Analysis of Inflammatory and Homeostatic Roles of Tissue-resident Macrophages in the Progression of Cholesteatoma by RNA-Seq.

BACKGROUND: Tissue-resident macrophages (TRMØs) can act as innate-immune sentinels to protect body against microbe invaders and stimulating materials such as cholesterol crystals in cholesteatoma, as well as to preserve tissue integrity by cleaning unwanted cellular debris. METHODS: TRMØs in the incised middle ear tissues were obtained from the patients with cholesteatoma as an experimental group and the patients without cholesteatoma as a control group. Differential gene expression profiling of TRMØs was conducted between two groups by analyzing GO processes, KEGG and GSEA pathways of inflammation, tissue repair and homeostasis. RESULTS: The current study showed that 145 of 7060 genes were significantly up-regulated (logFC>2 and FDR <0.05) when compared with the patients without cholesteatoma. GO process, GSEA and Cytoscape analysis of the over-expressed genes illustrated the boosted inflammatory and anti-infection functions of TRMØs existed neutrophil function, leukocyte migration, and adaptive immune response involved receptors and signaling pathways. Whereas the homeostasis and repair functions of TRMØs were affected from up-regulated genes, such as over-expressed keratin-13 that helped form the outer keratinising squamous epithelial layer, and over-expressed MMPs that activated the extracellular matrix molecules to promote inflammation and disturb tissue remodeling. Additionally, 74 down-regulated genes (logFC<-2 and FDR <0.05) also affected the homeostasis and repair functions by affecting extracelluar matrix structure and contractile fibres in TRMØs. CONCLUSIONS: The cellular and molecular levels in cholesteatoma is attributable to chronic infection and several disturbed cellular biological processes involving cell integrity and tissue remodeling.

Analysis of main pathogenic bacteria and drug sensitivity in patients with chronic suppurative otitis media and middle ear cholesteatoma in China.

BACKGROUND: Chronic suppurative otitis media (CSOM) and middle ear cholesteatoma (MEC) were classified as different diseases recently. Owing to the difference in pathogeneses, the pathogenic bacteria of the two diseases can be different. However, few studies have compared the two types of pathogenic bacteria. OBJECTIVE: To analyze the main pathogenic bacteria and drug sensitivities in patients of Southwest China with CSOM and MEC and compare the difference of ear dryness rate between empirical and sensitive medication. METHODS: According to the data of culture of ear discharge and postoperative pathological examination, the patients were divided into CSOM and MEC groups. A cohort study was carried out in 1087 hospitalized patients with CSOM and MEC in the Department of Otolaryngology head and neck surgery, West China Hospital from January 2015 to June 2019. RESULTS: Pathogenic bacteria were detected in 467 of 726 cases of CSOM (64.46%) and in 190 of 361 cases of MEC (52.63%). Of the positive cases, 53.96% involved gram-positive (G+) bacteria and 46.04% involved gram-negative (G-) bacteria in the CSOM group; 41.05% involved G+ bacteria and 58.95% involved G- bacteria in the MEC group (P < 0.05). In the CSOM group, the top four pathogens were Staphylococcus aureus (33.62%), Pseudomonas aeruginosa (23.98%), Proteus (16.92%), and Coagulase-negative Staphylococci (10.28%). The top four pathogens in the MEC group were Proteus (22.11%), S. aureus (21.05%), P. aeruginosa (15.26%), and coagulase-negative staphylococci (8.42%). S. aureus was more sensitive to ciprofloxacin and moxifloxacin in the MEC group than in the CSOM group (= 14.286, P < 0.001; = 8.244, P < 0.01). P. aeruginosa was more sensitive to neomycin and tobramycin in the MEC group than in the CSOM group (= 21.285, P < 0.001; = 4.060, P < 0.05). The sensitivity rate of coagulase-negative staphylococci to neomycin in the MEC group was higher than that in the CSOM group (= 5.126, P < 0.05). The sensitivity of Proteus to piperacillin tazobactam in the CSOM group was higher than that in the MEC group (= 8.881, P < 0.05). The dry ear rate of patients with sensitive drug was significantly higher than the patients with empirical drug (= 19.431, P < 0.001). CONCLUSION: The detection rate of G+ bacteria in the CSOM group was higher than that in the MEC group. The detection rate of G- bacteria in the CSOM group was lower than that in the MEC group. The main pathogens in the two groups included S. aureus, P. aeruginosa, Proteus and coagulase negative Staphylococcus. The dry ear rate of patients who used sensitive drugs was significantly higher than the patients who used empirical drugs. Reducing the rate of empirical use of antibiotics as much as possible and selecting antibiotics reasonably were beneficial to the improvement of dry ear rate after surgery.

