Search

Case Series Summary: The efficacy of medical treatment associated with single myringotomy in cats with suppurative otitis media (OM) and intact tympanum was retrospectively evaluated. Patients' records were retrieved from cats with suppurative OM. Cats were included in this retrospective study based on fluid content within the tympanic cavity on imaging, intact tympanum on otoendoscopic examination and neutrophils on ear bulla exudate cytological examination. A total of 26 cats with middle ear suppurative otitis were included. Just over half of the cats (54%) presented bilateral involvement. Clinical signs included head tilt (13/26), otalgia (9/26), Horner's syndrome (7/26), external ear discharge (5/26), and nystagmus and facial paralysis (1/26). Cocci were identified on cytological examination in 18/40 samples and rods in 2/40. Bacterial culture results were positive in 15/40 samples, with Pseudomonas species (4/15), Pasteurella multocida (3/15), Staphylococcus felis (3/15), Staphylococcus schleiferi (2/15), Staphylococcus canis (2/15), Escherichia coli (2/15), Staphylococcus pseudintermedius (1/15) and Serratia marcesens (1/15) isolation. After myringotomy and gentle flushing of middle ear bullae (0.5-2 ml saline per ear), all cats were treated with oral corticosteroids and a 1-month course of systemic antibiotics according to sensitivity testing. In total, 19 (73%) cats were clinically healed 60-240 days after treatment. One cat was euthanased because of failure to eradicate a Pseudomonas species infection. Another cat died a few hours after the procedure. The five remaining cats were cured after ventral bulla osteotomy., Relevance and Novel Information: OM without an aural polyp is a frequent diagnosis in cats, but data on medical treatment are scarce. This simple therapeutic technique, single myringotomy procedure and mild ear flushing, offers a practical, safe and efficient first-line treatment of suppurative OM with an intact tympanum in cats with or without neurological signs., Competing Interests: Conflict of interestThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: 17β-estradiol (E2) is a steroidal hormone with immunomodulatory functions that play a role in infectious and inflammatory diseases. E2 was recently identified as the leading upstream regulator of differentially expressed genes in a comparative RNA sequencing study of pediatric patients with otitis media (OM) versus OM-free counterparts and may therefore play a role in the inflammatory response to bacterial otopathogens during pediatric OM. This study examined the effect of E2 on bacterial-induced inflammatory cytokine expression in an in vitro pediatric OM model., Methods: An immortalized middle ear (ME) epithelial cell line, ROM-SV40, was developed from a pediatric recurrent OM patient. The culture was exposed to E2 at physiological levels for 1-48 h prior to 6 h-stimulation with nontypeable Haemophilus influenzae (NTHi) whole cell lysate. TNFA, IL1B, IL6, and IL8 were assayed by qPCR and ELISA., Results: E2 pretreatment (24 h) abrogated NTHi induction of IL6; a longer pretreatment (1-10 nM, 48 h) abrogated IL1B induction (p < 0.05). E2 pretreatment (5 nM, 48 h) abrogated NTHi-induced IL8 secretion (p = 0.017)., Conclusion: E2 pretreatment partially rescued NTHi-induced cytokine production by ME epithelia. These data support a role for E2 in moderating the excessive inflammatory response to middle ear infection that contributes to OM pathophysiology., Levels of Evidence: NA Laryngoscope, 134:3815-3819, 2024., (© 2024 The American Laryngological, Rhinological and Otological Society, Inc.)

Background: Rapid identification of causative bacteria in treatment of acute otitis media (AOM) is of paramount importance for appropriate antibiotic use., Materials and Methods: This prospective observational study was conducted in 15 hospitals and clinics in Japan between 2018 and 2020. A new rapid antigen test kit (AOS-116), which simultaneously detects antigens for Streptococcus pneumoniae (Sp) and Haemophilus influenzae (Hi), was applied for middle ear fluids (MEFs) and nasopharyngeal secretions (NPSs) in patients with moderate to severe AOM. We investigated relationship between the results of rapid test, severity at initial visit, and clinical course., Results: Regarding performance accuracy based on culture results, AOS-116 showed 1) high (>80%) sensitivity, specificity, and negative predictive value (NPV) in MEFs for both antigens, 2) high sensitivity, specificity, and positive predictive value (PPV) in NPSs for Hi antigen, and 3) high specificity, and PPV in NPSs for Sp antigen. Regarding predictive value of nasopharyngeal culture and antigen detection for causative middle ear pathogens, similar results were observed between AOS-116 and culture, which was characterized with high sensitivity and NPV for both pathogens. MEFs/NPSs positive for Hi antigen were significantly associated with eardrum findings, and severity. MEFs/NPSs positive for pneumococcal antigen were significantly associated with severity of otalgia, fever, and otorrhea. Among patients with prior antimicrobial treatment, improvement tended to be slower in cases positive for Hi than in cases negative., Conclusion: The rapid antigen detection test is useful as a decision-making tool for prescribing antimicrobial agents and may play an important role in promoting appropriate antimicrobial use., Competing Interests: Declaration of competing interest The authors declare financial support from ASAHI KASEI Corporation. Takuji Miyata and Norikazu Okamura are employees of Asahi Kasei Corporation. The funder had no role in the experimental design, implementation of the study, interpretation of the data, or decision to submit the manuscript for publication. This does not alter the authors’ adherence to any publication policies on sharing data and materials., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

Objectives: To evaluate the bacterial biofilm's role in mucosal chronic suppurative otitis media (CSOM) utilizing scanning electron microscopy (SEM)., Methods: This study involved 123 participating patients with active and inactive mucosal CSOM who underwent tympanomastoid surgery. SEM was used to examine middle ear mucosa biopsies for the development of biofilms. Middle ear discharge or mucosal swabs from patients were cultured to detect any bacterial growth. The biofilm formation was correlated to the culture results., Results: The biofilm was present in 69.9 % of patients (59% of them were with active mucosal CSOM) and absent in 30.1% of the patients (70% of them were with inactive mucosal CSOM), being more statistically significant in active mucosal CSOM (p-value = 0.003). A correlation that was statistically significant was found between active mucosal CSOM and higher grades (3 and 4) of biofilms (p-value <0.05). The mucosal CSOM type and the results of the culture had a relationship that was statistically significant (p-value <0.001). 60% of patients had positive culture (70% of them were with active mucosal CSOM). There was a statistically significant relation between Pseudomonas aeruginosa bacterial growth and active mucosal CSOM (p-value = 0.004) as well as higher grades of biofilms in mucosal CSOM., Conclusion: Mucosal CSOM, especially the active type, is a biofilm-related disease. There is a significant relation between the state of mucosal CSOM (active or inactive) and culture results with predominance of Pseudomonas aeruginosa bacterial growth in active mucosal CSOM and in higher grades of biofilms in mucosal CSOM., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Objective: It has been estimated that about 5 million people of those affected with otitis media have cholesteatoma, however, its pathophysiology is unclear. In this study we aimed to detect Helicobacter pylori via polymerase chain reaction and real-time polymerase chain reaction in young patients with chronic otitis media., Methods: Patients included in our prospective cross-sectional study had middle-ear/mastoid inflammation and underwent surgical procedures. Middle-ear mucosa samples were collected, and genomic DNA was extracted for H pylori detection by polymerase chain reaction and real-time polymerase chain reaction analyses. Sociodemographic data and gastroesophageal reflux symptoms were analysed., Results: We included 49 patients with mean age of 12.7 ± 3.8 years. Twenty per cent of the patients were diagnosed with cholesteatoma. No increase in H pylori -amplified fluorescence was observed, indicating absence of H pylori ., Conclusion: Due to the absence of amplification for H pylori and the fact that albumin was amplified in all samples, we conclude that H pylori does not appear to be a causal factor.

