Chapter 4 Biofilms in Health and Medicine
Section 5 Evidence of Biofilms in Chronic Wounds
Page 3 Evidence from a CBE study

What is the evidence that biofilm forms in chronic wounds?

Gram-stained diabetic foot ulcer tissue
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 1. Diabetic foot ulcer tissue.
Diabetic Foot Ulcer
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 2. Diabetic foot ulcer tissue.
Venous leg ulcer tissue
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 3. Venous leg ulcer tissue.
Pressure ulcer tissue
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 4. Pressure ulcer tissue.
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Figure 5. (Forthcoming) Biofilm in Wounds.
Light micrographs of Gram-stained tissue sections from chronic wounds showing biofilms of Gram-positive cocci.

Evidence from a CBE study

In collaboration with the Southwest Regional Wound Care Center, in Lubbock, Texas, the CBE examined acute and chronic wound samples from human volunteers for the presence of biofilms. The primary questions addressed by this research included:

Chronic wound samples were obtained from patients undergoing sharp debridement of persistent wounds as part standard wound care management. Samples were also collected from patients with acute wounds using standard surgical procedures to collect elliptical tissue sections with a minimum diameter of approximately 1 centimeter. The orientation of the tissue (i.e. top and bottom) was indicated using tissue dye. The samples were preserved in a phosphate-buffered formalin solution, transferred to a 70% ethanol solution, and shipped overnight to CBE. A total of 50 samples from persistent wound infections and 16 samples from acute wounds were obtained. Analysis of the samples included scanning electron microscopy (SEM) and light microscopic evaluation of Gram-stained tissue thin sections. Criteria for biofilm classification the included number and density of cells, large cellular aggregates were classified as biofilm while individual or small clusters of cells were not, and the presence of extracellular polymer substance (EPS) around the bacterial cells.

The predominant type of biofilm bacteria observed using light microscopy were Gram positive cocci (Figure 5). These results were in agreement with culture data which showed a predominance of Staphylococcus and Enterococcus from these patients. SEM examination of the samples confirmed the predominance of coccoid cells, which often appeared to be coated with EPS (Figure 6). Similar coatings were observed in pure culture biofilms of Staphylococcus aureus grown using the colony biofilm model (Figure 7).

Biofilms composed of Gram-negative rods (Figure 8) and mixed-species biofilms (Figure 9) were also observed. These results were confirmed by results of culture and molecular analysis, discussed below, indicating that wound biofilms are indeed polymicrobial.

Overall, 30 out of 50 chronic wounds and one in 16 acute wounds were characterized as containing biofilm. Using Fisher’s exact test, this is a statistically significant difference (P>0.001) and indicates biofilms appear to be prevalent in chronic and rare in acute wounds.

Characterization of microorganisms present in wounds. From the patients involved in the biofilm study, described above, culture data was available for 37 of the chronic wounds and 5 of the acute wounds. Based on these data, bacteria from eight genera were frequently (>10%) isolated from wound samples (Table 1).

Genus Chronic % Acute %
Staphylococcus
65
60
Enterococcus
62
80
Pseudomonas
35
20
Proteus
24
20
Citrobacter
24
20
Enterobacter
24
20
Streptococcus
22
0
Escherichia
14
0
Morganella
8
0
Klebsiella
5
0
Acinetobacter
5
0
Serratia
3
0
Xanthomonas
3
0
Table 1. Characterization of microorganisms present in wounds

Scanning Electron micrographs of tissue specimens from chronic wounds
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R. Wolcott, Southwest Regional Wound Care Center, Lubbuck, Texas

Figure 6.
Scanning Electron micrographs of tissue specimens from chronic wounds showing biofilms of coccoid cells blanked by extracellular polymer matrix; A, pressure ulcer; B, venous leg ulcer.

 

Scanning Electron micrograph of Staphylococcus aureus biofilm
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 7.
Scanning Electron micrograph of Staphylococcus aureus biofilm grown in-vitro using the colony biofilm model. The biofilm of coccoid cells is blanked by extracellular polymer matrix.

 

Light Micrograph of Gram-stained tissue
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 8.
Light micrograph of Gram-stained tissue section from a diabetic foot ulcer showing a biofilm composed of Gram-negative rods.

 

tissue section from a diabetic foot ulcer showing the presence of both Gram-positive cocci and Gram-negative rods
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E. Swogger, Center for Biofilm Engineering, Montana State University, Bozeman

Figure 9.
Light micrograph of Gram-stained tissue section from a diabetic foot ulcer showing the presence of both Gram-positive cocci and Gram-negative rods.

In agreement with microscopic analysis, coccoid cells (Staphylococcus, Enterococcus) predominated in the wounds, although bacilli were also present. Strictly anaerobic bacteria implicated in previous studies of chronic wounds (Bowler and Davies, 1999) not cultured from chronic wounds despite specific attempts by two clinical laboratories to culture these organisms. However, the presence of facultative anaerobes, such as coliforms and fecal streptococci, in chronic wounds and not in acute wounds corroborates the findings of the previous studies. Culture-based methods for the identification of microorganisms have been the mainstay of clinical microbiology and will likely remain an important tool for the diagnosis and treatment of disease. For example, the isolation of specific strains from patients can provide important antibiotic sensitivity information to help guide treatment regimens. Nonetheless, the presence of viable but non-culturable bacteria and inability to culture many bacterial species under standard laboratory conditions necessitates the use of non-culture-based methods for the accurate diagnosis of disease.