The Laryngoscope
C 2011 The American Laryngological,
V
Rhinological and Otological Society, Inc.
Contemporary Review
Do Biofilms Contribute to the Initiation and Recalcitrance
of Chronic Rhinosinusitis?
Andrew Foreman, BMBS (Hons); Joshua Jervis-Bardy, MBBS; Peter-John Wormald, MD
Chronic rhinosinusitis is a common disease whose underlying aetiopathogenesis has not been completely understood. Amongst a range of other potential environmental triggers in this disease, a role has recently been proposed
for bacterial biofilms. Adopting the biofilm paradigm to explain the initiation and maintenance of this disease may
help to clarify previous inconsistencies in this disease that have resulted in the role of bacteria being questioned. Of
particular interest is the association of bacterial biofilms with recalcitrant disease states. Over the last five years,
research has progressed rapidly since biofilms were first identified on the surface of diseased sinonasal mucosa.
Their presence there has now been associated with more severe disease that is often recalcitrant to current management paradigms. Technological advances are allowing accurate characterization of the bacterial and fungal species
within these biofilms, which would appear to be an important step in improving our understanding of how these
bacterial communities might interact with the host to cause disease. This is an unanswered, yet highly important,
question in this field of research that will undoubtedly be an area of investigation in the near future. As the body of
evidence suggesting biofilms may be involved in this disease grows, research interest has switched to the development of antibiofilm therapies. Given the unique properties of bacteria existing in this form, biofilm eradication strategies will need to incorporate novel medical therapies into established surgical practices as we attempt to improve
the outcomes of our most difficult patients.
Key Words: Chronic rhinosinusitis (CRS), biofilms, S. aureus, fungi, bacteria, treatment.
Laryngoscope, 121:1085–1091, 2011
INTRODUCTION
Chronic rhinosinusitis (CRS) is a common and debilitating illness affecting up to 16% of the adult population.1
Despite CRS’s prevalence and a sustained research effort,
a unifying underlying aetiology for this condition has not
been identified. Indeed this seems unlikely considering
the diverse clinical manifestations of the disease and its
variable response to current therapies. As a disease, CRS
has been unable to fulfill Koch’s postulates and thus is
rarely considered to be a classical bacterially mediated infectious disease. This, however, does not exclude a potential role for bacteria and fungi in the development of this
From the Department of Surgery-Otorhinolaryngology, Head and
Neck Surgery, University of Adelaide and Flinders University, Adelaide,
Australia.
Editor’s Note: This Manuscript was accepted for publication
November 9, 2010.
Financial support was provided by the Garnett Passe and Rodney
Williams Memorial Foundation.
The authors have no conflicts of interest to disclose.
Send correspondence to Dr. Peter-John Wormald, Department of
Otorhinolaryngology, Head and Neck Surgery, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville, SA 5011, Australia.
E-mail: [email protected]
DOI: 10.1002/lary.21438
Laryngoscope 121: May 2011
disease, and the application of the biofilm hypothesis to
CRS may help to clarify such inconsistencies.
Bacteria are known to be able to exist in two states
that are genotypically and phenotypically distinct. The
planktonic form has been well studied throughout medical history and is clearly responsible for acute infectious
diseases such as pneumonia and pyelonephritis. Conversely, despite being first recognized in the 1700s on
dental scrapings performed by Anton van Leeuwenhoek,2
the importance of the biofilm phenotype was largely overlooked until quite recently.3 Over the last 30 years, the
biofilm concept has reemerged in the scientific literature
as it has been realized that most bacteria actually exist
in this sessile state. Furthermore, the role of bacterial
biofilms in a large range (possibly up to 80%4) of chronic
diseases has been recognized. This includes diseases of
the ear, nose, and throat such as otitis media with effusion,5 chronic tonsillitis,6 and cholesteatoma.7 Recent
literature has proposed a role for biofilms in CRS.
What Is a Biofilm?
A biofilm is defined as a microbially derived sessile
community characterized by cells that are irreversibly
attached to a surface. These cells are embedded in a
Foreman et al.: Do Biofilms Contribute to CRS?
1085
matrix of extracellular polymeric substances that they
have produced and they exhibit an altered phenotype
with respect to growth rate and gene transcription.8
Bacteria move through a well-described life cycle from
their planktonic form to an established biofilm community, within which they communicate via complex intercellular signalling pathways. During transformation, the
bacteria acquire multiple genetic alterations, inducing a
phenotypic change that contributes to persistence of the
biofilm. Biofilms have an enhanced ability to evade the
host’s defenses and demonstrate a reduced susceptibility
to traditional antimicrobial agents.9 This occurs through
a number of mechanisms including the physical protection afforded by the matrix, the slow-growing, sessile
state of the nutrient-deprived bacteria, and genotypic
changes that alter drug targets.
There is a consistent set of clinical features that
characterize diseases caused by biofilms. Biofilm infections typically evolve slowly with an initial infection
that may be subtle and slow to produce overt symptoms.10 Antibiotics often alleviate symptoms during
acute exacerbations but fail to remove the biofilm nidus.
