Review
Chronic Wound Biofilms: Pathogenesis and
Potential Therapies
Allie Clinton, PhD, M(ASCP)CM,1* Tammy Carter, PhD, MT(ASCP)2
Lab Med FFalalll 2015;46:277-284
DOI: 10.1309/LMBNSWKUI4JPN7SO
ABSTRACT
Chronic wounds are a growing medical problem that cause high rates
of morbidity and mortality, costing the healthcare industry in the
United States millions of dollars annually. Chronic wound healing is
hampered by the presence of bacterial infections that form biofilms,
in which the bacteria are encased in exopolysaccharide (EPS) and
are less metabolically active than their free-living counterparts.
Bacterial biofilms make chronic wounds more refractory to treatment
and slow tissue repair by stimulating chronic inflammation at the
Chronic wounds are a severe, worldwide problem.
Wounds are considered chronic when healing fails to
proceed normally and the anatomic and functional
integrity of the skin is not achieved in approximately 1
month. Vascular insufficiency and infection contribute to
nonhealing wounds; infection occurs from microorganism
multiplication in the wound bed, leading to a prolonged
excessive inflammatory response, delays in collagen
synthesis, epithelialization, and tissue damage.1
Chronic wounds include diabetic foot ulcers, pressure
or decubitus ulcers, venous leg ulcers, and nonhealing
surgical-site infections. The annual incidence of foot
ulcers in diabetic patients is 1% to 4% in the United
States, with a lifetime risk of occurrence of between 15%
Abbreviations:
EPS, exo-polysaccharide; QS, quorum sensing; CDC, Centers for Disease
Control and Prevention; AIs, autoinducers; AI-2, autoinducer 2; AIPs,
autoinducing peptide pheromones; AHLs, acyl-homoserine lactones;
HSLs, homoserine lactones; PQS, Pseudomonas quinolone signal
Departments of 1Immunology and Infectious Diseases and 2Laboratory
Sciences and Primary Care, Texas Tech University Health Sciences
Center, Lubbock, Texas
*To whom correspondence should be addressed.
allie.clinton@ttuhsc.edu
www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  277
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
wound site. Bacterial species communicate through a mechanism
known as quorum sensing (QS) to regulate and coordinate the gene
expression that is important for virulence-factor production, including
biofilm formation. This review focuses on the relationships between
chronic wounds, biofilms, and QS in the virulence of chronic-wound
pathogens.
Keywords: bacteria, biofilms, chronic wounds, exopolysaccharide,
polymicrobial, quorum sensing
and 25%. In 2006, the total cost of treatment, amputation,
rehabilitation, and long-term care of diabetic foot ulcers
in the U.S. totaled $10.9 billion.2 Approximately 85% of
amputations are preceded by these types of ulcers. These
figures will increase as the number of diabetes diagnoses
is expected to rise.2 Pressure/decubitus ulcers are a
common problem in nursing home, rehabilitation clinics
and home-care populations; venous leg ulcers affect
as many as 1% of the worldwide population.3 Surgical-
site infections occur in as many as 5% of procedures
and are an increasingly common type of postoperative
complication; an average of 0.5% of the total hospital
budget in the United States is allocated to manage these
infections in affected patients.4
Currently, acute and chronic wounds are treated using
a multistep approach known as TIME, as described by
Schultz et al.5 First, the nonviable tissues (T) from within
and around the wound are removed by debridement.
Next, infection and inflammation (I) are minimized by
administering antibiotics and anti-inflammatory drugs;
then, moisture (M) imbalance is corrected, generally with
carefully selected dressings. Last, epithelialization (E)
and tissue formation are promoted by the application
of specific therapies, such as growth factors.5 Wound
cleansing and debridement of chronic wounds have been
shown to improve healing rates.6 Debridement can be
performed surgically (mechanically removing necrotic
Review
tissue), enzymatically (using naturally occurring matrix-
degrading enzymes such as papain or collagenase), and
biologically (using debriding organisms such as maggots).7
Hyperbaric oxygen can also be used to treat certain types
of wounds such as clostridial myonecrosis, necrotizing
soft-tissue infections, and selected nonhealing problem
wounds. Hyperbaric oxygen stimulates tissue regeneration
by increasing oxygen tension to elicit cellular responses
such as angiogenesis and collagen production. Increased
oxygen has also been shown to inhibit the growth of
anaerobes while increasing the respiration of aerobes,
which increases the uptake and efficacy of broad-
spectrum antibiotics by aerobic respiration.7
Despite diligent care given to patients with acute wounds,
treatment often fails in chronically infected wounds. One
of the major reasons for treatment failure is that acute
infections can lead to the formation of biofilms.8 It has
been shown9 that less than 10% of acute wounds contain
biofilm, whereas as many as 60% of chronic wounds
produce this barrier to treatment.