Background: Otitis media (OM) is defined as middle ear (ME) inflammation that is usually due to infection. Globally, OM is a leading cause of hearing loss and is the most frequently diagnosed disease in young children. For OM, pediatric patients with Down syndrome (DS) demonstrate higher incidence rates, greater severity, and poorer outcomes. However, to date, no studies have investigated the bacterial profiles of children with DS and OM. Method: We aimed to determine if there are differences in composition of bacterial profiles or the relative abundance of individual taxa within the ME and nasopharyngeal (NP) microbiotas of pediatric OM patients with DS ( n  = 11) compared with those without DS ( n  = 84). We sequenced the 16S rRNA genes and analyzed the sequence data for diversity indices and relative abundance of individual taxa. Results: Individuals with DS demonstrated increased biodiversity in their ME and NP microbiotas. In children with OM, DS was associated with increased biodiversity and higher relative abundance of specific taxa in the ME. Conclusion: Our findings suggest that dysbioses in the NP of DS children contributes to their increased susceptibility to OM compared with controls. These findings suggest that DS influences regulation of the mucosal microbiota and contributes to OM pathology.

Objective: This study aimed to describe the microbiology of the middle ear and nasopharynx, determining the prevalence of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in a group of children vaccinated with pneumococcal conjugate vaccine (PCV) who underwent ventilation tube insertion for recurrent acute otitis media., Methods: We analyzed 278 middle ear effusion and 139 nasopharyngeal samples obtained from 139 children who underwent myringotomy and ventilation tube insertion for recurrent acute otitis media between June 2017 and June 2021. The children's ages ranged from 9 months to 9 years, 10 months, with a median of 21 months. The patients had no signs of acute otitis media or respiratory tract infection and were not on antibiotic therapy at the time of the procedure. The middle ear effusion and nasopharyngeal samples were collected with an Alden-Senturia aspirator and a swab, respectively. Bacteriological studies and multiplex PCR were performed for the detection of the three pathogens. Direct molecular determination of pneumococcal serotypes was performed by real-time PCR. The chi-square test was used to verify associations between categorical variables and measures of strength of association based on prevalence ratios, considering a 95% confidence interval a 5% significance level., Results: Vaccination coverage was 77.7% with the basic regimen plus booster dose and 22.3% with the basic regimen alone. Middle ear effusion culture identified H. influenzae in 27 children (19.4%), S. pneumoniae in 7 (5.0%), and M. catarrhalis in 7 (5.0%). PCR detected H. influenzae in 95 children (68.3%), S. pneumoniae in 52 (37.4%), and M. catarrhalis in 23 (16.5%), a three-to seven-fold increase compared to culture. In the nasopharynx, culture isolated H. influenzae in 28 children (20.1%), S. pneumoniae in 29 (20.9%), and M. catarrhalis in 12 (8.6%). PCR identified H. influenzae in 84 children (60.4%), S. pneumoniae in 58 (41.7%), and M. catarrhalis in 30 (21.5%), a two-to three-fold increase in detection. The most common pneumococcal serotype was 19A, both in the ears and the nasopharynx. In the ears, of the 52 children who had pneumococcus, 24 (46.2%) had serotype 19A. In the nasopharynx, of the 58 patients who had pneumococcus, 37 (63.8%) had serotype 19A. Of all 139 children, 53 (38.1%) had polymicrobial samples (more than 1 of the 3 otopathogens) in the nasopharynx. Of the 53 children who had polymicrobial samples in the nasopharynx, 47 (88.7%) also had 1 of the 3 otopathogens in the middle ear, mainly H. influenzae (40%-75.5%), especially when it was found in the nasopharynx in conjunction with S. pneumoniae., Conclusion: The prevalence of bacteria in a group of Brazilian children immunized with the PCV who required ventilation tube insertion for recurrent acute otitis media was similar to that reported in other parts of the world after the advent of PCV. H. influenzae was the most frequent bacteria, both in the nasopharynx and the middle ear, while S. pneumoniae serotype 19A was the most common pneumococcus in the nasopharynx and middle ear. Polymicrobial colonization of the nasopharynx was strongly associated with detection of H. influenzae in the middle ear., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chronic otitis media (COM) is the long-term infection and inflammation of the middle ears typically caused by upper respiratory tract pathogens that are able to ascend the Eustachian tube. Our understanding of contributing factors is limited because human otopathogens cannot naturally colonize or persist in the middle ears of mice. We recently described a natural COM in mice caused by Bordetella pseudohinzii and proposed this as an experimental system to study bacterial mechanisms of immune evasion that allow persistent infection of the middle ear. Here we describe a novel pertussis toxin (PTx)-like factor unique to B. pseudohinzii , apparently acquired horizontally, that is associated with its particularly efficient persistence and pathogenesis. The catalytic subunit of this toxin, PsxA, has conserved catalytic sites and substantial predicted structural homology to pertussis toxin catalytic subunit PtxA. Deletion of the gene predicted to encode the catalytic subunit, psxA , resulted in a significant decrease in persistence in the middle ears. The defect was not observed in mice lacking T cells, indicating that PsxA is necessary for persistence only when T cells are present. These results demonstrate the role of a novel putative toxin in the persistence of B. pseudohinzii and its generation of COM. This PsxA-mediated immune evasion strategy may similarly be utilized by human otopathogens, via other PTx-like toxins or alternative mechanisms to disrupt critical T cell functions necessary to clear bacteria from the middle ear. This work demonstrates that this experimental system can allow for the detailed study of general strategies and specific mechanisms that otopathogens use to evade host immune responses to persist in the middle ear to cause COM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ma, Sedney, Su, Dewan, Linz and Harvill.)

Background: Otitis media (OM) is one of the most common infections in young children, arising from bacterial and/or viral infection of the middle ear. Globally, Streptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) are the predominant bacterial otopathogens. Importantly, common upper respiratory viruses are increasingly recognized contributors to the polymicrobial pathogenesis of OM. This study aimed to identify predominant bacteria and viruses in the nasopharynx, adenoids and middle ears of peri-urban/urban South-East Queensland Australian children, with and without clinical history of chronic otitis media with effusion (COME) and/or recurrent acute otitis media (RAOM)., Methods: Sixty children, 43 diagnosed with OM and 17 controls with no clinical history of OM from peri-urban/urban South-East Queensland community were recruited to the study. Respiratory tract bacterial and viral presence were examined within nasopharyngeal swabs (NPS), middle ear effusions (MEE) and adenoids, using real-time polymerase chain reaction (RT-PCR) and bacterial culture., Results: At least one otopathogen present was observed in all adenoid samples, 86.1% and 82.4% of NPS for children with and without OM, respectively, and 47.1% of the MEE from the children with OM. NTHi was the most commonly detected bacteria in both the OM and control cohorts within the adenoids (90.0% vs 93.8%), nasopharynx (67.4% vs 58.8%) respectively, and in the MEE (OM cohort 25.9%). Viruses were detected in all adenoid samples, 67.4% vs 47.1% of the NPS from the OM and control cohorts, respectively, and 37% of the MEE. Rhinovirus was the predominant virus identified in the adenoids (85.0% vs 68.8%) and nasopharynx (37.2% vs 41.2%) from the OM and control cohorts, respectively, and the MEE (19.8%)., Conclusions: NTHi and rhinovirus are predominant otopathogens within the upper respiratory tract of children with and without OM from peri-urban and urban South-East Queensland, Australia. The presence of bacterial otopathogens within the middle ear is more predictive of concurrent URT infection than was observed for viruses, and the high otopathogen carriage within adenoid tissues confirms the complex polymicrobial environment in children, regardless of OM history., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ngo, Massa, McMonagle, Perry, Nissen, Sloots, Thornton and Cripps.)

Acute otitis media (AOM) is commonly caused by bacterial pathobionts of the nasopharynx that ascend the Eustachian tube to cause disease in the middle ears. To model and study the various complexities of AOM, common human otopathogens are injected directly into the middle ear bullae of rodents or are delivered with viral co-infections which contribute to the access to the middle ears in complex and partially understood ways. Here, we present the novel observation that Bordetella bronchiseptica , a well-characterized respiratory commensal/pathogen of mice, also efficiently ascends their Eustachian tubes to colonize their middle ears, providing a flexible mouse model to study naturally occurring AOM. Mice lacking T and/or B cells failed to resolve infections, highlighting the cooperative role of both in clearing middle ear infection. Adoptively transferred antibodies provided complete protection to the lungs but only partially protected the middle ears, highlighting the differences between respiratory and otoimmunology. We present this as a novel experimental system that can capitalize on the strengths of the mouse model to dissect the molecular mechanisms involved in the generation and function of immunity within the middle ear., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dewan, Sedney, Caulfield, Su, Ma, Blas-Machado and Harvill.)

