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Biofilms in chronic suppurative otitis media and cholesteatoma: scanning electron microscopy findings
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American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32 – 37 www.elsevier.com/locate/amjoto
Biofilms in chronic suppurative otitis media and cholesteatoma: scanning electron microscopy findings☆,☆☆,★ James Saunders, MDa,⁎, Michael Murray, MDb , Anthony Alleman, MDc a
Section of Otolaryngology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA b Camino Ear, Nose and Throat Clinic, San Jose, CA, USA c Department of Radiology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA Received 10 September 2009
Abstract
Background: Biofilms play a role in the pathogenesis of a variety of otorhinolaryngologic diseases, including otitis media and cholesteatoma. Despite this, relatively few studies have undertaken to demonstrate the presence of biofilms tissues from patients with chronic otitis media or infected cholesteatoma. Objective/hypothesis: Our objective is to detect evidence of biofilms human chronic ear infections with scanning electron microscopy (SEM). We hypothesized that bacterial biofilms are present in patients with chronic otitis media. Study design: We performed prospective collection of tissue collected during middle ear surgery from 16 patients undergoing middle ear or mastoid surgery with chronic ear infections. Methods: A total of 31 middle and mastoid tissue samples were harvested at the time of surgery and processed with critical point drying for SEM analysis. Samples were then searched for evidence of biofilms. Results: Bacterial-shaped objects were identified that displayed both surface binding and the presence of a glycocalyx in 4 patients, findings consistent with bacterial biofilms. Most of these (3 of 4) were in patients with infected cholesteatoma, and biofims were identified in 60% of cholesteatoma cases (3 of 5). On the other hand, only 1 of 7 cases with chronic suppurative otitis media had evidence of biofilms. Conclusion: SEM supports the hypothesis that bacterial biofilms are common in chronic infections associated with cholesteatoma and are present in some cases of chronic suppurative otitis media without cholesteatoma. © 2011 Elsevier Inc. All rights reserved.
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
☆
Presented at the Middle Section of the Triological Society 2005. Funded by a Resident Research Grant from the Triological society. ★ The authors have no conflicting interests in the publication of this paper, and funding was internal within the Otolaryngology Department of The University of Oklahoma Health Science Center. ⁎ Corresponding author. Section of Otolaryngology, DartmouthHitchcock Medical Center, Lebanon, NH, USA. Tel.: +1 603 650 8124; fax: +1 603 650 0052. E-mail address: [email protected] (J. Saunders). ☆☆
0196-0709/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2009.09.010
behave differently once bound to a surface. Surface-bound bacteria grow into biofilms, which are colonies of slowgrowing bacteria that surround themselves in a coat of glycopolysaccharides called a glycocalyx [1-3]. Bacteria in biofilms have been shown to be highly resistant to antibiotics and are nearly impossible to detect with standard culture techniques [4]. There is mounting evidence of the role of biofilms in otolaryngologic infections [5,6]. Tissue samples from patients with chronic rhinosinusitis have been shown to have biofilm formation [7,8]. 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 [9-12]. Many of the bacteria common to both acute and chronic otitis media are known to exist in a
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
biofilm state under favorable conditions. Biofilms have also been demonstrated in a nonhuman primate model of chronic otitis media [13]. There is, however, only 1 previous clinical study demonstrating direct clinical evidence of biofilms in chronically infected ears. In this study, Chole et al [14] 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, 3dimensional 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
33
microscopy to visualize the presence of bacteria. Various techniques have been used including scanning electron microscopy (SEM) [14,15], transmission electron microscopy [14], scanning laser confocal microscopy [16,17], and 3dimensional magnetic resonance imaging [18]. SEM has been previously used to show the presence of biofilms medical implants, such as tympanostomy tubes [19]. 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
Table 1 Tissue characteristics and SEM findings Pt
Diagnosis/condition
Description of specimen
SEM findings
1 2 3
1. Mucosa 1. Incus (control tissue) 1. Mucosa
1. Cocci, glycocalyx, and surface binding (Fig. 5) 1. No evidence of biofilm/bacteria 1. No evidence of biofilm/bacteria
1. Mucosa
1. No evidence of biofilm/bacteria
5
Infected cholesteatoma Trauma Chronic mucoid otorrhea with TM perforation Chronic suppurative otitis media with TM perforation Attic cholesteatoma
6
Infected cholesteatoma
7
Infected craniotomy site after translabyrinthine removal of acoustic neuroma Infected cholesteatoma
1. Long arm of incus 2. Body of incus 3. Mucosa and cholesteatoma 4. Mucosa and cholesteatoma 1. Edematous tissue next to cholesteatoma capsule 2. Cholesteatoma capsule and matrix 3. Cholesteatoma capsule and matrix 1. Hydroxyapatite cement 2. Hydroxyapatite cement
1. 2. 3. 4. 1. 2. 3. 1. 2.