Biofilms
A bacterial biofilm is a structured community of
microorganisms encased in an exo-polysaccharide (EPS)
or exo-polymeric substance, which adheres to an inert
or living surface.10 Biofilms are polymicrobial and may
consist of not only bacterial cells but also fungi, viruses,
proteins, extracellular DNA, and other biogenic factors.11,12
Biofilm growth helps bacteria because it is protective and
increases survival in a hostile environment. The majority
of bacteria in most natural13 and pathogenic10 ecosystems
compose biofilms. A hallmark of biofilm producing
infections is that they are highly polymicrobial; it is thought
colloquially that naturally occurring biofilms are never
caused by a single organism. The polymicrobial nature
of biofilm increases infection virulence and complicates
treatment.
Biofilm Formation
Biofilm formation is dynamic and typically involves the
following 5 steps, as outlined by Stoodley et al.14 The first
step is reversible attachment of the microbe to a surface
mediated by pili, flagella, or other surface appendages or
specific receptors; the second is irreversible attachment
278  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
Figure 1
Schematic of biofilm formation. Numbers indicate stages of
formation
mediated by the secretion of EPS. The third step is cell
proliferation, resulting in the formation of a microcolony;
the fourth step is growth and differentiation, culminating in
a mature biofilm community with characteristic structural
features such as water channels and towering clusters of
cells. The final step is dispersion of biofilm cells, actively
or by passive detachment (Figure 1).14
The hallmark of a biofilm is its secretion of EPS.15 Biofilm
EPS serves a variety of purposes to the colony, the most
important of which is bacterial attachment to biotic or
abiotic surfaces. The EPS layer is a type of house in which
the bacteria live, which features a strong foundation and
offers protection from the outside environment. EPS is
highly refractory to penetration by antimicrobial agents
and the immune system. Bacterial EPS is also thought
to protect biofilm bacteria from desiccation; to assist
in ion exchange; to house and maintain degradation
enzymes; and to carry nutrients such as carbon, nitrogen,
and phosphorous.15 Fully developed biofilms (stage 4 of
development) exhibit characteristic structures such as
mushroom towers and water channels that bring nutrients
and water to, and waste away from, the lower layers of
the biofilm.16 The production of many virulence factors,
including biofilm formation, has been attributed to cell-to-
cell communication via quorum sensing (QS).
Mechanisms of Biofilm Virulence
Bacterial cells encased in biofilm EPS are different
than free-living, planktonically growing bacteria in that
the former are sessile (non-motile) and have reduced
Review
tissue), enzymatically (using naturally occurring matrix-
degrading enzymes such as papain or collagenase), and
biologically (using debriding organisms such as maggots).7
Hyperbaric oxygen can also be used to treat certain types
of wounds such as clostridial myonecrosis, necrotizing
soft-tissue infections, and selected nonhealing problem
wounds. Hyperbaric oxygen stimulates tissue regeneration
by increasing oxygen tension to elicit cellular responses
such as angiogenesis and collagen production. Increased
oxygen has also been shown to inhibit the growth of
anaerobes while increasing the respiration of aerobes,
which increases the uptake and efficacy of broad-
spectrum antibiotics by aerobic respiration.7
Despite diligent care given to patients with acute wounds,
treatment often fails in chronically infected wounds. One
of the major reasons for treatment failure is that acute
infections can lead to the formation of biofilms.8 It has
been shown9 that less than 10% of acute wounds contain
biofilm, whereas as many as 60% of chronic wounds
produce this barrier to treatment.
Biofilms
A bacterial biofilm is a structured community of
microorganisms encased in an exo-polysaccharide (EPS)
or exo-polymeric substance, which adheres to an inert
or living surface.10 Biofilms are polymicrobial and may
consist of not only bacterial cells but also fungi, viruses,
proteins, extracellular DNA, and other biogenic factors.11,12
Biofilm growth helps bacteria because it is protective and
increases survival in a hostile environment. The majority
of bacteria in most natural13 and pathogenic10 ecosystems
compose biofilms. A hallmark of biofilm producing
infections is that they are highly polymicrobial; it is thought
colloquially that naturally occurring biofilms are never
caused by a single organism. The polymicrobial nature
of biofilm increases infection virulence and complicates
treatment.
Biofilm Formation
Biofilm formation is dynamic and typically involves the
following 5 steps, as outlined by Stoodley et al.14 The first
step is reversible attachment of the microbe to a surface
mediated by pili, flagella, or other surface appendages or
specific receptors; the second is irreversible attachment
278  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
Figure 1
Schematic of biofilm formation. Numbers indicate stages of
formation
mediated by the secretion of EPS. The third step is cell
proliferation, resulting in the formation of a microcolony;
the fourth step is growth and differentiation, culminating in
a mature biofilm community with characteristic structural
features such as water channels and towering clusters of
cells. The final step is dispersion of biofilm cells, actively
or by passive detachment (Figure 1).14
The hallmark of a biofilm is its secretion of EPS.15 Biofilm
EPS serves a variety of purposes to the colony, the most
important of which is bacterial attachment to biotic or
abiotic surfaces. The EPS layer is a type of house in which
the bacteria live, which features a strong foundation and
offers protection from the outside environment. EPS is
highly refractory to penetration by antimicrobial agents
and the immune system. Bacterial EPS is also thought
to protect biofilm bacteria from desiccation; to assist
in ion exchange; to house and maintain degradation
enzymes; and to carry nutrients such as carbon, nitrogen,
and phosphorous.15 Fully developed biofilms (stage 4 of
development) exhibit characteristic structures such as
mushroom towers and water channels that bring nutrients
and water to, and waste away from, the lower layers of
the biofilm.16 The production of many virulence factors,
including biofilm formation, has been attributed to cell-to-
cell communication via quorum sensing (QS).