Microbiome Analysis of Cholesteatoma by Gene Sequencing.

OBJECTIVE: To compare the microbial flora of cholesteatoma and normal middle ears using gene-based sequencing analysis. STUDY DESIGN: Controlled ex vivo human study. SETTING: Academic, tertiary medical center. SUBJECTS AND METHODS: Brush, swab, and tissue samples were each taken from cholesteatoma matrix and uninvolved tissue in patients with previously untreated, acquired cholesteatoma (n = 19) or middle ear mucosa from patients undergoing cochlear implantation with no history of cholesteatoma or previous middle ear surgery (control; n = 12). DNA was isolated from specimens then 16S rRNA gene sequencing was performed. RESULTS: There was no difference in microbial yield between the sampling methods. Cholesteatoma specimens had lower relative abundance of 14 bacterial species compared with controls including Acidovorax sp., Bacillus sp., Masillia sp., Moraxella osloensis, Phenylobacterium conjunctum, Sphingomonas sp., and Staphylococcus epidermidis (all p < 0.05). Alternaria sp. were present on nearly all the specimens. Alternaria sp. and Cladosporium herbarum (both p ≤ 0.05) were lower in the cholesteatoma compared with control group. There was no difference in the relative abundance of any bacteria or fungi between the cholesteatoma matrix and uninvolved middle ear mucosa. CONCLUSIONS: Microbiome of cholesteatoma matrix is largely similar to adjacent mucosa. This differs from healthy ears. Further study is needed to understand if middle ear microbiome may impact cholesteatoma pathogenesis or treatment.

Changes in biofilm in chronic cholesteatomatous otitis media in children following the application of sodium 2-mercaptoethanesulfonate (MESNA).

BACKGROUND: Pediatric cholesteatoma is a clinically challenging disease entity. Its biological behavior in the pediatric population differs from its behavior in adult population in terms of aggressiveness and recurrence. Several studies have shown the presence of biofilms associated with cholesteatoma that hinder the management and eradication of the infection. This led is to study the use of non-antimicrobial treatments impacting on the structure or composition of biofilms. OBJECTIVE: To evaluate the changes that occur in the biofilm of cholesteatoma in pediatric patients after the application of sodium 2-mercaptoethanesulfonate (MESNA). METHODS: A pilot study of 10 pediatric patients, with a median age of 10 years and a diagnosis of cholesteatomatous chronic otitis media, who underwent surgery for primary or revision mastoidectomy in the Otorhinolaryngology Service of the Hospital Infantil de México Federico Gómez between January 2016 and May 2017. During the surgery, basal samples of cholesteatoma and tissue were taken after topical application of 4% MESNA for 10 min. The samples were then processed for confocal laser microscopy. RESULTS: In all samples structures compatible with bacterial biofilms were identified. The most relevant finding was the changes in the structure of the biofilm after the application of MESNA, such as disintegration and separation of the underlying tissue. CONCLUSIONS: This is the first study that showing changes associated with cholesteatoma in the structure of the bacterial biofilm after the application of MESNA. The observed disintegration of cholesteatoma biofilm ultrastructure could aid in the management of the chronic infection associated with cholesteatoma.

Extended-spectrum Beta-lactamase-producing Escherichia coli Meningitis That Developed from Otitis Media with Cholesteatoma.

A 78-year-old man had a fever and exhibited disordered consciousness, which led to his transportation to our hospital. On arrival, he exhibited discharge from the ear. Because extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli was detected in the ear discharge and cerebrospinal fluid specimens, it was inferred to be the causal bacteria. Pulsed-field gel electrophoresis indicated the same ESBL-producing E. coli pattern in the patient's ear discharge, external auditory canal granulation, cerebrospinal fluid, and stool, indicating their common molecular epidemiological origin. Although ESBL-producing E. coli is an extremely rare cause of bacterial meningitis, it should be considered as a potential causal bacteria for community-acquired meningitis.

