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Background: Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Currently, there is an urgent need for developing novel therapeutic agents for treating OM based on full understanding of molecular pathogenesis in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM., Objective: To provide a state-of-the-art review concerning recent advances in OM in the areas of molecular biology, biochemistry, genetics, and animal model studies and to discuss the future directions of OM studies in these areas., Data Sources and Review Methods: A structured search of the current literature (since June 2007). The authors searched PubMed for published literature in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM., Results: Over the past 4 years, significant progress has been made in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. These studies brought new insights into our understanding of the molecular and biochemical mechanisms underlying the molecular pathogenesis of OM and helped identify novel therapeutic targets for OM., Conclusions and Implications for Practice: Our understanding of the molecular pathogenesis of OM has been significantly advanced, particularly in the areas of inflammation, innate immunity, mucus overproduction, mucosal hyperplasia, middle ear and inner ear interaction, genetics, genome sequencing, and animal model studies. Although these studies are still in their experimental stages, they help identify new potential therapeutic targets. Future preclinical and clinical studies will help to translate these exciting experimental research findings into clinical applications.

Bacteria can grow as free-floating, planktonic bacteria or complex communities called biofilms. Biofilms promote bacterial growth and diversity and offer bacteria unique environments, including aerobic and anaerobic layers, that facilitate resistance to antimicrobial therapies. Respiratory and related structures provide ideal environments for the development of bacterial biofilms, which predispose patients to recurrent and chronic infections. Biofilms are important for the persistence of chronic rhinosinusitis, pulmonary infections in cystic fibrosis, chronic otitis media, and device-related infections. Antimicrobial therapy that is proven effective against planktonic bacteria is often insufficiently effective against the defenses of biofilms. Furthermore, biofilms modify themselves following exposure to antimicrobial therapy, thus developing increased resistance. Understanding the nature of biofilms in common pediatric infections is essential to comprehending the expected course of bacterial illness and identifying treatments that are most likely to be beneficial against more resistant biofilms.

Background: Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced., Methodology/principal Findings: For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity., Conclusions/significance: As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.

Purpose of Review: Biofilms have been shown to play a role in otitis media, sinusitis, cholesteatoma, tonsillitis, adenoiditis, and device infections. This article is written to review recent advances in the field., Recent Findings: The role of biofilms in the persistence of chronic, mucosal-based ENT-related infections was first recognized in otitis media. Definitive proof was lacking until the demonstration of bacterial biofilms on the middle-ear mucosa of children, not only with chronic otitis media with effusion, but also with recurrent otitis media. Strains of Pseudomonas aeruginosa isolated from cholesteatoma are avid biofilm formers. Biofilms have been reported in the adenoids of children with chronic rhinosinusitis, helping to explain the clinical observation that adenoidectomy can be beneficial to children with chronic otitis or chronic rhinosinusiti. Additional studies have confirmed the presence of biofilms in chronic tonsillitis. Biofilms have also been shown to be involved in infected cochlear implants and tracheotomy tubes., Summary: The recognition that chronic otolaryngologic bacterial infections are biofilm related has been the impetus for the development of new technologies for the study of biofilms and their prevention and treatment. Understanding that chronic bacterial infections are biofilm related is fundamental to developing rationale strategies for treatment and prevention.

Background: The nontypeable Haemophilus influenzae (NTHi) are associated with a spectrum of respiratory mucosal infections including: acute otitis media (AOM); chronic otitis media with effusion (COME); otorrhea; locally invasive diseases such as mastoiditis; as well as a range of systemic disease states, suggesting a wide range of virulence phenotypes. Genomic studies have demonstrated that each clinical strain contains a unique genic distribution from a population-based supragenome, the distributed genome hypothesis. These diverse clinical and genotypic findings suggest that each NTHi strain possesses a unique set of virulence factors that contributes to the course of the disease., Results: The local and systemic virulence patterns of ten genomically characterized low-passage clinical NTHi strains (PittAA - PittJJ) obtained from children with COME or otorrhea were stratified using the chinchilla model of otitis media (OM). Each isolate was used to bilaterally inoculate six animals and thereafter clinical assessments were carried out daily for 8 days by blinded observers. There was no statistical difference in the time it took for any of the 10 NTHi strains to induce otologic (local) disease with respect to any or all of the other strains, however the differences in time to maximal local disease and the severity of local disease were both significant between the strains. Parameters of systemic disease indicated that the strains were not all equivalent: time to development of the systemic disease, maximal systemic scores and mortality were all statistically different among the strains. PittGG induced 100% mortality while PittBB, PittCC, and PittEE produced no mortality. Overall Pitt GG, PittII, and Pitt FF produced the most rapid and most severe local and systemic disease. A post hoc determination of the clinical origins of the 10 NTHi strains revealed that these three strains were of otorrheic origin, whereas the other 7 were from patients with COME., Conclusion: Collectively these data suggest that the chinchilla OM model is useful for discriminating between otorrheic and COME NTHi strains as to their disease-producing potential in humans, and combined with whole genome analyses, point the way towards identifying classes of virulence genes.