Background: Otitis media (OM) is a major disease burden in Australian Aboriginal children, contributing to serious long-term health outcomes. We report a pilot analysis of OM in children attending an outreach ear and hearing clinic in a remote south Australian community over a two-year period. Our study focuses on longitudinal relationships between ear canal microbiota characteristics with nasopharyngeal microbiota, and clinical and treatment variables., Results: Middle ear health status were assessed in 19 children (aged 3 months to 8 years) presenting in remote western South Australia and medical interventions were recorded. Over the two-year study period, chronic suppurative OM was diagnosed at least once in 7 children (37%), acute OM with perforation in 4 children (21%), OM with effusion in 11 children (58%), while only 1 child had no ear disease. Microbiota analysis of 19 children (51 sets of left and right ear canal swabs and nasopharyngeal swabs) revealed a core group of bacterial taxa that included Corynebacterium, Alloiococcus, Staphylococcus, Haemophilus, Turicella, Streptococcus, and Pseudomonas. Within-subject microbiota similarity (between ears) was significantly greater than inter-subject similarity, regardless of differences in ear disease (p = 0.0006). Longitudinal analysis revealed changes in diagnosis to be associated with more pronounced changes in microbiota characteristics, irrespective of time interval. Ear microbiota characteristics differed significantly according to diagnosis (P (perm) = 0.0001). Diagnoses featuring inflammation with tympanic membrane perforation clustering separately to those in which the tympanic membrane was intact, and characterised by increased Proteobacteria, particularly Haemophilus influenzae, Moraxella catarrhalis, and Oligella. While nasopharyngeal microbiota differed significantly in composition to ear microbiota (P (perm) = 0.0001), inter-site similarity was significantly greater in subjects with perforated tympanic membranes, a relationship that was associated with the relative abundance of H. influenzae in ear samples (r s  = - 0.71, p = 0.0003). Longitudinal changes in ear microbiology reflected changes in clinical signs and treatment., Conclusions: Children attending the ear and hearing clinic in a remote Aboriginal community present with a broad spectrum of OM conditions and severities, consistent with other remote Aboriginal communities. Ear microbiota characteristics align with OM diagnosis and change with disease course. Nasopharyngeal microbiota characteristics are consistent with the contribution of acute upper respiratory infection to OM aetiology., (© 2022. The Author(s).)

This study aimed to investigate the bacteriome in microscopically healthy middle ear mucosa using Next-generation sequencing (NGS) technology. A total of 60 middle ear washing fluids of pediatric and adult were obtained from 47 patients (35 children and 12 adults). Both children and adults with normal middle ears harbored diverse bacteriome. Seventeen different genera with a mean relative abundance of more than 1% were detected in all samples. Both in adult and children, the most abundant genus was Propionibacterium followed by Streptococcus, Staphylococcus, and Ralstonia. The species Propionibacterium acnes and Corynebacterium tuberculostearicum were significantly more abundant in the adult group. Although there were differences in the prevalence and relative abundance of some bacteria observed from adult and child groups, no specific genus or species was detected only in children or adults., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Otitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility., Methods: We performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues., Results: A large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma., Conclusion: SPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear., Competing Interests: Competing interests: AR is a cofounder of, shareholder in and uncompensated consultant to Otonomy, Inc, a relationship that was approved by the University of California San Diego., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To investigate the distribution of pathogenic bacteria in patients with tympanic membrane perforation after chronic suppurative otitis media (CSOM) in dry ear and its influence on the success rate of tympanoplasty and postoperative infection., Methods: 740 patients with tympanic membrane perforation after CSOM underwent endoscopic tympanoplasty were selected. The mucosal surface secretion of middle-ear was collected for bacterial culture and drug sensitivity test. The patients were followed up several times from 1 week to 3 months after the surgery., Results: 740 cases of ear secretions samples, raise the pathogens of 208 cases (28.1%), the success rate of surgery with microorganism grown and with no grown was 93.8% and 91.5%. fungus (14.6%) was the most species among the patients with the positive result, followed by methicillin-sensitive Staphylococcus aureus (4.1%), Pseudomonas (2.0%), Staphylococcus epidermidis (1.9%), methicillin-resistant Staphylococcus aureus (1.6%) and so on. There was no statistical difference in the proportion of perforation and infection in each group. There were no statistically significant differences in gender, age and duration of disease among the groups., Conclusion: There were still microbial colonization in patients with tympanic membrane perforation after CSOM in dry ear, include fungus, Staphylococcus aureus and Pseudomonas aeruginosa. Different microbial colonization had no influence on the success rate of tympanoplasty and postoperative infection., (Copyright © 2021. Published by Elsevier Inc.)

Objective: Streptococcus pneumoniae ( S.pn ) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. However, little is known about the immunometabolism during AOM. This study was to assess the presence of glucose metabolic reprogramming during AOM and its underlying mechanism affecting inflammatory response and middle ear injury., Methods: The levels of glycolytic metabolism were evaluated by measuring the expression of glycolysis-related genes and the production of metabolites. HE stain, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to measure the effect of glucose metabolic reprogramming on inflammatory response, pneumococcal clearance, hypoxia-inducible factor 1 alpha (HIF-1α) expression and cytokine secretion during AOM, respectively., Results: The analysis of microarray revealed an increase of the expression of glycolysis-related genes during S.pn -induced AOM, which was verified by real-time PCR. Increased glycolysis promoted the production of IL-1β and TNF-α and facilitated the clearance of S.pn by enhancing phagocytosis and killing capability of neutrophils, but also aggravated the middle ear injury. Furthermore, these pathogenic effects could be reversed after glycolytic inhibitor 2DG treatment. Additionally, HIF-1α was observed to involve in glycolytic metabolism during AOM., Conclusion: S.pn infection induced increased glycolysis conversion during AOM, which promoted inflammatory responses and bacterial clearance, but also aggravated tissue damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fan, Ma, Ai, Ding, Li, Zhu, He, Zhang, Dong and He.)

Studying the impact of antibiotic treatment on otitis media (OM), the leading cause of primary care office visits during childhood, is critical to develop appropriate treatment strategies. Tracking dynamic middle ear conditions during antibiotic treatment is not readily applicable in patients, due to the limited diagnostic techniques available to detect the smaller amount and variation of middle ear effusion (MEE) and middle ear bacterial biofilm, responsible for chronic and recurrent OM. To overcome these challenges, a handheld optical coherence tomography (OCT) system has been developed to monitor in vivo response of biofilms and MEEs in the OM-induced chinchilla model, the standard model for human OM. As a result, the formation of MEE as well as biofilm adherent to the tympanic membrane (TM) was longitudinally assessed as OM developed. Various types of MEEs and biofilms in the chinchilla model were identified, which showed comparable features as those in humans. Furthermore, the effect of antibiotics on the biofilm as well as the amount and type of MEEs was investigated with low-dose and high-dose treatment (ceftriaxone). The capability of OCT to non-invasively track and examine middle ear conditions is highly beneficial for therapeutic OM studies and will lead to improved management of OM in patients.

