Otitis Media in Children with Down Syndrome Is Associated with Shifts in the Nasopharyngeal and Middle Ear Microbiotas.

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.

Otopathogens in the middle ear and nasopharynx of children with recurrent acute otitis media.

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.

Predominant Bacterial and Viral Otopathogens Identified Within the Respiratory Tract and Middle Ear of Urban Australian Children Experiencing Otitis Media Are Diversely Distributed.

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.

Contribution of a Novel Pertussis Toxin-Like Factor in Mediating Persistent Otitis Media.

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.

Ear microbiota and middle ear disease: a longitudinal pilot study of Aboriginal children in a remote south Australian setting.

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 (rs = - 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.

Generalized tympanism in a horse and its possible association with Sarcina-like microorganism: A case report.

Background: Sarcina spp. is a Gram-positive, coccoid microorganism that forms tetrads or octets, and is observed with a characteristic "bundle" arrangement. The most recognized species are Sarcina ventriculi and Sarcina maxima. It has been described as part of the normal microbiota in horses and cats, but it has also been linked to abomasal bloat in goats, lambs, and calves, although its causality has not been proven yet. Case Description: This work presents the case of a 3-months-old female horse that died of generalized tympanism. Macroscopic findings showed mild cyanosis and abundant gas in the lumen of the stomach, and small and large bowel. Microscopically, high numbers of Gram-positive microorganisms compatible with Sarcina spp. in the gastric lumen and on the surface of the small and large bowel were observed, along with mild inflammation. Conclusion: The severe tympanism was the only relevant lesion observed and could explain the death of the animal. Although it is not possible to determine a relationship between these lesions and Sarcina spp., it is interesting to highlight that the high amount of these bacteria could be associated with gas production and tympanism. It is important to continue investigating the role of Sarcina spp. in horses, and its possible link with tympanism.

Microbiological investigation of samples collected from healthy middle ears during cochlear implant surgery.

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.

Glycolytic Metabolism Is Critical for the Innate Antibacterial Defense in Acute Streptococcus pneumoniae Otitis Media.

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-1ß 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.

Longitudinal optical coherence tomography to visualize the in vivo response of middle ear biofilms to antibiotic therapy.

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.

The Possible Role of Sclerostin in the Pathogenesis of Tympanosclerosis.

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, ß-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, ß-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.

Evaluation and comparison of tympanoplasty efficacy with tympanic membrane perforation after chronic suppurative otitis media in dry ear with different microorganisms.

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.

A single nucleotide polymorphism in an IgA1 protease gene determines Streptococcus pneumoniae adaptation to the middle ear during otitis media.

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.

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice.

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.

Otitis media susceptibility and shifts in the head and neck microbiome due to SPINK5 variants.

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.

Phase variation with altering phosphorylcholine expression of nontypeable Haemophilus influenzae affects bacteria clearance and mucosal immune response in the middle ear and nasopharynx.

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.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

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-ß (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-1ß, 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.

Site-Specific Mutations of GalR Affect Galactose Metabolism in Streptococcus pneumoniae.

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.

Patent evaluation of US2019338018 (A1) 2019-11-07 (antibody fragments for the treatment of biofilm-related disorders).

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 mIhfB4NTHI 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.

Multi-omic studies on missense PLG variants in families with otitis media.

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.

Niche- and Gender-Dependent Immune Reactions in Relation to the Microbiota Profile in Pediatric Patients with Otitis Media with Effusion.

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.