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How Biofilms Evade Host Defenses

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DOI: 10.1128/microbiolspec.MB-0012-2014 · Source: PubMed

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 How Biofilms Evade
Host Defenses
EMMANUEL ROILIDES,1 MARIA SIMITSOPOULOU,1
ASPASIA KATRAGKOU,1,2 and THOMAS J. WALSH2
1
Infectious Diseases Unit, 3rd Department of Pediatrics, Faculty of Medicine,
Aristotle University School of Health Sciences, Hippokration Hospital, 54642 Thessaloniki, Greece;
2
Transplantation-Oncology Infectious Diseases Program, Weill Cornell Medical Center of
Cornell University, New York, NY 14850

ABSTRACT The steps involved during the biofilm growth cycle
include attachment to a substrate followed by more permanent
adherence of the microorganisms, microcolony arrangement,
and cell detachment required for the dissemination of single or
clustered cells to other organ systems. Various methods have
been developed for biofilm detection and quantitation.
Biofilm-producing microorganisms can be detected in tissue
culture plates, using silicone tubes and staining methods,
and by visual assessment using scanning electron microscopy or
confocal scanning laser microscopy. Quantitative measurement
of biofilm growth is determined by using methods that include
dry cell weight assays, colony-forming-unit counting,
DNA quantification, or XTT 2,3-bis (2-methoxy-4-nitro-5-
sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium
hydroxide reduction assay. Upon infection, innate immune
defense strategies are able to establish an immediate response
through effector mechanisms mediated by immune cells,
receptors, and several humoral factors. We present an overview
of the life cycle of biofilms and their diversity, detection methods
for biofilm development, and host immune responses to
pathogens. We then focus on current concepts in bacterial
and fungal biofilm immune evasion mechanisms. This appears
to be of particular importance because the use of host
immune responses may represent a novel therapeutic
approach against biofilms.
INTRODUCTION
Historically, microbial organisms have been grown in
pure liquid cultures as free-floating “planktonic” cells,
promoting the general theory of the unicellular lifestyle.
However, in the late 1970s, Costerton et al. (1) dem-
onstrated that groups of bacteria were embedded in a
highly hydrated polysaccharide matrix that mediated
adhesion to solid aquatic surfaces. Several years later,
the same research team called these cellular communi-
ties “biofilms,” defined as a functionally heterogeneous
aggregate of microcolonies or single cells encased in a
matrix of self-produced extracellular polymeric mole-
cules that could adhere either to organic, abiotic surfaces
or to each other (2). Microbial biofilms can develop
into highly organized structures containing channels
in which water, nutrients, and metabolic waste can be
transported. Adhesion to substrates or surfaces induces
expression of a large number of genes, while cell aggre-
gates in different regions in a biofilm exhibit different
gene expression profiles that regulate biofilm develop-
ment and maturation processes (3, 4). A large amount
of research since the 1980s has brought to light the
theory that most, if not all, bacteria and fungi can form
biofilms as a survival mechanism in hostile environ-
ments, providing protection from biotic and abiotic
stresses (5–8). Prime candidates for cell attachment and
biofilm growth are surfaces exposed to or containing
moisture and some nutrients. Natural or man-made
Received: 13 August 2014, Accepted: 18 February 2015,
Published: 19 June 2015
Editors: Mahmoud Ghannoum, Case Western Reserve University,
Cleveland, OH; Matthew Parsek, University of Washington, Seattle,
WA; Marvin Whiteley, University of Texas at Austin, Austin, TX; and
Pranab Mukherjee, Case Western Reserve University, Cleveland, OH
Citation: Roilides E, Simitsopoulou M, Katragkou A, Walsh TJ. 2015.
How biofilms evade host defenses. Microbiol Spectrum 3(3):MB-
0012-2014. doi:10.1128/microbiolspec.MB-0012-2014.
Correspondence: Emmanuel Roilides, roilides@med.auth.gr
© 2015 American Society for Microbiology. All rights reserved.

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Roilides et al.
substrates for cell attachment and biofilm growth in-
clude river stones, oil and gas installations, ship hulls,
water pipes, food-processing surfaces, contaminated
surgical instruments, indwelling medical devices, human
teeth, and infected wounds (9–11). In this chapter, we
will present an overview of the life cycle of biofilms and
their diversity, detection methods for biofilm develop-
ment, and host immune responses to pathogens. We will
then focus on current concepts in bacterial and fungal
biofilm immune evasion mechanisms.