Mechanisms of Biofilm Virulence
Bacterial cells encased in biofilm EPS are different
than free-living, planktonically growing bacteria in that
the former are sessile (non-motile) and have reduced

metabolic activity. This reduced activity increases
antimicrobial tolerance because many classes of
antibiotics are only effective against actively dividing
cells by targeting peptidoglycan produced in the cell wall
(β-lactams), protein (aminoglycoside) synthesis, or DNA
replication (quinolones).17 EPS is a mechanical barrier to
antimicrobials and immune system cells, which decreases
their effectiveness.9 Stimulation of the immune system
without effectively eradicating the infection causes
collateral damage to surrounding tissue and chronic
inflammation, which aggravates the wound, and further
slows the healing process.
Biofilms increase the opportunity for the transfer of
antimicrobial-resistance genes carried on mobile genetic
elements, such as plasmids. Genetic transfer can occur
between bacteria of the same species or among cells of
different species, which increases the potential for virulent
and persistent infections.9 Acquisition of antibiotic-
resistance genes causes irreversible genotypic changes
in the bacteria (with the exception of resistance genes
harbored on mobile genetic elements). Antibiotics are
able to eradicate susceptible bacteria that have not
acquired resistance genes; however, once antibiotic use
is suspended, the remaining cells can cause a recurrent
infection with microbes that retain antibiotic resistance.
Distinct from antibiotic resistance is the issue of
antimicrobial tolerance unique to the biofilm environment.
Antibiotic tolerance is a transient phenotype that is highly
refractory to antimicrobial therapy, allowing a subpopulation
of cells, termed persister cells, to be maintained in
the wound environment. Tolerance is not mediated by
acquisition of genetic modifications18 but rather is thought
to result from metabolically inactive biofilm cells. When
antimicrobial therapy is suspended, remaining persister
cells can regenerate the biofilm with a microbial population
that retains a similar susceptibility profile as the original
biofilm, and so persister cells are maintained.19
A vital step in wound care is to surgically remove necrotic
issue and microbial bioburden by debridement.20 Bacterial
EPS promotes strong attachment of the biofilm to the
wound bed, which makes full debridement difficult.
Despite having most of the biofilm mass removed by
debridement, a small amount of cells remains tightly
attached to the wound bed, allowing remaining cells to
regenerate the biofilm, leading to a high rate of recurrent
infections commonly associated with biofilms.21
www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  279
Review
Biofilm Susceptibility Testing
Clinical microbiology uses several isolated colonies of a
single bacterial species from an infection, determining the
organism identification and antimicrobial susceptibility.
The discovery of biofilms has revealed that a number of
infections, particularly chronic wounds, are polymicrobial.
A bacterial species in polymicrobial infections can
have a different and higher antibiotic susceptibility—a
phenomenon known as antimicrobial synergism—than
that it would have if that species was causing an infection
by itself.22-24 Also, current methods of antimicrobial
susceptibility testing are performed on free-living,
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
metabolically active planktonic bacteria, whereas biofilm
bacteria are sessile and relatively inactive. This can
lead to misleading results from current antimicrobial-
susceptibility testing methods. In a study performed by
Keays et al,25 60% of bacterial isolates tested were treated
with antibiotic combinations that successfully inhibited
all planktonically grown bacterial isolates. However, when
the same isolates were grown as biofilms, only 22% of all
biofilm-grown isolates remained susceptible to antibiotics.
For chronic wounds, a new method of antibiotic
susceptibility testing is needed.
Many in vitro biofilm experimental models have been
developed.26 These models are now generating interest
in the clinical microbiology community for biofilm-
susceptibility testing (Table 1). These methods promote
biofilm growth by supporting bacteria that adhere to a
surface while washing away their planktonic counterparts.
Bacteria can be grown statically (microtiter plate, Lubbock
chronic wounds biofilm), resembling the conditions
observed in fixed, chronic biofilm infections, or under
dynamic, moving conditions (Centers for Disease Control
and Prevention [CDC] biofilm reactor, drip-flow reactor)
to mimic infections subjected to blood flow, such as
endocarditis. Using these methods, investigators can
more effectively study polymicrobial infections; the
resulting bacteria are much more representative of
biofilm infections, making susceptibility profiles more
representative of what occurs in chronic wounds.