As a result, a chronic infection is established and
patients will report a relapsing and remitting course as
the biofilm periodically sheds planktonic organisms. The
failure of medical therapy to clear the biofilm means disease eradication is unlikely without surgical intervention.
In addition, culture rates in biofilm-mediated diseases are
variable, perhaps dependent on the timing of culturing in
relation to dispersal of free-floating bacteria from the biofilm, a fact that has contributed to the conjecture surrounding the role bacteria might play in these diseases.
It is now clear that biofilm diseases are different to the
world of microbiology Koch opened up with his famous
postulates, necessitating a paradigm shift in our thinking
of chronic disease. Understanding and applying the biofilm hypothesis will undoubtedly assist us in advancing
our knowledge in a number of microbial processes, in particular bacterial infections. As the features of other biofilm diseases are strikingly similar to those experienced
by many CRS patients, it is not surprising that the biofilm paradigm was applied to CRS. The subsequent
research is the focus of this review.
DISCUSSION
Applying the Biofilm Hypothesis to CRS
The research into the role of biofilms in CRS can be
divided into a number of different subtopics. Our department utilizes the framework outlined in Fig. 1 to reflect
the requisite steps in building an argument that biofilms
do indeed play a role in the initiation and recalcitrance
of CRS. In varying degrees, all of these questions have
been addressed already in the current literature, but
none have been definitively answered. This model is useful for summarizing and understanding current research
in this field and also highlights deficiencies in the available literature, thus providing a guide for future
research. Until all of these areas have been robustly
addressed, the role of biofilms in CRS will remain both
theoretical and controversial.
Laryngoscope 121: May 2011
1086
Fig. 1. Biofilms in chronic rhinosinusitis (CRS). Research direction
can be divided into a number of subtopics to enable appreciation
of both the state of current literature and the questions that
remain unanswered.
Biofilm Determination
The first report of biofilms in CRS patients was of
their isolation from frontal recess stents placed at the
time of endoscopic sinus surgery.11 This was not surprising given the avidity with which bacteria assume the
biofilm form when associated with inert surfaces. Cryer
et al.12 then made a significant advance in this area by
identifying biofilms on sinus mucosal biopsies from medically and surgically recalcitrant CRS patients. Since
this initial report, numerous centers have substantiated
this initial finding using a range of microscopic imaging
techniques (Table I).12–22
The specificity of scanning electron microscopy
(SEM) as a technique remains debatable. Although
excellent at identifying biofilms on inert surfaces,23 difficulty in differentiating the biofilm matrix from surface
mucus limits the utility of SEM as a diagnostic modality
in the sinonasal tract. Using a sheep model of CRS, Ha
et al.24 demonstrated that BacLight LIVE DEAD (Invitrogen Corp., Carlsbad, CA) staining imaged on the confocal scanning laser microscope was more sensitive and
specific for biofilm determination than both SEM and
transmission electron microscopy (TEM). An advantage
of the confocal microscope is its ability to image the
three-dimensional biofilm structure with either the nonspecific BacLight LIVE/DEAD stain or species-specific
fluorescence in situ hybridization (FISH) probes. These
two techniques can be complementary in the determination and characterization of biofilms in CRS.25
A point of continued debate in this field is whether
biofilms are present in the sinuses of healthy controls.
There have been seven controlled studies in this field
(Table I). Five of these studies failed to demonstrate biofilms in the control arm,14,15,18,20,22 whereas the remaining two studies did identify biofilms in the control
patients.16,21 Interestingly, both of these studies are
from the same institution and utilized FISH probes for
biofilm determination. This has not been our experience,
Foreman et al.: Do Biofilms Contribute to CRS?
TABLE I.
Biofilm Determination in Chronic Rhinosinusitis (CRS).
Author
Cryer et al.
Journal
12
Imaging Modality
CRS
4/16 (25%)
Controls
Journal for ORL and Its Related Specialties, 2004
SEM
Ramadan et al.19
Otolaryngology Head and Neck Surgery, 2005
SEM
Sanclament et al.20
Ferguson et al.13
Laryngoscope, 2005
American Journal of Rhinology, 2005
SEM/TEM
TEM
24/30 (80%)
2/4 (50%)
0/4
NA
Sanderson et al.21
Laryngoscope, 2006
FISH
14/18 (78%)
2/5 (40%)
Psaltis et al.18
Healy et al.16
Laryngoscope, 2007
Otolaryngology Head and Neck Surgery, 2008
BacLight/CSLM
FISH
17/38 (44%)
12/13 (92%)
0/9
3/3 (100%)
Galli et al.15
Annals of Otology, Rhinology & Laryngology, 2008
SEM
10/24 (41.7%)
0/20
Hekiert et al.17
Foreman et al.14
Otolaryngology Head and Neck Surgery, 2009
American Journal of Rhinology and Allergy, 2009
SEM
FISH/CSLM
17/60 (28%)
36/50 (72%)
NA
0/10
Singhal et al.22
American Journal of Rhinology and Allergy, 2009
BacLight/CSLM
36/51 (71%)
0/5
5/5 (100%)
NA
NA
Various microscopic techniques have been used to identify biofilms on the mucosa of CRS patients. This finding has been consistent, although the
reported prevalence varies. The identification of biofilms in healthy controls is inconsistent in these studies and remains a point of controversy.