Metagenomics Analysis of Bacterial Population of Tympanosclerotic Plaques and Cholesteatomas.

Objective Chronic otitis media can cause cholesteatomas or tympanosclerosis; however, the pathophysiology of such conditions is not completely known. The aim was to identify a bacterial genome that might be present in tympanosclerotic plaques and cholesteatomas using sequence analysis of the gene responsible for the transcription of 16 ribosomal RNA (rRNA). Study Design Metagenomics analysis of the samples. Setting Samples were collected and evaluated at tertiary care centers. Subjects and Methods Sixty-five tympanosclerotic plaques and 37 cholesteatomas were evaluated. The polymerase chain reaction (PCR) was performed using primers designed for the amplification of the gene responsible for the transcription of bacterial 16 rRNA. The PCR-positive samples were sequenced via Sanger method, and 46 selected samples were analyzed with next-generation sequencing (NGS). Results Sanger sequencing revealed the presence of bacterial genomes in a total of 18 of the 102 samples tested. Sequencing of these genomes indicated the presence of Alloiococcus otitis, Staphylococcus aureus, Achromobacter xylosoxidans, Escherichia coli, Staphylococcus sciuri, Staphylococcus caprae, Parvimonas spp., and Bacillus sp. in the tested samples. The NGS showed 1 or more different bacterial genomes in 44 (95.7%) of the 46 samples tested. Predominately, genome of Clostridiales (27 samples), Staphylococcaceae (24 samples), Peptoniphilaceae (12 samples), and Turicella otitidis (9 samples) were identified. Conclusion The middle ear is inhabited by a diverse microbial community than that previously known. With the use of molecular biology, it has become easier to identify the bacterial genomes and improve our understanding of the role of middle ear microbiota in the pathogenesis of chronic inflammatory ear diseases.

Mycology of chronic suppurative otitis media-cholesteatoma disease: An evaluative study.

AIMS & OBJECTIVES: To detect the prevalence of fungus in chronic suppurative otitis media-cholesteatoma disease and to evaluate its clinical significance. STUDY DESIGN: Prospective observational study conducted in a sample size of 46 patients at a tertiary care university teaching hospital. MATERIALS & METHODS: Forty six patients suffering from chronic suppurative otitis media-cholesteatoma disease were recruited in this prospective study. Data was duly recorded. Cholesteatoma sample was procured at the time of mastoid surgery and microbiologically analysed for fungal infestation. Clinical correlation to fungus infestation of cholesteatoma was statistically analysed. RESULTS: Out of the recruited 46 patients, post-operatively cholesteatoma was seen in 40 cases only. Seventeen i.e. 42.5% of these cases had fungal colonization of cholesteatoma. Further a statistically significant correlation between persistent otorrhoea and fungal infestation of cholesteatoma was observed. Three cases of fungal otomastoiditis were also recorded in this study, but a statistically significant correlation between complications and fungus infestation of cholesteatoma could not be clearly established. CONCLUSIONS: There is fungal colonization of cholesteatoma which is pathogenic and can cause persistent otorrhoea.

Chronological changes in microbial profiles in external and middle ear diseases: a 20-year study in Korea.

Microbial infection is one of the most significant causes of ear diseases, but microbial profiles are very diverse according to the diseases and change over time. The purpose of the study was to clarify differences and chronological changes in causative pathogens among infectious ear diseases over the last 20 years, and to identify antibiotic resistance. In total, 1191 isolates were included from patients diagnosed with chronic otitis media without cholesteatoma (COM), cholesteatomatous otitis media (Chole), middle ear effusion (MEE), including acute otitis media and otitis media with effusion, and external otitis (EO). Data were collected periodically for the years 1995, 2000, 2004, 2009, and 2013. Culture results and antibiotic resistance were assessed. The most common microorganism identified was S. aureus. The microbial profiles differed significant among the COM, Chole, and MEE groups (p < 0.001). In contrast, there was no distinct difference between COM and EO (p = 0.332). COM, Chole, and MEE also showed significant chronological changes in microbial profiles over time. The frequency of CNS increased markedly in COM and Chole (p = 0.029 and 0.028, respectively); however, S. pneumoniae infection decreased significantly in MEE (p = 0.016). Methicillin-resistant S. aureus (MRSA) demonstrated a constant trend (p = 0.564), whereas ciprofloxacin-resistant P. aeruginosa increased over time (p < 0.001). Microbial profiles have changed over a 20-year period. Increases in the frequency of coagulase-negative Staphylococcus (CNS) and bacterial resistance to ciprofloxacin, used widely in treating ear infections, are noteworthy.