Most studies examining the efficacy of biofilm
susceptibility testing versus traditional planktonic
susceptibility testing have been performed in orthopedic
infections;27 comparative studies involving Pseudomonas
aeruginosa have been performed in patients with cystic
fibrosis. In these studies, patients treated with antibiotics
Review

QS signaling has been discovered in more than 100

Table 1. Examples of In Vitro Biofilm-Grown
Models Used in Research
Method
96-well microtiter plate 60
Calgary biofilm device61
Drip-flow reactor62
Lubbock chronic wound
Description
Biofilm grows statically within wells of the
plate
The device is a lid with 96 pegs that fit into
the wells of a standard microtiter plate;
biofilm grows statically on the pegs
An inclined chamber continuously drips
nutrient medium onto a microscope
slide; biofilm is grown on the slide. The
medium runs down the surface of the
slide, allowing for dynamic growth
Biofilm is grown statically in a test tube in
microbial species and is associated with dozens of
different receptors and effector molecules. QS systems
consist of constitutively expressed signal molecules, or
autoinducers (AIs), and a corresponding receptor that
regulates gene expression when signal concentration has
reached threshold concentration. AIs passively diffuse
between cells to activate intracellular receptors, which
alter gene expression directly, or are actively pumped
out of cells to bind to transmembrane receptors (usually
2-component systems), resulting in signal transduction
and downstream changes in gene expression (Figure 2).

Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016

biofilm model63 a very rich medium containing nutrient
broth, plasma, and red blood cells. A
pipette tip can be used as a scaffold
CDC biofilm reactor64 Apparatus that contains chambers
submerged in a vessel; growth media is
continuously mixed and poured through
the chambers, creating dynamic growth
of biofilm
CDC, Centers for Disease Control and Prevention.
determined from biofilm antimicrobial susceptibility have
improved clinical outcomes compared with traditional
susceptibility testing.25,28
These protocols require standardization and further
tested in clinical settings. However, they seem to reveal
superior and more representative antibiotic-susceptibility
patterns, which can improve treatment efficacy of biofilm-
associated infections such as chronic wounds.29-31
Quorum Sensing
Gram-positive and Gram-negative bacteria use
QS to accomplish cell-to-cell communication. This
communication is mediated by small molecules that can
pass freely through membranes or through a membrane
channel or protein. This process depends on cell density;
when a species of bacteria reaches a critical population
mass, the signaling molecules are produced at high
enough concentration to alter gene expression, with as
many as 5% of the genes in a given genome potentially
affected by QS systems.32 This allows bacteria to
coordinate their activity based on population size. Many
proteins and virulence factors produced by bacteria are
differentially expressed in response to QS, including the
genes responsible for biofilm formation.
The universal QS molecule, termed the autoinducer 2
(AI-2), is produced by more than 50 species of gram-
positive and gram-negative bacteria and can influence
numerous activities such as bioluminescence, virulence-
factor production, and exoproduct secretion.33 E. coli and
Salmonella spp. are the model organisms for AI-2 QS. AI-2
is produced by the lsr genes in E. coli,34 which positively
mediates QS and directly increases biofilm production.35
Gram-positive bacterial QS systems generally use small
peptides, termed autoinducing peptide pheromones
(AIPs), as signaling molecules. These AIP peptides are
actively exported from the cell and bind to transmembrane
receptors or intracellular regulatory proteins, which
regulate the transcription of target genes. An example
is the accessory gene regulator (arg) QS system of
Staphylococcus aureus.36
QS in Gram-negative bacteria is controlled by homologs
of 2 regulatory proteins, LuxI and LuxR. LuxI is an
autoinducer synthase, which produces AIs, and LuxR
is the transcriptional activator protein that, once bound
to AI, promotes transcription of downstream genes in
neighboring bacterial cells and itself. AI small-chemical
signals in Gram-negative bacteria are termed acyl-
homoserine lactones (AHLs) and are synonymous with
homoserine lactones (HSLs).37 The model organism
for Gram-negative QS is Pseudomonas aeruginosa, an
opportunistic pathogen that is one of the most commonly
isolated organisms in chronic wounds.38 P. aeruginosa
has two 2-component systems, LasI/R and RhlI/R, as
well as a third, quinolone-based, system, that utilizes
the Pseudomonas quinolone signal (PQS).39 Many P.
aeruginosa exoproducts that are thought to induce
virulence and pathogenesis, including staphyolysin,40
elastase, pyocyanin, and rhamnolipids, as well as genes
associated with biofilm formation,36 are controlled by QS.

280  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com


Gram-Positive Gram-Negative
Quorum Sensing Quorum Sensing
SIgnal peptide Chemical
signal
Kinase
P
LuxR
P
LuxI
PR
Expression PR Expression
Figure 2
Gram-positive and Gram-negative quorum-sensing systems.
