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Short Scientific Communication—Otology and Neurotology
Biofilm’s Role in ChronicCholesteatomatous Otitis Media:A Pilot Study
Otolaryngology–Head and Neck Surgery2016, Vol. 154(5) 914–916� American Academy ofOtolaryngology—Head and NeckSurgery Foundation 2016Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/0194599816630548http://otojournal.org
Jacopo Galli, MD, PhD1, Lea Calo, MD, PhD1, Monica Giuliani, MD1,Bruno Sergi, MD, PhD1, Daniela Lucidi, MD1,Duino Meucci, MD1, Ezio Bassotti3,Maurizio Sanguinetti, MD, PhD2, andGaetano Paludetti, MD, PhD1
No sponsorships or competing interests have been disclosed for this article.
Abstract
Cholesteatoma is a destructive lesion involving the temporalbone, which may induce severe complications due to itsexpansion and erosion of adjacent structures. Bacterial bio-film plays a crucial role in the pathogenesis of many otolar-yngologic inflammatory/infectious chronic diseases. In thispilot study, we investigated, by means of cultural examina-tion and with scanning electron microscope, the presence ofbacterial biofilm in a series of samples from the epitympanicand mastoid region in patients affected by cholesteatomaand from the promontory region in patients with healthymucosa who were undergoing to stapes surgery. The pre-liminary data support the association between biofilm andcholesteatoma (81.3% of the cases) and allow us tohypothesize that keratinized matrix of cholesteatoma mayrepresent the ideal substrate for biofilm colonization andsurvival; this finding is consistent with the clinical course ofaural cholesteatoma, characterized by recurrent exacerba-tions and recalcitrant course.
Keywords
cholesteatoma, biofilm, chronic otitis media, pseudomonasaeruginosa.
Received October 7, 2015; revised December 14, 2015; accepted
January 13, 2016.
The first report of bacterial biofilm in cholesteatomas,
by Chole and Faddis1 in 2002, described the massive
colonization by bacterial biofilm within human and
experimental matrix of infected cholesteatoma. Gram-
positive, gram-negative, and fungal pathogens have been
isolated from cholesteatoma tissue; in particular, numer-
ous biofilm-forming Pseudomonas aeruginosa colonies
have been isolated.2-4
The objective of this study was to evaluate the presence
of bacterial biofilm by means of scanning electron micro-
scopy (SEM) in our series of patients undergoing surgery
for cholesteatomatous otitis media and to identify changes
in middle ear epithelium favoring microbial aggregation as
compared with normal middle ear mucosa samples.
Patients and Methods
We examined cholesteatoma samples from the epitympanic
and mastoid region, obtained from 15 patients (mean age,
46 years) admitted to our otorhinolaryngology unit under-
going to ear surgery (12 canal wall down and 3 canal wall
up tympanoplasty; cholesteatoma group) and 10 specimens
of healthy middle ear mucosa (promontory region) of 10
patients undergoing to stapes surgery (control group). This
study was approved by the Ethics Committee of the
Medical Faculty of the Catholic University of the Sacred
Heart in Rome; all patients provided informed consent
before participation.
All patients showed recurrent otorrhea associated with
hearing loss for .6 months, resistant to repeated systemic
antibiotic therapy. Computed tomography scan of the tem-
poral bone demonstrated inflammatory tissue in the middle
ear, with partial erosion of temporal bone. Each specimen
was divided in 2 fragments. One fragment was submitted to
histologic examination by means of optical microscope, and
1Department of Head and Neck Surgery, Institute of Otorhinolaryngology,
Catholic University of the Sacred Heart School of Medicine and Surgery,
Rome, Italy2Department of Diagnostic and Laboratory Medicine, Institute of
Microbiology, Catholic University of the Sacred Heart School of Medicine
and Surgery, Rome, Italy3Institute of Odontostomatology, Catholic University of the Sacred Heart
School of Medicine and Surgery, Rome, Italy
Corresponding Author:
Lea Calo, Catholic University School of Medicine and Surgery, Department
Head and Neck Surgery, Institute of Otorhinolaryngology, A. Gemelli
Hospital, Largo A. Gemelli n. 1, 00168, Rome, Italy.
Email: [email protected]
at SOCIEDADE BRASILEIRA DE CIRUR on May 6, 2016oto.sagepub.comDownloaded from
the other fragment was fixed in Karnosvky buffer, treated,
and coated with colloidal gold for SEM examination.