BIOFILM FORMATION
Although different model systems have been described
to define biofilm development in bacteria and fungi,
the steps involved in the biofilm growth cycle are fairly
universal with many common characteristics (Table 1).
In the human body, attachment to accessible human host
proteins is the first step of biofilm formation and is de-
pendent either on the hydrophobic nature of microbial
surfaces or on specific cell surface molecules that enable
adherence to host proteins. For example, Staphylococ-
cus epidermidis and Staphylococcus aureus express
MSCRAMMs (microbial surface components recogniz-
ing adhesive matrix molecules) that have domains for
noncovalent attachment to peptidoglycan moieties and
also harbor binding sites for fibronectin, fibrinogen,
laminin, collagen IV, and other human matrix proteins
(12, 13). For bacteria with flagellar motility, swimming
cells attach to the surface first transiently and then
permanently, expressing conditionally synthesized ad-
hesive proteins to form a monolayer biofilm (14, 15).
Like the exopolysaccharide (EPS) matrix of bacterial
biofilms, the polysaccharide capsule of Cryptococcus
neoformans, a human pathogenic fungus, promotes
the attachment process to prosthetic medical devices,
whereas cell-surface glycoproteins facilitate adhesion
of Candida albicans and Candida glabrata (16–19).
Attachment is promoted by several environmental sig-
nals, such as changes in nutrient concentrations, pH,
flow velocity of surrounding body fluids (urine, blood,
saliva), temperature, oxygen concentration, osmolality,
and iron.
TABLE 1 Steps in the biofilm growth cycle
Attachment of microorganisms to surfaces
Adherence
Microcolony arrangement
Detachment and dissemination of single or clustered cells to other
organ systems
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The next phase of biofilm formation is characterized
by a microcolony arrangement on the attached surface
produced by the multiplication of free-floating cells;
motility is reduced and exopolysaccharide production is
activated to trap nutrients and planktonic cells, whereas
signal molecules are secreted in a cell-density-dependent
manner to communicate and coordinate the cellular
responses through a process called quorum sensing. The
presence of farnesol, a quorum sensing molecule first
described in C. albicans, has been found to inhibit the
yeast-to-mycelium conversion, enhancing active bud-
ding yeast production without compromising cellular
growth rates (20). In Pseudomonas aeruginosa, rhamno-
lipids maintain biofilm architecture by affecting the
attachment of bacterial cells to substrates and cell-to-
cell-interactions. When cell density increases during
the later stages of the maturation phase, rhamnolipids
maintain open channels between cellular aggregates to
distribute certain types and amounts of nutrients to
support cell growth (21).
Another way to trap both nutrients and planktonic
cells once a critical biofilm mass has been achieved is
the production and secretion of extracellular matrix
predominantly composed of polysaccharides, proteins,
and extracellular DNA (eDNA). This matrix promotes
initial cell adhesion, triggers polysaccharide formation,
and serves as a support that links molecules together in
the biofilm matrix, thus influencing the structure and
organization of mature biofilms. Extracellular DNA is
an important component of Aspergillus fumigatus bio-
films that originates from either fungal autolysis (22) or
is externally supplied from human neutrophils attracted
to the infected site (23, 24).
The last phase of biofilm formation is cell detachment,
which is required for the dissemination of single or
clustered cells to other organ systems. Factors that could
contribute to cell dispersal include sheer mechanical
forces (blood flow), enzymes that digest the extracellular
matrix, and nutrient limitation in a mature biofilm. The
dispersed cell population of C. albicans displays yeast
morphology with different phenotypic characteristics
from their planktonic counterparts, including increased
adherence capacity, filamentation, biofilm formation,
and increased pathogenicity to establish new foci of in-
fection (25). In staphylococci, quorum sensing–controlled
phenol-soluble modulins (PSMs) participate in establish-
ing biofilm architecture as well as detachment processes
using a mechanism similar to that used by P. aeruginosa
but with different effector molecules. PSMs are part of a
novel toxin family with multiple roles in staphylococcal
pathogenesis, because they promote formation of biofilm

channels and control biofilm expansion by detachment
and regrowth, contributing to bacterial dissemination.