QS has been shown to be important during all 5 stages
of biofilm development; however, the specific QS-
controlled stages differ between microbes that use
different mechanisms of QS. QS was first linked with
biofilm formation in 1998 by Davies et al,41 who showed
that P. aeruginosa QS-negative mutants formed extremely
weak biofilms with altered architecture and were not as
resistant to treatment as biofilms made by QS-competent
P. aeruginosa.41 P. aeruginosa contains at least 39 genes
that are regulated by QS systems.42 QS-deficient mutants
of P. aeruginosa are highly attenuated and less virulent
in mouse pneumonia43 and burn44 models, presumably
because they cannot communicate or make robust
biofilms. QS has also been linked to biofilm formation in
bacterial species other than P. aeruginosa, such as S.
aureus45 and Vibrio cholera.46
Development of Antibiofilm
Therapies
A major hallmark of biofilm is the thick layer of EPS
that protects the infecting microbial population from
the immune system and antimicrobials. An area of
current therapeutic research is the development of
agents that degrade the EPS layer so already-developed
antimicrobials will be effective. These dispersing
agents show promise as a future therapy to be used in
combination with antibiotics. An example is the enzyme
β-amylase, an enzyme produced by oral bacteria that
break down polysaccharide bonds; this compound has
been reformulated as a dispersing agent to target the
polysaccharide bonds of the EPS with in vitro success
in degrading biofilm.47 Other EPS matrix-degrading
enzymes include deoxyribonuclease I, a glycoside
www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  281
Review
hydrolases dispersin B,48,49 and DNAse.50 Synthetic
dispersing agents have also been developed, such as
2-aminoimidazole, that show activity against S. aureus
biofilms.51
QS is considered to be important for the transition
between antimicrobial-sensitive planktonic cells to
antimicrobial-resistant aggregates of biofilm cells.
Researchers have developed agents designed to inhibit
or prevent QS, with the strategy that if QS does not
occur, biofilms will not be formed, and the bacteria will
remain sensitive to therapy. Some examples of these
QS inhibitors include furanone, which has been effective
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
against P. aeruginosa,52 Streptococcus mutans,53 E.coli,
and Salmonella spp.;54 thiophenone, which shows activity
against Staphylococcus epidermidis;55 and S. aureus
virulence inhibitor (savarin).56
Another approach to antibiofilm therapy is to use
diagnostic procedures based in molecular biology, instead
of culture-based methods, for the bacterial species
in chronic wounds.20 Molecular-pathogen diagnostic
applications allow comprehensive evaluation of the
microbial bioburden in chronic wounds, which leads
to a more comprehensive and targeted therapeutic
approach to wound care. Molecular diagnostics of
these wounds use a rapid screen of known, common
bacterial genes (including selected known resistance
markers) to diagnose common wound pathogens or
general bacterial primers, such as ribosomal 16S, to
sequence all bacterial species in a specimen and detect
species that are difficult to grow or that represent only
a small fraction of the infecting bacteria.57 Rhoads et
al58 demonstrated that molecular diagnostics detect
organisms that do not commonly grow under standard
wound-culture techniques (especially anaerobes) and
that the most common organisms detected via molecular
diagnostics versus traditional culture are often extremely
different.58 For example, in decubitus ulcers, culture-
based diagnostics detected Enterococcus spp. most
frequently, whereas molecular-based diagnostic methods
detected Corynebacterium spp. most frequently. Also, for
trauma/abscess wounds, culture methods most commonly
reported no growth, whereas molecular-based methods
detected Staphylococcus spp. most frequently. Of the
105 specimens tested, culture and molecular results
agreed only 63% of the time, and in 13% of culture-
negative specimens, bacteria were detected via molecular
methods.58
Review
Completeness of chronic wound healing can differ
among treatment methods. In a study performed by
Wolcott et al, 20 48.5% of patients with chronic wounds
treated based on traditional culture-based methods
healed completely, versus 62.4% of patients with
chronic wounds who were treated based on molecular
diagnostics and were fully healed; the latter patients
also had significantly shorter healing times. Additionally,
in response to implementing molecular diagnostics,
use of first-line antibiotics declined in lieu of targeted
antibiotics, a strategy that has the potential to attenuate
antibiotic resistance. 20 Topical antibiotics, based on
the results of molecular diagnostics, have also showed
increased effectiveness in wound care. In a study
performed by Dowd et al,59 culture-driven standard-of-
care treatments were compared with a commercially
available treatment guided by molecular diagnostics
(instead of culture) and personalized topical therapy
developed in a compound pharmacy specific to the
microbial burden of each patient, as determined by
molecular diagnostic testing. A total of 90.4% of patients
who received personalized topical therapies experienced
complete healing of their wounds and had more than
200% improved odds of healing compared to patients
receiving care based on other protocols.59 The results
of these studies demonstrate that personalized topical
therapeutic and molecular diagnostic strategies yield
statistically improved outcomes for patients afflicted with
chronic wounds and that these protocols provide direct,
targeted approaches to wound care.