SEM ¼ scanning electron microscopy; NA ¼ not assessed; TEM ¼ transmission electron microscopy; FISH ¼ fluorescence in situ hybridization;
CSLM ¼ confocal scanning laser microscopy.
and we have been unable to identify biofilms in nonsinusitis subjects using a range of imaging techniques
including FISH, BacLight, SEM, and TEM.
The data from biofilm determination studies does
give some insights into CRS pathogenesis. The presence
of biofilms in diseased patients and their relative absence in health circumstantially implies a role for them
in the initiation of the disease, although further
research is definitely required to identify a more robust
association or pathogenic role for them in CRS. Secondly,
the fact that most studies have thus far only identified
biofilms in a subset of patients (generally 40% to 80%)
reinforces the multifactorial nature of this disease. A limitation of this research, when considered as a whole, is
the difference in biofilm identification rates that exist
between studies. This may be due to differing detection
methods, different patient populations, or errors in sampling and data analysis. The true prevalence of biofilms
in CRS probably lies somewhere between the figures
reported by the studies in Table I. In any case, more
work is required to clarify their true prevalence and contribution to disease, possibly by employing more sensitive
techniques that do not rely on morphologic identification
of the biofilm communities.
Biofilm Characterization
Initial attempts to characterize the biofilm-forming
species in CRS used ex vivo biofilm-forming assays
(Crystal Violet) of bacteria recovered from CRS patients.
Prince et al.26 studied 157 consecutive patients in whom
they found 28.6% had bacteria with moderate or severe
biofilm-forming capacity. Pseudomonas aeruginosa and
Staphylococcus aureus were prominent organisms in this
population with S. aureus, in particular, frequently present within a polymicrobial bacterial mix. This study, however, did not assess biofilms in vivo. All strains of a given
bacteria exist along a continuum of biofilm-forming
capacity, and it is quite likely that biofilm infections in
CRS are also polyclonal, in addition to being polymicrobial.
Laryngoscope 121: May 2011
As such the cultured bacteria may not be representative of
the organisms that actually compose the biofilms in these
patients with the swab just sampling the planktonic clones
within the sinuses. Furthermore, studies have found that
the presence of biofilms on the mucosa does not correlate
with bacterial recovery from standard cultures.18 Therefore, a number of biofilm-positive patients may have been
overlooked in the analysis, and cultures that grew biofilms
in vitro may not form biofilms in vivo, making it difficult
to analyze the true meaning of these results.
Several further advances in our understanding of
the role of biofilms in CRS were made when FISH
probes were employed for in vivo biofilm characterization. The first two FISH papers identified Haemophilus
influenzae as the most common biofilm-forming organism.16,21 However, our work subsequently characterized
S. aureus as the most common organism within the biofilms of CRS patients, present in 50% of the study group
(Table I).14 The points of difference between these studies are debatable but may include geographical and
methodological variations as well as differences in the
studied populations. H. influenzae is a much-debated organism when considered in the context of CRS disease initiation. It is a fastidious organism that is difficult to culture
using standard techniques, so its true incidence and potential role in CRS pathogenesis has been difficult to accurately quantify. The work of Sanderson et al.21 reignited
debate about the role of H. influenzae because it used a
molecular detection technique (i.e., FISH) that could circumvent some of the previous barriers to H. influenzae
identification. Unfortunately these results have not been
replicated in other FISH work or in a recent 16S rRNA
gene sequencing study,27 which highlighted both S. aureus
and anaerobes as the most frequently identified bacteria
in the disease sinus. Interestingly, the rate of S. aureus
detection in the Stephenson et al.27 study was also 50%.
Taken together, these studies highlight some key
concepts. Firstly, CRS is a polymicrobial disease. This has
been previously demonstrated using standard culture and
gene sequencing techniques, but biofilm characterization
Foreman et al.: Do Biofilms Contribute to CRS?
1087
studies confirm that biofilms are also polymicrobial in
the diseased sinus.14,26 Identifying which organisms are
important in determining disease course and which
are merely bystanders in the pathologic process is an important current investigative pathway that will have
therapeutic implications by enabling us to more effectively target the relevant organisms.
Secondly, an association exists between bacterial
biofilms and fungi. Fungi have been associated with
CRS pathogenesis for some time now. Although this
association is at times controversial, most rhinologists
would accept they play a role in at least some CRS
patients. Biofilm characterization with FISH probes has
enabled us to identify potential links between these two
aetiologic agents. Our work confidently identified fungal
biofilms and found them to be associated with S. aureus
in 8 of 11 fungal biofilm patients.14 Healy et al.16 did not
confidently identify fungal biofilms; however, this may
just represent a difference in definitions, structural
understanding, and, ultimately, reporting. Their work
found fungal elements in 11 of 12 CRS patients (including the allergic fungal sinusitis and eosinophilic-mucus
CRS subgroups), of which 8 were associated with bacterial biofilms. Improving our understanding of fungal biofilms and their interactions with bacteria such as S.
aureus as well as elucidating their pathologic relevance
will be key steps in future biofilm research.
