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Article Reference Novel concepts in nontypeable Haemophilus influenzae biofilm formation.
Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative microbe that frequently colonizes the human host without obvious signs of inflammation, but is also a frequent cause of otitis media in children and exacerbations in chronic obstructive pulmonary disease patients. Accumulating data suggest that NTHi can reside in biofilms during both colonization and infection. Recent literature proposes roles for phosphorylcholine, sialic acid, bacterial DNA, but also eukaryotic DNA in the development of NTHi biofilms. However, many questions remain. Until now, there are insufficient data to explain how NTHi forms biofilms. Here, we review the recent advances in NTHi biofilm formation with particular focus on the role that neutrophils may play in this process. We propose that recruitment of neutrophils facilitates NTHi biofilm formation on mucosal sites by the initiation of neutrophil extracellular traps.
Article Reference Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis.
Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.
Article Reference Non typable-Haemophilus influenzae biofilm formation and acute otitis media.
Non-typable Haemophilus influenzae (NT-Hi) infection is frequently associated with acute otitis media (AOM) treatment failure, recurrence or chronic otitis media. Persistence of otopathogens in a biofilm-structured community was implicated in these situations. Here, we compared biofilm production by H. influenzae strains obtained by culture of middle ear fluid (MEF) from children with AOM treatment failure and by strains isolated from nasopharyngeal (NP) samples from healthy children or those with AOM (first episode or recurrence). We aimed to evaluate an association of clinical signs and in vitro biofilm formation and establish risk factors of carrying a biofilm-producing strain.
Article Reference Extracellular DNA within a nontypeable Haemophilus influenzae-induced biofilm binds human beta defensin-3 and reduces its antimicrobial activity.
Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are associated with multiple chronic infections of the airway, including otitis media. Extracellular DNA (eDNA) is part of the biofilm matrix and serves as a structural component. Human β-defensin-3 (hBD-3) is a cationic antimicrobial host defense protein (AMP) critical to the protection of the middle ear. We hypothesized that anionic eDNA could interact with and bind hBD-3 and thus shield NTHI in biofilms from its antimicrobial activity. We demonstrated that recombinant hBD-3 [(r)hBD-3] bound eDNA in vitro and that eDNA in biofilms produced by NTHI in the chinchilla middle ear co-localized with the orthologue of this AMP. Incubation of physiological concentrations of (r)hBD-3 with NTHI genomic DNA abrogated the ability of this innate immune effector to prevent NTHI from forming robust biofilms in vitro. Establishment of NTHI biofilms in the presence of both DNase I and (r)hBD-3 resulted in a marked reduction in the overall height and thickness of the biofilms and rescued the antimicrobial activity of the AMP. Our results demonstrated that eDNA in NTHI biofilms sequestered hBD-3 and thus diminished the biological activity of an important effector of innate immunity. Our observations have important implications for chronicity of NTHI-induced diseases.
Article Reference The biofilm matrix destabilizers, EDTA and DNaseI, enhance the susceptibility of nontypeable Hemophilus influenzae biofilms to treatment with ampicillin and ciprofloxacin.
Nontypeable Hemophilus influenzae (NTHi) is a Gram-negative bacterial pathogen that causes chronic biofilm infections of the ears and airways. The biofilm matrix provides structural integrity to the biofilm and protects biofilm cells from antibiotic exposure by reducing penetration of antimicrobial compounds into the biofilm. Extracellular DNA (eDNA) has been found to be a major matrix component of biofilms formed by many species of Gram-positive and Gram-negative bacteria, including NTHi. Interestingly, the cation chelator ethylenediaminetetra-acetic acid (EDTA) has been shown to reduce the matrix strength of biofilms of several bacterial species as well as to have bactericidal activity against various pathogens. EDTA exerts its antimicrobial activity by chelating divalent cations necessary for growth and membrane stability and by destabilizing the matrix thus enhancing the detachment of bacterial cells from the biofilm. In this study, we have explored the role of divalent cations in NTHi biofilm development and stability. We have utilized in vitro static and continuous flow models of biofilm development by NTHi to demonstrate that magnesium cations enhance biofilm formation by NTHi. We found that the divalent cation chelator EDTA is effective at both preventing NTHi biofilm formation and at treating established NTHi biofilms. Furthermore, we found that the matrix destablilizers EDTA and DNaseI increase the susceptibility of NTHi biofilms to ampicillin and ciprofloxacin. Our observations indicate that DNaseI and EDTA enhance the efficacy of antibiotic treatment of NTHi biofilms. These observations may lead to new strategies that will improve the treatment options available to patients with chronic NTHi infections.
Article Reference Kinetic analysis and evaluation of the mechanisms involved in the resolution of experimental nontypeable Haemophilus influenzae-induced otitis media after transcutaneous immunization.