Conclusions
Interest is growing in the clinical microbiology community
for adapting established biofilm analysis techniques
used in research to determine antimicrobial susceptibility
patterns of biofilm bacteria for use in chronic wound
treatment. Preliminary data suggest that using these
adapted methods to determine antimicrobial susceptibility
will increase treatment efficacy. Antibiofilm research has
generated many avenues of potential novel therapies,
including QS inhibitors and dispersing agents, as well as
the use of molecular diagnostic techniques to improve
current therapies. Combining biofilm research with
therapeutic development has the potential to significantly
increase the ability of healthcare providers to effectively
treat biofilm infections.  LM
282  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com
References
1. Rondas AALM, Schols JMGA, Stobberingh EE, Halfens RJG.
Prevalence of chronic wounds and structural quality indicators of
chronic wound care in Dutch nursing homes. Int Wound J. 2013;
e-pub ahead of print. doi: 10.1111/iwj.12172.
2. Driver VR, Blume PA. Evaluation of wound care and health-care
use costs in patients with diabetic foot ulcers treated with negative
pressure wound therapy versus advanced moist wound therapy. J
Am Podiatr Med Assoc. 2014;104(2):147-153.
3. Trent JT, Falabella A, Eaglstein WH, Kirsner RS. Venous ulcers:
pathophysiology and treatment options. Ostomy Wound Manage.
2005;51(5):38-54; quiz 55-56.
4. Kathju S, Nistico L, Hall-Stoodley L, Post JC, Ehrlich GD, Stoodley P.
Chronic surgical site infection due to suture-associated polymicrobial
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
biofilm. Surgic Infect (Larchmont). 2009;10(5):457-461.
5. Schultz GS, Sibbald RG, Falanga V, et al. Wound bed preparation: a
systematic approach to wound management. Wound Repair Regen.
2003;11 Suppl 1:S1-S28.
6. Spear M. The necessity of wound debridement. Plastic Surg Nurs.
2010;30(1):54-56.
7. Bertesteanu S, Triaridis S, Stankovic M, et al. Polymicrobial wound
infections: pathophysiology and current therapeutic approaches. Int J
Pharm. 2014;463(2):119-126.
8. Bjarnsholt T. The role of bacterial biofilms in chronic infections.
APMIS Suppl. 2013(136):1-58.
9. James GA, Swogger E, Wolcott R, et al. Biofilms in chronic wounds.
Wound Repair and Regen. 2008;16(1):37-44.
10. Costerton JW, Stewart PS, Greenberg EP. Bacterial
biofilms: a common cause of persistent infections. Science.
1999;284(5418):1318-1322.
11. Fuxman Bass JI, Russo DM, Gabelloni ML, et al. Extracellular DNA:
a major proinflammatory component of Pseudomonas aeruginosa
biofilms. J Immunol. 2010;184(11):6386-6395.
12. Watters C, Everett JA, Haley C, Clinton A, Rumbaugh KP. Insulin
treatment modulates the host immune system to enhance
Pseudomonas aeruginosa wound biofilms. Infect Immun.
2014;82(1):92-100.
13. Costerton JW, Geesey GG, Cheng KJ. How bacteria stick. Sci Am.
1978;238(1):86-95.
14. Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex
differentiated communities. Annu Rev Microbiol. 2002;56:187-209.
15. Flemming H-C, Neu TR, Wozniak DJ. The EPS matrix: the “house of
biofilm cells”. J Bacteriol. 2007;189(22):7945-7947.
16. O’Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial
development. Ann Rev Microbiol. 2000;54:49-79
17. Peterson LR. Squeezing the antibiotic balloon: the impact of
antimicrobial classes on emerging resistance. Clin Microbiol Infect.
2005;11 Suppl 5:4-16.
18. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister
cells and tolerance to antimicrobials. FEMS Microbiol Lett.
2004;230(1):13-18.
19. Fauvart M, De Groote VN, Michiels J. Role of persister cells in chronic
infections: clinical relevance and perspectives on anti-persister
therapies. J Med Microbiol. 2011;60(Pt 6):699-709.
20. Wolcott RD, Cox SB, Dowd SE. Healing and healing rates of chronic
wounds in the age of molecular pathogen diagnostics. J Wound
Care. 2010;19(7):272-278,280-281.
21. Falanga V, Brem H, Ennis WJ, Wolcott R, Gould LJ, Ayello EA.
Maintenance debridement in the treatment of difficult-to-heal chronic
wounds. Recommendations of an expert panel. Ostomy Wound
Manage. 2008;Suppl:2-13;quiz 14-15.

22. DeLeon S, Clinton A, Fowler H, Everett J, Horswill AR, Rumbaugh
KP. Synergistic interactions of Pseudomonas aeruginosa and
Staphylococcus aureus in an in vitro wound model. Infect Immun.
2014;82:4718-4728.
