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

BackgroundApproximately 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.ResultsUsing 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.ConclusionThe 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.

Traumatic Wound Microbiome Workshop

“…However, surgical site infections posed a unique problem; frequently, the Bacteroides which dominated those wounds required co-growth in a polymicrobial wound biofilm model [37]. In 99 % of wounds, 1 % of the population is easy to grow and is characterized by routine clinical cultures [38].…”

Influence of Microorganisms on the Healing of Skin Grafts from Chronic Venous Leg Wounds

“…It differs from the classic Sanger sequencing, in that it relies on the detection of pyrophosphate release upon nucleotide incorporation, rather than chain termination with dideoxynucleotides (Ronaghi et al, 1998). This deep sequencing technique has been used to evaluate the diversity of microbial populations where thousands of reads/sequences can be www.intechopen.com Skin Grafts -Indications, Applications and Current Research 280 obtained from each sample (Andersson et al, 2007;Bogaert et al;Dowd et al, 2008;Price et al, 2010;Smith et al, 2010;Stewart et al, 2010;Wolcott et al, 2009). …”

“…This means that different microorganisms have more or less favorable growth conditions at different locations which often results in a heterogeneous microbiological distribution throughout the wound (Price et al, 2010). Wolcott (2009) concluded that individual wounds have distinct ecological footprints, and within the individual wounds there can be both significant site specific differences and relative uniformity in the bacterial ecology.…”

“…Adding detection of known virulence genes to the molecular methods would be helpful in the process of interpretation. Ideally, each wound should be carefully evaluated and in addition to traditional biofilmbased wound care strategies, an antimicrobial/antibiofilm treatment program with individualized therapeutic approaches should be identified for each patient's respective wound microflora (Smith et al, 2010;Wolcott et al, 2009). However, we do not have the tools for interpretation yet.…”

Association Between Microbial Bioburden and Healing Outcomes in Venous Leg Ulcers: A Review of the Evidence

“…[41][42][43] In general, S. aureus has been visualized more toward the surface of the ulcers and biofilms, particularly compared with Pseudomonas aeruginosa, potentially explaining why S. aureus is most commonly isolated from swab samples of chronic wounds and P. aeruginosa is likely frequently underestimated. 41,43,44 Studies that have used bacterial 16S rRNA sequencing to investigate intrawound variation in chronic wounds include Price et al, 45 and Wolcott et al 46 In both studies, curette samples were collected from different geographic regions of chronic wounds, including the leading edge, opposing leading edge, and/or center of chronic wounds. Wolcott et al concluded there was substantial variation across the wound surface without further statistical analysis.…”