Objectives: Nontypeable Haemophilus influenzae (NTHi) is a chief pathogen in both acute otitis media and otitis media with effusion. Phosphorylcholine (ChoP) is expressed on lipooligosaccharides, and ChoP has phase variation, which is related to its adhesion to and invasion of epithelial cells in the upper airway. However, little is known about the role of ChoP expression. We examined the kinetics of the mucosal clearance of NTHi from the nose and middle ear and the mucosal immune response to NTHi infection by comparing ChoP(+) and ChoP(-) strains in a mouse model of middle ear and nasal challenge., Methods: Six-week-old male BALB/c mice were subjected to bacterial challenge in the middle ear and nasopharynx. Mice were inoculated with a suspension of a ChoP(+) strain or ChoP(-) strain of NTHi. On days 1, 3, and 7 after inoculation, the middle ear wash (MEW) and nasal wash (NW) were harvested from each group. The samples were used for bacterial counts and the supernatant was used to measure the level of cytokines and C-reactive protein (CRP)., Results: MEWs in the ChoP(+) strain group had significantly higher bacterial counts than those in the ChoP(-) strain group on day 1. However, bacteria were eradicated in the ChoP(+) strain group on day 7. NWs in the ChoP(+) strain group had higher bacterial counts than those in the ChoP(-) strain group during the experiment, however, there was no significant difference between the two strains. The levels of cytokines were significantly higher in the ChoP(-) strain group than in the ChoP(+) strain group in MEWs, but these cytokine levels were low in NWs. The CRP concentration in the ChoP(-) group was high on day 7 in the MEWs. In NWs, the CRP concentration was low in all groups during the experiment., Conclusion: ChoP expression of NTHi changes the organism susceptible to killing by CRP, and the ChoP(+) strain might be gradually eradicated from the middle ear via the CRP-complement cascade, but not from nasopharynx. Based on our findings, phase variation by altering Phosphorylcholine expression of nontypeable Haemophilus influenzae affects bacteria clearance and mucosal immune response in the middle ear and nasopharynx., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2020. Published by Elsevier B.V.)

Factors facilitating the chronicity of otitis media (OM) in children are, to date, not fully understood. An understanding of molecular factors aiding bacterial persistence within the middle ear during OM could reveal pathways required for disease. This study performed a detailed analysis of Streptococcus pneumoniae populations isolated from the nasopharynx and middle ear of one OM case. Isolates were assessed for growth in vitro and infection in a mouse intranasal challenge model. Whole genome sequencing was performed to compare the nasopharyngeal and middle ear isolates. The middle ear isolate displayed a reduced rate of growth and enhanced potential to transit to the middle ear in a murine model. The middle ear population possessed a single nucleotide polymorphism (SNP) in the IgA1 protease gene igA, predicted to render its product non-functional. Allelic exchange mutagenesis of the igA alleles from the genetic variant middle ear and nasopharyngeal isolates was able to reverse the niche-adaptation phenotype in the murine model. These results indicate the potential role of a SNP in the gene encoding the IgA1 protease, in determining S. pneumoniae adaptation to the middle ear during chronic OM. In contrast, a functional IgA1 protease was associated with increased colonisation of the nasopharynx., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Objective: The aim of this study was to investigate sclerostin (SOST) expression in a rat model of experimental tympanosclerosis (TS) and its possible role in the formation of TS., Materials and Methods: Thirty-four SD rats were randomly divided into 2 groups: experimental group (n = 17) and normal group (n = 17). The left tympanic cavities in the experimental group were inoculated with methicillin-resistant Staphylococcus aureus. The changes of tympanic membranes were examined and recorded under otoendoscope. Haematoxylin-eosin staining was adopted to detect the morphological changes in the tympanic membrane and middle ear mucosa. Immunohistochemistry and Western blot analysis were used to observe the expression of SOST, Wnt3a, β-catenin, and P-ERK1/2., Results: In the experimental group, sclerotic lesions were observed in 54.5% ears in the end of 6 weeks. Morphological changes such as mucosa incrassation, inflammatory cells infiltration, fibrous tissue proliferation, and interstitial tissue incrassation prominently appeared in the tympanic membrane and middle ear mucosa. SOST protein was mainly distributed in the cytoplasm of epithelial cells and gland cells, the expression of which increased significantly in the calcified experimental ears. In addition, expression levels of Wnt3a, β-catenin, and P-ERK1/2 increased significantly in the calcified group too., Conclusion: The upregulated expression level of SOST may be involved in the formation of TS, first, through the pro-phosphorylation of ERK1/2 in the inflammatory stage, and then through the enhancement of Wnt3a in the osteogenic stage., (© 2021 S. Karger AG, Basel.)

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonizing the nasopharynx. Progression from colonization to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We previously reported that cell-cell signaling via the autoinducer 2 (AI-2)/LuxS quorum-sensing system boosts the capacity of S. pneumoniae to utilize galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesized that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites, S317, T319, and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear, and lungs in a murine intranasal challenge model. IMPORTANCE Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle are closely linked to the organism's ability to utilize specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose 6-phosphate pathways are necessary for pneumococcal growth in galactose and demonstrated GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear, and lungs., (Copyright © 2020 American Society for Microbiology.)

Introduction: To date, microbial infections are also difficult to eradicate due to the increasing capability of bacteria to form a biofilm. In the era of antibiotic resistance, antibody-based approaches represent great promise in curing infective pathogens. The authors of US2019338018 patent propose a method for the treatment of biofilm-related disorders by using specific antibody fragments., Areas Covered: The US2019338018 patent reports antibody fragments, pharmaceutical composition that contains it, and their application for the treatment of biofilm-linked disorders. Proof concept and preclinical results show that mAb mIhfB4 NTHI Fab caused robust eradication of the biofilm in the middle ear lumen of chinchillas affected by Hemophilus influenzae infection., Expert Opinion: Fab fragments of the US2019338018 patent are new in a general concept to treat bacterial biofilms and biofilm-linked disorders. However, pre-clinical data are only shown for the treatment with Fab fragments of infections caused by H. influenzae in the middle ear of chinchillas. There are no clinical trials that demonstrate that the treatment with Fab fragments may induce a disruption of biofilm produced by H. influenzae or other pathogens and an anti-inflammatory response in infected patients.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media . Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-β (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1β, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Otitis media with effusion (OME) is a common inflammatory disease that primarily affects children. OME is defined as a chronic low-grade inflammation of the middle ear (ME), without any signs of infection and with effusion persisting in the ME for more than 3 months. The precise pathogenesis is, however, not fully understood. Here, we comprehensively characterized and compared the host immune responses (inflammatory cells and mediators) and the overall microbial community composition (microbiota) present in matched middle ear effusion (MEE) samples, external ear canal (EEC) lavages, and nasopharynx (NPH) samples from children with OME. Female patients had significantly increased percentages of T lymphocytes and higher levels of a wide array of inflammatory mediators in their MEE compared to that of male patients, which were unrelated to microbiota composition. The relative abundances of identified microorganisms were strongly associated with their niche of origin. Furthermore, specific inflammatory mediators were highly correlated with certain bacterial species. Interestingly, some organisms displayed a niche-driven inflammation pattern in which presence of Haemophilus spp. and Corynebacterium propinquum in MEE was accompanied by proinflammatory mediators, whereas their presence in NPH was accompanied by anti-inflammatory mediators. For Turicella and Alloiococcus , we found exactly the opposite results, i.e., an anti-inflammatory profile when present in MEE, whereas their presence in the the NPH was accompanied by a proinflammatory profile. Together, our results indicate that immune responses in children with OME are highly niche- and microbiota-driven, but gender-based differences were also observed, providing novel insight into potential pathogenic mechanisms behind OME., (Copyright © 2020 Enoksson et al.)

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045 T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045 T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

Objective: The purpose of the present study was to assess the rate of tympanic membrane perforation in patients with otomycosis and to discuss the literature regarding the difficulties in managing this condition., Data Sources: Literature review from 1999 to 2019, Web of Science, PubMed, and Medline., Study Selection: We searched for eligible articles concerning the clinical entity of tympanic membrane perforation secondary to otomycosis. Case series and clinical trials were the types of articles included for this review., Data Extraction: All the articles described in the study selection were used for this review., Data Synthesis: Statistical techniques were not used., Conclusion: Based on the available literature, it seems that tympanic membrane perforation secondary to otomycosis is not uncommon. The presence of this complication is associated with 2 problems: Antimycotic solutions are irritant to middle ear and may be ototoxic to the cochlea. Although most cases of fungus caused tympanic membrane (TM) perforation resolve with proper medical treatment, in a few patients a tympanoplasty may be required.

