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Molecular analysis
Culture based methodologies for the identification and
quantification of microorganisms form the basis of most clinical lab
studies. However, many microorganisms have been overlooked in these
studies due to the inability to grow them in culture. Denaturing
gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes
was used to characterize biofilm communities from wound samples
provided by the Southwest Regional Wound Care Center. DGGE is a method for separating similar sized
DNA fragments based on sequence. Bacterial community diversity (i.e.
number of different species) can be estimated from the number of
bands in gels from each species.
Tissue samples were obtained during standard sharp debridement of
wounds during normal wound care management. Each sample was
homogenized prior to extraction. Total DNA was extracted using the
Bio101 FAST DNA Spin Kit for soil (QBioGene). Extracted total DNA
was stored as -20º C prior to shipment to CBE-MSU for PCR and DGGE.
The DNA sent to MSU-CBE was used as a template for polymerase chain
reaction (PCR).
Bacterial DNA from the wound samples was initially amplified using
two sets of universal Eubacterial forward and reverse primers
containing a GC clamp. These primer sets included: 1070F (5’ ATG GCT
GTC GTC AGC T 3’) combined with 1392R + GC Clamp (5’ CGC CCG CCG
CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC C ACG GGC GGR GRG TAC
3’) and 518R + GC Clamp (5'GTA TTA CCG CGG CTG CTG G 3') combined
with a 357F (5" CGC CCG CCG CGC CCC GCG CCC GGC CCG CCGC CCC CGC
CCC C CTA CGG GAG GCA GCA G 3') (Integrated DNA Technologies).
Primer reactions and DNA amplification were performed using a
PTC-100 Programmable Thermal Controller (MJ Research). Verification
of the presence of DNA was assessed in 1.5% agarose gels.
Amplified DNA was separated by DGGE using a 40% to 70% denaturing
gradient in 8% to 12% polyacrylamide gels following recommended
manufacturer protocols (BioRad). DGGE can detect differences in
single base changes anywhere along the strand of amplified DNA. The
double stranded, amplified DNA is subjected to an increasing
gradient of denaturant during gel electrophoresis. The DNA strands
then melts, creating a branched molecule which stops its migration
within the gel. The temperature at which the DNA melts (TM)
is a function of the sequence of the DNA molecule.
In initial studies, the combination of 518R + GC Clamp and 375F
primers resulted in the highest number of bands and was selected for
subsequent amplifications. DGGE results for a number of wound
samples are shown in Figure 6. Each wound sample displayed a unique
banding pattern within the DGGE gels, although many samples
contained bands at similar locations within the gels. Similar
findings have been reported from DGGE of samples from venous leg
ulcers (Davies et. al., 2004). In contrast to the venous leg ulcer
study, an identical band was not present in all of the samples.
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Figure 6. Denaturing Gradient
Gel Electrophoresis (DGGE) profiles of polymerase Chain Reaction (PCR)
amplified bacterial DNA from chronic wound samples. Each wound
examined has a unique banding pattern, although many wounds have
common bands. The wound type and bacteria cultured for each sample
is shown in Table 3.
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Information on wound types and culture results for the above DGGE
gels are presented in Table 3. In one case, (A) no microorganisms
were cultured, but a distinct band was apparent after PCR and DGGE
of extracted DNA from the chronic wound sample. In many cases, one
to three organisms were cultured from the wound samples but many
bands are present in the DGGE analysis indicating that multiple
species were present. These findings underscore the utility of
molecular methods for detecting unculturable microorganisms. These
preliminary studies also suggest that the samples from the diabetic
foot ulcers had the most species diversity (i.e. bands) and venous
leg ulcers had the least species diversity. However, a detailed
analysis of more samples will be necessary to confirm this
suspicion. |
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Gel Lane* |
Wound type |
Genera/species cultured |
A
|
Non-healing surgical wound
|
Enterobacter, Pseudomonas
|
B
|
Venous leg ulcer
|
None
|
C
|
Calciphylaxis
|
Pseudomonas, Staphylococcus
|
D
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Venous leg ulcer
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Escherichia coli, Staphylococcus aureus
|
E
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Diabetic foot ulcer
|
Streptococcus
(Group B),
Citrobacter, Staphylococcus
|
F
|
Venous leg ulcer
|
Pseudomonas, Staphylococcus
|
G
|
Chronic wound
|
Enterococcus
(Group B),
Escherichia coli, Staphylococcus aureus
|
H
|
Venous leg ulcer
|
Pseudomonas
|
I
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Diabetic foot ulcer
|
Enterococcus
(Group D)
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J
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Diabetic foot ulcer
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Citrobacter freundii, Staphylococcus aureus
|
K
|
Decubitus ulcer
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Staphylococcus aureus
|
L
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Diabetic foot ulcer
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Staphylococcus
|
M
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Non-healing surgical wound
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Pseudomonas, Staphylococcus
|
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Overall, our preliminary microbiological studies of wounds indicated
that biofilms were prevalent in chronic wounds and rare in acute
wounds. These biofilms were polymicrobial communities, and each
wound sample appeared to have unique community structure. Although
the mere presence of complex microbial biofilms in wounds does not
provide evidence that they are a barrier to healing, clinical
observations of chronic wounds are consistent with those of other
biofilm-related diseases and further research is warranted to assess
the role of biofilms in the prevention of wound healing. |
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On the next
page: Clinical
observations from biofilm-based treatment approaches |
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