23. Burmølle M, Webb JS, Rao D, Hansen LH, Sørensen SJ, Kjelleberg
S. Enhanced biofilm formation and increased resistance to
antimicrobial agents and bacterial invasion are caused by synergistic
interactions in multispecies biofilms. Appl Environ Microbiol.
2006;72(6):3916-3923.
24. Dalton T, Dowd SE, Wolcott RD, et al. An in vivo polymicrobial biofilm
wound infection model to study interspecies interactions. PloS One.
2011;6(11):e27317. doi: 10.1371/journal.pone.0027317.
25. Keays T, Ferris W, Vandemheen KL, et al. A retrospective analysis
of biofilm antibiotic susceptibility testing: a better predictor of
clinical response in cystic fibrosis exacerbations. J Cyst Fibros.
2009;8(2):122-127.
26. James GA, Agostinho AM, Pulcini E de L. In vitro models for the
growth and analysis of chronic wound biofilms. Adv Wound Care.
2010;1:293-298.
27. Molina-Manso D, del Prado G, Ortiz-Pérez A, et al. In vitro
susceptibility to antibiotics of staphylococci in biofilms isolated from
orthopaedic infections. Int J Antimicrob Agents. 2013;41(6):521-523.
28. Waters V, Ratjen F. Standard versus biofilm antimicrobial
susceptibility testing to guide antibiotic therapy in cystic
fibrosis. Cochrane Database Syst Rev. 2012;11:CD009528. doi:
10.1002/14651858.CD009528.pub2.
29. Pratten J, Ready D. Use of biofilm model systems to study
antimicrobial susceptibility. Methods Mol Biol. 2010;642:203-215.
30. Santopolo L, Marchi E, Frediani L, Decorosi F, Viti C, Giovannetti L. A
novel approach combining the Calgary Biofilm Device and Phenotype
MicroArray for the characterization of the chemical sensitivity of
bacterial biofilms. Biofouling. 2012;28(9):1023-1032.
31. Kim S, Kim MJ, Kang HY, Seol SY, Cho DT, Kim J. A simple
colorimetric method for testing antimicrobial susceptibility of
biofilmed bacteria. J Microbiol. 2010;48(5):709-711.
32. Schuster M, Lostroh CP, Ogi T, Greenberg EP. Identification, timing,
and signal specificity of Pseudomonas aeruginosa quorum-controlled
genes: a transcriptome analysis. J Bacteriol. 2003;185(7):2066-2079.
33. Taga ME, Semmelhack JL, Bassler BL. The LuxS-dependent
autoinducer AI-2 controls the expression of an ABC transporter that
functions in AI-2 uptake in Salmonella typhimurium. Mol Microbiol.
2001;42(3):777-793.
34. Li J, Attila C, Wang L, Wood TK, Valdes JJ, Bentley WE. Quorum
sensing in Escherichia coli is signaled by AI-2/LsrR: effects on small
RNA and biofilm architecture. J Bacteriol. 2007;189(16):6011-6020.
35. González Barrios AF, Zuo R, Hashimoto Y, Yang L, Bentley WE,
Wood TK. Autoinducer 2 controls biofilm formation in Escherichia coli
through a novel motility quorum-sensing regulator (MqsR, B3022). J
Bacteriol. 2006;188(1):305-316.
36. Williams P. Quorum sensing, communication and cross-kingdom
signalling in the bacterial world. Microbiology. 2007;153(Pt 12):3923-
3938.
37. Bassler BL. How bacteria talk to each other: regulation of gene
expression by quorum sensing. Curr Opin Microbiol. 1999;2(6):582-587.
38. Gjødsbøl K, Christensen JJ, Karlsmark T, Jørgensen B, Klein BM,
Krogfelt KA. Multiple bacterial species reside in chronic wounds: a
longitudinal study. Int Wound J. 2006;3(3):225-231.
39. Miller MB, Bassler BL. Quorum sensing in bacteria. Ann Rev
Microbiol. 2001;55:165-199.
40. Kessler E, Safrin M, Olson JC, Ohman DE. Secreted LasA of
Pseudomonas aeruginosa is a staphylolytic protease. J Biol Chem.
1993;268(10):7503-7508.
41. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton
JW, Greenberg EP. The involvement of cell-to-cell signals in the
development of a bacterial biofilm. Science. 1998;280(5361):295-298.
www.labmedicine.com Fall 2015  |  Volume 46, Number 4  Lab Medicine  283
Review
42. Whiteley M, Lee KM, Greenberg EP. Identification of genes controlled
by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci
U S A. 1999;96(24):13904-13909.
43. Tang HB, DiMango E, Bryan R, et al. Contribution of specific
Pseudomonas aeruginosa virulence factors to pathogenesis of
pneumonia in a neonatal mouse model of infection. Infect Immun.
1996;64(1):37-43.
44. Rumbaugh KP, Griswold JA, Iglewski BH, Hamood AN. Contribution
of quorum sensing to the virulence of Pseudomonas aeruginosa in
burn wound infections. Infect Immun. 1999;67(11):5854-5862.