During the dispersal phase, PSMs act as surfactant-
like peptides that inhibit cell-to-cell interactions at the
surface of the biofilm, leading to the detachment of bac-
terial cells at the fluid-biofilm interphase and the subse-
quent systemic spread of biofilm-associated infections
(26, 27).
Natural biofilms in most environments, including
human disease, tend to coexist forming polymicrobial
communities (28, 29). The interactions that take place
between fungal and/or bacterial species can either be
synergistic or antagonistic in nature (30). An example
of a mutually beneficial interaction is a phenomenon
called “coaggregation symbiosis” that is observed be-
tween C. albicans and S. aureus, whereby Candida hy-
phal penetration through epithelial layers provides a
route of entry for staphylococci. Moreover, the observed
hyphal-mediated increased pathogenicity of S. aureus
may be attributed not only to the physical interactions
but also to the differential regulation of virulence fac-
tors produced during polymicrobial growth (31). Dif-
ferent microbial species in a single biofilm community
could offer passive resistance, metabolic cooperation,
quorum sensing systems, and genotypic variability
that give an advantage to counteract adverse environ-
mental conditions (32). In contrast, the interactions be-
tween A. fumigatus and P. aeruginosa (both present in
the cystic fibrosis [CF] lung microbiome) is described as
being antagonistic. A. fumigatus biofilm formation is
inhibited by direct contact with a P. aeruginosa–secreted
heat-stable soluble factor, suggesting that small diffus-
ible molecules can interfere with filamentous fungal
growth in polymicrobial environments (33). Although
in recent years investigations have shifted to polymicro-
bial biofilms, there is considerable knowledge yet to
be gained in our understanding of microbial cohabita-
tion and microbes’ interaction with the host to con-
trol the impact of polymicrobial biofilm-associated
diseases.
DETECTION AND QUANTIFICATION
METHODS OF BIOFILM FORMATION
Various methods have been developed for biofilm de-
tection and quantitation (Table 2). They include staining
and microscopic visualization of biofilm structure as
well as quantitation of the numbers of biofilm-associated
cells in situ or after detachment of microbial cells from
the substrate. Biofilm-producing microorganisms are
also detected by methods that include growth in tissue
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How Biofilms Evade Host Defenses
TABLE 2 Methods for biofilm detection and quantitation
Direct methods Indirect methods
Tissue culture plating Dry cell weight assays
Tube method Colony-forming-unit counting
Congo red agar method DNA quantification
Crystal violet staining XTT reduction assay
Visual assessment by scanning
electron microscopy
Visual assessment by confocal
scanning laser microscopy
culture wells or silicone tube biofilm reactors, followed
by staining with Congo red or crystal violet stains.
These stained biofilms can be visually assessed by scan-
ning electron microscopy or confocal scanning laser
microscopy, which is most effective for studying an
intact biofilm’s three-dimensional architecture (34–36).
Quantitative measurement of biofilm growth is deter-
mined by using methods that include determining dry cell
weight assays, assessing colony forming unit by plating
on solid media, DNA quantification, or an XTT 2,3-
bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)
carbonyl]-2H-tetrazolium hydroxide reduction assay
(34, 37, 38). The latter method is colorimetric and uses
XTT, a colorless or slightly yellow compound that when
reduced to a formazan product becomes bright orange.
The oxidation-reduction process is initiated by mito-
chondrial dehydrogenases of metabolically active cells.
The colorimetric change is proportional to the number of
living cells and can be quantified spectrophotometrically.
The biodiversity and abundance of biofilm-associated mi-
croorganisms in polymicrobial communities is detected
using a combination of molecular diagnostics based on
PCR, sequencing technologies, and advanced mathe-
matical algorithms to identify the biofilm-producing
microorganisms present and to analyze the copy number
of each organism relative to the total number of copies
for all organisms (39–41).
HOST DEFENSES AGAINST BIOFILMS
Upon infection, innate immune defense strategies are
able to establish an immediate response through effec-
tor mechanisms mediated by immune cells, receptors,
and several humoral factors (Table 3). The main role
of humoral factors, such as mannose binding lec-
tins, collectins, complement, and antibodies, is to bind
invading microbes and promote receptor-mediated rec-
ognition and phagocytosis by cells of the innate im-
mune system. Pathogen-associated molecular structures
found on the surface of microbes are recognized by
3
Roilides et al.