Context: Chronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection., Objective: To test the hypothesis that chronic OM in humans is biofilm-related., Design, Setting, and Patients: Middle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice., Main Outcome Measures: Confocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively., Results: Of the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation., Conclusion: Direct detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.

Context: Otitis media (OM) is the most common reason that a child undergoes a general anesthetic, with the total costs of treating this disease exceeding five billion dollars annually. Concerns regarding the development of antibiotic-resistant organisms in response to medical treatment for OM have fueled the demand for surgical intervention. However, reimbursements are decreasing. Non-traditional settings for children requiring bilateral myringotomy and tube (BMT) placement for ear disease have the potential to offer the same degree of patient safety and improved efficiency but at lower cost., Objective: To develop a non-traditional setting for BMT surgery that is safe, cost effective, and well received by patients, families and staff., Design: Prospective design of an outpatient treatment suite (OTS) for BMT placement; prospective evaluation of safety and family satisfaction; analysis of costs., Setting: A 778 bed US urban area level one trauma center and teaching hospital, with a 2160 ft(2) electro-convulsive therapy suite that was underutilized and non-revenue generating on Tuesdays and Thursdays., Participants: A design task force of health care providers, administrators and operations personnel; 794 healthy children between the ages of 6 months to 16 years requiring BMT surgery; 100 families of patients., Main Outcome Measures: Financial comparison was made between the traditional operating room (OR) setting, the outpatient surgery center (SC) and the OTS comparing overhead and indirect costs to run each site. A prospective survey was conducted of 100 consecutive patients undergoing surgery between November 2000 and June 2001. The survey was conducted at the 2 weeks postoperative check and was composed of 18 questions divided into five sections, with a 5-point rating scheme, with one being very poor, and five being very good., Results: Designing a new treatment venue was successful because of teamwork and a willingness to think creatively. The OTS was found to be far more cost-effective than both the main OR and SC for BMTs. The contribution to margin for the SC was US$ 280 per case and for the main OR was US$ 2130 per case. By operating on 794 patients in OTS, the hospital was able to generate additional contribution to margin of US$ 197,100 when compared to the cost of performing these cases in the SC and US$ 1,499,500 when compared to performing all cases in the main OR. No adverse consequences were noted. Patient/Family satisfaction was also rated very high, with an overall rating of 4.85 and markedly reduced time in hospital., Conclusion: Operating rooms (ORs) today are busier than in years past, but revenues barely meet or in some cases fall below expenses due to insurers' decreased reimbursement. This innovative approach to BMT placement has been shown to be safe and results in excellent family satisfaction, with a substantial contribution to margin. As over one million BMT cases are performed annually in the US, adoption of this approach nationally has the potential to markedly reduce the treatment costs of this common disease.

Unlabelled: Haemophilus influenzae is one of the most important respiratory pathogens of man. It has been etiologically associated with otitis media, otorrhea, and chronic obstructive pulmonary disease. Identification of new genomic elements will provide novel targets to fight chronic infections caused by this organism., Objective: The new paradigm that chronic infections are caused by bacterial biofilms prompted us to study the relationship between bacterial pathogenicity, biofilm formation and bacterial communal cooperation. To do this, it is essential to determine the virulence gene sets that are involved in the above processes and whether they are present in every bacterial cell or distributed in a "communal gene-pool", the distributed genome hypothesis (DGH). We designed, constructed and characterized a highly redundant genomic DNA library comprised of the genomes of ten low passage clinical isolates of H. influenzae carrying large numbers of genes that are not present in the laboratory strains of H. influenzae., Methods: Genomic DNA fragments of the ten clinical strains were hydro-dynamically sheared to produce a mean fragment size of 1.5-2.5 kb. The ten sheared DNAs were than pooled and used in the construction of a genomic library with 76800 clones., Results: Our restriction endonuclease and sequence analyses of 800 clones demonstrate that 75% of the clones carry an insert larger than 0.5 kb. The library has an approximately 1.5 kb average insert size, and therefore, better than 4.5x redundancy for each of the genomes of the ten clinical isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that a high percentage of genes (75 clones, 11% of the 686 sequenced clones) present in this library are not represented in the genome of the reference strain H. influenzae Rd., Conclusions: The library, based on the above results, has a better than 4.5x coverage for each of the ten constituent genomes. On the basis of our preliminary sequencing data ( approximately 1 million nucleotides) the library lacks of highly repeated sequences, therefore, the expected genome coverage (4.5x) is not degraded. Using the prevalence of non-Rd like sequences (11%) detected during characterization of the genomic library, we estimated that the library contains DNA sequences equivalent to approximately 2 million bp, which are not represented in the reference genome of the H. influenzae Rd strain and that is greater in size than the genome of this reference strain, providing ample targets for innovative drug design.