1. Incus 2. Granulation tissue 3. Anterior remnant of TM with pocket of cholesteatoma 4. Matrix 5. Cholesteatoma capsule 1. Squamous debris 2. Edematous mastoid mucosa with clear mucous
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 1. Bacterial bacilli, glycocalyx, surface binding on squamous debris (Fig. 2) 2. No evidence of biofilm/bacteria 1. Single bacterium 2. No evidence of biofilm/bacteria 3. No evidence of biofilm/bacteria 1. No evidence of biofilm/bacteria
4
8
9
Recurrent cholesteatoma in chronically infected mastoid cavity
10
Chronic mucoid effusion with polypoid mucosa found on myringotomy Revision tympanoplasty with ossicular chain reconstruction, previous prosthesis removed (for control), TM atelectasis Chronic suppurative otitis media Chronic suppurative otitis media
11
12 13 14 15 16
Chronic suppurative otitis media Chronic suppurative otitis media, TM perforation with aural polyp Chronic suppurative otitis media
TM indicates tympanic membrane.
1. 2. 3. 1.
Polypoid mucosa Polypoid mucosa Edematous mucosa Hapex prosthesis covered with normal appearing mucosa
1. 1. 2. 1. 1. 2. 1.
Incus Granulation tissue with associated mucosa Mucosa Mucosa Mucosa Mucosa Granulation tissue
1. 1. 2. 1. 1. 2. 1.
No evidence of biofilm/bacteria No evidence of biofilm/bacteria Single bacterium No evidence of biofilm/bacteria No evidence of biofilm/bacteria Single bacterium No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria
Single bacterium No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria Two bacilli, glycocalyx, surface binding
34
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
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
Fig. 1. This image shows the surface of a incus removed during an ossicular chain reconstruction. The specimen was used as a control. Note the relatively smooth surface and lack of organisms (magnification = 5000×).
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
Fig. 2. (A) A colony of cocci bacteria can be seen attached to the inflamed mucosal surface adjacent an infected cholesteatoma from patient 1. The image shows a central area where the bacteria are uncovered and a peripheral area where the cocci are covered by an amorphous substance, consistent with glycocalyx (magnification = 1000×). (B) Higher magnification of the lower left corner of image A (magnification = 3000×, bar length = 5.0 μm).
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
35
whereas the remainder was found on adjacent mucosa or granulation tissue. Culture results were not available on many of these patients; but 2 of the biofilm cases were found to have rod-shaped bacteria consistent with Pseudomonas sp., and 2 cases were noted to have associated bacterial cocci. Bacteria not bound to a surface or associated with an amorphous substance were noted in 4 patients. These images were consistent with planktonic bacteria.
4. Discussion
Fig. 3. High-magnification image of granulation tissue from patient 8 shows bacterial-sized objects bound to the underlying surface and immersed in an amorphous substance, consistent with glycocalyx (magnification = 10 000×).