45. Otto M. Staphylococcal infections: mechanisms of biofilm maturation
and detachment as critical determinants of pathogenicity. Annu Rev
Med. 2013;64:175-188.
46. Hammer BK, Bassler BL. Quorum sensing controls biofilm formation
in Vibrio cholerae. Mol Microbiol. 2003;50(1):101-104.
47. Kalpana BJ, Aarthy S, Pandian SK. Antibiofilm activity of β-amylase
Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016
from Bacillus subtilis S8-18 against biofilm forming human bacterial
pathogens. Appl Biochem Biotechnol. 2012;167(6):1778-1794.
48. Kaplan JB. Therapeutic potential of biofilm-dispersing enzymes. Int J
Artif Organs. 2009;32(9):545-554.
49. Gawande PV, Clinton AP, LoVetri K, Yakandawala N, Rumbaugh
KP, Madhyastha S. Antibiofilm efficacy of DispersinB® wound spray
used in combination with a silver wound dressing. Microbiol Insights.
2014;7:9-13.
50. Nijland R, Hall MJ, Burgess JG. Dispersal of biofilms by secreted,
matrix degrading, bacterial DNase. PloS One. 2010;5(12):e15668.
doi:10.1371/journal.pone.0015668.
51. Rogers SA, Huigens RW 3rd, Cavanagh J, Melander C. Synergistic
effects between conventional antibiotics and 2-aminoimidazole-
derived antibiofilm agents. Antimicrob Agents Chemother.
2010;54(5):2112-2118.
52. Choi S-C, Zhang C, Moon S, Oh Y-S. Inhibitory effects of 4-hydroxy-
2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-
mediated virulence factor production and biofilm formation in
Pseudomonas aeruginosa PAO1. J Microbiol. 2014;52:734-742.
53. He Z, Wang Q, Hu Y, et al. Use of the quorum sensing inhibitor
furanone C-30 to interfere with biofilm formation by Streptococcus
mutans and its luxS mutant strain. Int J Antimicrob Agents.
2012;40(1):30-35.
54. Vestby LK, Johannesen KCS, Witsø IL, et al. Synthetic brominated
furanone F202 prevents biofilm formation by potentially human
pathogenic Escherichia coli O103:H2 and Salmonella ser. Agona on
abiotic surfaces. J Appl Microbiol. 2014;116:258-268.
55. Lönn-Stensrud J, Naemi A-O, Benneche T, Petersen FC, Scheie
AA. Thiophenones inhibit Staphylococcus epidermidis biofilm
formation at nontoxic concentrations. FEMS Immunol Med Microbiol.
2012;65(2):326-334.
56. Sully EK, Malachowa N, Elmore BO, et al. Selective chemical
inhibition of agrquorum sensing in Staphylococcus aureus promotes
host defense with minimal impact on resistance. PLoS Pathog.
2014;10(6):e1004174. doi:10.1371/journal.ppat.1004174.
57. Research and Testing Laboratory. Microbial Diversity Analysis. 2014.
Available at: http://www.researchandtesting.com/microbial-diversity-
analysis.html. Accessed on: April 12, 2015.
58. Rhoads DD, Wolcott RD, Sun Y, Dowd SE. Comparison of culture
and molecular identification of bacteria in chronic wounds. Int J Mol
Sci. 2012;13(3):2535-2550.
59. Dowd SE, Wolcott RD, Kennedy J, Jones C, Cox SB. Molecular
diagnostics and personalised medicine in wound care: assessment
of outcomes. J Wound Care. 2011;20(5):232, 234-239.
60. Christensen GD, Simpson WA, Younger JJ, et al. Adherence of
coagulase-negative staphylococci to plastic tissue culture plates:
a quantitative model for the adherence of staphylococci to medical
devices. J Clin Microbiol. 1985;22(6):996-1006.
Review

61. Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. The
Calgary Biofilm Device: new technology for rapid determination
of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol.
1999;37(6):1771-1776.
62. Xu KD, Stewart PS, Xia F, Huang C-T, McFeters GA. Spatial
physiological heterogeneity in Pseudomonas aeruginosa biofilm
is determined by oxygen availability. Appl Environ Microbiol.
1998;64(10):4035-4039.
63. Sun Y, Dowd SE, Smith E, Rhoads DD, Wolcott RD. In vitro
multispecies Lubbock chronic wound biofilm model. Wound Repair
Regen. 2008;16(6):805-813.
64. Gilmore BF, Hamill TM, Jones DS, Gorman SP. Validation of the CDC
biofilm reactor as a dynamic model for assessment of encrustation
formation on urological device materials. J Biomed Mater Res B Appl
Biomater. 2010;93B(1):128-140.

Downloaded from http://labmed.oxfordjournals.org/ by guest on January 12, 2016

284  Lab Medicine  Fall 2015  |  Volume 46, Number 4 www.labmedicine.com