JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2009, p. 4084–4089 Vol. 47, No.

12
0095-1137/09/$12.00 doi:10.1128/JCM.01395-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Nonrandom Distribution of Pseudomonas aeruginosa and

Staphylococcus aureus in Chronic Wounds䌤
Mustafa Fazli,1 Thomas Bjarnsholt,1 Klaus Kirketerp-Møller,2 Bo Jørgensen,2
Anders Schou Andersen,2,3 Karen A. Krogfelt,3 Michael Givskov,1
and Tim Tolker-Nielsen1*
Department of International Health, Immunology, and Microbiology, Faculty of Health Sciences, University of Copenhagen,
Copenhagen, Denmark1; Copenhagen Wound Healing Center, Bispebjerg Hospital, Copenhagen, Denmark2; and
Statens Serum Institut, ABMP, Copenhagen, Denmark3
Received 17 July 2009/Returned for modification 24 August 2009/Accepted 30 September 2009

The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was
investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as
having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by
the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning
microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five
wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the
bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound
surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of
the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent
finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds
and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic
wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory
processes.

Chronic wounds, such as diabetic foot ulcers, pressure
ulcers, and venous leg ulcers, are an increasing problem
worldwide. One to 2% of the population in developed coun-
tries develops chronic wounds, a condition associated with
severe patient suffering, the loss of employment, a reduced
quality of life, and high costs to the health care system (13).
Detailed knowledge about chronic wounds is required in
faecalis (71.7%), Pseudomonas aeruginosa (52.2%), coagu-
lase-negative staphylococci (45.7%), Proteus species
(41.3%), and anaerobic bacteria (39.1%) (12). S. aureus and
P. aeruginosa are opportunistic pathogenic bacteria and are
widely known to cause chronic biofilm-based infections in
their hosts. S. aureus is most commonly isolated from
chronic wounds (8, 12, 15, 17) and, in certain situations, can

Downloaded from https://journals.asm.org/journal/jcm on 12 January 2023 by 147.78.30.170.

order to develop better wound treatment and management
strategies.
A normal wound healing process involves four main phases:
(i) coagulation, (ii) inflammation, (iii) cell proliferation and
repair of the matrix, and (iv) epithelialization and remodeling
of the scar tissue (23). However, chronic wounds are believed
to be captured in the inflammatory phase, where persistent
influx and elevated activity of polymorphonuclear neutrophils
(PMNs) occur (1). Although PMNs play a critical role in the
host defense and wound healing, they release cytolytic en-
zymes, free oxygen radicals, inflammatory mediators, and ma-
trix metalloproteases, which cause local tissue damage in the
host (22, 23, 26).
It is known that the microflora of chronic wounds com-
prises multiple species. In a bacterial profiling study, Gjøds-
bol et al. found that chronic venous leg ulcers harbored
Staphylococcus aureus (in 93.5% of the ulcers), Enterococcus
* Corresponding author. Mailing address: Department of Inter-
national Health, Immunology and Microbiology, Faculty of Health
Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen N
DK-2200, Denmark. Phone: 45 353 26656. Fax: 45 353 27853. E-mail:
ttn@sund.ku.dk.
䌤
Published ahead of print on 7 October 2009.
express a number of potential virulence factors and surface
proteins which promote its adherence to the damaged tissue
and decrease neutrophil functions and immune responses of
the host (10, 11). P. aeruginosa often causes biofilm-based
chronic infections and expresses virulence factors, in partic-
ular, rhamnolipid, that can eliminate the activity of PMNs
(4, 16). A number of studies have demonstrated that P.
aeruginosa is frequently present in chronic wounds (12, 17)
and have provided evidence that the bacteria are located in
aggregates enclosed in extracellular polymeric matrix mate-
rial as found in biofilms (17). Furthermore, chronic wounds
that harbored P. aeruginosa were larger than those that did
not, and the healing process also seemed to be more se-
verely hindered for those wounds (12, 14, 20).
Biofilms are bacterial aggregates enclosed in a self-produced
extracellular polymeric matrix (6, 21, 25). In clinical environ-
ments biofilms can form on dead or living tissues, mucosal
surfaces, or the surfaces of medical devices in the host. The
bacteria in biofilms often display characteristics different from
those of their planktonic counterparts, such as increased resis-
tance to the activities of the host immune system and tolerance
to antimicrobial treatments (7). Such characteristics are im-
portant, since biofilms are involved in many chronic bacterial
infections. Recent studies have shown the presence of bacterial