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). The incus control tissue (Fig. 1) and noninfected hydroxyapatite prosthesis showed no evidence of bacteria or biofilm formation. Likewise, there was no biofilm identified on the infected alloplastic cranioplasty cement. Four patients had at least 1 specimen with bacterial-sized and -shaped object visualized with a surrounding amorphous substance and surface binding consistent with a biofilm (Figs. 2-5). Three of the 4 specimens identified with biofilms were in patients with infected cholesteatomas. Cholesteatoma was by far the most common diagnosis associated with a biofilm (3 of 5, 60%), and only 1 of the 7 patients (14%) with chronic suppurative otitis media demonstrated findings of a biofilm (Fig. 6). In only one of the cholesteatoma cases was the biofilm noted to be on squamous debris (Fig. 5),
The role of biofilm in chronic otitis media with effusion has been well documented and studied. In contrast, to date, there is only 1 prior clinical study of biofilm in cholesteatoma [14]. Chole et al [14] described the light and transmission electron microscopy findings in 24 patients with cholesteatoma and 22 experimental cholesteatomas in gerbils. Their criteria for the diagnosis of biofilm were similar to those used in our study, and they identified findings of biofilm in 16 of 24 clinical cases (66%). Interestingly, this is nearly identical to the 60% rate of biofilm in cholesteatomas in our smaller study. Like the previous study, we included both infected and noninfected cholesteatomas; and 1 of the 2 cholesteatomas that did not have evidence of biofilm was an attic cholesteatoma with no obvious sign of infection (case 5). On the other hand, no evidence of biofilm was seen in case 6 despite the fact that this patient had an active infection with P aeruginosa, a known biofilm former [20]. It is of course possible that
Fig. 4. This image shows a biofilm on chronically infected mucosa. The contrast between the smooth surface and the biofilm collection in the central portion of the image (magnification = 1000×).
36
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
Fig. 6. High magnification of rod-shaped bacteria bound to the surface of granulation tissue from a patient with chronic suppurative otitis media (patient 16). Note the background of collagen fibers and the presence of glycocalyx between the 2 visible bacteria and the surface (magnification = 10 000×).
biofims were present in these samples, but undetected. These observations, however, would support those of Chole et al [14] that biofilms are a common but not ubiquitous feature of cholesteatoma. We find it interesting that a relatively low yield was found in specimens from chronic suppurative otitis media without cholesteatoma. Only 1 in 7 (14%) of such cases had evidence of biofilm. The only other study that documented biofilm in chronic suppurative otitis media was an experimental study in a nonhuman primate [13]. In this experimental study, the details of the infected state were not specified. Still, the clinical condition of chronic suppurative otitis media seems to fit that expected for a biofilm disease. As above, it is possible that biofilms were present in these specimens, but remained undetected. One reason may be due to the relative difficulty in surveying the entire specimen with SEM techniques. The labor-intensive process of searching for biofilms with SEM may, therefore, lead to less sensitivity compared with other methods. In addition, there may be structural features that make the disruption of biofilm more
Fig. 5. (A) This specimen was taken from an infected cholesteatoma (patient 9). Sheets of squamous debris with several rod-shaped bacteria associated with the string of amorphous material (magnification = 3000×, bar = 8 μm). (B) Higher magnification of the upper left corner of image A. The center of the image clearly shows rods encased in glycocalyx (magnification = 10 000×, bar length = 1 μm). (C) The upper left-hand portion of the previous image is shown in greater detail. Numerous rod-shaped objects are noticed throughout the image.
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
likely in the surgical treatment of suppurative otitis media than in cholesteatoma. It is certainly possible that biofilms are relatively uncommon in chronic suppurative otitis media; however, this seems unlikely given the preponderance of evidence in chronic otitis media with effusion and cholesteatoma. Further clinical studies will be required to clarify whether biofilms are indeed prevalent in chronic suppurative otitis media. The visual identification of biofilms represents a surrogate end point in proving the existence of biofilms in chronic ear infections. We adhered, therefore, to fairly strict criteria to consider the specimen positive. Several hallmark features of biofilms were identified including surface binding, 3-dimensional aggregates of bacteria, and the presence of glycocalyx. This evidence suggests that biofilms were present in these patients with cholesteatoma and chronic suppurative otitis media. However, one cannot assume from the visual images that these bacteria were the source of the infections or that these bacteria possessed other characteristics of biofilms such as antibiotic resistance. Further study is needed to better clarify the relationships between these apparent biofilms and clinical behavior. Although researchers in the field generally agree on the morphologic features that constitute a biofilm, the identification of biofilms with imaging techniques remains somewhat subjective, time consuming, and of limited direct clinical value. Laser confocal microscopy has the advantage of labeling the biofilm, allowing for a much more rapid identification of suspicious areas of the specimen; but this method still requires tissue acquisition. Perhaps the most promising method of detecting biofilms would be the identification of the unique genomic changes associated with surface binding or quorum sensing [21]. Ultimately, the utilization of this technology may resolve the inherent difficulties of the imaging methods.