Almost every child will suffer at least one episode of otitis media (OM). Therefore, it is not immediately obvious that there is a genetic predisposition to the development of the disease. However, evidence from a variety of studies has shown that there is a clear genetic component to susceptibility to OM. Methodologies for discovering these genes are described within this article. The identification of OM susceptibility genes allows the development of molecular diagnostic assays that will inform the clinician as to which child is at increased risk and warrants more aggressive intervention. Delineating the OM genes not only provides a deeper understanding of the pathophysiology of this common disease, but also of acute and chronic bacterial disease in general.

Context: Chronic otitis media with effusion (OME) has long been considered to be a sterile inflammatory process. The previous application of molecular diagnostic technologies to OME suggests that viable bacteria are present in complex communities known as mucosal biofilms; however, direct imaging evidence of mucosal biofilms associated with OM is lacking., Objective: To determine whether biofilm formation occurs in middle-ear mucosa in an experimental model of otitis media., Design and Materials: A total of 48 research-grade, young adult chinchillas weighing 500 g were used for 2 series of animal experiments: one to obtain specimens for scanning electron microscopy and the other to obtain specimens for confocal laser scanning microscopy using vital dyes. In each series, 21 animals were bilaterally injected with viable Haemophilus influenzae bacteria and 1 was inoculated to account for expected mortality. Three served as negative controls. Effusions and mucosal specimens were collected from 2 infected animals that were euthanized at 3, 6, 12, and 24 hours and at days 2, 4, 5, 10, 16, and 22 after inoculation., Main Outcome Measures: Images were analyzed for biofilm morphology, including presence of microcolony formation and for presence of bacteria on tissue surfaces., Results: Scanning electron microscopy demonstrated that biofilm formation was evident in all specimens from animals beginning 1 day after infection and was present through 21 days. Confocal laser scanning microscopy indicated that bacteria within the biofilms are viable., Conclusion: These preliminary findings provide evidence that mucosal biofilms form in an experimental model of otitis media and suggest that biofilm formation may be an important factor in the pathogenesis of chronic otitis media with effusion.

Multiplex PCR analyses for both bacterial and viral pathogens were conducted in a blinded manner on 33 archival specimens, of known culture status, procured from chinchilla models of both single- and mixed-pathogen-induced otitis media and from a pediatric patient. These specimens had been maintained at -70 degrees C for up to 6 years. Experimental specimens evaluated included middle-ear effusions, nasopharyngeal lavage fluids and middle-ear lavage fluids from animals which were immunologically naive, sham-immunized or actively immunized with nontypeable Haemophilus influenzae antigens. Sampling times used ranged from the day of bacterial or viral challenge to 42 days after challenge. Initial PCR analyses of the 33 specimens matched the traditional culture data in 24 instances (73%), correctly identifying nontypeable H. influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, or adenovirus as the causative agent. A PCR-positive signal for the microbe(s) inoculated was also obtained in four animal model specimens (12%) which were culture negative. One of two culture-negative human effusions was also PCR positive. Thus, overall, results obtained by blinded PCR were 85% concordant with traditional culture methods or correctly indicated the specific pathogen introduced in four specimens that were sterile. In no instance was a false-positive signal obtained for any of the five etiologic agents being evaluated. We conclude that the multiplex PCR analyses are rapid and accurate methodologies when they are used to retrospectively evaluate diverse archival specimens of limited volume from experimental models of otitis media.