Efficacy of antibiotic prophylaxis prior to tympanoplasty for contaminated cholesteatoma.

OBJECTIVES: To evaluate the efficacy of combined antistaphylococcal and antipseudomonal preoperative antibiotics for preventing surgical site infections following tympanoplasty and mastoidectomy with contaminated cholesteatoma. STUDY DESIGN: Retrospective chart review. METHODS: Medical records of patients who underwent tympanoplasty ± mastoidectomy for cholesteatoma were reviewed. Only cases considered to have contaminated or dirty surgical fields were included. The primary outcome measure was occurrence of postoperative surgical site infections, perichondritis, pinna abscess, periotic cellulitis, or periotic abscess requiring systemic antibiotic therapy or surgical intervention. RESULTS: The charts of 326 patients who underwent tympanoplasty ± mastoidectomy were reviewed. Of those, 195 met inclusion criteria. Preoperative antibiotics included clindamycin and ceftazidime or gentamicin. Patients treated with no perioperative antibiotics had a surgical site infection rate of 11%, and those treated with perioperative antibiotics had a rate of 1% (P = 0.02). CONCLUSION: Administration of preoperative antibiotics to cover staphylococcal and pseudomonal species may prevent surgical site infections with tympanoplasty ± mastoidectomy for contaminated cholesteatoma. LEVEL OF EVIDENCE: 4. Laryngoscope, 126:2363-2366, 2016.

FleQ, a Transcriptional Activator, Is Required for Biofilm Formation In Vitro But Does Not Alter Virulence in a Cholesteatomas Model.

HYPOTHESIS: Bacterial biofilm formation within cholesteatomas is responsible for increased persistence and tissue destruction and Pseudomonas aeruginosa deficient in biofilm formation (PAO1 ΔfleQ) are less virulent than the parent bacteria. BACKGROUND: Infected aural cholesteatomas have been demonstrated to be more destructive than uninfected cholesteatomas and infections are more persistent. The chronicity and persistence of infections within cholesteatomas may be because of the presence of biofilm formation. METHODS: Twenty-seven mutant strains of PAO1 were screened for surface adherence. These strains were also screened for static biofilm formation. The biofilms were quantified by staining with crystal violet. Aural cholesteatomas were then induced in Mongolian gerbils by ligation of the ear canal. At the time of ligation, the ear canals were inoculated with wild-type PAO1 and a biofilm deficient PAO1 ΔfleQ strain of P. aeruginosa. A 7 weeks course of ciprofloxacin (20 mg/kg/day) was started on postoperative day 7. Eight weeks after induction of cholesteatomas, the cholesteatoma size, levels of bone destruction, and levels of bone remodeling were evaluated using microCT imaging. RESULTS: PAO1 ΔfleQ was identified as a poorly adherent and deficient biofilm forming mutant strain of P. aeruginosa. Infected cholesteatomas had more growth, bone destruction and bone remodeling than uninfected cholesteatomas. However, there was no difference observed between cholesteatomas infected with PAO1 (biofilm competent strain) and PAO1 ΔfleQ (biofilm deficient strain). CONCLUSION: We demonstrate that the biofilm phenotype is not an important virulence factor in cholesteatomas infected with P. aeruginosa.

Biofilm's Role in Chronic Cholesteatomatous Otitis Media: A Pilot Study.