4084
VOL. 47, 2009
FIG. 1. Sampling region on a chronic venous leg ulcer. Biopsy
specimens were taken from a central region within the wounds. The
arrows point to a representative sampling region.
biofilms in chronic wounds (9, 15, 17). Although the role of
biofilms in chronic wounds is not yet fully understood, it is
believed that their existence may be one of the reasons for
impaired wound healing (4, 16).
We previously demonstrated that there is a lack of correla-
tion between the bacteria detected by standard culturing and
those detected directly by peptide nucleic acid (PNA)-based
fluorescence in situ hybridization (FISH) in chronic wound
samples (17). While S. aureus was detected more frequently
by swab sample cultivation than by PNA-FISH, the opposite
was true for P. aeruginosa. This lack of correlation between
detection by swab sample cultivation and PNA-FISH may be
due to the ability of the different bacterial species to colo-
nize different regions of chronic wounds. Swab sample cul-
tivation identifies the microorganisms present in the surface
region of the wound but may not detect microorganisms
located inside the wound bed. Accordingly, in the present
report, we present evidence that S. aureus primarily colo-
nizes the region of chronic wounds which is close to the
surface, whereas P. aeruginosa primarily colonizes the
deeper regions of chronic wounds. The ability of P. aerugi-
nosa to colonize the deeper regions of chronic wounds may
be due to the ability of this organism to produce virulence
factors which destroy PMNs (4, 16), and it may play an
important role in keeping the wounds arrested in a stage
dominated by inflammatory processes.
MATERIALS AND METHODS
Tissue sample collection and preparation. Nine patients diagnosed with
chronic venous leg ulcers were included in the study. As described below, four
patients had S. aureus-containing wounds, four patients had P. aeruginosa-con-
taining wounds, and one patient had a wound that contained both S. aureus and
P. aeruginosa. Material (4-mm punch biopsy specimens) from chronic venous leg
ulcers (Fig. 1) was obtained with the acceptance of the patients and in accor-
dance with biomedical project protocols H-B-2008-023 and KA-20051011, which
were approved by the Danish Scientific Ethical Board. Wound biopsy material
was collected by a surgical team before cleansing and surgical preparation of the
wound (2). The material was immediately transferred to phosphate-buffered
saline with 4% paraformaldehyde and stored at 5°C before further preparation
for microscopic investigation. The biopsy material for microscopic investigation
was embedded in paraffin, cut into 4-␮m sagittal sections, and mounted on glass
slides. Prior to microscopic investigation, the paraffin was removed from the
tissue sections by immersing the glass slides twice in xylene (total, 10 min), twice
P. AERUGINOSA AND S. AUREUS IN CHRONIC WOUNDS 4085
in 99.9% ethanol (total, 6 min), twice in 96% ethanol (total, 6 min), and three
times in distilled sterile water (total, 9 min).
PNA-FISH and conventional tissue staining. The deparaffinized tissue sec-
tions were analyzed by means of conventional hematoxylin and eosin (H&E)
staining and FISH with PNA probes. The PNA probe in hybridization solution
(AdvanDx, Inc., Woburn, MA) was added dropwise to each tissue section, which
was then covered with a coverslip and hybridized in a PNA-FISH workstation
(AdvanDx, Inc.), which was covered with a lid, at 55°C for 90 min. Three separate
PNA probe solutions were used: (i) a Texas Red (TxR)-labeled P. aeruginosa-
specific probe, (ii) a fluorescein isothiocyanate (FITC)-labeled S. aureus-specific
probe, and (iii) a mixture of the TxR-labeled P. aeruginosa-specific probe and the
FITC-labeled S. aureus-specific probe. The slides with tissue sections were
washed in a wash solution (AdvanDx, Inc.) at 55°C for 30 min, air dried, mounted
with Vectashield mounting medium with 4⬘,6⬘-diamidino-2-phenylindole (DAPI;
Vector Laboratories), and covered with a coverslip. The tissue sections were
examined as described below.
Image acquisition and analysis. Microscopic observations of the tissue sec-
tions were performed with an epifluorescence microscope (Olympus, Hamburg,
Germany) or a TCS-SP5 confocal laser scanning microscope (Leica Microsys-
tems, Mannheim, Germany) equipped with an argon laser and a helium-neon
laser for excitation of the fluorophores. Multichannel simulated fluorescence
projection images were generated by using the IMARIS software package (Bit-
plane AG, Zurich, Switzerland) and were further processed for display by using
PhotoShop software (Adobe). Subtraction of the background from the images
was performed with the IMARIS software package to remove the host tissue
autofluorescence. The images were converted to eight-bit gray-scale images by
using ImageJ (version 1.41o) software (http://rsb.info.nih.gov/ij/index.html), and
the moment calculator tool (http://rsb.info.nih.gov/ij/plugins/moments.html) of
the same software was used to locate the center of mass of the bacterial popu-
lation displayed on the images.
Statistical evaluation. To evaluate whether the data obtained from the dis-
tance measurement of P. aeruginosa and S. aureus aggregates to the wound
surface were statistically significant, an unpaired t test was performed. P values
of ⱕ0.05 were considered significant. The statistical program Stat-View (SAS
Institute Inc., Cary, NC) was used to calculate P values.
RESULTS
Initially, we identified P. aeruginosa and S. aureus in biopsy
material from chronic wounds by the use of PNA-FISH with
species-specific probes. On the basis of those identities, we
selected five wounds containing P. aeruginosa and five wounds
Downloaded from https://journals.asm.org/journal/jcm on 12 January 2023 by 147.78.30.170.
containing S. aureus (one of the wounds selected contained
both P. aeruginosa and S. aureus). In order to study the spatial
distribution of P. aeruginosa and S. aureus in these wounds, we
cut five sections sagittally at 50-␮m intervals from each wound
biopsy specimen and performed PNA-FISH of these sections
with P. aeruginosa- and S. aureus-specific probes. We subse-
quently acquired images at three different regions on each
section by confocal laser scanning microscopy (CLSM). The
bacteria were predominantly present as large aggregates. To
get a measure of the distance of the bacteria to the wound
surface, we located the center of mass of the bacterial aggre-
gates identified on each image by using the moment calculator
tool of ImageJ software and measured its distance to the
wound surface. This analysis showed that the S. aureus aggre-
gates were located close to the wound surface, whereas the P.
aeruginosa aggregates were located deeper in the wound bed
(P ⬍ 0.0001) (Table 1). The centers of mass of the S. aureus
aggregates were primarily located at a distance of 20 to 30 ␮m
to the wound surface, whereas the centers of mass of the P.
aeruginosa aggregates were primarily located at a distance of
50 to 60 ␮m to the wound surface (Fig. 2). Figure 3 shows
representative CLSM images of the locations of P. aeruginosa
and S. aureus in the chronic wounds. The range of the distri-
bution of P. aeruginosa and S. aureus was limited, so colocal-
4086 FAZLI ET AL. J. CLIN. MICROBIOL.