5. Conclusion Our research supports the hypothesis that biofilms are involved in chronic otitis media and cholesteatoma and, to a lesser degree, in chronic suppurative otitis media. The recognition of the role of biofilms in these disease states is critical to our understanding of the disease and to developing more effective treatment strategies. Future research should continue to develop more sophisticated and clinically applicable ways to detect biofilms, as well as ways to improve treatment.
37
References [1] Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999;284:1318-22. [2] Costerton JW, Geesey GG, Cheng KJ. How bacteria stick. Sci Am 1978;238:86-95. [3] Whittaker CJ, Klier CM, Kolenbrander PE. Mechanisms of adhesion by oral bacteria. Annu Rev Microbiol 1996;50:513-52. [4] Costerton JW, Lewandowski Z, Caldwell DE, et al. Microbial biofilms. Annu Rev Microbiol 1995;49:711-45. [5] Post JC, Hiller NL, Nistico L, et al. The role of biofilms in otolaryngologic infections: update 2007. Curr Opin Otolaryngol Head Neck Surg 2007;15:347-51. [6] Vlastarakos PV, Nikolopoulos TP, Maragoudakis P, et al. Biofilms in ear, nose and throat infections: how important are they? Laryngoscope 2007;117:668-73. [7] Sanderson AR, Leid JG, Hunsaker D. Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis. Laryngoscope 2006;116:1121-6. [8] Ramadan HH. Chronic rhinosinusitis and bacterial biofilms. Curr Opin Otolaryngol Head Neck Surg 2006;14:183-6. [9] Post JC. Direct evidence of bacterial biofilms in otitis media. Laryngoscope 2001;111:2083-94. [10] Ehrlich GD, Veeh R, Wang X, et al. Mucosal biofilm formation in middle-ear mucosa in the chinchilla model of otitis media. JAMA 2002;287:1710-5. [11] Fergie N, Bayston R, Pearson JP, et al. Is otitis media with effusion a biofilm infection? Clin Otolaryngol Allied Sci 2004;29:38-46. [12] Hall-Stoodley L, Hu FZ, Gieseke A, et al. Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media. JAMA 2006;296:202-11. [13] Dohar JE, Hebda PA, Veeh R, et al. Mucosal biofilm formation on middle-ear mucosa in a nonhuman primate model of chronic suppurative otitis media. Laryngoscope 2005;115:1469-72. [14] Chole RA, Faddis BT. Evidence for microbial biofilms in cholesteatomas. Arch Otolaryngol Head Neck Surg 2002;128:1129-33. [15] Barakate M, Beckenham E, Curotta J, et al. Bacterial biofilm adherence to middle-ear ventilation tubes: scanning electron micrograph images and literature review. J Laryngol Otol 2007;121:993-7. [16] Palmer Jr RJ, Sternberg C. Modern microscopy in biofilm research: confocal microscopy and other approaches. Curr Opin Biotechnol 1999;10:263-8. [17] Lawrence JR, Neu TR. Confocal laser scanning microscopy for analysis of microbial biofilms. Methods Enzymol 1999;310:131-44. [18] Paterson-Beedle M, Nott KP, Macaskie LE, et al. Study of biofilm within a packed-bed reactor by three-dimensional magnetic resonance imaging. Methods Enzymol 2001;337:285-305. [19] Tater EC, Unal FO, Tatar I, et al. Investigation of surface changes in different types of ventilation tubes using scanning electron microscopy and correlation of findings with clinical follow-up. Int J Pediatr Otorhinolaryngol 2006;70:411-7. [20] Wang EW, Jung JY, Pashia ME, et al. Otopathogenic Pseudomonas aeruginosa strains as competent biofilm formers. Arch Otolaryngol Head Neck Surg 2005;131:983-9. [21] Lenz AP, Williamson KS, Pitts B, et al. Localized gene expression in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 2008;74: 4463-71.