Background: Studies have shown that Helicobacter pylori (H. pylori) infection may be associated with the occurrence of otitis media with effusion (OME) in children., Objective: This is a systematic review of the relevant published literature to explore the relationship between H. pylori infection and OME in children., Method: Articles published before October 30, 2019 in the PubMed, Web of Science, Ovid, the China National Knowledge Infrastructure (CNKI) database, and Wanfang databases were retrieved. Articles were screened based on prespecified inclusion and exclusion criteria. Quality assessment was applied to the included studies. Data in the included studies were extracted and classified for qualitative analysis., Results: Ten studies, which enrolled a total of 397 cases and 334 controls, were included; all were case-control studies of varying quality. We summarized and compared H. pylori infections in different specimens from pediatric patients with OME. Due to the apparent heterogeneity between the included studies, meta-analysis was not appropriate, hence we carried out only a qualitative analysis., Conclusion: The detection rate of H. pylori in the middle ear, tonsil and gastric juice in children with OME was higher than that in children without OME. There is no clear and reliable conclusion as to whether there is a difference in the detection rate of H. pylori in adenoid specimens of children with or without OME. Eradication of H. pylori may improve symptoms of drug-resistant OME. Nevertheless, more studies of higher quality are needed to improve the conclusions., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Otitis media with effusion (OME) is one of the most common pediatric diseases worldwide. Several studies have analyzed the diversity of the microbiomes found in the middle ear effusions (MEEs) of populations from developed countries. However, no microbiological studies of MEEs from Chinese children with OME have been reported. This study investigated the middle ear and adenoid microbiological profiles of children with OME, and compared the microbial flora of the adenoid between children with and without otitis media., Methods: MEEs and adenoid swabs were acquired from 15 children undergoing ventilation tube insertion and adenoidectomy. Adenoid swabs from 15 patients with no ear disease were used as controls. Samples were analyzed by 16S rRNA sequencing. Operational taxonomic units (OTUs) abundance information were normalized. Alpha diversity analyses were used to assess the richness and diversity of the microbial community for each sample. Beta diversity analyses were used to determine the inter-group variability between microbiome structure., Results: Based on the mean relative abundance, the MEEs were dominated by Haemophilus (14.75%), Staphylococcus (9.37%) and Halomonas (7.85%), and the bacterial compositions of the adenoids in the OME groups were dominated by Haemophilus (21.87%), Streptococcus (19.65%), and Neisseria (5.8%). The bacterial compositions in the adenoids of the controls were dominated by Haemophilus (15.96%), Streptococcus (13.33%), and Moraxella (12.28%). Alpha diversity analyses showed that there were no significant differences in microbiome richness or diversity between the middle ear effusions (TM) and adenoids (TA) of OME subjects. Adenoid samples from OME patients (TA) and control patients (CA) were also similar. Beta diversity analyses showed that the microbiomes of the adenoids in OME patients were also similar to that of controls. However, the microbiome structure of middle ear effusions was dissimilar to those of the adenoids in OME patients according to beta diversity analyses., Conclusions: Our results confirmed the microbial diversity of MEEs among Chinese children. However, the dissimilar microbiome composition between samples taken from the surface of the adenoids and from the middle ear effusions challenges the conventional theory that the adenoid serves as a microbial reservoir in children with otitis media with effusion., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The use of broad-spectrum antibiotics to treat diseases, such as the highly prevalent pediatric disease otitis media (OM), contributes significantly to the worldwide emergence of multiple-antibiotic-resistant microbes, and gut dysbiosis with diarrhea is a common adverse sequela. Moreover, for many diseases, like OM, biofilms contribute significantly to chronicity and recurrence, yet biofilm-resident bacteria are characteristically highly resistant to antibiotics. The most cost-effective way to both prevent and resolve diseases like OM, as well as begin to address the problem of growing antibiotic resistance, would be via the development of novel approaches to eradicate bacterial biofilms. Toward this goal, we designed a vaccine antigen that induces the formation of antibodies that prevent biofilm formation and, thereby, experimental OM in the middle ears of chinchillas by the predominant Gram-negative pathogen responsible for this disease, nontypeable Haemophilus influenzae These antibodies also significantly disrupt preexisting biofilms formed by diverse pathogens. Whereas preclinical data strongly support the continued development of this vaccine antigen, which targets an essential structural element of bacterial biofilms, a concern has been whether active immunization would also lead to unintended collateral damage in the form of an altered gut microbiome. To address this concern, we assessed changes in the microbiome of the chinchilla gut over time after the delivery of either amoxicillin-clavulanate, the standard of care for OM, or after immunization with our biofilm-targeted vaccine antigen either via a traditional subcutaneous route or via a novel noninvasive transcutaneous route. We show that differences in the abundance of specific taxa were found only in the stools of antibiotic-treated animals. IMPORTANCE The prevalence of chronic and recurrent diseases, combined with the overuse/abuse of antibiotics that has led to the sobering emergence of bacteria resistant to multiple antibiotics, has mandated that we develop novel approaches to better manage these diseases or, ideally, prevent them. Biofilms play a key role in the pathogenesis of chronic and recurrent bacterial diseases but are difficult, if not impossible, to eradicate with antibiotics. We developed a vaccine antigen designed to mediate biofilm disruption; however, it is also important that delivery of this vaccine does not induce collateral damage to the microbiome. The studies described here validated a vaccine approach that targets biofilms without the consequences of an altered gut microbiome. While delivery of the antibiotic most commonly given to children with ear infections did indeed alter the gut microbiome, as expected, immunization via traditional injection or by noninvasive delivery to the skin did not result in changes to the chinchilla gut microbiome., (Copyright © 2020 Bailey et al.)

Air-pollutants containing toxic particulate matters (PM) deposit in the respiratory tract and increases microbial infections. However, the mechanism by which this occurs is not well understood. This study evaluated the effect of urban particles (UP) on Streptococcus pneumoniae (pneumococcus) in vitro biofilm formation, colonization of human middle ear epithelium cells (HMEECs) as well as mouse nasal cavity and its transition to the middle ear and lungs. The in vitro biofilms and planktonic growth of S. pneumoniae were evaluated in metal ion free medium in the presence of UP. Biofilms were quantified by crystal violet (CV) microplate assay, colony forming unit (cfu) counts and resazurin staining. Biofilm structures were analyzed using a scanning electron microscope (SEM) and confocal microscopy (CM). Gene expressions of biofilms were evaluated using real time RT-PCR. Effects of UP exposure on S. pneumoniae colonization to HMEECs were evaluated using fluorescent in-situ hybridization (FISH), cell viability was detected using the Ezcyto kit, apoptosis in HMEECs were evaluated using Annexin-V/PI based cytometry analysis and reactive oxygen species (ROS) production were evaluated using the Oxiselect kit. Alteration of HMEECs gene expressions on UP exposure or pneumococci colonization was evaluated using microarray. In vivo colonization of pneumococci in the presence of UP and transition to middle ear and lungs were evaluated using an intranasal mice colonization model. The UP exposure significantly increased (*p < 0.05) pneumococcal in vitro biofilms and planktonic growth. In the presence of UP, pneumococci formed organized biofilms with a matrix, while in absence of UP bacteria were unable to form biofilms. The luxS, ply, lytA, comA, comB and ciaR genes involved in bacterial pathogenesis, biofilm formation and quorum sensing were up-regulated in pneumococci biofilms grown in the presence of UP. The HMEECs viability was significantly decreased (p < 0.05) and bacteria colonization was significantly elevated (p < 0.05) in co-treatment (UP + S. pneumoniae) when compared to single treatment. Similarly, increased apoptosis and ROS production were detected in HMEECs treated with UP + pneumococci. The microarray analysis of HMEECs revealed that the genes involve in apoptosis and cell death, inflammation, and immune response, were up-regulated in co-treatment and were unchanged or expressed in less fold in single treatments of UP or S. pneumoniae. The in vivo study showed an increased pneumococcal colonization of the nasopharynx in the presence of UP and a higher transition of bacteria to the middle ear and lungs in the presence of UP. The UP exposure elevated S. pneumoniae in vitro biofilm and colonization of HMEECs, and in vivo mouse nasopharyngeal colonization, and increased dissemination to mouse middle ear and lungs.