TABLE 3 Relation of innate host defenses and specific microbial biofilms

Organism Host defenses

Pseudomonas Activation of oxidative burst by PMN
Altered migratory behavior of phagocytes
Maintenance of phagocytic functions
Biofilm formation increases in the presence of PMNs
Function of lactoferrin is severely compromised by both bacteria and PMNs
Secreted rhamnolipids protect bacteria against PMN antibacterial activity
Dislodged biofilm cells are susceptible to immune cells
Enterococcus Effective phagocytosis of biofilm-embedded cells
Infected monocytes with biofilm cells display reduced cytokine, chemokine expression
Candida Monocytes exert a biofilm-enhancing effect associated with increased pro-inflammatory cytokine expression
Biofilms have an immunosuppressive effect
Mature biofilms fail to trigger a reactive oxygen species response
β-glucan in the extracellular matrix evades host defenses
Cytokine-primed PMNs do not enhance biofilm damage
Biofilm glycosyl phosphatidylinositol–anchored proteins confer resistance to PMN killing
Echinocandins have positive immunomodulatory effects on host cells against biofilms
Dislodged biofilm cells are susceptible to immune cells
Aspergillus Biofilms are resistant to the antifungal activity of phagocytes
Gliotoxin downregulates vitamin D and cytokine expression

specific receptors on natural killer cells or professional
phagocytes consisting of polymorphonuclear neutro-
phils (PMNs), mononuclear leukocytes (macrophages),
and dendritic cells (DCs). Toll-like receptors (TLRs),
the best-defined immune sensors of invading pathogens
on the surface of phagocytes, initiate a cascade of sig-
naling pathways that induce phagocytosis, secretion
of antimicrobial products generated in phagocytes by
oxidative and nonoxidative mechanisms, release of pro-
and anti-inflammatory cytokines, or other factors that
contribute to the activation, maturation, and immuno-
regulation of adaptive immune responses. DCs are the
main cell populations that bridge innate and adaptive
immunity, because recruitment by inflammatory signals
leads to DC accumulation at injured or infected sites
to capture, internalize, and present microbial cell frag-
ments to naïve T cells in the lymph nodes. TLRs and
C-type lectin receptors contribute to the recognition and
activation of specific DC programs to decode the struc-
tural information of the antigens captured and to con-
vert it into different T-cell immune responses (42–45).
Thus, early phases of immune response help to control
excessive propagation of pathogens, while activation
of proper T cell subsets leads to long-lived protective
immunity.
In most of the studies conducted to date, host-
pathogen interactions concern bacteria and fungi in their
free-living planktonic state. However, despite the pau-
city of information, recent studies have begun to inves-
tigate immune responses to biofilms. Antimicrobial
factors, such as lactoferrin and the human cationic host
defense peptide LL-37 found at mucosal surfaces or
in secondary granules of PMN, when used in vitro at
very low concentrations, strongly inhibit formation of
P. aeruginosa biofilms. Lactoferrin, as an iron-binding
glycoprotein, reduces the iron supply necessary for
biofilm growth and promotes twitching motility (46),
whereas LL-37 reduces the initial attachment phase,
increases surface motility, and interferes with the quo-
rum sensing system of P. aeruginosa (47). However,
bacteria are known to counteract such host defense
processes by secreting proteases able to degrade both
lactoferrin and LL-37 (48, 49). Although specific im-
mune receptors for the biofilm mode of growth have
not been identified, the active involvement of PMNs
against biofilms is demonstrated by the increased oxygen
depletion caused by enhanced oxidative burst, by the
increased glucose uptake by PMN in CF lungs, and
by the high concentration of L-lactate in the sputum of
CF patients with chronic lung infection (50). It has been
shown that upon contact of PMNs with S. aureus bio-
films, a decrease in biofilm mass is caused by phagocy-
tosis, rapid degranulation of lactoferrin and elastase,
and finally, DNA release (51). Time-lapse video mi-
croscopy has documented migration of PMNs into
S. aureus biofilms, and clearance of bacterial cells within
the biofilm by phagocytosis is shown to be dependent
on biofilm maturation, because young biofilms are more
susceptible to PMN antimicrobial functions compared
to mature biofilms (52). In contrast to planktonic cells
of S. epidermidis, adherence of PMNs to biofilms and
phagocytosis seems to be opsonization-independent,
suggesting that biofilms could contain PMN signaling
molecules (53).