Biofilm Pathogenicity
A direct role for biofilms in the pathogenesis of CRS
has not yet been proven, and this shapes as a key area
for future biofilm research. An answer to this question
will both validate previous research as well as provide a
new target for therapy in CRS. The establishment of a
biofilm within the sinuses likely requires defects in both
innate and adaptive immunity of the host coupled with
specific features of the invading microorganisms. Current research in this area is sparse.
Antimicrobial peptides form one part of the innate
immune system, and of these lysozyme and lactoferrin
are the two most common in airway secretions.28 Lactoferrin has been shown to be down-regulated in CRS
patients29 and Psaltis et al.30 have also found that the
presence of biofilms in CRS patients is associated with a
significant further down-regulation at the mRNA level.
Although a cause and effect could not be definitively
deduced from this association, it suggests that individuals may be predisposed to acquiring biofilm infections
because of deficiencies in their innate immune function.
A recent study of 60 CRS patients evaluated adaptive immune system cytokines and leukocyte subpopulations.17 Unfortunately, 41 of these patients were exposed
to corticosteroids at the time of sampling, leaving a small
group of only 19 steroid naive patients. Analysis of this
group revealed that the presence of biofilms, using SEM
techniques, was associated with elevated interferon-c,
granulocyte-colony stimulating factor, and macrophage
inflammatory protein 1-b, suggesting a skewing of the
T-cell response toward the T-helper1 pathway. This is not
consistent with the findings in other biofilm-mediated disLaryngoscope 121: May 2011
1088
eases such as chronic periodontitis, which have repeatedly been associated with a T-helper2 response.31–33 The
small sample size and detection modality may have contributed to these incongruous results.
Although host responses to the biofilm bacteria are
likely very important in the maintenance of ongoing disease, the actions of biofilms on the host may in part
explain disease initiation. SEM studies of biofilm presence and epithelial integrity found that biofilm-positive
patients have marked destruction of the epithelial layer
with complete absence of the cilia.15,19 In contrast, onethird of the CRS patients without biofilms had normal
ciliary structure and the remaining two-thirds had partial epithelial damage with the remaining mucosal surface still lined by normal cilia. This differential pattern
of ciliary damage may contribute to disease initiation
through mucociliary impairment at the ostiometal complex, leading to mucociliary stasis and distal spread of
bacterial biofilms.15
Clinical Relevance of Biofilms in CRS
Beyond identifying the presence of biofilms in CRS
patients, the first investigation that implied a clinically
significant role for biofilms in mediating disease persistence was that of Bendouah et al.34 In studying a cohort
of postendoscopic sinus surgery (ESS) patients 12 months
after their surgery, they concluded that the biofilm-forming capacity of both P. aeruginosa and S. aureus, but not
coagulase-negative Staphylococci, was associated with
poor clinical evolution following surgical intervention.
This study suffers from the same methodologic limitations
as that of Prince et al.26 in using crystal violet assay for
biofilm determination. Nevertheless, this information was
novel, interesting, and instigated more robust investigations into the clinical significance of biofilms in CRS,
some of which partly confirm these results.
Psaltis et al.35 performed a retrospective analysis of
a cohort of patients undergoing ESS in whom the biofilm
status was known. After a median of 8 months followup, those in the biofilm-positive group were significantly
more likely to have ongoing postoperative symptoms and
mucosal inflammation than those in the biofilm-negative
group. The presence of fungus at the time of surgery
(either on culture or staining) was the only other factor
that was significantly associated with an unfavorable
outcome. These results were confirmed in a recent prospective study utilizing validated, ordinal subjective and
objective measures.22 Fifty-one CRS patients were followed for a median of 16 months. The biofilm-positive
group had more severe disease preoperatively (objectively
and radiologically) and in the postoperative period
required more visits to their treating surgeon with more
persistent symptoms, poorer quality of life, and significantly more diseased mucosa on blinded endoscopic evaluation. This robust investigation of the impact of biofilms
on post-ESS outcomes consolidates their association with
recalcitrant CRS, a fact also suggested by Prince et al.,26
who identified that patients undergoing multiple surgeries
were more likely to harbor biofilm-forming bacteria.
Foreman et al.: Do Biofilms Contribute to CRS?
TABLE II.
Antibiofilm Treatments in Chronic Rhinosinusitis (CRS).