Objective: To perform a comprehensive review of otitis media microbiome literature published between 1 st July 2015 and 30th June 2019., Data Sources: PubMed database, National Library of Medicine., Review Methods: Key topics were assigned to each panel member for detailed review. Draft reviews were collated and circulated for discussion when the panel met at the 20th International Symposium on Recent Advances in Otitis Media in June 2019. The final draft was prepared with input from all panel members., Conclusions: Much has been learned about the different types of bacteria (including commensals) present in the upper respiratory microbiome, but little is known about the virome and mycobiome. A small number of studies have investigated the middle ear microbiome; however, current data are often limited by small sample sizes and methodological heterogeneity between studies. Furthermore, limited reporting of sample collection methods mean that it is often difficult to determine whether bacteria detected in middle ear fluid specimens originated from the middle ear or the external auditory canal. Recent in vitro studies suggest that bacterial interactions in the nasal/nasopharyngeal microbiome may affect otitis media pathogenesis by modifying otopathogen behaviours. Impacts of environmental pressures (e.g. smoke, nutrition) and clinical interventions (e.g. vaccination, antibiotics) on the upper respiratory and middle ear microbiomes remain poorly understood as there are few data., Implications for Practice: Advances in understanding bacterial dynamics in the upper airway microbiome are driving development of microbiota-modifying therapies to prevent or treat disease (e.g. probiotics). Further advances in otitis media microbiomics will likely require technological improvements that overcome the current limitations of OMICs technologies when applied to low volume and low biomass specimens that potentially contain high numbers of host cells. Improved laboratory models are needed to elucidate mechanistic interactions among the upper respiratory and middle ear microbiomes. Minimum reporting standards are critically needed to improve inter-study comparisons and enable future meta-analyses., Competing Interests: Declaration of competing interest RLM, CA, SB, JB, MB, AC, KPL and HCS-V have no declarations of interest. LSK has received investigator-initiated grants and travel support from Pfizer and GSK to attend conferences that are not related to this work. MPES has received personal fees from GSK, Pfizer, AstraZeneca and Sanofi Pasteur as a speaker at international meetings and as a member of advisory boards (unrelated to the submitted work)., (Copyright © 2019. Published by Elsevier B.V.)

Objective: To review the most recent advances in human and bacterial genomics as applied to pathogenesis and clinical management of otitis media., Data Sources: PubMed articles published since the last meeting in June 2015 up to June 2019., Review Methods: A panel of experts in human and bacterial genomics of otitis media was formed. Each panel member reviewed the literature in their respective fields and wrote draft reviews. The reviews were shared with all panel members, and a merged draft was created. The panel met at the 20th International Symposium on Recent Advances in Otitis Media in June 2019, discussed the review and refined the content. A final draft was made, circulated, and approved by the panel members., Conclusion: Trans-disciplinary approaches applying pan-omic technologies to identify human susceptibility to otitis media and to understand microbial population dynamics, patho-adaptation and virulence mechanisms are crucial to the development of novel, personalized therapeutics and prevention strategies for otitis media., Implications for Practice: In the future otitis media prevention strategies may be augmented by mucosal immunization, combination vaccines targeting multiple pathogens, and modulation of the middle ear microbiome. Both treatment and vaccination may be tailored to an individual's otitis media phenotype as defined by molecular profiles obtained by using rapidly developing techniques in microbial and host genomics., Competing Interests: Declaration of competing interest Dr. Allen F. Ryan is a Co-founder of and equity holder in Otonomy, Inc., a company that develops slow-release compounds for local delivery of medications to the middle and inner ear. The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To perform a comprehensive review of the literature from July 2015 to June 2019 on the pathogenesis of otitis media. Bacteria, viruses and the role of the microbiome as well as the host response are discussed. Directions for future research are also suggested., Data Sources: PubMed database of the National Library of Medicine., Review Methods: PubMed was searched for any papers pertaining to OM pathogenesis between July 2015 and June 2019. If in English, abstracts were assessed individually for their relevance and included in the report. Members of the panel drafted the report based on these searches and on new data presented at the 20th International Symposium on Recent Advances in Otitis Media., Conclusions: The main themes that arose in OM pathogenesis were around the need for symptomatic viral infections to develop disease. Different populations potentially having different mechanisms of pathogenesis. Novel bacterial otopathogens are emerging and need to be monitored. Animal models need to continue to be developed and used to understand disease pathogenesis., Implications for Practice: The findings in the pathogenesis panel have several implications for both research and clinical practice. The most urgent areas appear to be to continue monitoring the emergence of novel otopathogens, and the need to develop prevention and preventative therapies that do not rely on antibiotics and protect against the development of the initial OM episode., Competing Interests: Declaration of competing interest Ruth B Thornton: Nothing to disclose. Johanna Nokso-Koivisto: None. Kristian Riesbeck has received recent funding from Pfizer (Sweden), and has taken part in scientific advisory boards arranged by GSK (Belgium) and Merck (USA). Peter Richmond has served on vaccine scientific advisory boards and received institutional funding for investigator-initiated grants, from GlaxoSmithKline and Pfizer. Kenneth L Brockman: Nothing to disclose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: To update the medical literature on recent large-scale studies employing bioinformatics data analysis tools in otitis media (OM) disease models with a principal focus on developments in the past 5 years., Data Sources: Pubmed indexed peer-reviewed articles., Review Methods: Comprehensive review of the literature using the following search terms: 'genomics, inflammasome, microRNA, proteomics, transcriptome, bioinformatics' with the term 'otitis media', and 'middle ear'. Included articles published in the English language from January 1, 2015-April 1, 2019., Implications for Practice: Large scale bioinformatics tools over the past five years lend credence to the paradigm of innate immune response playing a critical role in host defense against bacteria contributing to Otitis Media (OM) progression from acute to chronic. In total, genomic, miRNAomic, and proteomic analyses all point to the need for a tightly regulated innate immune and inflammatory response in the middle ear. Currently, there is an urgent need for developing novel therapeutic strategies to control immunopathology and tissue damage, improve hearing and enhance host defense for both acute and chronic OM based on full understanding of the basic molecular pathogenesis of OM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Repeat ventilation tube insertion (VTI) is common in children with recurrent acute otitis media (rAOM). Identifying risk factors associated with repeat surgery will improve clinical management and prevent repeat VTI., Methods: Surgical records were assessed at 8 years following VTI surgery for rAOM in children 6-36 months of age. Children were grouped according to detection of bacterial otopathogen in their middle ear effusion (MEE) at the time of VTI, and outcomes for future otorhinolaryngology surgery compared., Results: Age, gender, pneumococcal vaccination status, antibiotic usage, day-care attendance, number of siblings and number of AOM episodes were similar between groups. Of the 63 children who had PCR +ve MEE, 58.7% required repeat VTI compared with 31.4% of the 51 children with no otopathogen detected in their MEE (odds ratio = 3.1, 95% confidence interval [1.4-6.8]; P = 0.004). Nontypeable Haemophilus influenzae (NTHi) was the predominant otopathogen in MEE (79% of all PCR +ve MEE). Respiratory virus detection was not associated with repeat VTI., Conclusions: Presence of bacterial otopathogen, specifically nontypeable H. influenzae, in the middle ear during VTI was a predictor of children at-risk of repeat VTI. Here, we identify a modifiable microbiologic factor for repeat VTI that can be targeted to improve clinical management of rAOM.

Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear. In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative (absence of bacterial otopathogen), n = 26; PCR-positive/culture-negative (presence of nonculturable bacterial otopathogen), n = 26; PCR-positive/culture-positive (presence of culturable bacterial otopathogen), n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFβ) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis., Competing Interests: Professor Peter Richmond has served on vaccine scientific advisory boards, and together with Dr Kirkham has received institutional funding for investigator-initiated grants from GlaxoSmithKline and Pfizer that are unrelated to this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Acute otitis media (AOM) is a common ear infection caused by respiratory viruses and bacteria of the nasopharynx. The present study aimed to detect various respiratory viruses and bacteria in middle ear fluid (MEF) and nasopharyngeal aspirates (NPA) using polymerase chain reaction (PCR)., Methods: We collected MEF and NPA samples from 122 pediatric patients with AOM. Real-time PCR detected 11 types of respiratory viruses (respiratory syncytial virus A/B, parainfluenza virus 1/2/3, human metapneumovirus, influenza virus A/B, adenovirus, human bocavirus and rhino virus) and 7 types of bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus pyogenes, Legionella pneumophila and Moraxella catarrhalis). MEF specimens were also examined using bacterial culture., Results: At least 1 respiratory viral or bacterial pathogen was detected in MEF of 120 cases (98%) by viral and bacterial PCR and of 93 cases (76%) by viral PCR and bacterial culture. Respiratory viruses were detected in NPA of 84 cases (69%) and MEF of 67 cases (55%). The most common virus detected in MEF was respiratory syncytial virus (21%), followed by parainfluenza virus (15%). All the viruses present in MEF were also detected in NPA specimens. Bacteria were detected by PCR in MEF of 109 cases (89%); H. influenzae was the most frequently detected (65%)., Conclusions: In many cases, pediatric AOM was found to constitute a respiratory polymicrobial infection. Multiplex PCR was useful to detect multiple respiratory viruses and bacteria in AOM. To understand intractable AOM, further studies regarding the clinical features of each viral and bacterial coinfection are required.

