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: lea.calo@rm.unicatt.it

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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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