TABLE 1. Average distance of bacterial aggregates to the surface can in some cases effectively control superficial bacterial bur-
of wound samples dens if the infection is localized but may not be appropriate for
Wound biopsy Bacterial species detected Avg distance to wound highly infected wounds. Systemic antibiotics may be effective in
specimen by PNA-FISH surface (␮m)b some cases of severe infection with tissue invasion (23). The
LGA02 S. aureus 28.3 (6.6) use of a nanocrystalline silver dressing was shown to decrease
BIJ04 S. aureus 8.8 (1.7) the superficial bacterial burden, as assessed by surface swab
HAH08 S. aureus 28.1 (5.0) investigation, but had no effect on the bacterial burden of the
M2a S. aureus 26.1 (5.1) deep wound compartment, as measured by tissue biopsy (24).
Pt17 S. aureus 23.7 (3.7)
M3a P. aeruginosa 57.5 (9.4)
Thus, it is of great importance to define the spatial organiza-
Pt11 P. aeruginosa 50.0 (13.4) tion of the bacterial species within a chronic wound for the
Pt20 P. aeruginosa 53.5 (9.9) most effective management of the infection. A relevant picture
Pt23B P. aeruginosa 68.7 (11.2) of the spatial organization of the bacteria in a chronic wound
Pt31 P. aeruginosa 46.1 (6.0) might be obtained by using molecular methods, such as dena-
a
Specimens M2 and M3 are biopsy specimens obtained from the same wound. turing gradient gel electrophoresis (2, 8) and FISH (16), in
Both S. aureus and P. aeruginosa were detected in these biopsy specimens. In the combination with traditional culturing of swab as well as biopsy
case of specimen M2, the distance of S. aureus to the wound surface was ana-
lyzed, whereas in the case of specimen M3, the distance of P. aeruginosa to the samples.
wound surface was analyzed. The biofilm mode of growth provides bacteria characteristics
b
The center of mass of the bacterial aggregates on each image was located,
and its distance to the wound surface was measured. The average distances of the
different from those of their planktonic counterparts, such as
center of mass to the wound surface were obtained from 15 images acquired for protection against the activities of the host immune system and
each wound sample. The values in parentheses are standard deviations. increased tolerance to antimicrobial treatments (7). P. aerugi-
nosa bacteria in biofilms express quorum-sensing-controlled
virulence factors that can kill or eliminate the activity of host
ization of the two bacterial species was rare. In order to visu- immune cells. It has been shown that rhamnolipid, a leukocidal
alize host cells and bacteria in the wound biopsy specimens, we toxin produced by P. aeruginosa, causes rapid necrosis of
performed combined PNA-FISH and DAPI staining as well as PMNs in vitro (16). Bjarnsholt and colleagues proposed that
H&E staining of the biopsy specimens from wounds containing rhamnolipid offers a protective shield against the activities of
P. aeruginosa or S. aureus. As shown in Fig. 4, the analyzed host immune cells and demonstrated that aggregates of P.
sections from wounds with P. aeruginosa had a higher number aeruginosa in chronic wounds were surrounded by host cells,
of PMNs than the sections from wounds with S. aureus, sug- possibly PMNs, but were not penetrated (5, 17), similar to what
gesting that wounds infected with P. aeruginosa may have a was observed in in vitro biofilms of P. aeruginosa overlaid with
higher degree of inflammation than wounds infected with S. freshly isolated PMNs (4). The bacteria in chronic wounds are
aureus. expected to compete with each other for the available nutri-
ents. The ability of P. aeruginosa to migrate via type IV pili and
DISCUSSION flagellum-mediated motility in biofilms (3, 18, 19) and to pro-
duce virulence factors that can eliminate the activity of host
Although the microflora of chronic wounds is polymicrobial defense systems (4, 16) may explain the presence of these