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The pathology of AOM increases during long-term infection in the middle ear (ME), but the host cellular immune response to bacterial infection in this inflamed environment is poorly understood. Using the Junbo mouse, a characterized NTHi infection model, we analyzed the cellular response to NTHi infection in the Junbo mouse middle ear fluid (MEF). NTHi infection increased the total cell number and significantly decreased the proportion of live cells in the MEF at day 1, and this further decreased gradually on each day up to day 7. Flow cytometry analysis showed that neutrophils were the dominant immune cell population in the MEF and that NTHi infection significantly increased their proportion whereas it decreased the monocyte, macrophage, and dendritic cell proportions. Neutrophil and macrophage numbers increased in blood and spleen after NTHi infection. The T-cell population was dominated by T-helper (Th) cells in noninoculated MEF, and the effector Th (CD44 + ) cell population increased at day 2 of NTHi infection with an increase in IL-12p40 levels. Sustained NTHi infection up to 3 days increased the transforming growth factor β levels, decreasing the effector cell population and increasing the T-regulatory (T-reg) cell population. In the preinflamed ME environment of the Junbo mouse, neutrophils are the first responder to NTHi infection followed by T-reg immune suppressive cells. These data indicate that sustained NTHi infection in the ME induces the immune suppressive response by inducing the T-reg cell population and reducing immune cell infiltration, thus promoting longer-term infection., (© Crown copyright 2019.)

Objective: The aim of this study was to evaluate the composition and the diversity of bacteriome in middle ear effusion (MEE) and adenoid specimens of pediatric patients having otitis media with effusion (OME)., Materials and Methods: Sample collection from children with OME followed by next generation sequencing. Seventeen adenoid and 43 middle ear effusion specimens from 25 children having OME were evaluated. Microbiome analysis was performed via Ion 16S rRNA metagenomics kit., Results: Twenty-two different bacterial species were identified from all of the samples analyzed. There were variations in the prevalence and relative abundance of the bacteriome observed between adenoid and MEE samples. MEE microbiome was significantly dominated by Alloicoccus otitis (44%), Turicella otitidis (6%), and Staphylococcus auricularis (3%). Whereas, Rothia mucilaginosa (39%), R. dentocariosa (11%), S. aureus (5%), Veillonella rogosae (2%), Granulicatella elegans (2%), Granulicatella adiacens (2%), Eikenella corrodens (1%), and Prevotella nanceiensis (1%) had significantly higher relative abundance in adenoid samples. Overall, there was no statistically significant difference in alpha diversity of MEE and adenoid samples, whereas adenoid samples constituted a cluster in the beta diversity graph., Conclusion: Bacteriome of MEE is mostly dominated by A. otitis yet accompanied by other bacteria with lower relative abundances suggests that OME is likely to be a polymicrobial process. Despite similarities, significant differences in relative abundances of several predominant species between bacteriome in the MEE and adenoid put the theory that OME in children is originated from the adenoids under question., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chronic otitis media with effusion (COME) is a common childhood disease characterized by an accumulation of fluid behind the eardrum. COME often requires surgical intervention and can also lead to significant hearing loss and subsequent learning disabilities. Recent characterization of the middle ear fluid (MEF) microbiome in pediatric patients has led to an improved understanding of the microbiota present in the middle ear during COME. However, it is not currently known how the MEF microbiome might vary due to other conditions, particularly respiratory disorders. Here, we apply an amplicon sequence variant (ASV) pipeline to MEF 16S rRNA high-throughput sequencing data from 50 children with COME (ages 3-176 months) undergoing tube placement. We achieve a more detailed taxonomic resolution than previously reported, including species and genus level resolution. Additionally, we provide the first report of the functional roles of the MEF microbiome and demonstrate that despite high taxonomic diversity, the functional capacity of the MEF microbiome remains uniform between patients. Furthermore, we analyze microbiome differences between children with COME with and without a history of lower airway disease (i.e., asthma or bronchiolitis). The MEF microbiome was less diverse in participants with lower airway disease than in patients without, and phylogenetic β-diversity (weighted UniFrac) was significantly different based on lower airway disease status. Differential abundance between patients with lower airway disease and those without was observed for the genera Haemophilus, Moraxella, Staphylococcus, Alloiococcus , and Turicella . These findings support previous suggestions of a link between COME and respiratory illnesses and emphasize the need for future study of the middle ear and respiratory tract microbiomes in diseases such as asthma and bronchiolitis., (Copyright © 2019 Kolbe, Castro-Nallar, Preciado and Pérez-Losada.)

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.

Otitis media (OM) is one of the most common ear diseases affecting humans. Children are at greater risk and suffer most frequently from OM, which can cause serious deterioration in the quality of life. OM is generally classified into two main types: acute and chronic OM (AOM and COM). AOM is characterized by tympanic membrane swelling or otorrhea and is accompanied by signs or symptoms of ear infection. In COM, there is a tympanic membrane perforation and purulent discharge. The most common pathogens that cause AOM are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis whereas Pseudomonas aeruginosa and Staphylococcus aureus are commonly associated with COM. Innate and adaptive immune responses provide protection against OM. However, pathogens employ a wide arsenal of weapons to evade potent immune responses and these mechanisms likely contribute to AOM and COM. Immunologic evasion is multifactorial, and involves damage to host mucociliary tract, genetic polymorphisms within otopathogens, the number and variety of different otopathogens in the nasopharynx as well as the interaction between the host's innate and adaptive immune responses. Otopathogens utilize host mucin production, phase variation, biofilm production, glycans, as well as neutrophil and eosinophilic extracellular traps to induce OM. The objective of this review article is to discuss our current understanding about the mechanisms through which otopathogens escape host immunity to induce OM. A better knowledge about the molecular mechanisms leading to subversion of host immune responses will provide novel clues to develop effective treatment modalities for OM., (©2019 Society for Leukocyte Biology.)

Otitis media (OM) is a cluster of diseases of the middle ear that commonly result from bacterial infection. OM subtypes in which the tympanic membrane is intact (acute otitis media and otitis media with effusion) are presumed to result from pathogen translocation through the eustachian tube. Recent molecular-based studies have suggested that a diverse middle ear microbiome exists in the absence of disease. These have been largely unsupported by culture and feature species that commonly contaminate low-biomass sequencing data. Combining culture-based and molecular techniques, we undertook a detailed investigation of the evidence for bacterial colonization of the healthy middle ear. Middle ear (ME), nasopharynx (NP), and external ear canal (EC) swabs were collected from a total of 25 adult patients undergoing cochlear implant, stapedotomy, or translabyrinthine vestibular schwannoma resection. Diagnostic culture, microscopy, quantitative PCR, and 16S rRNA gene amplicon sequencing were used to assess sample bacterial content. EC and NP microbiota were consistent with previous reports. In contrast, bacterial levels in ME samples were not significantly above those in unused control swabs. Commonly detected taxa were among recognized sequencing contaminants ( Methylobacterium , Pseudomonas , and Acinetobacter ). Linear regression of dominant ME taxa confirmed a negative relationship between relative abundance and bacterial load, consistent with contamination. No bacteria were detected by microscopy or diagnostic culture in any middle ear sample. Our findings cast substantial doubt on previous reports identifying a healthy middle ear microbiome using 16S amplicon sequencing. IMPORTANCE Recent molecular-based studies have suggested that a diverse middle ear microbiome in adults and children can exist in the absence of disease. These studies have been largely unsupported by culture and feature species that commonly contaminate low-biomass sequencing data. While 16S rRNA gene amplicon sequencing has proven to be a highly informative technique in many clinical contexts, it is susceptible to spurious signal arising from sequencing reagent contaminants where sample biomass is low. Combining culture-based and molecular techniques, we undertook a detailed investigation of the evidence for bacterial colonization of the healthy middle ear. In finding no evidence of viable bacterial cells in middle ear samples, our study further underlines the importance of careful consideration of amplicon sequence data derived from very-low-biomass contexts and the value of analytical approaches that combine culture and molecular techniques., (Copyright © 2019 Jervis-Bardy et al.)

