Evaluation of the bacterial diversity of pressure ulcers using bTEFAP pyrosequencing

Abstract

Background: Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories.

Methods: Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities.

Results: Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics.

Conclusions: Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.

Figure 1

Double dendogram of major genera in decubitus ulcers. Describes major genera detected among the 49 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.

Figure 2

Double dendogram of major species in decubitus ulcers. Describes major species detected among the 49 samples. The heat map indicates the relative percentage of the given species 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 species and the associated clustering are provided along the Y-axis and their associated distance scores indicated.

Figure 3

Double dendogram of phenotypes in decubitus ulcers. The heat map shows relative percentages of the given phenotypes in each of the 49 samples 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 displayed at the top of the figure along with a distance score. The bacterial phenotypes and the associated clustering are provided along the Y-axis and their associated distance scores are indicated. The bacteria predominantly expressed the gram-positive and cocci phenotypes, with aerotolerance being of relatively equitable distribution.

Figure 4

Three dimensional PCA plot of the unifrac distance in relation to diabetes. Principle component analysis based upon a unifrac analysis of the sequencing data was utilized. Based upon this PCA analysis the primary 3 vectors are plotted in 3 dimensions. The percent variability explained by each vector is indicated in parenthesis in the figure. Based upon the primary vector a t-test was utilized to determine if there was significant variation between diabetic samples. The separation across the primary vector was found to be significant (p = 0.003) indicating that the community structure of ulcers in subjects with diabetics may be different from those of non-diabetics.