Surface Motion of Tympanic Membrane in a Chinchilla Model of Acute Otitis Media

The conductive hearing loss caused by acute otitis media (AOM) is commonly related to a reduction of the tympanic membrane (TM) mobility in response to sound stimuli. However, spatial alterations of the TM surface motion associated with AOM have rarely been addressed. In this study, the TM surface motion was determined using scanning laser Doppler vibrometry (SLDV) in a chinchilla model of AOM. The AOM was established by transbullar injection of nontypeable Haemophilus influenzae. The TM surface vibration was measured in control (uninfected) animals and two AOM groups of animals: 4 days (4D) and 8 days (8D) post inoculation. To quantify the effect of middle ear pressure in those infected ears, the SLDV measurement was first conducted in unopened AOM ears and then in middle ear pressure released ears. Results showed that middle ear infection generally reduced the TM displacement across the entire surface, but the reduction in the umbo displacement over the time course, from 4 to 8 days post inoculation, was less than the reduction in the displacement at the center of each quadrant. The presence of middle ear fluid shifted the occurrence of traveling-wave-like motion on the TM surface to lower frequencies. The observation of the spatial variations of TM surface motion from this study will help refine our understanding of the middle ear sound transmission characteristics in relation to AOM.     

Experimental recurrent otitis media induced by Haemophilus influenzae: Protection and serum antibodies

Purpose: To study whether acute otitis media caused by encapsulated or nontypeable Haemophilus influenzae confers cross-reactive protective immunity in an animal model system and to explore the possible involvement of various humoral specific antibodies in protection.Materials and Methods: Rats were intrabullarly challenged with H influenzae type b and two different nontypeable H influenzae strains. One month after the initial infection, the animals were rechallenged ipsilaterally or contralaterally with either a homologous or heterologous strain, and the susceptibility to reinfection was investigated by otomicroscopy.Results: The animals challenged and rechallenged with the type b strain were well-protected ipsilaterally and contralaterally, while the protection after homologous rechallenge with a nontypeable strain was partial in the ipsilateral ear and very poor in the contralateral ear. Middle ears previously infected with a nontypeable strain remained fully susceptible to infections with heterologous strains, but there was an indication of cross-protection in the animal groups where the first episode of acute otitis media was caused by type b and the second by a nontypeable strain. Using the Western blot technique and an enzyme linked immunosorbant assay, the serological response to different outer membrane proteins, especially protein D, of H influenzae during and after middle ear infection were investigated. The serological response from the type b infected animals were generally more distinct, while the antibody levels against protein D were lower in these groups compared with the groups infected with nontypeable strains.Conclusions: These data indicate that H influenzae type-b-induced experimental otitis media results in a better protection than a nontypeable-induced, and H influenzae b confers a cross protection.     

Hearing Preservation With Labyrinthine Ablation in Otitis Media

Objectives Iatrogenic fenestration of the inner ear in the presence of otitis media is commonly associated with permanent hearing loss. Hearing can generally be preserved when the vestibular labyrinth is ablated in a controlled manner in noninflamed ears. The purpose of this study was to examine the feasibility of hearing preservation with violation of the inner ear in the presence of middle ear inflammation. Study Design Prospective and controlled animal model. Methods Otitis media was induced bilaterally in pigmented guinea pigs with transtympanic injection of Streptococcus pneumoniae, nontypeable Haemophilus influenzae, or formalin‐killed nontypeable H influenzae. Two to 4 days after injection, the horizontal canal of one ear was transected and sealed. Hearing was tested before and after labyrinthine ablation. Results Otitis media was induced in all ears. Bacterial cultures were positive in 19 of 20 S pneumoniae–injected ears, and in 10 of 16 nontypeable H influenzae–injected ears. One week after surgery, elevation of click thresholds (> 15 dB) was encountered in none of the fenestrated or unfenestrated S pneumoniae–infected ears, in two of six unfenestrated and three of six fenestrated nontypeable H influenzae–infected ears, and in one of five killed‐nontypeable H influenzae– injected ears both with and without fenestration. Conclusions These data suggest that ablation of a semicircular canal in the presence of middle ear inflammation or infection does not necessarily lead to profound sensorineural hearing loss. Hearing loss associated with iatrogenic violation of the semicircular canals may be more dependent on factors other than the presence of nonspecific middle ear inflammation.     

What are the leading causative pathogens in acute otitis media with tympanic membrane perforation?

