1

Introduction

The importance of biofilms in human infectious disease is becoming increasingly apparent. Over the past 20 years, a new appreciation has developed regarding how bacteria behave differently once bound to a surface. Surface-bound bacteria grow into biofilms, which are colonies of slow-growing bacteria that surround themselves in a coat of glycopolysaccharides called a glycocalyx . Bacteria in biofilms have been shown to be highly resistant to antibiotics and are nearly impossible to detect with standard culture techniques .

There is mounting evidence of the role of biofilms in otolaryngologic infections . Tissue samples from patients with chronic rhinosinusitis have been shown to have biofilm formation . Biofims have also been demonstrated to play a major role in otitis media with effusion and have been identified in direct biopsy specimens of the middle ear . Many of the bacteria common to both acute and chronic otitis media are known to exist in a biofilm state under favorable conditions. Biofilms have also been demonstrated in a nonhuman primate model of chronic otitis media . There is, however, only 1 previous clinical study demonstrating direct clinical evidence of biofilms in chronically infected ears. In this study, Chole et al identified biofilms in both an experimental animal model of cholesteatoma and clinical biopsies from patients with cholesteatoma. Our study looks at a collection of tissue samples taken at the time of otologic surgery in an effort to confirm and expand our understanding of the role of biofilms in these diseases.

No standard definition of biofilms exists, but several attributes prevail that are found in most descriptions of biofilms: surface binding, presence of glycocalyx, 3-dimensional structure, increased antimicrobial resistance, slow-growing low metabolic state making cultures difficult, and up-regulation of unique gene products not expressed in the planktonic form. Most biofilm research uses specialized microscopy to visualize the presence of bacteria. Various techniques have been used including scanning electron microscopy (SEM) , transmission electron microscopy , scanning laser confocal microscopy , and 3-dimensional magnetic resonance imaging . SEM has been previously used to show the presence of biofilms medical implants, such as tympanostomy tubes . SEM was used in this study because it gives a clear visualization of bacteria within a biofilm and is capable of demonstrating even a single bacterium and the relation of the biofilm to the underlying surface.

2

Experimental design and methods

Patients undergoing surgical treatment of chronic otitis media (with or without cholesteatoma) were asked to participate in the study. The study was approved by the Institutional Review Board of Oklahoma University Health Science Center, and informed consent was obtained. Specimens of middle ear tissue were collected during routine surgical treatment. A variety of locations were sampled including the middle ear, mastoid, ossicles, and alloplastic materials. Tissue was taken only if the surgical treatment called for debridement of the tissue. An incus removed during an ossicular chain reconstruction was used as noninfected control tissue in the absence of clinical evidence of inflammation. In addition, a previously placed ossicular replacement prosthesis without clinical signs of infection was removed and included in the study. Surgical irrigation was limited as much as possible, and an atraumatic technique was used. The harvested tissue was placed immediately in 2.5% gluteraldehyde in cacodylic acid (0.1 mol/L) titrated to a pH of 7.2.

The specimens were then dehydrated in serial solutions of ethanol (60–100%) for 15 minutes each, with a second soak in 100% ethanol. The specimens were then attached to a copper specimen container with carbon tape. Liquid carbon paint was applied to the edges of the sample to the copper container to facilitate conductivity. Each specimen was subjected to carbon dioxide critical point drying then sputter coated with 60/40 gold-palladium then visually inspected with a JEOL JSM-880 high-resolution scanning electron microscope (Peabody, MA). Several areas of each sample were systematically scanned. A sample was considered to have a biofilm if 3 criteria were met: (1) presence of bacterial-sized and -shaped objects; (2) presence of an amorphous material, consistent with glycocalyx around the bacteria; and (3) surface binding. If no evidence of biofilms was found within 4 hours of scanning time, the specimen was considered negative. Digital images were captured to TIF files, and the magnification was recorded.

2

Experimental design and methods

Patients undergoing surgical treatment of chronic otitis media (with or without cholesteatoma) were asked to participate in the study. The study was approved by the Institutional Review Board of Oklahoma University Health Science Center, and informed consent was obtained. Specimens of middle ear tissue were collected during routine surgical treatment. A variety of locations were sampled including the middle ear, mastoid, ossicles, and alloplastic materials. Tissue was taken only if the surgical treatment called for debridement of the tissue. An incus removed during an ossicular chain reconstruction was used as noninfected control tissue in the absence of clinical evidence of inflammation. In addition, a previously placed ossicular replacement prosthesis without clinical signs of infection was removed and included in the study. Surgical irrigation was limited as much as possible, and an atraumatic technique was used. The harvested tissue was placed immediately in 2.5% gluteraldehyde in cacodylic acid (0.1 mol/L) titrated to a pH of 7.2.