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Confocal scanning laser microscopy shows that
phagocytes enhance the ability of C. albicans to form
biofilms and that these phagocytes appear as unstimu-
lated rounded cells, inducing significantly less damage
to biofilms than their planktonic counterparts (54, 55).
C. neoformans, an encapsulated fungus that can cause
meningoencephalitis in immunocompromised patients,
shows resistance to oxidative stress, but it becomes
considerably more susceptible to cationic antimicrobial
peptides when switching to the biofilm mode of growth.
Defensins with higher net positive charges (β-defensin-1,
β-defensin-3) interact strongly with negatively charged
biofilm surfaces, while their hydrophobic characteristics
enable them to enter the cells’ membranes and create
temporary pores through which cell contents leak,
leading to cell death. In contrast, defensins with lower
net positive charges (α-defensin-3 and magainin-1) are
less efficient against biofilms, implying that the greater
affinity to biofilms results in increased biofilm suscepti-
bility (56).
C. neoformans capsule is primarily composed of
glucuronoxylomannan, a polysaccharide present in
large amounts in the extracellular biofilm matrix and
for this reason indispensible for biofilm formation.
Moreover, it has been shown that anti–C. neoformans
antibodies have a multitude of protective functions,
including enhancement of animal survival against cryp-
tococcosis, promotion of phagocytosis, antigen pre-
sentation, complement activation, and modulation of
immune protein expression. Testing the hypothesis that
antibodies could also interfere with biofilm production,
Martinez and Casadevall demonstrated that monoclonal
antibodies raised against glucuronoxylomannan inhibit
biofilm formation, suggesting that humoral immune
responses could be important to the prevention of bio-
film formation. In the clinical setting, administration
of protective antibodies could present a valuable alter-
native in the management of biofilm-associated diseases
(17).
HOW BIOFILMS OF BACTERIA AND
FUNGI EVADE HOST DEFENSES
Staphylococcus spp.
Staphylococcus spp. are among the leading causes of
health care facility– and community-associated infec-
tions. S. epidermidis and S. aureus are frequent etiologic
factors of biofilm-associated infections on indwelling
medical devices as well as chronic and recalcitrant infec-
tions such as endocarditis, periodontitis, rhinosinusitis,
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How Biofilms Evade Host Defenses
and osteomyelitis (57). The emergence of drug-resistant
strains, especially methicillin-resistant S. aureus, further
accentuates their therapeutic difficulty. While the bulk of
the available information on biofilms concerns biofilm
development, the host immune responses against biofilm
infections remain largely unidentified.
Initial studies of medically relevant S. aureus biofilms
showed that human leukocytes are able to effectively
penetrate the biofilm, possibly by using the nutrient
channels that exist in mature biofilms; however, leuko-
cytes exhibit impaired phagocytosis and show a decreased
ability to kill the bacteria (58). Subsequent studies using
more sophisticated methods demonstrated that PMNs
are able to clear Staphylococcus spp. biofilms by phago-
cytosis (51, 52, 59). Furthermore, immature biofilms were
more sensitive to phagocytosis compared to mature ones,
although mature biofilms are not entirely immune to
PMN attack (52). By comparison, macrophages did not
show appreciable phagocytosis of S. aureus biofilms. Of
note, macrophages were capable of engulfing disrupted
biofilm material, suggesting that the size and/or physical
complexity of biofilm ultrastructure are responsible for
their recalcitrance to phagocytosis (60).
The opsonization of S. aureus biofilms with immu-
noglobulin G did not affect the adherence of PMNs
to the biofilms; however, it increased the clearance of
biofilm, possibly through upregulated oxygen radical
production in PMNs (61). It seems that biofilm matrix
can protect bacteria from antibody-mediated phagocy-
tosis (62).
Another mechanism utilized by S. aureus biofilms to
evade host immunity, which may explain their in vivo
persistence, is the macrophage dysfunction and cell
death caused upon contact with the biofilm itself. It
was noted that macrophages showed a differential sen-
sitivity to cell death based on their physical distance
from the biofilm surface, with macrophages most inti-
mately associated with biofilm being dead, while mac-
rophages that remained above the biofilm surface
remained viable (60). Among the potential mechanisms
proposed to account for this phenomenon is the phe-
nomenon of metabolic “layering” within the biofilm
(63). Regions of anaerobic and aerobic microenviron-
ments within the biofilm, bacterial-influenced fluctua-
tions in pH, and macrophage survival may be affected
due to the release of bacterially produced toxic by-
products of metabolism. Furthermore, the biofilm may
contain lytic toxins, which may lead to cell death.