American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32 – 37 www.elsevier.com/locate/amjoto
Biofilms in chronic suppurative otitis media and cholesteatoma: scanning electron microscopy findings☆,☆☆,★ James Saunders, MDa,⁎, Michael Murray, MDb , Anthony Alleman, MDc a
Section of Otolaryngology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA b Camino Ear, Nose and Throat Clinic, San Jose, CA, USA c Department of Radiology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA Received 10 September 2009
Abstract
Background: Biofilms play a role in the pathogenesis of a variety of otorhinolaryngologic diseases, including otitis media and cholesteatoma. Despite this, relatively few studies have undertaken to demonstrate the presence of biofilms tissues from patients with chronic otitis media or infected cholesteatoma. Objective/hypothesis: Our objective is to detect evidence of biofilms human chronic ear infections with scanning electron microscopy (SEM). We hypothesized that bacterial biofilms are present in patients with chronic otitis media. Study design: We performed prospective collection of tissue collected during middle ear surgery from 16 patients undergoing middle ear or mastoid surgery with chronic ear infections. Methods: A total of 31 middle and mastoid tissue samples were harvested at the time of surgery and processed with critical point drying for SEM analysis. Samples were then searched for evidence of biofilms. Results: Bacterial-shaped objects were identified that displayed both surface binding and the presence of a glycocalyx in 4 patients, findings consistent with bacterial biofilms. Most of these (3 of 4) were in patients with infected cholesteatoma, and biofims were identified in 60% of cholesteatoma cases (3 of 5). On the other hand, only 1 of 7 cases with chronic suppurative otitis media had evidence of biofilms. Conclusion: SEM supports the hypothesis that bacterial biofilms are common in chronic infections associated with cholesteatoma and are present in some cases of chronic suppurative otitis media without cholesteatoma. © 2011 Elsevier Inc. All rights reserved.
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
☆
Presented at the Middle Section of the Triological Society 2005. Funded by a Resident Research Grant from the Triological society. ★ The authors have no conflicting interests in the publication of this paper, and funding was internal within the Otolaryngology Department of The University of Oklahoma Health Science Center. ⁎ Corresponding author. Section of Otolaryngology, DartmouthHitchcock Medical Center, Lebanon, NH, USA. Tel.: +1 603 650 8124; fax: +1 603 650 0052. E-mail address: [email protected] (J. Saunders). ☆☆
0196-0709/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2009.09.010
behave differently once bound to a surface. Surface-bound bacteria grow into biofilms, which are colonies of slowgrowing bacteria that surround themselves in a coat of glycopolysaccharides called a glycocalyx [1-3]. Bacteria in biofilms have been shown to be highly resistant to antibiotics and are nearly impossible to detect with standard culture techniques [4]. There is mounting evidence of the role of biofilms in otolaryngologic infections [5,6]. Tissue samples from patients with chronic rhinosinusitis have been shown to have biofilm formation [7,8]. 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 [9-12]. Many of the bacteria common to both acute and chronic otitis media are known to exist in a
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
biofilm state under favorable conditions. Biofilms have also been demonstrated in a nonhuman primate model of chronic otitis media [13]. There is, however, only 1 previous clinical study demonstrating direct clinical evidence of biofilms in chronically infected ears. In this study, Chole et al [14] 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, 3dimensional 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
33
microscopy to visualize the presence of bacteria. Various techniques have been used including scanning electron microscopy (SEM) [14,15], transmission electron microscopy [14], scanning laser confocal microscopy [16,17], and 3dimensional magnetic resonance imaging [18]. SEM has been previously used to show the presence of biofilms medical implants, such as tympanostomy tubes [19]. 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
Table 1 Tissue characteristics and SEM findings Pt
Diagnosis/condition
Description of specimen
SEM findings
1 2 3
1. Mucosa 1. Incus (control tissue) 1. Mucosa
1. Cocci, glycocalyx, and surface binding (Fig. 5) 1. No evidence of biofilm/bacteria 1. No evidence of biofilm/bacteria
1. Mucosa
1. No evidence of biofilm/bacteria
5
Infected cholesteatoma Trauma Chronic mucoid otorrhea with TM perforation Chronic suppurative otitis media with TM perforation Attic cholesteatoma
6
Infected cholesteatoma
7
Infected craniotomy site after translabyrinthine removal of acoustic neuroma Infected cholesteatoma
1. Long arm of incus 2. Body of incus 3. Mucosa and cholesteatoma 4. Mucosa and cholesteatoma 1. Edematous tissue next to cholesteatoma capsule 2. Cholesteatoma capsule and matrix 3. Cholesteatoma capsule and matrix 1. Hydroxyapatite cement 2. Hydroxyapatite cement
1. 2. 3. 4. 1. 2. 3. 1. 2.