The specimens were then dehydrated in serial solutions of ethanol (60–100%) for 15 minutes each, with a second soak in 100% ethanol. The specimens were then attached to a copper specimen container with carbon tape. Liquid carbon paint was applied to the edges of the sample to the copper container to facilitate conductivity. Each specimen was subjected to carbon dioxide critical point drying then sputter coated with 60/40 gold-palladium then visually inspected with a JEOL JSM-880 high-resolution scanning electron microscope (Peabody, MA). Several areas of each sample were systematically scanned. A sample was considered to have a biofilm if 3 criteria were met: (1) presence of bacterial-sized and -shaped objects; (2) presence of an amorphous material, consistent with glycocalyx around the bacteria; and (3) surface binding. If no evidence of biofilms was found within 4 hours of scanning time, the specimen was considered negative. Digital images were captured to TIF files, and the magnification was recorded.

3

Results

A total of 31 specimens were collected from 16 patients, including 2 patients without evidence of infection for control tissues. One of these samples was a hydroxyapatite prosthesis that was removed for correction of a conductive hearing loss with no evidence of infection, and the other was an incus removed in the process of posttraumatic ossicular chain reconstruction. Despite the absence of clinical infection, it was felt that a biofilm might be present on the alloplastic material. All other patients had a clinical history of a chronic infection. Various tissue types were harvested including edematous mucosa, polypoid tissue, granulation tissue, cholesteatoma matrix and capsule, ossicles, and ossicular prostheses. The clinical history, specimen descriptions, and SEM results are presented in Table 1 . The diagnoses of these patients included chronic suppurative otitis media (7 cases), cholesteatoma (5 cases), chronic serous otitis media (1 case), and an infected alloplastic cement at craniotomy site (1 case).

Table 1

Tissue characteristics and SEM findings

Pt	Diagnosis/condition	Description of specimen	SEM findings
1	Infected cholesteatoma	1. Mucosa	1. Cocci, glycocalyx, and surface binding ( Fig. 5 )
2	Trauma	1. Incus (control tissue)	1. No evidence of biofilm/bacteria
3	Chronic mucoid otorrhea with TM perforation	1. Mucosa	1. No evidence of biofilm/bacteria
4	Chronic suppurative otitis media with TM perforation	1. Mucosa	1. No evidence of biofilm/bacteria
5	Attic cholesteatoma	1. Long arm of incus 2. Body of incus 3. Mucosa and cholesteatoma 4. Mucosa and cholesteatoma	1. No evidence of biofilm/bacteria 2. No evidence of biofilm/bacteria 3. Single bacterium 4. No evidence of biofilm/bacteria
6	Infected cholesteatoma	1. Edematous tissue next to cholesteatoma capsule 2. Cholesteatoma capsule and matrix 3. Cholesteatoma capsule and matrix	1. No evidence of biofilm/bacteria 2. Single bacterium 3. No evidence of biofilm/bacteria
7	Infected craniotomy site after translabyrinthine removal of acoustic neuroma	1. Hydroxyapatite cement 2. Hydroxyapatite cement	1. No evidence of biofilm/bacteria 2. No evidence of biofilm/bacteria
8	Infected cholesteatoma	1. Incus 2. Granulation tissue 3. Anterior remnant of TM with pocket of cholesteatoma 4. Matrix 5. Cholesteatoma capsule	1. No evidence of biofilm/bacteria 2. Cluster of cocci, glycocalyx, and surface binding ( Fig. 3 ) 3. No evidence of biofilm/bacteria 4. No evidence of biofilm/bacteria 5. No evidence of biofilm/bacteria
9	Recurrent cholesteatoma in chronically infected mastoid cavity	1. Squamous debris 2. Edematous mastoid mucosa with clear mucous	1. Bacterial bacilli, glycocalyx, surface binding on squamous debris ( Fig. 2 ) 2. No evidence of biofilm/bacteria
10	Chronic mucoid effusion with polypoid mucosa found on myringotomy	1. Polypoid mucosa 2. Polypoid mucosa 3. Edematous mucosa	1. Single bacterium 2. No evidence of biofilm/bacteria 3. No evidence of biofilm/bacteria
11	Revision tympanoplasty with ossicular chain reconstruction, previous prosthesis removed (for control), TM atelectasis	1. Hapex prosthesis covered with normal appearing mucosa	1. No evidence of biofilm/bacteria
12	Chronic suppurative otitis media	1. Incus	1. Single bacterium
13	Chronic suppurative otitis media	1. Granulation tissue with associated mucosa 2. Mucosa	1. No evidence of biofilm/bacteria 2. No evidence of biofilm/bacteria
14	Chronic suppurative otitis media	1. Mucosa	1. No evidence of biofilm/bacteria
15	Chronic suppurative otitis media, TM perforation with aural polyp	1. Mucosa 2. Mucosa	1. No evidence of biofilm/bacteria 2. No evidence of biofilm/bacteria
16	Chronic suppurative otitis media	1. Granulation tissue	1. Two bacilli, glycocalyx, surface binding

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