Agent
Mechanism of Action
Study Design
Targeted Species
Outcome
51
Mupirocin
Antibiotic
In vitro
S. aureus
Greater cidality than Ciprofloxacin or Vancomycin
Mupirocin45
Antibiotic
Animal in vivo
S. aureus
96.4% reduction in biofilm biomass compared
with control following 5-day treament protocol
Mupirocin43
Antibiotic
Human in vivo
S. aureus
Pilot study; objectively, subjectively efficacious
and well tolerated. 15/16 patients S. aureus
culture-negative after treatment
Gallium Nitrate45
Antibiotic
Animal in vivo
S. aureus
Manuka Honey46
Antibiotic
In vitro
S. aureus
P. aeruginosa
68.5% reduction in biofilm biomass compared
with control following single dose
Biocidal against 16/22 S. aureus and 10/11
P. aeruginosa strains
Manuka Honey47
Antibiotic
Animal in vitro
*
No evidence of histologic injury following
repeated treatments
Moxifloxacin49
Antibiotic
In vitro
S. aureus
P. aeruginosa
Biocidal at very high concentrations
(103 MIC planktonic)
Tobramycin48
Antibiotic
Animalin vivo
P. aeruginosa
LL-37-derived
antimicrobial
peptide50
Baby shampoo52
Antibiotic
Animal in vivo
P. aeruginosa
Antibiotic/physical
removal
In vitro
P. aeruginosa
Recalcitrant biofilm despite eradication of
planktonic bacteria at 400 MIC planktonic
Significant reduction of sinus lavage planktonic
bacteria. No biofilm assessment;
ciliotoxic at high concentrations
Inhibits biofilm formation at 1%; no cidality
demonstrated
Baby shampoo52
Antibiotic/physical
removal
Human in vivo
S. aureus
Quality-of-life improvement in
7/15 patients following treatment
CAZS54
Antibiotic/physical
removal
Animal in vivo
*
Cilia acutely denuded following treatment,
subsequent reciliation after 6 days
CAZS 6 Hydrodynamic
force53
CAZS þ Hydrodynamic
force45
Laser57
Antibiotic/physical
removal
Antibiotic/physical
removal
Antibiotic/physical
removal
In vitro
Animal in vivo
S. aureus
P. aeruginosa
S. aureus
In vitro
S. aureus
Significant reduction in CFUs posttreatment
with and without hydrodynamic force
30.9% reduction in biofilm biomass
compared with control following single dose
34% reduction in biofilm biomass
following SW and NIR laser
Hydrodynamic force53
Physical removal
In vitro
S. aureus
P. aeruginosa
Significant reduction in CFUs posttreatment
Summary of the current literature investigating biofilm eradication strategies in both the in vitro and in vivo (both animal model and human) settings.
*In vitro safety study of treatment in noninfected animal model.
CFUs ¼ colony-forming units; SW ¼ shock wave; NIR ¼ near infra-red.
The most recent research in this area suggests that
not all biofilms are associated with the same clinical
characteristics.36 In a retrospective review, we identified
the presence of S. aureus biofilms in vivo (either alone or
in a polymicrobial biofilm) to be a predictor of both adverse
preoperative disease severity and postoperative disease resolution, in line with the work of Bendouah et al.34 in their
ex vivo investigation. Conversely, patients with unimicrobial H. influenzae biofilms have milder disease, which is
highly responsive to surgical therapy. This is a significant
extension of previous knowledge and suggests a need for
us to be targeted in our identification of biofilm-forming
species to select patients whose postoperative course may
be ameliorated with aggressive perioperative therapies.
Biofilm research in other specialties, particularly
device-related infections, has emphasized the importance
of surgical removal of biofilms to achieve eradication.
Complete exenteration of the sinonasal mucosa is neither
possible nor desirable in ESS, hence biofilm management
in CRS needs to be different from that proposed in other
diseases. Hai et al.37 and Zhang et al.38 have investigated
the effect of current surgical techniques for CRS on biofilm bacteria. Hai et al.37 used an ex vivo biofilm-forming
Laryngoscope 121: May 2011
assay for swabs taken before and 3 months after ESS.
Methodologically, this study suffers the shortcomings of
other crystal violet assay studies,26,39 in addition to the
fact that CRS subgroups associated with biofilm presence—nasal polyposis and surgically recalcitrant cases—
were excluded. At the end of the short follow-up period,
the preoperative biofilm-forming presence of 75% had
dropped to 50%, but this did not correlate with the symptomatic, quality of life, and objective improvements that
were also observed following surgery. These results cast
some doubt over both the effect of surgery on biofilms
and the role of biofilms in mediating recalcitrant disease.
Zhang et al.38 obtained mucosal biopsies of CRS patients
pre- and post-ESS and used SEM to document the in vivo
presence of biofilms. Of the 15 patients who had biofilms
present intraoperatively, 9 had biofilms present postoperatively as well. This study also implies ESS can reduce
biofilm prevalence but not completely eradicate them.
Although there are methodologic limitations of both of
these studies, it does seem to suggest that a combined
approach of targeted medical and surgical therapy will be
required to remove biofilms from the sinus cavity to
improve patient outcomes after ESS.
Foreman et al.: Do Biofilms Contribute to CRS?
1089
Biofilm Treatment
As the evidence mounts that biofilms do play a role
in CRS initiation and recalcitrance, the focus of research
will undoubtedly shift toward development of biofilm
eradication strategies. The paranasal sinuses are unique
in their open access for delivery of topical treatments,
making CRS a prototype disease to test new anti-biofilm
agents. The ideal agent should be active against formed
biofilms (biocidal) rather than merely inhibitory to biofilm
formation and/or growth. It must have a satisfactory local
and systemic side-effect profile and be delivered in a way
that optimizes delivery of topical treatments to the
sinuses.40,41 Safety and efficacy profiling in CRS patients
for agents with published in vitro antibiofilm activity
(Table II) is a current research priority.