Results
All samples from the cholesteatoma group and the control
group were negative at bacterial culture. At SEM examination,
no evidence of bacterial biofilm was found in samples from
the control group, whereas the presence of bacterial biofilms
was diagnosed in 14 of 15 samples from the cholesteatoma
group (81.3%). Bacterial colonies appeared as densely packed
microbial cells with rod-shaped and/or spherical profiles and a
variety of capsular staining patterns. Close inspection showed
that the cells were embedded in a homogeneous amorphous
background substance, which was well preserved in the
solvent-processed tissues (Figure 1). The epithelium observed
at SEM examination showed abundant anucleate keratin
squames, gradually transformed into ciliated pseudo-stratified
columnar epithelium, with no evidence of the simple squa-
mous epithelium usually lining the middle ear. In specimens in
which biofilm was detected, we found evident destruction of
the ciliated epithelium, minimum residual of intact cilia, and
goblet cells partially recognizable and disarrayed (Figure 2).
It was also observed that the biofilm was adhered to the
superficial layer of the squamous debris. Only in 1 case was
bacterial biofilm absent (Figure 3) and isolated spherical
microorganisms, consistent with planktonic bacteria, were
detected. Mucociliary structures appeared, in this unique
sample, less damaged, and a higher number of cilia and a
homogeneous distribution of goblet cells were assessed.
No statistical analysis was performed because no speci-
men showed positive bacterial culture. Moreover, in the
control group, biofilm was not detected; these data do not
allow any statistical comparison.
Figure 2. Cholesteatoma sample colonized by biofilm, with abundant anucleate keratin squames, with no evidence of the simple squamousepithelium usually lining the middle ear (scanning electron microscopy).
Figure 1. Evidence of bacterial biofilm adhered to the superficial layer of the squamous and densely packed microbial cells with rod-shapedand/or spherical profiles (scanning electron microscopy).
Figure 3. Single case with no bacterial biofilm (scanning electronmicroscopy).
Galli et al 915
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Discussion
Bacterial biofilm plays a crucial role in the pathogenesis of an
increasing number of otolaryngologic diseases.4-7 The role of bac-
terial biofilms in chronic otitis media with effusion has been
described by many studies.8,9 Acquired cholesteatoma frequently
becomes chronically infected, especially with P aeruginosa.10-12
Biofilm colonization of the middle ear seems to be responsible
for resistance to topical and systemic antimicrobial agents; chroni-
cally infected cholesteatomas are described as highly relapsing,
rapidly progressive, and more subject to multiple surgical treat-
ments.1,13-15 Increased bacterial retaining and biofilm formation
are histologically found in association to massive entrapment of
keratin and keratinocyte proliferation resulting in an expanding
matrix with osteoclasts recruitment and bone erosion.1
In our study, the high rate of bacterial biofilm (81.3%)
confirms the association between bacterial biofilm evidence
and cholesteatoma, even though the causal relationship
remains unclear. Few authors have analyzed the relationship
between disorder/absence of functioning mucociliary clear-
ance and biofilm detection in the sinonasal region,5,16 but
the behavior of mucociliary clearance in cholesteatomatous
tissue colonized by biofilm is controversial. Our data sup-
port the association among ciliary damage/loss, goblet cell
disarray, and biofilm detection.
Cultural results in all our samples were negative; we can
assume that this result was determined by pre- and intrao-
perative antibiotic prophylaxis, administered to all our
enrolled patients, which could invalidate cultural examina-
tion. These assumptions are also in agreement with previous
reports17 in which the presence of bacteria was positively
assessed by means of SEM and polymerase chain reaction
in children affected by chronic otitis media, showing nega-
tive results at bacterial culture. Our purpose is to prove, in a
future research, the direct pathogenic role in cholesteatoma
of virulent species such as P aeruginosa (wild type)4 by
means of immunohistochemistry and polymerase chain reac-
tion and its biofilm-forming capacity as a correlate to the
clinically aggressive pattern.
Our current research demonstrates the strong association
between biofilm and cholesteatoma (81.3% of the cases).
SEM examination, moreover, allowed us to demonstrate
that biofilm massively colonizes the keratinized matrix.
In our opinion, the keratinized matrix of cholesteatoma
and the destruction of the ciliated epithelium of the respira-
tory tract may represent an ideal substrate for biofilm colo-
nization and survival. The role of biofilm in maintaining
active chronic inflammation in the interface between matrix
and bone, its role in the active bone resorption and enlarge-
ment of cholesteatoma, and the characteristics of mucosa at
sites away from the matrix need to be clarified in the future.
Author Contributions
Jacopo Galli, designed study, revised article; Lea Calo, designed
study, revised article; Monica Giuliani, collected data, wrote article;
Bruno Sergi, analyzed data, wrote article; Daniela Lucidi, collected
data, wrote article; Duino Meucci, collected data, wrote article; Ezio
Bassotti, collected data, wrote article; Maurizio Sanguinetti, designed
study, revised article; Gaetano Paludetti, designed study, revised article
Disclosures
Competing interests: None.
Sponsorships: None.
Funding source: None.
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916 Otolaryngology–Head and Neck Surgery 154(5)
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