ObjectivesAcute otitis media (AOM) is a common infectious disease in children. Data regarding the distribution of causative pathogens are not universal. Tympanic perforation due to AOM may occur in 5–30% of AOM patients. The causative pathogens for AOM with tympanic perforation are limited.MethodsThis was a prospective study conducted at the Department of Otolaryngology, Faculty of Medicine, Chiang Mai University, Thailand. All consecutive children diagnosed as having AOM with tympanic perforation were enrolled. The age of the eligible patients was between 3 months and 5 years. Pus from the middle ear of each patient was swabbed and tested for culture/sensitivity.ResultsThere were 40 eligible patients diagnosed with AOM with tympanic perforation in this study. The mean age of all patients was 24.3 months and the patients were predominantly male (26 male; 65.0%). None of these patients received S. pneumoniae or H. influenzae vaccination. All specimens were culture positive (100%) and 13 organisms were identified. There were 53 identified pathogens; the most common pathogen was H. influenzae (19 times or 35.8%), followed by Staphylococcus aureus (14 times or 26.4%). H. influenzae was 100% sensitive to chloramphenicol, amoxicilllin/clavulanic acid, cefotaxime, and ciprofloxacin, while S. aureus was also 100% sensitive to oxacillin, vancomycin, and fusidic acid.ConclusionsThe two most common pathogens for AOM with tympanic perforation were H. influenzae and Staphylococcus aureus. Both pathogens were mostly sensitive to antibiotics.     

Animal models of acute otitis media – A review with practical implications for laboratory research

Considerable animal research has focused on developing new strategies for the prevention and treatment of acute otitis media (AOM). Several experimental models of AOM have thus been developed. A PubMed search of the English literature was conducted from 1975 to July 2016 using the search terms “animal model” and “otitis media” from which 91 published studies were included for analysis, yielding 123 animal models. The rat, mouse and chinchilla are the preferred animals for experimental AOM models with their individual advantages and disadvantages. The most common pathogens used to create AOM are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Streptococcus pneumoniae (types 3, 23 and 6A) and non-typeable Haemophilus influenzae (NTHi) are best options for inoculation into rat and mouse models. Adding viral pathogens such as RSV and Influenza A virus, along with creating ET dysfunction, are useful adjuncts in animal models of AOM. Antibiotic prophylaxis may interfere with the inflammatory response without a significant reduction in animal mortality.     

Polyp and Fibrous Adhesion Formation in Acute Otitis Media Caused by Non-typeable or Type b Haemophilus influenzae or Moraxella catarrhalis

Among a variety of other histopathologic changes, polyps and fibrous adhesions are readily formed in the middle ear mucosa during experimental acute otitis media (AOM) caused by Streptococcus pneumoniae. Quantitative studies on experimental AOM caused by other bacteria have shown that some of these histopathologic changes, such as adaptive bone modeling and increase in goblet cell density, differ according to the type of bacteria. This investigation surveys polyp and fibrous adhesion formation in experimental AOM caused by either non-typeable or type b Haemophilus influenzae, or Moraxella catarrhalis. Seventy-five rats were inoculated with 1 of these 3 bacteria (25 rats in each of 3 groups). Five rats from each group were sacrificed on days 4, 8, 16, 60 and 180 post-inoculation. The middle ear mucosae were dissected and histopathologic changes in whole-mount and section preparations were studied using light microscopy. Polyps were found in most ears and in the greatest numbers on the early days; fewer polyps were found on the later days, regardless of the type of bacteria. However, non-typeable and type b H. influenzae induced formation of significantly more polyps than M. catarrhalis. The polyps were primarily located in the epitympanum. Fibrous adhesions were primarily located in the hypotympanum and formed in almost all ears, on all days post-inoculation, regardless of the type of bacteria. Numbers increased to a peak on day 16 and then decreased. Non-typeable and type b H. influenzae induced formation of significantly more adhesions than M. catarrhalis, and the middle ears displayed a higher number of persisting adhesions in the animals inoculated with non-typeable H. influenzae. We conclude that polyps and adhesions are formed in experimental AOM regardless of bacterial type, confirming a pathogenesis based on inflammation. Both types of H. influenzae induce formation of greater numbers of polyps/adhesions than M. catarrhalis, and the non-typeable form causes more adhesive sequelae in the mucosa than the encapsulated type b.     

Chlamydia trachomatis in the etiology of acute otitis media

In an effort to show that Chlamydia trachomatis (CT) may be involved in the causation of acute otitis media (AOM), we performed three experiments. In the first, we inoculated the tympanic bullae of 6 chinchillas with CT. Five of the 6 inoculated animals developed CT AOM. In the second experiment, we sprayed the nasopharynx of 10 chinchillas with CT. Of these, 8 developed both pharyngitis and AOM, and in 6, live CT was cultured from the middle ear and pharynx. In the third experiment, 5 chinchillas had their conjunctiva inoculated with CT. Three developed CT conjunctivitis. Of these, 2 developed CT pharyngitis and 1 developed CT AOM. We concluded that CT will cause AOM in the chinchilla by direct inoculation into the middle ear as well as indirectly by infection of the nasopharynx and conjunctiva.     