Biofilm eradication strategies can be classified
depending on their mechanism of action. Broadly, the
agent may act against the individual bacterial (traditional antibiotic); deliver a physical force to disrupt the
surface attachment of the biofilm (physical removal), or
free bacteria from the biofilm (dispersal or dissociation).
Dispersal may occur either by degrading the matrix (passive dispersal) or by promoting a phenotypic shift from
biofilm-encased to purely planktonic bacteria (active dispersal). Some authors consider physical removal to be a
form of passive dispersal.42
Antibiotic agents specifically proposed for the treatment of biofilms in CRS have included Mupirocin,43 the iron
chelator Gallium Nitrate,44,45 Manuka Honey,46,47 Tobramycin,48 Moxifloxacin,49 as well as antimicrobial peptides.50
Mupirocin has an in vitro51 and in vivo (sheep model)45
activity against S. aureus biofilms that significantly exceeds
that of both Vancomycin and Ciprofloxacin. In a pilot clinical
study, it yielded impressive subjective, objective, and microbiologic outcomes following a four-week treatment protocol
in recalcitrant CRS patients who were culture-positive for
S. aureus, presumably due to its antibiofilm action.43
Biofilm dissociation with surfactant therapy52 is a
potential method for breaking the irreversible bond the
biofilm matrix forms with the mucosal surface. Although
citric acid/zwitterionic surfactant (CAZS) is effective at
achieving this,45,53 it has subsequently been shown to
cause deciliation in a rabbit model.54 Similar dissociation
compounds usually require coadministration of either an
antibiotic or physical removal to effectively treat planktonic bacteria released after disruption of the biofilm
matrix. A hand-held hydrodebrider (Medtronic, Jacksonville, FL) has been developed for this purpose. This device has demonstrated efficacy in conjunction with CAZS
in a sheep model of CRS;45 however, the ciliotoxic effect
of the solution was thought to be responsible for significant biofilm regrowth observed in the week after treatment. A less toxic biofilm dissociation preparation is
required if this strategy is to be employed in CRS.
Promotion of biofilm dispersal via endogenous enzymatic activity has not yet been explored in the rhinology
literature. The glycoside hydrolase Dispersin B, produced by the periodontopathogen A. actinomycetemcomitans, has been shown to degrade the polysaccharide
poly-N-acetylglucosamine (PNAG), a key component of
the S. aureus and S. epidermidis biofilm matrix. By this
Laryngoscope 121: May 2011
1090
mechanism, A. actinomycetemcomitans breaks down its
own matrix PNAG, allowing dispersal of individual bacteria, which in turn seed surrounding tissue and generate further biofilm under favorable conditions.55 This,
therefore, is a biofilm-driven, active dispersal process.
Given the potential relevance of S. aureus biofilms in CRS,
enzymes such as Dispersin B, when delivered as a treatment agent and coupled with an appropriate antibiotic to
kill dissociated planktonic bacteria, may provide novel
antibiofilm regimes for future clinical use in CRS. Interestingly, nitric oxide has been shown to induce P. aeruginosa
biofilm dispersal;56 whether a similar induction is seen in
S. aureus has not yet been studied. A better understanding of the mechanism involved in S. aureus biofilm dispersal may pave the way for exciting new biofilm eradication
strategies to be introduced into our armamentarium for
managing patients with recalcitrant CRS.49,52,57
CONCLUSIONS
CRS shares many similarities with other biofilmmediated diseases. Amongst an ever-expanding body of
research, biofilms have been demonstrated on the mucosa
of CRS patients, and their presence there has been associated with more severe disease clinically. This suggests
they may play a role in CRS initiation and maintenance.
However, there remain a number of unanswered questions
and these will be the focus of future research. Specifically,
a definitive link between biofilms and the host is required
to prove they are intimately involved in the pathogenesis
of CRS, and targeted antibiofilm treatments need to be
formally tested to enable an evidence-based approach to
biofilm eradication. Only then can we hope to improve the
clinical outcomes of our most recalcitrant patients.
BIBLIOGRAPHY
1. Benninger MS, Ferguson BJ, Hadley JA, et al. Adult chronic rhinosinusitis: definitions, diagnosis, epidemiology, and pathophysiology. Otolaryngol Head Neck Surg 2003;129:S1–32.
2. Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002;8:
881–890.
3. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common
cause of persistent infections. Science 1999;284:1318–1322.
4. Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol 2007;5:48–56.
5. Ehrlich GD, Veeh R, Wang X, et al. Mucosal biofilm formation on middle-ear
mucosa in the chinchilla model of otitis media. JAMA 2002;287:1710–1715.
6. Chole RA, Faddis BT. Anatomical evidence of microbial biofilms in tonsillar tissues: a possible mechanism to explain chronicity. Arch Otolaryngol
Head Neck Surg 2003;129:634–636.
7. Chole RA, Faddis BT. Evidence for microbial biofilms in cholesteatomas.
Arch Otolaryngol Head Neck Surg 2002;128:1129–1133.
8. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically
relevant microorganisms. Clin Microbiol Rev 2002;15:167–193.
9. El-Azizi M, Rao S, Kanchanapoom T, Khardori N. In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci. Ann Clin Microbiol Antimicrob 2005;4:2.
10. Wolcott RD, Ehrlich GD. Biofilms and chronic infections. JAMA 2008;299:
2682–2684.
11. Perloff JR, Palmer JN. Evidence of bacterial biofilms on frontal recess stents
in patients with chronic rhinosinusitis. Am J Rhinol 2004;18:377–380.
12. Cryer J, Schipor I, Perloff JR, Palmer JN. Evidence of bacterial biofilms in
human chronic sinusitis. ORL J Otorhinolaryngol Relat Spec 2004;66:
155–158.
13. Ferguson BJ, Stolz DB. Demonstration of biofilm in human bacterial
chronic rhinosinusitis. Am J Rhinol 2005;19:452–457.
14. Foreman A, Psaltis AJ, Tan LW, Wormald PJ. Characterization of bacterial
and fungal biofilms in chronic rhinosinusitis. Am J Rhinol Allergy 2009;
23:556–561.
15. Galli J, Calo L, Ardito F, et al. Damage to ciliated epithelium in chronic
rhinosinusitis: what is the role of bacterial biofilms? Ann Otol Rhinol
Laryngol 2008;117:902–908.
Foreman et al.: Do Biofilms Contribute to CRS?
16. Healy DY, Leid JG, Sanderson AR, Hunsaker DH. Biofilms with fungi in
chronic rhinosinusitis. Otolaryngol Head Neck Surg 2008;138:641–647.
17. Hekiert AM, Kofonow JM, Doghramji L, et al. Biofilms correlate with TH1
inflammation in the sinonasal tissue of patients with chronic rhinosinusitis. Otolaryngol Head Neck Surg 2009;141:448–453.
18. Psaltis AJ, Ha KR, Beule AG, Tan LW, Wormald PJ. Confocal scanning
laser microscopy evidence of biofilms in patients with chronic rhinosinusitis. Laryngoscope 2007;117:1302–1306.
19. Ramadan HH, Sanclement JA, Thomas JG. Chronic rhinosinusitis and
biofilms. Otolaryngol Head Neck Surg 2005;132:414–417.
20. Sanclement JA, Webster P, Thomas J, Ramadan HH. Bacterial biofilms in
surgical specimens of patients with chronic rhinosinusitis. Laryngoscope
2005;115:578–582.
21. Sanderson AR, Leid JG, Hunsaker D. Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope 2006;
116:1121–1126.
22. Singhal D, Psaltis AJ, Foreman A, Wormald PJ. The impact of biofilms on
outcomes after endoscopic sinus surgery. Am J Rhinol Allergy 2010;24:
169–174.
23. Keen M, Foreman A, Wormald PJ. The clinical significance of nasal irrigation bottle contamination. Laryngoscope 2010;120:2110–2114.
24. Ha KR, Psaltis AJ, Tan L, Wormald PJ. A sheep model for the study of
biofilms in rhinosinusitis. Am J Rhinol 2007;21:339–345.
25. Foreman A, Singhal D, Psaltis AJ, Wormald PJ. Targeted imaging modality selection for bacterial biofilms in chronic rhinosinusitis. Laryngoscope 2010;120:427–431.
26. Prince AA, Steiger JD, Khalid AN, et al. Prevalence of biofilm-forming
bacteria in chronic rhinosinusitis. Am J Rhinol 2008;22:239–245.
27. Stephenson MF, Mfuna L, Dowd SE, et al. Molecular characterization of
the polymicrobial flora in chronic rhinosinusitis. J Otolaryngol Head
Neck Surg 2010;39:182–187.
28. Ganz T. Antimicrobial polypeptides in host defense of the respiratory tract.
J Clin Invest 2002;109:693–697.
29. Psaltis AJ, Bruhn MA, Ooi EH, Tan LW, Wormald PJ. Nasal mucosa
expression of lactoferrin in patients with chronic rhinosinusitis. Laryngoscope 2007;117:2030–2035.
30. Psaltis AJ, Wormald PJ, Ha KR, Tan LW. Reduced levels of lactoferrin in
biofilm-associated chronic rhinosinusitis. Laryngoscope 2008;118:895–901.
31. Berglundh T, Donati M. Aspects of adaptive host response in periodontitis.
J Clin Periodontol 2005;32(suppl 6):87–107.
32. Ohlrich EJ, Cullinan MP, Seymour GJ. The immunopathogenesis of periodontal disease. Aust Dent J 2009;54(suppl 1):S2–10.
33. Seymour GJ, Gemmell E, Reinhardt RA, Eastcott J, Taubman MA. Immunopathogenesis of chronic inflammatory periodontal disease: cellular
and molecular mechanisms. J Periodontal Res 1993;28:478–486.
34. Bendouah Z, Barbeau J, Hamad WA, Desrosiers M. Biofilm formation by
Staphylococcus aureus and Pseudomonas aeruginosa is associated with
an unfavorable evolution after surgery for chronic sinusitis and nasal
polyposis. Otolaryngol Head Neck Surg 2006;134:991–996.