Using a mouse model of catheter-associated biofilm
infection, it was demonstrated that several inflamma-
tory signals responsible for macrophage and neutrophil
5
Roilides et al.
recruitment (CCL2 and CXXL2, respectively) and acti-
vation (tumor necrosis factor alpha and interleukin
[IL]-1β) were significantly reduced in biofilm-infected
tissues compared to the wound healing response elicited
by sterile catheters (60). The role of IL-1β is potentially
mediated through the MyD88-dependent pathway and
can reduce biofilm development but is not sufficient
to eradicate staphylococcal biofilms (63). Additionally,
inducible nitric oxide synthase (iNOS) expression was
decreased and arginase-1, a key enzyme involved in
collagen biosynthesis, was increased in macrophages
surrounding the biofilm. These findings would be ex-
pected to skew the cellular response away from bacterial
killing to favor fibrosis and could represent another
mechanism to account for the ability of biofilms to evade
clearance (60, 63). Further in vivo data showed that
S. aureus biofilms could evade TLR2 and TLR9 recog-
nition (60). While the mechanism responsible for TLR2/
TLR9 evasion is not known, it could be attributed to
ligand inaccessibility.
Current data collectively indicate that the persist-
ence of staphylococcal biofilm could be attributed
to its ability to skew the immune response to favor
anti-inflammatory and pro-fibrotic pathways. As such,
it has been shown that targeting macrophage pro-
inflammatory activity can represent a novel therapeutic
strategy to overcome the local immune inhibitory envi-
ronment created during biofilm infections (64).
From the currently available data, one can deduce
that innate host defense cells may be able to recognize
Staphylococcus spp. biofilms, migrate toward biofilms,
and degrade biofilms in vitro. However, the latter de-
pends greatly on the experimental conditions, namely
the Staphylococcus strain, biofilm maturation phase,
opsonization with immunoglobulin, and the immune
cell population used. It is likely that the unique proper-
ties of Staphylococcus spp. biofilms circumvent tradi-
tional antimicrobial pathways. Additional studies are
warranted to investigate the mechanisms that lead to
host defense impairment upon contact with Staphylo-
coccus spp. biofilms.
Pseudomonas spp.
The opportunistic pathogen P. aeruginosa has drawn
special attention in the biofilm field due to its role in
chronic and recalcitrant infections, particularly lung in-
fection of CF patients, ventilator-associated pneumonia,
chronic wounds, otitis media, and medical device–
related infections. In humans, infection by P. aeruginosa,
and subsequent biofilm formation, invariably occurs in
association with an exuberant inflammatory response.
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Pseudomonas biofilm–related infections seem to follow
the current dogma regarding host defense mechanisms
where immune responses fail in eradicating biofilms
(65).
Initial studies using a chemiluminescence assay
showed that while biofilms of P. aeruginosa were ca-
pable of activating the oxidative burst response of
PMNs, this response was reduced by about 25% com-
pared to planktonic cells (66). In vitro microscopic evi-
dence suggests that the normal migratory behavior
of phagocytes is disrupted by P. aeruginosa biofilms.
The innate cells settle into biofilms but do not appear
to be capable of migrating from the point of contact
even though they appear to mount a respiratory burst,
degranulate, and retain their phagocytic and secretory
activity. The mechanism by which the biofilms immo-
bilize neutrophils (yet they are still capable of phago-
cytosing bacteria) is not known (67). It is possible
that exotoxins or other components produced by
P. aeruginosa and immune cells, respectively, may play
a role (46, 67). Others suggest that P. aeruginosa bio-
film tolerance to PMNs is quorum sensing dependent
(68).
It has also been reported that P. aeruginosa biofilm
formation is increased by 2- to 3.5-fold in the presence of
PMNs (69, 70). The mechanism of biofilm enhancement
by PMNs was attributed to PMN-generated polymers
comprised of actin and DNA (69). This is consistent with
the clinical finding of higher numbers of necrotic PMNs
detected in CF lungs infected with P. aeruginosa com-
pared with other bacterial infections (71). Lactoferrin, a
common secretory component of human neutrophil
granules, has been shown to inhibit P. aeruginosa bio-
film production (46). However, lactoferrin inhibitory
effect was not evident when the total content of neu-
trophil granules was combined with P. aeruginosa (69).