Downloaded from https://journals.asm.org/journal/jcm on 12 January 2023 by 147.78.30.170.

and heterogeneous, S. aureus and P. aeruginosa are among the bacteria in the deeper regions of chronic wounds. The destruc-
bacteria that are most frequently isolated from these wounds tion of PMNs by virulence factors produced by P. aeruginosa
(8, 12, 15). In the present study, we characterized the distribu-
tion of P. aeruginosa and S. aureus in nine chronic wounds: four
wounds with S. aureus, four wounds with P. aeruginosa, and one
wound with both S. aureus and P. aeruginosa. Analysis of the
images obtained by PNA-FISH and CLSM indicated that P.
aeruginosa was located significantly deeper in the wound bed
than S. aureus.
We previously investigated samples from 22 chronic venous
leg ulcers for the presence of bacteria by standard culturing
and PNA-FISH (17). By swab sample cultivation, we found
that 12 of the wounds were colonized with S. aureus, whereas
5 of them were colonized with P. aeruginosa. Conversely, by
using PNA-FISH, P. aeruginosa was detected in nine of the
wounds, while S. aureus was detected in only two of them. Our
present finding that P. aeruginosa is located in the deeper
regions of the wound bed offers an explanation for the different
results obtained by swab sample cultivation and PNA-FISH.
Because the swab sample culture technique detects bacteria in
the upper region of the wounds, bacteria that primarily colo-
FIG. 2. Distribution of the distances from the wound surface to the
nize the deeper regions may not be detected. center of mass of S. aureus aggregates (light gray shading) or P. aerugi-
For a good healing response, the bacterial load of chronic nosa aggregates (dark gray shading). The distances are average values
wounds needs to be optimally managed. Topical antimicrobials obtained from the analysis of 15 images for each wound sample.
 Downloaded from https://journals.asm.org/journal/jcm on 12 January 2023 by 147.78.30.170.

FIG. 3. Representative CLSM images of S. aureus (A and B), P. aeruginosa (C and D), and both organisms (E) in chronic wounds. The bacteria
were detected by PNA-FISH with an FITC-labeled S. aureus-specific probe (green) or a TxR-labeled P. aeruginosa-specific probe (red), or a
mixture of the two probes. Arrows point to the wound surfaces. Bars, 30 ␮m.