1. Incus 2. Granulation tissue 3. Anterior remnant of TM with pocket of cholesteatoma 4. Matrix 5. Cholesteatoma capsule 1. Squamous debris 2. Edematous mastoid mucosa with clear mucous
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 1. Bacterial bacilli, glycocalyx, surface binding on squamous debris (Fig. 2) 2. No evidence of biofilm/bacteria 1. Single bacterium 2. No evidence of biofilm/bacteria 3. No evidence of biofilm/bacteria 1. No evidence of biofilm/bacteria
4
8
9
Recurrent cholesteatoma in chronically infected mastoid cavity
10
Chronic mucoid effusion with polypoid mucosa found on myringotomy Revision tympanoplasty with ossicular chain reconstruction, previous prosthesis removed (for control), TM atelectasis Chronic suppurative otitis media Chronic suppurative otitis media
11
12 13 14 15 16
Chronic suppurative otitis media Chronic suppurative otitis media, TM perforation with aural polyp Chronic suppurative otitis media
TM indicates tympanic membrane.
1. 2. 3. 1.
Polypoid mucosa Polypoid mucosa Edematous mucosa Hapex prosthesis covered with normal appearing mucosa
1. 1. 2. 1. 1. 2. 1.
Incus Granulation tissue with associated mucosa Mucosa Mucosa Mucosa Mucosa Granulation tissue
1. 1. 2. 1. 1. 2. 1.
No evidence of biofilm/bacteria No evidence of biofilm/bacteria Single bacterium No evidence of biofilm/bacteria No evidence of biofilm/bacteria Single bacterium No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria
Single bacterium No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria No evidence of biofilm/bacteria Two bacilli, glycocalyx, surface binding
34
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
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
Fig. 1. This image shows the surface of a incus removed during an ossicular chain reconstruction. The specimen was used as a control. Note the relatively smooth surface and lack of organisms (magnification = 5000×).
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
Fig. 2. (A) A colony of cocci bacteria can be seen attached to the inflamed mucosal surface adjacent an infected cholesteatoma from patient 1. The image shows a central area where the bacteria are uncovered and a peripheral area where the cocci are covered by an amorphous substance, consistent with glycocalyx (magnification = 1000×). (B) Higher magnification of the lower left corner of image A (magnification = 3000×, bar length = 5.0 μm).
J. Saunders et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery 32 (2011) 32–37
35
whereas the remainder was found on adjacent mucosa or granulation tissue. Culture results were not available on many of these patients; but 2 of the biofilm cases were found to have rod-shaped bacteria consistent with Pseudomonas sp., and 2 cases were noted to have associated bacterial cocci. Bacteria not bound to a surface or associated with an amorphous substance were noted in 4 patients. These images were consistent with planktonic bacteria.
4. Discussion
Fig. 3. High-magnification image of granulation tissue from patient 8 shows bacterial-sized objects bound to the underlying surface and immersed in an amorphous substance, consistent with glycocalyx (magnification = 10 000×).
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). The incus control tissue (Fig. 1) and noninfected hydroxyapatite prosthesis showed no evidence of bacteria or biofilm formation. Likewise, there was no biofilm identified on the infected alloplastic cranioplasty cement. Four patients had at least 1 specimen with bacterial-sized and -shaped object visualized with a surrounding amorphous substance and surface binding consistent with a biofilm (Figs. 2-5). Three of the 4 specimens identified with biofilms were in patients with infected cholesteatomas. Cholesteatoma was by far the most common diagnosis associated with a biofilm (3 of 5, 60%), and only 1 of the 7 patients (14%) with chronic suppurative otitis media demonstrated findings of a biofilm (Fig. 6). In only one of the cholesteatoma cases was the biofilm noted to be on squamous debris (Fig. 5),
The role of biofilm in chronic otitis media with effusion has been well documented and studied. In contrast, to date, there is only 1 prior clinical study of biofilm in cholesteatoma [14]. Chole et al [14] described the light and transmission electron microscopy findings in 24 patients with cholesteatoma and 22 experimental cholesteatomas in gerbils. Their criteria for the diagnosis of biofilm were similar to those used in our study, and they identified findings of biofilm in 16 of 24 clinical cases (66%). Interestingly, this is nearly identical to the 60% rate of biofilm in cholesteatomas in our smaller study. Like the previous study, we included both infected and noninfected cholesteatomas; and 1 of the 2 cholesteatomas that did not have evidence of biofilm was an attic cholesteatoma with no obvious sign of infection (case 5). On the other hand, no evidence of biofilm was seen in case 6 despite the fact that this patient had an active infection with P aeruginosa, a known biofilm former [20]. It is of course possible that
Fig. 4. This image shows a biofilm on chronically infected mucosa. The contrast between the smooth surface and the biofilm collection in the central portion of the image (magnification = 1000×).