The effect of isotretinoin on propylene glycol-induced cholesteatoma in chinchilla middle ears

Previous studies have shown that propylene glycol causes inflammatory changes and cholesteatoma when applied to chinchilla middle ears. Vitamin A and synthetic analogues are essential for the normal differentiation of epithelial tissues. The purpose of this study was to determine whether the administration of isotretinoin to chinchillas would prevent propylene glycol exposure from inducing middle ear cholesteatomas. Sixteen chinchillas received 90% propylene glycol to the left middle ear and normal saline to the right. Half the animals were placed in the experimental group and received a daily dose of isotretinoin of 2 mg/kg for 7 days prior to propylene glycol administration and then for 6 weeks until killed. At 6 weeks, cholesteatoma was found in six of eight ears treated with propylene glycol in animals receiving isotretinoin. Two animals in the control group died. Three of the remaining eight had cholesteatoma. No ears treated with saline had cholesteatoma. We conclude that isotretinoin, in our chinchilla model, does not prevent propylene glycol-induced cholesteatoma formation.     

Otic preparations altered permeability and thickness of the round window membrane of the chinchilla

The effects of topical otic preparations (Cortisporin, Coly-Mycin, Aristocort, and Bestron) upon the permeability of the round window membrane (RWM) in chinchillas were investigated. Using K(+)-selective microelectrodes, the concentration of tetraethylammonium (TEA) ions was measured. Changes in the thickness of the RWM were measured using light microscopy. The RWM permeability was reduced significantly in Cortisporin- and Coly-Mycin-treated ears. Moreover, these two drugs resulted in a marked thickening of the RWM. In contrast, Aristocort or Bestron resulted in no alteration of the RWM permeability.     

Effect of lipooligosaccharide mutations of Haemophilus influenzae on the middle and inner ears

Objective

The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear.

Results

Fifteen chinchillas were divided into three equal groups and their bullas inoculated bilaterally with 0.5 ml of 10² CFU/ml of parent NTHi 2019, B29 or DK1 mutant strains. Two days after inoculation all animals had otitis media and inflamed middle ear mucosa. There was a trend of greater thickness and infiltration of the round window membrane in animals inoculated with the wild-type NTHi strain compared to the mutant strains and a significant increase in both inflammatory cell infiltration and bacteria presence in the scala tympani area of the inner ear. Strial edema was only observed in the wild-type-inoculated group.

Conclusions

LOS mutants of NTHi appear to have a reduced ability to pass through the round window membrane resulting in less inner ear inflammation and pathological changes.     

Individual-level effects of antibiotics on colonizing otitis pathogens in the nasopharynx

BackgroundAlthough there is evidence of an association between antibiotic consumption and resistant bacteria on a population level, the relationship on an individual level has been less well studied, particularly in terms of nasopharyngeal colonization. We have therefore analysed this association, using data from a closely followed cohort of children taking part in a vaccination trial.Methods109 children with early onset of acute otitis media (AOM) were randomised to heptavalent pneumococcal conjugate vaccine (PCV7) or no vaccination. They were followed for three years with scheduled appointments as well as sick visits. Nasopharyngeal cultures were obtained at all visits. Antibiotic treatments were recorded, as were risk factors for AOM, including siblings, short breast-feeding and parental smoking. Data were entered into a Cox regression model, and the findings of Streptococcus pneumoniae and Haemophilus influenzae with reduced susceptibility to the penicillin group were related to the number of previous courses of antibiotics.ResultsThere was evidence of an association between the amount of previously consumed betalactams and colonization with beta-lactamasenegative ampicillin-resistant (BLNAR) H. influenzae (RR 1.21; 95% CI 1.03–1.43; p = 0.03), and also with the most commonly prescribed drug; amoxicillin (RR 1.39; 95% CI 1.09–1.76; p = 0.01). There was no evidence for an association between antibiotic consumption and betalactamase producing H. influenzae or S. pneumoniae with reduced susceptibility to penicillin. Furthermore, there was no evidence of an association between resistant bacteria and AOM risk factors or PCV7.ConclusionIn this subgroup of children, most of whom were given several courses of antibiotics in early childhood, there was evidence of an association between betalactam/amoxicillin consumption and nasopharyngeal colonization with BLNAR strains, bacteria that have increased in prevalence during the last 10–15 years, and that are notoriously difficult to treat with oral antibiotics.     