Cholesteatoma is a destructive lesion involving the temporal bone, which may induce severe complications due to its expansion and erosion of adjacent structures. Bacterial biofilm plays a crucial role in the pathogenesis of many otolaryngologic inflammatory/infectious chronic diseases. In this pilot study, we investigated, by means of cultural examination and with scanning electron microscope, the presence of bacterial biofilm in a series of samples from the epitympanic and mastoid region in patients affected by cholesteatoma and from the promontory region in patients with healthy mucosa who were undergoing to stapes surgery. The preliminary data support the association between biofilm and cholesteatoma (81.3% of the cases) and allow us to hypothesize that keratinized matrix of cholesteatoma may represent the ideal substrate for biofilm colonization and survival; this finding is consistent with the clinical course of aural cholesteatoma, characterized by recurrent exacerbations and recalcitrant course.

Mycological study on cholesteatoma keratin obtained during primary mastoid surgery.

OBJECTIVE: Established middle-ear cleft cholesteatoma is associated with keratinous debris, which is likely to be an ideal medium for saprophytic fungal colonisation. This prospective case study aimed to explore the incidence and nature of fungal elements in cholesteatoma keratin samples obtained during primary mastoid surgery. METHODS: All cases of middle-ear cleft cholesteatoma treated with primary mastoid surgery at the El-Sahel Teaching Hospital over a seven-month period were included. Keratinous debris obtained from the mastoid antrum was subjected to mycological analysis at the Department of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University. A literature search was performed to determine the clinical and pathological relevance of fungal colonisation in cholesteatoma. RESULTS: Eighteen patients underwent primary mastoid surgery for cholesteatoma (nineteen ears in total) in a seven-month period starting 30 March 2013. Patients included 13 males and 5 females, with an age range of 9 to 45 years (mean 23 years). Fungal cultures were obtained from 17 keratin samples (89 per cent). Of these, five fungal isolates belonged to the dermatophyte group (21 per cent). CONCLUSION: Fungal colonisation in middle-ear cleft cholesteatoma probably plays a significant role in disease progression. Moreover, saprophytic fungal colonisation in cholesteatoma keratin may be responsible for the fetor commonly associated with the ear discharge.

[Correlation analysis of bacterial biofilm formation and bacterial culture in chronic otitis media].

OBJECTIVE: To study the correlation between the bacterial biofilm formation and bacterial culture in chronic otitis media. METHOD: As a prospective reserch, we used scanning electron microscopy to examinate patients samples which collected from 32 cases of patients with chronic suppurative otitis media and middle ear cholesteatoma in the operations, and performed the middle ear secretions bacterial culture. According to the different types of chronic otitis media group, we analysised the relationship between chronic otitis media bacterial biofilm formation and the bacterial culture results. RESULT: Chronic suppurative otitis media (activity) and middle ear cholesteatoma bacterial biofilm formation rate were 87.5%, 81.3%, chi-square (P > 0.05). Compared bacterial biofilm results with the results of bacterial cultured in chronic otitis media, sensitivity was 70.37%, specificity was 60.00%, the misdiagnosis rate was 40.00%, the missed diagnosis was 29.63%, positive predictive value was 90. 46%, negative predictive value was 27.27%, accuracy was 68.75%. Youden index was 30. 37%, and Pearson correlation coefficient was 0.232 (P > 0.05). CONCLUSION: Chronic suppurative otitis media (activity) and middle ear cholesteatoma bacteria had a higher biofilm formation rate. The routine bacterial culture results can't reflecte bacterial biofilm formation in chronic otitis media. We need to explore more reliable experimental methods to accurately reveal the infection status of chronic otitis media.

Inactivation of specific Pseudomonas aeruginosa biofilm factors does not alter virulence in infected cholesteatomas.

HYPOTHESIS: When experimental cholesteatomas are infected with Pseudomonas aeruginosa (PA) mutants lacking factors associated with the formation of biofilms, host defenses are more effective against these strains when compared with wild-type strains (PAO1 and OPPA8) in preventing tissue destruction. BACKGROUND: Previous studies have identified biofilms within chronically infected aural cholesteatomas. These infected cholesteatomas are associated with increased tissue destruction. Because biofilms are highly resistant to host defenses leading to prolonged infection, we propose that the biofilm phenotype of P. aeruginosa may be a virulence factor leading to persistence of infection and increased tissue destruction. METHODS: Aural cholesteatomas were induced in Mongolian gerbils. At the time of induction, the ear canals were inoculated with wild-type (PAO1 and OPPA8) and biofilm-deficient (PAO1 ΔpilA, PAO1 algD::aacC1 and PAO1 galU::aacC1) strains of P. aeruginosa. After 8 weeks, the size of the cholesteatomas and levels of bone destruction and deposition were measured using microCT scanning and double fluorochrome bone labeling. RESULT: Infected cholesteatomas resulted in increased growth, bone destruction, and bone deposition when compared with vehicle-only controls. We observed no differences between the wild-type (biofilm forming) and the biofilm-deficient strains of P. aeruginosa. CONCLUSION: Our hypothesis that biofilm formation is a virulence factor in cholesteatomas infected with P. aeruginosa was not supported. A number of interpretations of these data are reasonable. It is possible that biofilms are not critical in infected cholesteatomas. Alternatively, the mutants that are deficient in generating biofilms in vitro may be able to form effective biofilms in vivo using alternative pathways.

