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Evidence for Microbial Biofilms in Cholesteatomas
Richard A. Chole, MD, PhD;
Brian T. Faddis, PhD
Arch Otolaryngol Head Neck Surg. 2002;128:1129-1133.
ABSTRACT
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Background Sessile bacteria within biofilms are highly resistant to eradication
by antimicrobial agents. Previously, we have shown that the most common organisms
cultured from experimentally induced cholesteatomas are biofilm formers. Additionally,
the keratin "matrix" of a cholesteatoma is an ideal environment for the support
of biofilm formation.
Objective To determine if microbial biofilms occur within the keratin matrix of
infected cholesteatomas.
Design We evaluated the histomorphologic characteristics of 24 human and 22
experimental cholesteatomas for evidence of biofilm formation using light
and transmission electron microscopy.
Subjects Human tissues were collected during surgical eradication of existing
cholesteatomas. Twenty-two gerbil cholesteatomas were either spontaneously
occurring or induced by external auditory canal ligation and harvested several
months later.
Results Gram-positive and gram-negative bacteria were seen within acellular
deposits among the keratin accumulations in 21 of 22 gerbil and 16 of 24 human
cholesteatomas. Regions of accumulated bacteria possessed the ultrastructural
appearance of typical amorphous polysaccharide biofilm matrix.
Conclusions There is strong anatomic evidence for the presence of bacterial biofilms
in experimental and human cholesteatomas. The existence of bacterial biofilms
within cholesteatomas may explain the clinical characteristics of infected
cholesteatomas, that is, persistence and recurrence of infection, with surgical
eradication being the only effective treatment.
INTRODUCTION
IN NATURE, bacteria most commonly exist as microbial communities known
as biofilms. These biofilms provide an environment in which the bacteria are
protected from external deleterious conditions. Bacteria can become free from
the biofilm and become motile, free-swimming organisms. Hence, bacteria exist
in 2 principal forms, as motile, replicating cells (planktonic form) or as
quiescent cells, within a hydrated matrix of polysaccharide and protein (sessile
form). Many bacteria, typically Pseudomonas species, Staphylococcus species, and Haemophilus
influenzae, have the capacity to adhere to inanimate as well as living
surfaces. Once adherent, the bacteria secrete a complex polysaccharide matrix
in which the bacteria become embedded. These microcolonies gradually enlarge
and then through a process called "quorum sensing," form large colonies of
sessile bacteria. Bacteria in these biofilms are resistant to antibiotics
by mechanisms that are different than those used by planktonic bacteria.1 The exact mechanism of antibiotic resistance of bacteria
within biofilms is unknown but probably involves a number of factors including
the direct protection afforded by the biofilm itself,2 alterations
in the local environment,3-4and
changes in bacterial phenotype.5
Mixed microbial biofilms form on many environmental surfaces and are
found throughout nature.6 Biofilms also form
on medical devices such as voice prostheses7-8 and
tympanostomy tubes9 as well as many other implant
materials.10 Once established, portions of
biofilms may detach and, under favorable conditions, become infective.11
Microbial biofilms have been shown to be important factors in a number
of human infections.12 The chronic pulmonary
colonization of Pseudomonas in cystic fibrosis is
the archetypal biofilm infection.13 Over the
last several years, evidence has accumulated suggesting that otitis media
with effusion is a biofilm disease.9, 14-15 Recently,
Post16 showed anatomical evidence for bacterial
biofilms in experimental otitis media.
One of the hallmarks of aural cholesteatoma is chronic and recurrent
infection, which is highly resistant to eradication by topical and systemic
antimicrobial agents. Once a cholesteatoma is infected, chronic otorrhea usually
occurs. The otorrhea is often suppressed by topical and systemic antibiotics,
but recurrences of infection, often with the same organism, are common. We
propose that the matrix of cholesteatomas is an ideal environment for the
development of mixed microbial biofilms, and we hypothesize that biofilms
exist within the matrix of chronically infected cholesteatomas. In the present
study we evaluated matrix samples from human cholesteatomas and spontaneously
occurring and experimentally induced gerbil cholesteatomas for evidence of
biofilm formation.
