Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP)

Abstract

Background: Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections.

Methods and findings: Using a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp.

Conclusions: In this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP.

Figure 1. Dendrogram of Functional Equivalent Pathogroups (FEPs).

The most prevalent bacterial genera were used to perform multivariate hierarchical clustering. Using a geographic X scale and a color map we represent the 40 different wounds along the Y-axis and the predominant genera along the X-axis in this dendogram, which shows 8 primary clusters associated with possible functional equivalent pathogroups (FEPs). Thus, in cluster 1 (red dots) we see that the predominant genera are Serratia spp. and anaerobes (Finegoldia, Peptoniphilus and Anaerococus spp.). Together these genera contribute to FEP in cluster 1. Cluster 2 (yellow Y's) is made up of Corynebacterium and the same anaerobes as cluster 1. The most predominant cluster, cluster 3 (orange squares), involves Streptococcus and anaerobes including the previously mentioned genera from clusters 1 and 2 as well as Bacteroides. Cluster 4 (green diamond) is only a single sample but involves co-occurrence of Pseudomonas, Streptococcus and Porphyomonas spp. Cluster 5 (blue x's) is heavily populated by anaerobes particularly Bacteroides, Anaerococcus, Fingoldia, and Peptoniphilus spp. Cluster 6 (green crosses) is only made up of two samples and includes Enterococcus as the primary organism with significant signatures from Anaerococcus, Finegoldia, and Peptoniphilus spp. Cluster 7 (blue Z's) has the strongest color map signatures associated with anaerobes, especially Clostridium, Fingoldia, Porphyromonas and Peptoniphilus spp. Finally, cluster 8 is strongly associated with the anaerobes Anaerococcus and Fingoldia spp. with additional contributions from Streptococcus spp. The location of each extremity ulcer is also encoded into this figure along the Y-axis. The codes for the wound locations are lateral foot ulcer (L), dorsal foot ulcer (D), plantar foot ulcer (P), ankle ulcer (A), 5th metatarsal head ulcer (A), 4th metatarsal head ulcer (4), 2nd Metatarsal head ulcer (2), trans metatarsal ulcer (T), 1st metatarsal head ulcer (1), Great toe ulcer (G), and heel ulcer (H).