Expression of toll-like receptors 2, 4 and nuclear factor kappa B in mucosal lesions of human otitis: pattern and relationship in a clinical immunohistochemical study.

OBJECTIVES: The objectives were to detect and compare the expression of toll-like receptors (TLRs) 2, 4 and nuclear factor kappa B in mucosal lesions of chronic otitis. METHODS: Fifty-five tissue samples obtained from children and adults operated on for otitis were investigated by semiquantitative immunohistochemical methods using polyclonal antibodies for TLR 2, 4 and NFkappaB. Kruskal-Wallis, Mann-Whitney, and Kendall's tau rank correlation tests were used. RESULTS: Stronger expression of TLR2, 4 was found in inflamed mucosa than in the control for children and adults (TLR2: H = 23.86, P < .0011; TLR4: H = 22.80, P < .00 1) (TLR2: H = 17.53, P < .001; TLR4: H = 11.99, P < .001); in cholesteatoma perimatrix compared to tubotympanic lesions in children (TLR2: H = 11.06, P = .004; TLR4: H = 10.61, P = .005) and adults (TLR2: H = 10.73, P = .013; TLR4: H = 9.65, P = .021). No differences were found in NFkB expression (H = 0.042, P = .99). Significant correlations were found for all pairs of molecules in cholesteatoma and tubotympanic mucosa of adults (TLR2, 4: P = .002, P < .001; TLR2-NfkappaB: P = .032, P = .021; TLR4-NFkB: P = .035, P = .0013), only TLR4-NFkappaB in tubotympanic otitis of children (P = .026). CONCLUSIONS: Toll-like receptors 2,4 and NFkB mediate inflammation in cholesteatoma and mucosal lesions oftubotympanic otitis in children and adults. Significant correlations betweenall pairs of molecules in all samples were detected in adults, but only TLR4-NFkappaB in children.

Investigation of the presence of biofilms in chronic suppurative otitis media, nonsuppurative otitis media, and chronic otitis media with cholesteatoma by scanning electron microscopy.

OBJECTIVE: Biofilms have been shown to play a major role in the pathogenesis of otolaryngologic infections. However, very limited studies have been undertaken to demonstrate the presence of biofilms in tissues from patients with chronic otitis media (COM) with or without cholesteatoma. Our objective is to study the presence of biofilms in humans with chronic suppurative and nonsuppurative otitis media and cholesteatoma. Study Design. In all, 102 tissue specimens (middle ear, mastoid tissue, and ossicle samples) were collected during surgery from 34 patients. METHODS: The samples were processed for the investigation of biofilms by scanning electron microscopy (SEM). RESULTS: Our research supports the hypothesis in which biofilms are involved in chronic suppurative otitis media, cholesteatoma, and, to a lesser degree, chronic nonsuppurative otitis media. There were higher rates in hypertrophic and granulated tissue samples than in normal mucosa. In addition, the presence of biofilms was significantly higher in the middle ear mucosa compared with the mastoid and ossicle samples. CONCLUSION: In the clinic, the careful use of topical or systemic antimicrobials is essential, and, during surgery, hypertrophic tissue must be carefully removed from normal tissue.

Autologous meatal skin graft implantation and intratympanic injection of Pseudomonas aeruginosa: a new experimental mouse model of acquired middle ear cholesteatoma.