MATERIALS AND METHODS
CHOLESTEATOMA SPECIMENS
Cholesteatoma matrix was obtained from human cholesteatomas during tympanomastoid
surgery and placed in 10% buffered formalin. Specimens were obtained from
chronically infected cholesteatomas as well as noninfected cholesteatomas
behind an intact tympanic membrane. The Human Studies Review Committee of
the University of California, Davis, and Washington University in St Louis,
Mo, approved the human subjects portion of this study, and subjects provided
written consent to donate tissue to the study.
Histologic evaluation was also performed on matrix samples from spontaneously
occurring and experimentally induced cholesteatomas in gerbils. These were
specimens that were obtained and sectioned for previous studies.17-20 The
animal use protocol was approved by the Institutional Animal Care and Use
Committee (IACUC) of the University of California, Davis. All animal studies
were performed in accordance with the Public Health Service Policy on Humane
Care and Use of Laboratory Animals, the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, and the
Animal Welfare Act (7 USC  2131 et seq).
LIGHT MICROSCOPY
Human matrix samples were transferred from 10% buffered formalin to
a fixative consisting of 4% paraformaldehyde and 0.05 % glutaraldehyde in
0.1M phosphate buffer for 24 hours at 4°C. Tissue specimens were then
postfixed in 1% osmium, dehydrated in graded solutions of acetone and embedded
in Epon-Araldite. Several semithin sections (1.0 µm) were collected
at a variety of depths of the sample and counterstained with toluidine blue
and basic fuchsin. An alternate group of sections was gram stained using the
Protocol Gram Stain Set (Biochemical Sciences, Inc, Swedesboro, NJ). Gerbil
specimens had been fixed, processed, and sectioned in similar fashion. Additional
sections were taken from some of the blocks for gram staining. Sections were
examined with an upright Olympus BH-2 light microscope (Olympus Corp, Lake
Success, NY), and images were captured with the DKC-5000 digital photo system
(Sony, Tokyo, Japan).
TRANSMISSION ELECTRON MICROSCOPY
Tissue samples from gerbils and humans were fixed and embedded as described
above and thin sectioned for transmission electron microscopy. Thin sections
for transmission electron microscopy were then taken from regions containing
suspected biofilms and counterstained with uranyl acetate and lead citrate.
Sections were examined and photographed on a Hitachi H-7500 transmission electron
microscope (Hitachi, Tokyo, Japan) with digital imaging capabilities.
RESULTS
HUMAN CHOLESTEATOMA MATRIX
Of the 24 human cholesteatomas, 16 had anatomical findings consistent
with bacterial biofilms (Figure 1A
and B). We considered a dense colony of bacteria within an amorphous matrix,
in the absence of inflammatory cells, to be a microbial biofilm. Gram-positive
and gram-negative bacteria were evident in the human specimens, which could
be subjected to gram staining. Biofilms showed varied signs of degradation
of the acellular polysaccharide matrix.
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Figure 1. Light microscopic views of a bacterial
biofilm within the matrix of cholesteatomas. A and B, Human cholesteatoma;
C-F, cholesteatomas from gerbils. A, A low-power view of a human cholesteatoma
shows layers of keratin debris with a bacterial biofilm between keratin layers;
B, a higher magnification of the area indicated in A shows a bacterial biofilm,
which appears to be adherent (arrows) to keratin; C, clumps of bacteria between
layers of a gerbil cholesteatoma near an epithelial surface; D, adherent bacteria
within a cholesteatoma; E, clumps of gram-negative and gram-positive bacteria
within an amorphous matrix near keratin debris; and F, bacterial colonies
that appear to be adherent to keratin.
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GERBIL CHOLESTEATOMA MATRIX
Of 22 cholesteatoma specimens from gerbils, 21 showed evidence of biofilm
formation, using the same criteria of that for biofilms in human cholesteatomas
(Figure 1C-F). Gram-positive and
gram-negative bacteria were seen in many of the experimentally induced cholesteatomas
(Figure 2). Bacterial colonies were
consistently seen adhering to keratin debris in areas devoid of inflammatory
cells. Ultrastructural studies using transmission electron microscopy revealed
remnants of the polysaccharide biofilm matrix, which appeared as regions of
amorphous material surrounding the bacteria (Figure 3). The amorphous material surrounding bacteria is consistent
with a polysaccharide biofilms. This highly hydrated matrix contracts in aqueous
solutions during tissue processing.
