contribute to higher mortality among patients with diabetes mellitus. Traditional culture-based diagnostic methods often fail to identify the full diversity of wound-associated microorganisms, particularly fastidious and anaerobic species. This study aimed to characterize the microbial composition of DFIs through 16S rRNA gene sequencing and to examine its relationship with disease severity and wound healing outcomes. A cross-sectional comparative study was conducted on 300 participants divided into three groups of equal numbers: healthy controls, patients with mild DFIs, and patients with severe DFIs. Superficial and deep wound swabs were collected under aseptic conditions. Bacterial genomic DNA was extracted, and the V3 to V4 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. Sequence processing and quality control were performed through the QIIME2 DADA2 pipeline, while taxonomic classification was assigned using the SILVA 138 database. Microbial diversity analyses included Shannon and Simpson indices for alpha diversity, however,  Bray-Curtis and weighted UniFrac metrics for beta diversity. Significant differences in microbial community composition were observed among the study groups (PERMANOVA, p < 0.001), although overall amplicon sequence variant richness showed no significant variation (p = 0.12). Alpha diversity was highest in mild DFIs and declined significantly in severe infections (p < 0.001), indicating progressive microbial dysbiosis with increasing disease severity. Proteobacteria dominated all cohorts (> 98.2%), with Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Delftia acidovorans among the most prevalent taxa. Beta diversity analyses demonstrated partial clustering according to clinical severity, suggesting subtle but biologically meaningful microbial restructuring. Reduced microbial diversity and increased abundance of opportunistic pathogens are associated with poor healing outcomes in DFIs. These findings highlight the clinical relevance of microbiome profiling to enhance understanding of disease progression and optimize therapeutic strategies. Severe DFIs are associated with reduced microbial diversity and enrichment of opportunistic Gram-negative pathogens, particularly within the order Pseudomonadales. These findings highlight the limitations of conventional culture methods and support the integration of microbiome-based diagnostics for improved risk stratification and targeted antimicrobial management in diabetic foot infections.