In this regard, it is likely that the role of lactoferrin
in microbial biofilms is modulated by scavenging and
protease- or oxygen radical–mediated degradation by
P. aeruginosa and neutrophils (46, 67).
The pathogen-beneficial effects of the biofilm matrix
have been demonstrated in other studies (65, 72). The
exopolysaccharide alginate protects P. aeruginosa bio-
film bacteria from leukocyte phagocytosis. In vitro
studies demonstrate that in the presence of the potent
leukocyte activator interferon-γ, human phagocytes
killed P. aeruginosa biofilm bacteria lacking the ability
to produce the alginate exopolysaccharide (72). Of note,
the inability of innate cells to eliminate biofilms is abol-
ished as soon as biofilm cells are mechanically disrupted
into individual cells (65, 73).

There is increasing evidence supporting the role
of rhamnolipids (a class of glycolipids produced by
P. aeruginosa that are considered a key virulence de-
terminant acting under the control of the quorum sens-
ing system) in the host defense against PMNs, especially
in CF lung infection or other chronic infections (65, 74).
The activity of rhamnolipids was shown in vitro and
in vivo in pulmonary models, where P. aeruginosa bio-
films produce a shield of excreted rhamnolipids that
protects from the bactericidal activity of PMNs (65,
75, 76). Accordingly, disabling the biofilm shield of
rhamnolipids either by mutation or by treatment with
quorum sensing inhibitors leads to increased PMN-
mediated clearance of the biofilm (65, 76). Rhamno-
lipids appear to be a crucial component of a vicious
self-enhancing cycle in which rhamnolipids induce ne-
crotic lysis of PMNs, and the lysed PMNs subsequently
cause more inflammation and in turn attract more
PMNs (65). The hypothesis of a “biofilm shield” of
rhamnolipids which offers protection from the antibac-
terial activity of PMNs seems to contribute to biofilm
infections of P. aeruginosa (65).
Candida spp.
While the interactions between the host immune system
and planktonic Candida cells have been extensively ex-
amined, the corresponding biofilm interactions are
largely unstudied (77). Initial studies showed that mono-
cytes influence the ability of C. albicans to form biofilms.
Monocytes exert a biofilm-enhancing effect, which has
been associated with a modulated immune response of
increased levels of pro-inflammatory cytokines such as
IL-1β and decreased levels of IL-6, IL-10, MCP-1, I-309,
and tumor necrosis factor alpha (54). Overall, Candida
biofilms appear to have an immunosuppressive effect,
because monocytes are unable to phagocytose fungal
cells in the maturing biofilms and appear to be entrapped
within the biofilm ultrastructure (54). Lack of mono-
cyte-mediated phagocytosis within biofilms was also
observed for different stages of C. albicans biofilm de-
velopment. Confocal scanning microscopy showed that
human phagocytes resemble unstimulated cells, present-
ing a rounded shape when in the presence of biofilms
(55). This was also confirmed by reduced cytokine
production in the biofilm-phagocyte co-culture com-
pared to a planktonic cell-phagocyte mixture (55). How-
ever, the same phenomenon was not observed with
dislodged biofilm cells (55). Consistently, mature Can-
dida biofilms were more resistant to killing by leukocytes
than early biofilms. Further, mature biofilms failed
to trigger a reactive oxygen species response (78). The
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How Biofilms Evade Host Defenses
architecture of mature biofilms and the presence of
β-glucans in the extracellular matrix represent an im-
portant innate immune evasion mechanism of C. albicans
biofilms (55, 78).