Purpose: Bacterial deoxyribonucleic acid (DNA) has been previously detected by polymerase chain reactions (PCR) in a significant percentage of culturally-sterile pediatric middle-ear effusions. The current study was designed to determine whether this represents the existence of viable bacteria or the persistence of residual DNA in the middle-ear cleft., Materials and Methods: The middle-ear cavities of two sets of chinchillas were inoculated with either: 1) 100 colony-forming units (CFU) of live Haemophilus influenzae, 2.2 x 10(6) CFU of pasteurized Moraxella catarrhalis, and 1000 ng of DNA (>10(8) genomic equivalents) from Streptococcus pneumoniae; or 2) 100 CFU of live S pneumoniae, 2.2 x 10(6) CFU of pasteurized M catarrhalis and 1000 ng of purified DNA from H influenzae. Animals were treated with ampicillin for 5 days beginning on day 3. A single-point longitudinal study design was used for sampling to eliminate the possibility of contamination., Results: No DNA was detectable from the heat-killed bacteria or the purified DNA after day 3. However, DNA from the live bacteria persisted through day 21, even though all specimens were culture-negative following the initiation of antimicrobial therapy., Conclusion: These findings indicate that purified DNA and DNA from intact but nonviable bacteria do not persist in the middle-ear cleft in the presence of an effusion, even following high copy inoculation. In contrast, antibiotic-treated bacteria persist in some viable state for weeks as evidenced by the differential ability of the PCR-based assay systems to detect the live bacteria, but not detect the heat-killed organisms.

To better understand the significance of viral upper respiratory tract infections in the pathogenesis of acute otitis media (OM), 27 adults underwent intranasal inoculation with influenza A virus. Monitoring consisted of antibody titer determination, tympanometry, and otoscopy. Microbiologic analysis consisted of cultures and polymerase chain reaction (PCR)-based detection for influenza A virus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. All subjects became infected with the challenge virus. By day 4, 16 (59%) developed middle ear pressures of -100 mm H2O or below and 4 (25%) of them developed OM. One subject (4%) developed purulent OM requiring myringotomy for pain relief. Middle ear effusion cultures were negative. PCR analysis of that subject's middle ear effusion and nasal washes were positive for influenza A virus and S. pneumoniae. These findings support a causal role for viral upper respiratory tract infections in the pathogenesis of OM, possibly mediated by middle ear underpressures and viral and bacterial middle ear infection.

Background: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates. Results: We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons. Conclusions: These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences. [ABSTRACT FROM AUTHOR]

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

Summary: Objective: To create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis. Methods: High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of ∼1.5kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with ∼250,000 clones that was arrayed and subjected to quality control analyses. Results: Restriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5kb with the desired mean insert size of 1.4kb. Thus, this library provides better than 4.5× coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort (∼1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01. Conclusions: Our data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome. [Copyright &y& Elsevier]

Summary: Objectives: To identify mucin genes in chinchilla middle ear epithelium and characterize complimentary deoxyribonucleic acid (cDNA) sequences to facilitate further investigations into mucin physiology and pathophysiology on a molecular level using the chinchilla model. Methods: Chinchilla mucin gene exploration and cDNA characterization was accomplished using reverse transcriptase-polymerase chain reactions (RT-PCR). Forward and reverse primer pairs were designed using consensus sequences available for human and rodent species. Chinchilla middle ear epithelium was harvested and primary cell cultures (CMEEC) were established. The CMEEC were explored for the expression of chinchilla mucin genes 1, 2, 4 and 5AC (cMuc1, cMuc2, cMuc4 and cMuc5AC). Identified cDNA amplicons for each of these genes was sequenced and homology compared to previously published human and rodent sequences. Results: CMEEC express all four of the mucin genes cMuc1, cMuc2, cMuc4 and cMuc5AC. cDNA amplicons for each of the genes were able to be sequenced with lengths ranging from 66 to 362 base pairs. Each of the chinchilla cDNA sequences expressed significant homology with published human and rodent cDNA for these mucin genes. A cDNA sequence for the housekeeping gene, β-actin, was also identified. Conclusions: Chinchilla middle ear epithelium grown in culture expresses the mucin genes 1, 2, 4 and 5AC, which have been identified as important in mucin regulation in the middle ear. cDNA sequences corresponding to these mucin genes were identified and may serve as important molecular tools in future studies of otitis media using the chinchilla model. [Copyright &y& Elsevier]

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching ∼8 x 108 cells and ∼15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed. [ABSTRACT FROM AUTHOR]