Nasopharyngeal colonization of otopathogens in South Indian children with acute otitis media – A case control pilot study

BackgroundAcute otitis media (AOM) is an inflammatory disease of the middle ear causing significant morbidity in early childhood. A pilot study was undertaken to identify the role of various risk factors South Indian children with AOM, especially the role of nasopharyngeal otopathogens.MethodologyA prospective case control pilot study was conducted in children aged below six years, presenting to a single tertiary care from 2018 to 2019. Fifty cases with AOM and 45 age and gender matched controls were recruited. Two nasopharyngeal swabs were collected, one was processed for bacterial culture. The other swab was processed according to the CDC recommended broth enrichment method to identify carriage of S. pneumoniae. Subsequent serotyping was done by Quellung method and conventional sequential multiplex PCR.ResultOtalgia was the major presentation seen in 92% of the children with AOM. None of the clinical and demographic characteristics were found to be statistically significant between the cases and controls. The most common otopathogen was S. pneumoniae (55%) followed by H. influenza (29%). The common S. pneumoniae serotypes encountered were 11A and 19F.Nasopharyngeal colonization with S. pneumoniae [OR 6.57, p < 0.003] and H. influenzae [OR14.18, p < 0.003] were significant risk factors for AOM in children. The risk increased with co-colonization (OR 13.89,p < 0.003).ConclusionThis study strengthens the significant association between nasopharyngeal colonization of otopathogens and AOM as a risk factor that is enhanced by co-colonization.S. pneumoniae was the main otopathogen in this population, serotypes 11A and 19F being the most common.     

Round window membrane visibility related to success of hearing preservation in cochlear implantation

Objectives: This study was designed to evaluate the relationship between the degree of round window membrane (RWM) exposure and hearing outcome.

Materials and methods: Forty-six ears with cochlear implantation (CI) were enrolled. The degree of RWM exposure was divided into Grade I (<25%), Grade II (25–50%), and Grade III (>50%). The hearing outcomes were evaluated at 1.5 and 12 months postoperatively.

Results: Twenty-seven ears were Grade I, 13 were Grade II, and 6 were Grade III. RW approach was used in all ears of Grades II and III and 20 ears of Grade I and cochleostomy was used in 7 ears of Grade I. The pattern of bony overhang was multidirectional in 41 ears. Threshold shift significantly decreased proportional to the increase of RWM exposure after CI. The mean RWM exposure was 32.1?±?24.4% in ears with more than partial preservation (n?=?17), and 13.3?±?11.7% in the other ears (n?=?6) at 12 months post-CI (p?=?.061). Age at CI differed significantly between ears that had more than partial preservation and the other ears at 1.5 months post-CI.

Conclusions and significance: Degree of RWM exposure and age at CI might be factors predicting hearing outcome after CI using the RW approach.     

Inhibition of lymphoproliferation by middle ear effusion in experimental otitis media.

In this study, experimental otitis media was created in the chinchilla by direct middle ear challenge with Escherichia coli endotoxin, Streptococcus pneumoniae, Haemophilus influenzae, or Pseudomonas aeruginosa. The effusions recovered from the chinchillas in all four challenge groups were shown to inhibit the lymphoproliferative response of chinchilla peripheral blood lymphocytes to stimulation with phytohemagglutinin. The effect was dose dependent, and for effusions of infectious origin, the degree of inhibition was directly related to the duration of infection. Presence of the inhibitor in plasma was undocumented, suggesting a local production within the middle ear. Lymphocytes from middle ears infected with bacteria but not middle ears challenged with endotoxin were hyporesponsive or nonresponsive to stimulation with phytohemagglutinin. These results confirm the presence of an inhibitor of the lymphoproliferative response in experimental otitis media of different etiologies.     

Mucosal trauma induced apoptosis in guinea pig middle ear: Comparision of hemostatic agents

ObjectiveThe aim of this study is to compare the effects of the absorbable gelatin sponge (AGS), microporous polysaccharide hemospheres (MPH), and Ankaferd on wound healing after middle ear trauma and to evaluate their ototoxicity in an experimental guinea pig model.MethodsMiddle ear mucosal trauma was created in 21 healthy adult guinea pigs. MPH, Ankaferd, and AGS were applied into the right tympanic bulla of the guinea pigs (7 ears for each treatment modality). The left ears of the seven animals were used as the sham group. At the fourth postoperative week (28–30 days), the guinea pigs were decapitated. Apoptosis was investigated, and the expression of Bcl-xl, Apaf, p53, cytochrome 3, and caspase 3 were evaluated.ResultsThe Ankaferd and AGS groups demonstrated significantly lower epithelial thickness, inflammation, and capillary dilatation than did the control group (p < 0.001, <0.001, /0.001, <0.001/, 0.005, and 0.005, respectively). A statistically significant decrease in Bcl-xl staining was observed in the middle ears of animals treated with MPH (p = 0.003). There was significantly higher caspase 3 expression in the Ankaferd and AGS groups than in the control group (p < 0.001 and p = 0.002, respectively).ConclusionLight microscopy indicates that Ankaferd and AGS create less inflammation and increased caspase expression, which seems to induce inflammatory cell apoptosis. Ankaferd seems to be a promising hemostatic agent in otology.     

