Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and titanium amplicon pyrosequencing and metagenomic approaches

Abstract

Background: Approximately 1 out of every 100 individuals has some form of venous insufficiency, which can lead to chronic venous disease and Venous Leg Ulcer (VLU). There are known underlying pathologies which contribute to the chronic nature of VLU including biofilm phenotype infections.

Results: Using pyrosequencing based approaches we evaluated VLU to characterize their microbial ecology. Results show that VLU infections are polymicrobial with no single bacterium colonizing the wounds. The most ubiquitous and predominant organisms include a previously uncharacterized bacteroidales, various anaerobes, Staphylococcus, Corynebacterium, and Serratia. Topological analysis of VLU show some notable differences in bacterial populations across the surface of the wounds highlighting the importance of sampling techniques during diagnostics. Metagenomics provide a preliminary indication that there may be protozoa, fungi and possibly an undescribed virus associated with these wounds.

Conclusion: The polymicrobial nature of VLU and previous research on diabetic foot ulcers and surgical site infections suggest that the future of therapy for such wounds lies in the core of the logical and proven multiple concurrent strategy approach, which has been termed "biofilm-based wound care" and the use of individualized therapeutics rather than in a single treatment modality.

Figure 1

Double dendogram of major genera in Venous Leg Ulcers. This figure is a double dendogram describing the major genera detected among the 40 VLU samples. The heat map indicates the relative percentage of the given genera within each sample ID with a color legend and scale provided. The distance of the samples based upon weighted pair linkage and Manhattan distance methods with no scaling is provided at the top of the figure along with a distance score. The bacterial genera and the associated clustering are provided along the Y-axis and their associated distance scores indicated. The most determinative genera for clustering, based upon this analysis, are Staphylococcus, Bacteroides, Serratia, and Corynebacterium spp.

Figure 2

Visual representation of venous leg ulcer sampling strategy. Panels A-D. These figures provide examples of VLU with the transposed sampling locations for the topological bacterial diversity evaluation. The letters (e.g. A, B, C,...) indicate where each sample was gathered from each of these VLU. The detected bacterial diversity for each of these wounds is provided in Tables 3, 4, and 5.