AIM: To create an experimental model for the biomedical research of middle ear cholesteatoma. METHODS: Cholesteatoma was induced in the right ears of mice. An autologous meatal skin graft was implanted into the middle ear via the tympanic membrane followed by an intratympanic injection of Pseudomonas aeruginosa. Six weeks after surgery, the formation of acquired cholesteatoma was evaluated by macroscopic examination, CT scan, and histological analysis. The expressions of TNF-α, IL-1ß, and IL-6 were measured with real-time PCR. Auditory-evoked brain stem response was used for assessing the changes in hearing levels. RESULTS: None of the mice died during the modeling time. By the sixth postoperative week, cholesteatoma had successfully formed in 23 out of 25 mice, which equals a success rate of 92%. A CT scan revealed that the cholesteatoma was eroding the cochlea. Furthermore, histological analysis demonstrated a cystic structure covered by stratified squamous epithelium and keratin desquamation in the lamellae inside the cystic cavity in the bullae. All mice with experimentally induced cholesteatoma showed hearing impairment and an upregulated expression of TNF-α, IL-1ß, and IL-6. CONCLUSION: The present study successfully developed a mouse model of acquired middle ear cholesteatoma, which provides an extremely valuable tool for auditory and biomedical research. The modeling approach is technically easy and has a high success rate.

Mastoid biofilm in chronic otitis media.

HYPOTHESIS: We designed a study to determine the role of mastoid mucosal biofilm in chronic otitis media (COM). BACKGROUND: Biofilm formation has been found in several chronic airway infections. COM is associated with chronic, recalcitrant infection of the mastoid mucosa, and surgery often is required. METHODS: COM patients were divided into 2 groups: one with chronic suppurative otitis media (CSOM) and one with cholesteatoma presence. All COM patients had mastoid involvement in a preoperative computed tomographic scan. The control group consisted of patients undergoing cochlear implantation, with no previous history of chronic otitis media. Mastoid mucosa samples were harvested during mastoidectomy. The samples were studied with multiplex-polymerase chain reaction and with CSLM using BacLight Live/Dead stain. Routine bacterial culture was performed in selected cases. RESULTS: A total of 29 COM patients underwent mastoidectomy. Mastoid mucosal biofilm formation could be found in 19 (66%) of these patients. In the control group, there were 11 cases of cochlear implantation, and 1 patient (9%) presented mastoid mucosal biofilm. In the cholesteatoma group, there were 17 patients, of which, 14 (82%) presented biofilm, whereas in the CSOM group, 5 (42%) of 12 patients presented biofilm. The correlation between COM and biofilm was statistically significant (Fisher's exact test, p = 0.003), as was the correlation between cholesteatoma and biofilm, in comparison with the CSOM group (Fisher's exact test, p = 0.046). CONCLUSION: Mastoid mucosal biofilm could be seen in patients with COM with or without cholesteatoma. The role of mastoid biofilm in the development of cholesteatoma should be studied further.

Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients.

BACKGROUND: High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates. METHODOLOGY: Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium. Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy. PRINCIPAL FINDINGS: A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms. High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.

The pathogenesis of acquired cholesteatoma of the human middle ear: support for the migration hypothesis.

HYPOTHESIS: That acquired cholesteatoma of the human middle ear and mastoid process can be caused by migration of squamous epithelium from the tympanic membrane. OBJECTIVE: To provide histologic evidence in humans of the potential for medial epithelial migration toward the middle ear. BACKGROUND: The origin of cholesteatomas of the middle ear complex of humans is still not clearly understood. The most popular theory, the medial migration of squamous epithelium from the external auditory canal/tympanic membrane through a perforation of the tympanic membrane to form a cholesteatoma, was first proposed over a century ago independently by Bezold (1899) and Habermann (1899) based on their observations during surgery. Neither author had histologic proof. Despite much experimental efforts in animals, histologic evidence in the human continues to be the missing link. METHOD: We examined the histologic sections of the temporal bones of 60 children aged 1 day to 12 years with evidence of acute otitis media. We present our findings in the temporal bones of 3 infants. RESULTS: We find evidence of the propensity of the squamous epithelium of the tympanic membrane particularly in the pars flaccida to become very active and to migrate medially toward the middle ear. CONCLUSION: Our concept is that this material supports the migration theory that cholesteatomas are formed by the medial migration of the stimulated squamous epithelium of the tympanic membrane.