Previous studies have unequivocally shown that in-
terferon-γ and granulocyte colony-stimulating factor
enhance the antifungal activity of human PMNs against
planktonic Candida spp. (77). On the contrary, expo-
sure of C. albicans biofilms to cytokine-activated PMNs
does not enhance biofilm damage (77, 79). The increased
biofilm damage induced by cytokine-activated PMNs
can be explained by the upregulatory activity of the
two cytokines to the number and/or to the affinity of
the mannose and FcγR receptors on the surface of
the phagocytes leading to a more efficient interaction
between PMNs and fungal targets. Knowing that kill-
ing of C. albicans requires ligation of the various host
immune receptors by C. albicans surface structures
(mannoproteins, mannans, or β-glucans) and knowing
the role of extracellular matrix to biofilm immune eva-
sion, the lack of cytokine effectiveness was attributed
to the inability of the effector cells to recognize the
cells within the biofilm (79). Nevertheless, the notion
of biofilm recognition by the immune system appears to
be important. Large amounts of (1→3)-β-D-glucans are
found in the extracellular matrix of Candida biofilms
in vitro and in vivo (80–82). In addition, Hyr1, which
encodes cell surface glycosyl phosphatidylinositol–
anchored proteins, confers resistance to neutrophil kill-
ing in vitro and in an oral mucosal tissue biofilm model
(81, 83).
In this regard, exposure of C. albicans biofilms to
anidulafungin, an antifungal agent that causes height-
ened β-glucan exposure, was associated with a signifi-
cant increase in phagocyte-mediated damage and a
larger pro-inflammatory response from phagocytes (55).
Furthermore, Candida spp. biofilms were more suscep-
tible to the combined effect of anidulafungin with phago-
cytes compared to voriconazole with phagocytes (55,
79). These data underline the notion that echinocandins
(i.e., caspofungin, micafungin, and anidulafungin) have
immunomodulatory effects on host cells against biofilms.
Other Organisms
Enterococci constitute an important cause of hospital-
acquired infections and become particularly pathogenic
in intensive care settings, in debilitated patients with
impaired immune systems, and in the elderly. Despite
the clinical importance of enterococci, little is known
about their interactions with immune cells (84, 85).
The prevailing notion that biofilm cells encased in an
7
Roilides et al.
exopolysaccharide matrix are resistant to phagocytosis
by immune cells seems to contradict recent findings (86,
87). In vitro studies showed that immune cells are able to
phagocytose enterococcal cells recovered from biofilms
as effectively as their planktonic counterparts (86, 87).
In addition, enterococcal biofilms seem to elicit a dif-
ferential immune response relative to planktonic cells, in
that infected monocytes with enterococcal biofilm cells
invoke less pro-inflammatory cytokine and chemokine
expression (86, 87). However, in vivo it is likely that the
complex involvement of multiple host factors may allow
biofilm cells to circumvent host defenses, thus explaining
the persistence of biofilm-related infections.
Aspergillus spp. are frequently isolated from the res-
piratory tract of patients with CF (88), where it causes
typical biofilms and may be associated with deteriora-
tion of lung function, invasive infection, or allergic
bronchopulmonary aspergillosis. Host defenses against
Aspergillus in the respiratory tract of CF patients have
been reviewed (89, 90). Like all biofilms, Aspergillus
biofilms are quite resistant to the antifungal activity
of PMNs and macrophages, the two major airway
phagocytes. Of note, Aspergillus downregulates vitamin
D receptor gene and protein expression in vitro and
in vivo. This is mediated by the fungal virulence factor
gliotoxin, an immune-evader and promoter of cellular
apoptosis. Reduced gliotoxin and IL-5 as well as IL-13
(Th2 cytokines) concentrations were detectable post–
itraconazole treatment in patients with CF and Asper-
gillus with concomitant increases in vitamin D airway
receptor expression (91).
HIGHLIGHTS OF RECENT FINDINGS
AND FUTURE QUESTIONS
A large range of biofilm model systems has been devel-
oped; however, the technical challenges associated with
the growth and assessment of biofilm development fur-
ther complicate the generalization of the results of var-
ious studies. In assessing the effects of host defenses
on microbial biofilms, the experimental conditions, the
thickness, and the complexity of biofilms should be
considered. A major challenge in biofilm research is the
development of suitable models that effectively repro-
duce the host defense conditions at different infection
sites and that permit evaluation of the efficacy of novel
antifungal treatment strategies under in vivo conditions
(92). Animal models of biofilm formation represent an
invaluable alternative for improving our understanding
of biofilm development inside the host, their resilience
to immune cells, and their interaction with the host
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immune defense system. This appears to be of particular
importance because the use of host immune responses
may represent a novel therapeutic approach against
biofilms (64).
ACKNOWLEDGMENTS
Conflicts of interest: We disclose no conflicts.
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