Direct evidence of bacterial biofilms in otitis media

OBJECTIVES/HYPOTHESIS: Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations. STUDY DESIGN: Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients. METHODS: Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes. RESULTS: Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes. CONCLUSIONS: Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in children's ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.     

Assessment of ototoxicity of tea tree oil in a chinchilla animal model

ObjectiveThe aim of the present study is to examine the effects of tea tree oil on hearing function and cochlear morphology after intratympanic administration in a chinchilla animal model.MethodsNine chinchillas received intratympanic injection of 3% tea tree oil dissolved in olive oil in one ear, whereas the contralateral control ear received olive oil only. Outcome measures included auditory brainstem responses conducted before treatment and at 10 days and 30 days following the injection. Post-mortem cochlear morphology was assessed using scanning electron microscopy.ResultsAt 10 and 30 days following the injection, there was no significant change in auditory brain response thresholds at 8, 16, 20 or 25 kHz. Scanning electron microscopy imaging showed no damage to auditory hair cells.ConclusionTea tree oil (3%) does not appear to be ototoxic in a chinchilla animal model. Future preclinical and clinical studies are required to establish the effectiveness of TTO in treating otitis.     

Intracochlear infusion of buthionine sulfoximine potentiates carboplatin ototoxicity in the chinchilla

The aim of this experiment was to determine if buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, enhances the ototoxicity of carboplatin. Osmotic pumps were used to infuse BSO into the right cochleas of 12 adult chinchillas for 14 days. The left cochleas served as controls. Animals were assigned to three groups: a drug control group that did not receive carboplatin, a group that received a single dose of carboplatin (25 mg/kg i.p.), and a group that received a double dose of carboplatin (25 mg/kg i.p. x 2), with 4 days between injections. Carboplatin was administered after three days of BSO pre-treatment. Ototoxicity was assessed with evoked potentials recorded from electrodes implanted in the inferior colliculi (ICPs), distortion product otoacoustic emissions (DPOAEs), and cochleograms. BSO infusion itself caused no long-term functional or morphological changes. One of four animals treated with it single dose of carboplatin showed a significant loss of inner hair cells (IHCs), with greater loss in the BSO-treated ear. All animals in the double-dose carboplatin group showed marked differences between BSO-treated and control ears. Average IHC losses were 59% in BSO-treated ears vs. 18% in control ears. Moreover, BSO-treated ears sustained significantly greater outer hair cell (OHC) losses than control ears (37% vs. 2%, respectively). ICP and DPOAE response amplitudes were reduced slightly in BSO-treated ears relative to control ears, consistent with their greater hair cell loss. The results clearly show that BSO can enhance carboplatin ototoxicity in the chinchilla, supporting a role of GSH and reactive oxygen species in platinum ototoxicity.     

Formation of biofilm by Haemophilus influenzae isolated from pediatric intractable otitis media

Objectives

The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease.

Methods

Biofilm formations by NTHi clinical isolates from pediatric AOM patients were evaluated by a crystal violet microtiter plate and a 98 well pin-replicator assay with a confocal laser scanning microscopy (CLSM). Optical density values of clinical isolates were compared with a positive control and the ratio of clinical isolates to a positive control was defined as biofilm formation index (BFI).

Results

84.3% clinical isolates of NTHi were biofilm forming strains (BFI ≥ 0.4). The BFI represented the levels of biofilm formation and adherence on the surface. The identical strains isolated from both middle ear fluids (MEFs) and nasopharynx showed biofilm formation at the same level. The prevalence of biofilm forming isolates was significantly higher among the susceptible strains than resistant strains. The level of biofilm formation of NTHi isolated from AOM cases who was not improved by amoxicillin (AMPC) was significantly higher than that of NTHi isolated from AOM cases who was improved by AMPC.

Conclusion

We clearly showed the biofilm formation of clinical NTHi isolates from AOM children. In addition, the biofilm formed by NTHi would play an important role in persistent or intractable clinical course of AOM as a result of lowered treatment efficacy of antibiotics.