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Fig. 6. High magnification of rod-shaped bacteria bound to the surface of granulation tissue from a patient with chronic suppurative otitis media (patient 16). Note the background of collagen fibers and the presence of glycocalyx between the 2 visible bacteria and the surface (magnification = 10 000×).
biofims were present in these samples, but undetected. These observations, however, would support those of Chole et al [14] that biofilms are a common but not ubiquitous feature of cholesteatoma. We find it interesting that a relatively low yield was found in specimens from chronic suppurative otitis media without cholesteatoma. Only 1 in 7 (14%) of such cases had evidence of biofilm. The only other study that documented biofilm in chronic suppurative otitis media was an experimental study in a nonhuman primate [13]. In this experimental study, the details of the infected state were not specified. Still, the clinical condition of chronic suppurative otitis media seems to fit that expected for a biofilm disease. As above, it is possible that biofilms were present in these specimens, but remained undetected. One reason may be due to the relative difficulty in surveying the entire specimen with SEM techniques. The labor-intensive process of searching for biofilms with SEM may, therefore, lead to less sensitivity compared with other methods. In addition, there may be structural features that make the disruption of biofilm more
Fig. 5. (A) This specimen was taken from an infected cholesteatoma (patient 9). Sheets of squamous debris with several rod-shaped bacteria associated with the string of amorphous material (magnification = 3000×, bar = 8 μm). (B) Higher magnification of the upper left corner of image A. The center of the image clearly shows rods encased in glycocalyx (magnification = 10 000×, bar length = 1 μm). (C) The upper left-hand portion of the previous image is shown in greater detail. Numerous rod-shaped objects are noticed throughout the image.
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likely in the surgical treatment of suppurative otitis media than in cholesteatoma. It is certainly possible that biofilms are relatively uncommon in chronic suppurative otitis media; however, this seems unlikely given the preponderance of evidence in chronic otitis media with effusion and cholesteatoma. Further clinical studies will be required to clarify whether biofilms are indeed prevalent in chronic suppurative otitis media. The visual identification of biofilms represents a surrogate end point in proving the existence of biofilms in chronic ear infections. We adhered, therefore, to fairly strict criteria to consider the specimen positive. Several hallmark features of biofilms were identified including surface binding, 3-dimensional aggregates of bacteria, and the presence of glycocalyx. This evidence suggests that biofilms were present in these patients with cholesteatoma and chronic suppurative otitis media. However, one cannot assume from the visual images that these bacteria were the source of the infections or that these bacteria possessed other characteristics of biofilms such as antibiotic resistance. Further study is needed to better clarify the relationships between these apparent biofilms and clinical behavior. Although researchers in the field generally agree on the morphologic features that constitute a biofilm, the identification of biofilms with imaging techniques remains somewhat subjective, time consuming, and of limited direct clinical value. Laser confocal microscopy has the advantage of labeling the biofilm, allowing for a much more rapid identification of suspicious areas of the specimen; but this method still requires tissue acquisition. Perhaps the most promising method of detecting biofilms would be the identification of the unique genomic changes associated with surface binding or quorum sensing [21]. Ultimately, the utilization of this technology may resolve the inherent difficulties of the imaging methods.
5. Conclusion Our research supports the hypothesis that biofilms are involved in chronic otitis media and cholesteatoma and, to a lesser degree, in chronic suppurative otitis media. The recognition of the role of biofilms in these disease states is critical to our understanding of the disease and to developing more effective treatment strategies. Future research should continue to develop more sophisticated and clinically applicable ways to detect biofilms, as well as ways to improve treatment.
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