35. Psaltis AJ, Weitzel EK, Ha KR, Wormald PJ. The effect of bacterial biofilms on post-sinus surgical outcomes. Am J Rhinol 2008;22:1–6.
36. Foreman A, Wormald PJ. Different biofilms, different disease? A clinical
outcomes study. Laryngoscope 2010;120:1701–1706.
37. Hai PV, Lidstone C, Wallwork B. The effect of endoscopic sinus surgery on
bacterial biofilms in chronic rhinosinusitis. Otolaryngol Head Neck Surg
2010;142:S27–32.
Laryngoscope 121: May 2011
38. Zhang Z, Han D, Zhang S, et al. Biofilms and mucosal healing in postsurgical
patients with chronic rhinosinusitis. Am J Rhinol Allergy 2009;23:506–511.
39. Bendouah Z, Barbeau J, Hamad WA, Desrosiers M. Use of an in vitro
assay for determination of biofilm-forming capacity of bacteria in
chronic rhinosinusitis. Am J Rhinol 2006;20:434–438.
40. Grobler A, Weitzel EK, Buele A, et al. Pre- and postoperative sinus penetration of nasal irrigation. Laryngoscope 2008;118:2078–2081.
41. Beule A, Athanasiadis T, Athanasiadis E, Field J, Wormald PJ. Efficacy of
different techniques of sinonasal irrigation after modified Lothrop procedure. Am J Rhinol Allergy 2009;23:85–90.
42. Kaplan JB. Biofilm dispersal: mechanisms, clinical implications, and
potential therapeutic uses. J Dent Res 2010;89:205–218.
43. Uren B, Psaltis A, Wormald PJ. Nasal lavage with mupirocin for the treatment of surgically recalcitrant chronic rhinosinusitis. Laryngoscope
2008;118:1677–1680.
44. Baldoni D, Steinhuber A, Zimmerli W, Trampuz A. In vitro activity of gallium maltolate against Staphylococci in logarithmic, stationary, and biofilm
growth phases: comparison of conventional and calorimetric susceptibility
testing methods. Antimicrob Agents Chemother 2010;54:157–163.
45. Le T, Psaltis A, Tan LW, Wormald PJ. The efficacy of topical antibiofilm
agents in a sheep model of rhinosinusitis. Am J Rhinol 2008;22:560–567.
46. Alandejani T, Marsan J, Ferris W, Slinger R, Chan F. Effectiveness of
honey on Staphylococcus aureus and Pseudomonas aeruginosa biofilms.
Otolaryngol Head Neck Surg 2009;141:114–118.
47. Kilty SJ, Almutari D, Duval M, Groleau MA, De Nanassy J, Gomes MM.
Manuka honey: histological effect on respiratory mucosa. Am J Rhinol
Allergy 2010;24:e63–66.
48. Chiu AG, Antunes MB, Palmer JN, Cohen NA. Evaluation of the in vivo
efficacy of topical tobramycin against Pseudomonas sinonasal biofilms.
J Antimicrob Chemother 2007;59:1130–1134.
49. Desrosiers M, Bendouah Z, Barbeau J. Effectiveness of topical antibiotics
on Staphylococcus aureus biofilm in vitro. Am J Rhinol 2007;21:149–153.
50. Chennupati SK, Chiu AG, Tamashiro E, et al. Effects of an LL-37-derived
antimicrobial peptide in an animal model of biofilm Pseudomonas sinusitis. Am J Rhinol Allergy 2009;23:46–51.
51. Ha KR, Psaltis AJ, Butcher AR, Wormald PJ, Tan LW. In vitro activity of
mupirocin on clinical isolates of Staphylococcus aureus and its potential
implications in chronic rhinosinusitis. Laryngoscope 2008;118:535–540.
52. Chiu AG, Palmer JN, Woodworth BA, et al. Baby shampoo nasal irrigations for the symptomatic post-functional endoscopic sinus surgery
patient. Am J Rhinol 2008;22:34–37.
53. Desrosiers M, Myntti M, James G. Methods for removing bacterial biofilms: in vitro study using clinical chronic rhinosinusitis specimens. Am
J Rhinol 2007;21:527–532.
54. Tamashiro E, Banks CA, Chen B, et al. In vivo effects of citric acid/zwitterionic surfactant cleansing solution on rabbit sinus mucosa. Am J Rhinol Allergy 2009;23:597–601.
55. Kaplan JB, Ragunath C, Ramasubbu N, Fine DH. Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous betahexosaminidase activity. J Bacteriol 2003;185:4693–4698.
56. Barraud N, Hassett DJ, Hwang SH, Rice SA, Kjelleberg S, Webb JS.
Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J Bacteriol 2006;188:7344–7353.
57. Krespi YP, Kizhner V, Nistico L, Hall-Stoodley L, Stoodley P. Laser disruption and killing of methicillin-resistant Staphylococcus aureus biofilms.
Am J Otolaryngol 2010 [epub ahead of print].
Foreman et al.: Do Biofilms Contribute to CRS?
1091