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Figure 2. A high-power light micrograph
of a gram-stained specimen showing gram-positive (darkly staining) and gram-negative
(lightly staining) bacteria.
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Figure 3. A transmission electron micrograph
of a bacterial biofilm from a gerbil cholesteatoma. The low-power photomicrograph
shows a large bacterial colony near keratin debris. There are no inflammatory
cells in this region, and bacteria appear to be embedded in an amorphous,
acellular matrix (inset).
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COMMENT
Bacteria exist either as planktonic, mobile, replicating organisms or
as surface-attached, sessile colonies of bacteria within a polysaccharide
matrix known as a biofilm. Organisms within biofilms, while actively metabolizing,
do not replicate. These bacteria cannot be cultured using standard bacteriological
techniques and are highly resistant to eradication by antibiotics and disinfectants.21 In the present study, we demonstrate anatomical evidence
that biofilms form within the matrix of infected human as well as spontaneous
and experimental gerbil cholesteatomas. Bacteria were seen in microcolonies
embedded within an acellular polymeric matrix. In many cases, both gram-negative
and gram-positive bacteria were seen within the same microcolony, suggesting
that these aggregations are mixed bacterial biofilms.
BIOFILMS ON MEDICAL DEVICES
Biofilms have been shown to be present on a variety of medical devices
such as urinary catheters, central venous catheters, fracture fixation devices,
joint prostheses, tympanostomy tubes, and voice prostheses.7, 10 An
understanding of the mechanisms underlying the environmental promotion of
biofilm formation and the increased resistance to antibiotic treatment are
now essential for optimizing our standards of patient care.
BIOFILMS IN HUMAN DISEASE
Microbial biofilms have been shown to be an important factor in a number
of human diseases.22 Dental plaque, formed
on dental enamel surfaces, is a well-known biofilm disease leading to periodontitis.
In addition to its formation on nonliving surfaces, biofilms have been shown
to form on living mucosal surfaces. Pseudomonas biofilms
form within the lungs of individuals with cystic fibrosis leading to chronic
disease.23 Singh and colleagues24 showed
that Pseudomonas microcolonies consistent with biofilms
were recovered from sputum from cystic fibrosis patients. Post,16 Post
et al,25 and Rayner et al14 were
the first to demonstrate evidence for bacterial biofilms in ear disease. They
have shown indirect evidence in humans and direct evidence in an animal model
that H influenzae infections form biofilms within
the middle ear, causing chronic otitis media with effusion.
BIOFILMS AND MICROBIAL RESISTANCE
Bacteria within biofilms have been found to be highly resistant to common
disinfectants. Takeo and colleagues21 found
that 0.1% chlorhexidine and 0.5% alkyldiaminoethyl glycine would not eradicate Pseudomonas aeruginosa after 1 hour of exposure and that
eradication of this organism within a biofilm requires higher concentrations
and longer exposures. Poor biofilm-killing performance of common disinfectants
is likely due to bacterial resistance rather than poor or ineffective penetration
of the antimicrobial agent.1
Bacterial biofilms protect bacteria by physically shielding them from
UV-C, UV-B and UV-A radiation. Ultraviolet light appears to be absorbed by
the alginate matrix of a biofilm.26 Bacteria
within biofilms also show increased resistance to antibiotics. For example,
bacteria in aqueous solution were shown to have a minimum inhibitory concentration
of 2 g/mL of ampicillin, while the same bacteria grown as a biofilm were only
marginally inhibited by 4 hours of exposure to 5000 g/mL. The following 3
possible mechanisms of this bacterial resistance have been suggested: (1)
slow penetration of the biofilm matrix; (2) development of a resistant phenotype
within the matrix; and (3) altered microenvironment.1 There
is evidence for all 3 mechanisms, but each may not be operant in all biofilms.
For example, ampicillin can penetrate biofilms formed by Klebsiella pneumoniae but not others.27
The existence of biofilms within cholesteatomas may explain the clinical
nature of this disease. Like other biofilm diseases, infections within cholesteatomas
are resistant to eradication by antibiotics. Antibiotics may temporarily control
active infection by the planktonic bacteria within a cholesteatoma, but the
bacteria within biofilms persist only to reassume their planktonic state when
conditions are suitable, hence the recurrent and recalcitrant nature of these
infections. In fact, the cholesteatoma may prove to be a uniquely resistant
example in biofilm biology if the cholesteatoma matrix provides an additional
layer of protection for the bacteria. In addition, bacteria within biofilms
are actively metabolizing and producing endotoxin as well as other factors,
which may perpetuate an inflammatory host response even in the absence of
culturable (freely mobile planktonic) bacteria (Figure 4).