Summary To study the clinical features, diagnosis and treatments of tuberculous otomastoiditis. One case is reported and literatures are reviewed. In this report, the specimen of the middle ear revealed the presence of acid fast bacilli（AFB）, grew mycobacterium tuberculosis on a culture. Lung CT scans demonstrated tuberculosis. After anti-tuberculosis therapy, the symptoms disappeared and did not recur in 3 years. Hydrotympanum can be the early symptom of tuberculous otomastoiditis. When persistent otorrhea can not be improved by conventional therapy, tuberculous otomastoiditis should be considered as one of the differential diagnosis., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) bacteria play an important role in multiple respiratory tract diseases. Visual inspection of the morphology of biofilms formed during chronic infections shows distinct differences from biofilms formed in vitro To better understand these differences, we analyzed images of NTHI biofilms formed in the middle ears of Chinchilla lanigera and developed an in silico agent-based model of the formation of NTHI biofilms in vivo We found that, as in vitro , NTHI bacteria are organized in self-similar patterns; however, the sizes of NTHI clusters in vivo are more than 10-fold smaller than their in vitro counterparts. The agent-based model reproduced these patterns and suggested that smaller clusters occur due to elimination of planktonic NTHI cells by the host responses. Estimation of model parameters by fitting simulation results to imaging data showed that the effects of several processes in the model change during the course of the infection. IMPORTANCE Multiple respiratory illnesses are associated with formation of biofilms within the human airway by NTHI. However, a substantial amount of our understanding of the mechanisms that underlie NTHI biofilm formation is obtained from in vitro studies. Our in silico model that describes biofilm formation by NTHI within the middle ears of Chinchilla lanigera will help isolate processes potentially responsible for the differences between the morphologies of biofilms formed in vivo versus those formed in vitro Thus, the in silico model can be used to glean mechanisms that underlie biofilm formation in vivo and connect those mechanisms to those obtained from in vitro experiments. The in silico model developed here can be extended to investigate potential roles of specific host responses (e.g., mucociliary clearance) on NTHI biofilm formation in vivo The developed computational tools can also be used to analyze and describe biofilm formation by other bacterial species in vivo ., (Copyright © 2019 Brown et al.)

Chronic suppurative otitis media (CSOM) is one of the most common infectious diseases of the middle ear especially affecting children, leading to delay in language development and communication. Although Staphylococcus aureus is the most common pathogen associated with CSOM, its interaction with middle ear epithelial cells is not well known. In the present study, we observed that otopathogenic S. aureus has the ability to invade human middle ear epithelial cells (HMEECs) in a dose and time dependent manner. Scanning electron microscopy demonstrated time dependent increase in the number of S. aureus on the surface of HMEECs. We observed that otopathogenic S. aureus primarily employs a cholesterol dependent pathway to colonize HMEECs. In agreement with these findings, confocal microscopy showed that S. aureus colocalized with lipid rafts in HMEECs. The results of the present study provide new insights into the pathogenesis of S. aureus induced CSOM. The availability of in vitro cell culture model will pave the way to develop novel effective treatment modalities for CSOM beyond antibiotic therapy.

Vaccinations with the 10-valent pneumococcal conjugated vaccine (PHiD-CV) started in Iceland in 2011. Protein D (PD) from H. influenzae , which is coded for by the hpd gene, is used as a conjugate in the vaccine and may provide protection against PD-positive H. influenzae We aimed to evaluate the effect of PHiD-CV vaccination on H. influenzae in children, both in carriage and in acute otitis media (AOM). H. influenzae was isolated from nasopharyngeal swabs collected from healthy children attending 15 day care centers in 2009 and from 2012 to 2017 and from middle ear (ME) samples from children with AOM collected from 2012 to 2017. All isolates were identified using PCR for the hpd and fucK genes. Of the 3,600 samples collected from healthy children, 2,465 were culture positive for H. influenzae (68.5% carriage rate); of these, 151 (6.1%) contained hpd -negative isolates. Of the 2,847 ME samples collected, 889 (31.2%) were culture positive for H. influenzae ; of these, 71 (8.0%) were hpd negative. Despite the same practice throughout the study, the annual number of ME samples reduced from 660 in 2012 to 330 in 2017. The proportions of hpd -negative isolates in unvaccinated versus vaccinated children were 5.6% and 7.0%, respectively, in healthy carriers, and 5.4% and 7.8%, respectively, in ME samples. The proportion of hpd -negative isolates increased with time in ME samples but not in healthy carriers. The number of ME samples from children with AOM decreased. The PHiD-CV had no effect on the proportion of the hpd gene in H. influenzae from carriage, but there was an increase in hpd -negative H. influenzae in otitis media. The proportions of hpd -negative isolates remained similar in vaccinated and unvaccinated children., (Copyright © 2019 Sveinsdóttir et al.)

Background: Acute otitis media (AOM) is one of the most common childhood infections, generally thought to be caused by ascension of bacteria from the nasopharynx (NP) to the middle ear. Using 16S ribosomal RNA-based sequencing, we evaluated the relationship between the NP and middle ear fluid (MEF) microbiota in children with AOM with tympanostomy tubes (AOMT) as a proxy for AOM and explored whether microbiota profiling predicts natural disease course., Methods: Microbiota profiles of paired NP and MEF samples of 94 children below 5 years of age with uncomplicated AOMT were determined., Results: Local diversity (P < 0.001) and overall microbiota composition (P < 0.001) of NP and MEF samples differed significantly, although paired NP and MEF samples were much more similar than unpaired samples (P < 0.001). High qualitative agreement between the presence of individual bacteria in both niches was observed. Abundances of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Turicella otitidis, Klebsiella pneumoniae and Haemophilus spp. were strongly correlated between the 2 niches. Additionally, P. aeruginosa, S. aureus, T. otitidis and Streptococcus pneumoniae abundance in NP were predictive of the presence of a range of oral types of bacteria in MEF. Interestingly, there was no association between Moraxella catarrhalis in NP and MEF samples, which was highly present in NP but virtually absent in MEF. Finally, the NP microbiota composition could predict duration of AOMT, even better than MEF microbiota., Conclusions: We observed substantial correlations between paired NP and MEF microbiota in children with AOMT. Our data also suggest that NP microbiota profiling deserves further exploration as tool for future treatment decisions.

Background: Biofilms are communities of bacteria embedded in a self-produced glycocalyx matrix. Adenoids have been shown to harbor bacterial biofilms. Aim/objectives: To compare the prevalence of biofilms in adenoid of children with chronic otitis media (COM) (group1) versus a control group without any COM (group 2) having adenoids removed because of hypertrophy., Material and Methods: One hundred and three children were prospectively enrolled in this case-control study, group 1 (n = 52) and group 2 (n = 51). The main outcome measurement was the prevalence of biofilm in adenoidectomy specimens analyzed using confocal laser scanning microscopy. Children in group 1 who had middle ear (ME) effusion and requiring the insertion of a tympanostomy tube underwent biopsy of the ME mucosa and effusion sampling., Results: Biofilms were found in adenoids' specimens of both groups and in the ME biopsy and effusion. The biofilm prevalence in adenoids was 63.5% (33/52) in group 1 and 47.1% (24/51) in group 2. Day nursery and previous antibiotics intake were significantly more frequent in group 1 than in group 2., Conclusions and Significance: This case-control study demonstrates that adenoid tissue in children with COM contains more mucosal biofilms than adenoid tissue removed for hypertrophy. Biofilm was seen in ME biopsies and effusion.