4087
4088 FAZLI ET AL. J. CLIN. MICROBIOL.

Downloaded from https://journals.asm.org/journal/jcm on 12 January 2023 by 147.78.30.170.

FIG. 4. Epifluorescence micrographs (A and B) and bright-field micrographs (C and D) showing red PNA-FISH-stained P. aeruginosa cells (A),
green PNA-FISH-stained S. aureus cells (B), blue DAPI-stained host cells (A and B), H&E-stained host cells and P. aeruginosa cells (C), and
H&E-stained host cells and S. aureus cells (D). Some of the bacteria and host cells are encircled and labeled b and h, respectively. Arrows point
to the wound surfaces. Bars, 35 ␮m.

bacteria located in the deeper regions of chronic wounds may REFERENCES

be one of the factors that causes a persistent influx of PMNs
and that keeps the wound in an inflammatory stage. However,
more research is required before specific bacterial species in
specific modes of growth can be identified as the causative
agents in chronic wounds.
ACKNOWLEDGMENTS
We thank Jette Pedersen from the Bartholin Institute, University of
Copenhagen, and Anne Jørgensen from the Department of Pathology,
University of Copenhagen, for help with preparing and microscopy of
the tissue sections. AdvanDx, Inc., is gratefully acknowledged for pro-
viding the PNA probes.
M.G. received financial support for the present study from the
Danish Strategic Research Council. T.B. received financial support
from the Carlsberg Foundation and the Lundbeck Foundation).
1. Agren, M. S., W. H. Eaglstein, M. W. J. Ferguson, K. G. Harding, K. Moore,
U. K. Saarialo-Kere, and G. S. Schultz. 2000. Causes and effects of the
chronic inflammation in venous leg ulcers. Acta Dermatol. Venerol. Suppl.
210:3–17.
2. Andersen, A., K. E. Hill, P. Stephens, D. W. Thomas, B. Jorgensen, and K. A.
Krogfelt. 2007. Bacterial profiling study using skin grafting, standard culture
and molecular bacteriological methods. J. Wound Care 16:171–175.
3. Barken, K. B., S. J. Pamp, L. Yang, M. Gjermansen, J. J. Bertrand, M.
Klausen, M. Givskov, C. B. Whitchurch, J. N. Engel, and T. Tolker-Nielsen.
2008. Roles of type IV pili, flagellum-mediated motility and extracellular
DNA in the formation of mature structures in Pseudomonas aeruginosa
biofilms. Environ. Microbiol. 10:2331–2343.
4. Bjarnsholt, T., P. Ø. Jensen, M. Burmølle, M. Hentzer, J. A. Haagensen,
H. P. Hougen, H. Calum, K. G. Madsen, C. Moser, S. Molin, N. Høiby, and
M. Givskov. 2005. Pseudomonas aeruginosa tolerance to tobramycin, hydro-
gen peroxide and polymorphonuclear leukocytes is quorum-sensing depen-
dent. Microbiology 151:373–383.
5. Bjarnsholt, T., K. Kirketerp-Møller, P. O. Jensen, K. G. Madsen, R. Phipps,
K. Krogfelt, N. Hoiby, and M. Givskov. 2008. Why chronic wounds will not
heal: a novel hypothesis. Wound Repair Regen. 16:2–10.
VOL. 47, 2009 P. AERUGINOSA AND S. AUREUS IN CHRONIC WOUNDS
6. Costerton, J. W., Z. Lewandowski, D. E. Caldwell, D. R. Korber, and H. M.
Lappin-Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711–745.
7. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms:
a common cause of persistent infections. Science 284:1318–1322.
8. Davies, C. E., K. E. Hill, M. J. Wilson, P. Stephens, C. M. Hill, K. G.
Harding, and D. W. Thomas. 2004. Use of 16S ribosomal DNA PCR and
denaturing gradient gel electrophoresis for analysis of the microfloras of
healing and nonhealing chronic venous leg ulcers. J. Clin. Microbiol. 42:
3549–3557.
9. Davis, S. C., C. Ricotti, A. Cazzaniga, E. Welsh, W. H. Eaglstein, and P. M.