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Figure 4. This illustration depicts a number
of signaling events that could occur from biofilm formation within cholesteatomas.
Bacterial biofilms within the keratin matrix of cholesteatomas may produce
endotoxin and other products that lead to elaboration of inflammatory cytokines
within the subepithelium. In addition, bacterial interaction (adhesion) to
keratinocytes may induce the elaboration of proinflammatory cytokines into
the surrounding extracellular space. Cytokines such as tumor necrosis factor
and interleukin 1 and interleukin 6 may in turn lead to the recruitment and
activation of bone remodeling cells.
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BIOFILMS AND CELLULAR SIGNALING
The presence of sessile bacteria with biofilm communities in cholesteatomas
may mediate the host responses seen in this disease, including chronic inflammation,
epithelial proliferation, and bone resorption. For example, bacteria within
biofilm communities can produce endotoxin, leading to inflammatory host responses.
Dingman and colleagues15 showed that bacteria
within the middle ear, detected by polymerase chain reaction but undetectable
by culture, produced endotoxin in middle ear effusions.
In addition to the effects of bacterial endotoxin, biofilms may have
direct effects on epithelial cell signaling. The initial process of biofilm
formation is bacterial adherence to a surface; in human disease, bacteria
adhere to epithelial surfaces. For example, Escherichia
coli express type I fimbriae that contains FimH, an adhesion molecule
that binds to epithelium.28 Adherence of bacteria
to epithelial surfaces can induce cellular signaling, affecting the host.
In a gene expression study of adherent E coli, Mysorekar
and colleagues29 found altered regulation of
a wide variety of host genes. They found that adherence of uropathic E coli to bladder epithelium leads to regulation of signals,
which can result in epithelial differentiation (down-regulation of bone morphogenetic
protein 4) and proliferation (induction of epidermal growth factor family
members).29 In addition, they found that interleukin
6, a proinflammatory cytokine, was also up-regulated. Hence, sessile bacteria
within biofilms in the cholesteatoma matrix may mediate host responses by
direct elaboration of bacterial products, such as endotoxin, or induce host
cellular signaling by adherence to epithelial surfaces.
CONCLUSIONS
Aural cholesteatomas vary in progression and aggressiveness; the presence
of bacterial biofilms in some cholesteatomas may explain their activity. Infections
within cholesteatomas often defy eradication by topical and systemic antibiotics.
It is likely that the presence of bacterial biofilms within the cholesteatoma
matrix explain persistent infection within cholesteatomas. Antimicrobial agents
fail to eradicate the sessile bacteria within biofilms; when conditions are
favorable, the sessile bacteria within these biofilms become motile and planktonic,
leading to active infection. Direct signaling of bacterial products, such
as endotoxin, and indirect signaling by bacterial adherence may lead to the
chronic inflammation and epithelial proliferation, which is characteristic
of this disease. Aside from physical removal of the cholesteatoma and its
biofilm laden matrix, no effective measures are available to eradicate these
microbial biofilms.
AUTHOR INFORMATION
Accepted for publication March 21, 2002.
This study was supported by grant DC00263-12 and P30 DC04665 from the
National Institute on Deafness and Other Communication Disorders, Bethesda,
Md.
We thank Steve Tinling, MA, of the University of California, Davis,
and Ruth Hughes, MA, in the Department of Otolaryngology, Washington University
in St Louis, for assistance with the preparation of some of the materials
used in this study.
Corresponding author: Richard A. Chole, MD, PhD, Department of Otolaryngology,
Campus Box 8115, Washington University School of Medicine, 660 S Euclid Ave,
St Louis, MO 63110 (e-mail: choler{at}msnotes.wustl.edu).
From the Departments of Otolaryngology–Head & Neck Surgery
(Drs Chole and Faddis) and Molecular Pharmacology (Dr Chole), Washington University
in St Louis, St Louis, Mo.
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