Mertz. 2008. Microscopic and physiologic evidence for biofilm-associated
wound colonization in vivo. Wound Repair Regen. 16:23–29.
10. Fedtke, I., F. Gotz, and A. Peschel. 2004. Bacterial evasion of innate host
defences: the Staphylococcus aureus lesson. Int. J. Med. Microbiol. 294:189–
194.
11. Foster, T. J. 2005. Immune evasion by staphylococci. Nat. Rev. Microbiol.
3:948–958.
12. Gjødsbol, K., J. J. Christiensen, T. Karlsmark, B. Jørgensen, B. M. Klein,
and K. A. Krogfelt. 2006. Multiple bacterial species reside in chronic wounds:
a longitudinal study. Int. Wound J. 3:225–231.
13. Gottrup, F. 2004. A specialized wound healing center concept: importance of
a multidisciplinary department structure and surgical treatment facilities in
the treatment of chronic wounds. Am. J. Surg. 187:38S–43S.
14. Halbert, A. R., M. C. Stacey, J. B. Rohr, and A. Jopp-McKay. 1992. The
effect of bacterial colonization on venous leg ulcer healing. Australas. J.
Dermatol. 33:75–80.
15. James, G. A., E. Swogger, R. Wolcott, E. Pulcini, P. Secor, J. Sestrich, J. W.
Costerton, and P. S. Stewart. 2008. Biofilms in chronic wounds. Wound
Repair Regen. 16:37–44.
16. Jensen, P. Ø., T. Bjarnsholt, R. Phipps, T. B. Rasmussen, H. Calum, L.
Christoffersen, C. Moser, P. Williams, T. Pressler, M. Givskov, and N.
Høiby. 2007. Rapid necrotic killing of polymorphonuclear leukocytes is
4089
caused by quorum sensing controlled production of rhamnolipid by Pseudo-
monas aeruginosa. Microbiology 153:1329–1338.
17. Kirketerp-Møller, K., P. O. Jensen, M. Fazli, K. G. Madsen, J. Pedersen, C.
Moser, T. Tolker-Nielsen, N. Hoiby, M. Givskov, and T. Bjarnsholt. 2008.
Distribution, organization, and ecology of bacteria in chronic wounds.
J. Clin. Microbiol. 46:2717–2722.
18. Klausen, M., A. Heydorn, P. Ragas, L. Lambertsen, A. Aaes-Jørgensen, S.
Molin, and T. Tolker-Nielsen. 2003. Biofilm formation by Pseudomonas
aeruginosa wild type, flagella and type IV pili mutants. Mol. Microbiol.
48:1511–1524.
19. Klausen, M., A. Aaes-Jørgensen, S. Molin, and T. Tolker-Nielsen. 2003.
Involvement of microbial migration in the development of complex multi-
cellular structures in Pseudomonas aeruginosa biofilms. Mol. Microbiol. 50:
61–68.
20. Madsen, S. M., H. Westh, L. Danielsen, and V. T. Rosdahl. 1996. Bacterial
colonization and healing of venous leg ulcers. APMIS 104:895–899.
21. Matsukawa, M., and E. P. Greenberg. 2004. Putative exopolysaccharide
synthesis genes influence Pseudomonas aeruginosa biofilm development. J.
Bacteriol. 186:4449–4456.
22. Nagase, H., and J. F. Woessner, Jr. 1999. Matrix metalloproteinases. J. Biol.
Chem. 274:21491–21494.
23. Schultz, G. S., R. G. Sibbald, V. Falanga, E. A. Ayello, C. Dowsett, K.
Harding, M. Romanelli, M. C. Stacey, L. Teot, and W. Vanscheidt. 2003.
Wound bed preparation: a systematic approach to wound management.
Wound Repair Regen. 11:S1–28.
24. Sibbad, R. G., A. C. Browne, P. Coutts, and D. Queen. 2001. Screening
evaluation of an ionized nanocrystalline silver dressing in chronic wound
care. Ostomy Wound Manage. 47:38–43.
25. Whitchurch, C. B., T. Tolker-Nielsen, P. C. Ragas, and J. S. Mattick. 2002.
Extracellular DNA required for bacterial biofilm formation. Science 295:
1487.
26. Yager, D. R., and B. C. Nwomeh. 1999. The proteolytic environment of
chronic wounds. Wound Repair Regen. 7:433–441.
Downloaded from https://journals.asm.org/journal/jcm on 12 January 2023 by 147.78.30.170.