Transcutaneous immunization (TCI) is a simple and needle-free method with which to induce protective immune responses. Using a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM), we examined the efficacy afforded by TCI with a novel chimeric immunogen called 'chimV4' which targets two critical adhesins expressed by NTHI, outer membrane protein P5 and the majority subunit of NTHI Type IV pilus, PilA. Experimental OM was first established in cohorts of animals, and then TCI performed via a therapeutic immunization regime by rubbing vaccine formulations on hydrated pinnae. The kinetics of resolution of established experimental disease was evaluated by clinically-relevant assessments of OM, bacterial culture of planktonic and adherent NTHI within the middle ear and gross examination of the relative amount of NTHI mucosal biofilms within the middle ear space. Within seven days after primary TCI, a significant reduction in the signs of OM, significantly fewer NTHI adherent to the middle ear mucosa and significant resolution of mucosal biofilms was detected in animals that received chimV4+ the adjuvant LT(R192G-L211A), compared to animals administered LT(R192G-L211A) alone or saline by TCI (p<0.05) with eradication of NTHI within an additional seven days. The mechanism for rapid disease resolution involved efflux of activated dermal dendritic cells from the pinnae after TCI, secretion of factors chemotactic for CD4(+) T-cells, induction of polyfunctional IFNγ- and IL-17-producing CD4(+) T-cells and secretion of host defense peptide within the middle ear. These data support TCI as a therapeutic intervention against experimental NTHI-induced OM and begin to elucidate the host response to immunization by this noninvasive regimen.
Article Reference Comparison study of single and concurrent administrations of carbapenem, new quinolone, and macrolide against in vitro nontypeable Haemophilus influenzae mature biofilms.
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen and a common cause of otitis media in children, chronic bronchitis, and pneumonia in patients with chronic obstructive pulmonary disease. Many studies have reported that NTHi is capable of producing biofilms, which may be one of the important factors involved in chronic diseases and accelerating antimicrobial resistance. Unfortunately, there is still no consensus about the elimination of biofilms. In this study, concurrent administrations of levofloxacin (LVFX)-imipenem (IPM) and clarithromycin (CAM)-IPM, as well as the single administration of IPM, LVFX, and CAM, were performed to treat the mature biofilms produced by NTHi, respectively. Biofilm inhibition was quantified using microtiter biofilm assay (MBA), and relative biomass was calculated as the ratio compared to that of untreated control biofilms. The relative biomasses of biofilms treated with IPM, LVFX-IPM, and CAM-IPM against a β-lactamase-negative ampicillin-resistant strain was 1.10, 0.08, and 0.13 at 1× minimum inhibitory concentration (MIC), 0.90, 0.05, and 0.07 at 10× MIC, and 0.80, 0.06, and 0.07 at 100× MIC, respectively. Biofilms were also visually observed by scanning electron microscopy, and a focused ion-beam system showed that high concentrations of combined administration strongly inhibited the biofilms, which was consistent with the results of MBA. Our data demonstrated the antibiofilm effect of concurrent administration against mature NTHi biofilms, which indicated a rationale for the potential use of concurrent administrations in diseases involving chronic NTHi biofilms.
Article Reference Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection.
Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.
Article Reference Incorporation of phosphorylcholine into the lipooligosaccharide of nontypeable Haemophilus influenzae does not correlate with the level of biofilm formation in vitro.
Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. The objective of this report was to determine the relationship between the presence of phosphorylcholine in the lipooligosaccharide of NTHi and the level of biofilm formation. The study was performed on 111 NTHi clinical isolates collected from oropharyngeal samples of healthy children, middle ear fluid of children with otitis media, and sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. NTHi clinical isolates presented a large variation in the level of biofilm formation in a static assay and phosphorylcholine content. Isolates collected from the oropharynx and middle ear fluid of children tended to have more phosphorylcholine and made denser biofilms than isolates collected from sputum samples of patients with chronic obstructive pulmonary disease or community-acquired pneumonia. No correlation was observed between biofilm formation and the presence of phosphorylcholine in the lipooligosaccharide for either planktonic or biofilm growth. This lack of correlation was confirmed by abrogating phosphorylcholine incorporation into lipooligosaccharide through licA gene deletion, which had strain-specific effects on biofilm formation. Altogether, we present strong evidence to conclude that there is no correlation between biofilm formation in a static assay and the presence of phosphorylcholine in lipooligosaccharide in a large collection of clinical NTHi isolates collected from different groups of patients.
Article Reference Improving patient care via development of a protein-based diagnostic test for microbe-specific detection of chronic rhinosinusitis.
The hypothesis is that signature bacterial proteins can be identified in sinus secretions via high-throughput, proteomic based techniques. Nontypeable Haemophilus influenzae (NTHI) is the most common bacterial pathogen associated with sinusitis and serves as proof of principle pathogen for identifying biomarkers.
Article Reference Beta- lactam antibiotics stimulate biofilm formation in non-typeable haemophilus influenzae by up-regulating carbohydrate metabolism.
Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.
Article Reference The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae.
Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) often share a common niche within the nasopharynx, both associated with infections such as bronchitis and otitis media. This study investigated how the association between NTHi and S. pneumoniae and the host affects their propensity to form biofilms. We investigated a selection of bacterial strain and serotype combinations on biofilm formation, and the effect of contact with respiratory epithelial cells. Measurement of biofilm showed that co-infection with NTHi and S. pneumoniae increased biofilm formation following contact with epithelial cells compared to no contact demonstrating the role of epithelial cells in biofilm formation. Additionally, the influence of phosphorylcholine (ChoP) on biofilm production was investigated using the licD mutant strain of NTHi 2019 and found that ChoP had a role in mixed biofilm formation but was not the only requirement. The study highlights the complex interactions between microbes and the host epithelium during biofilm production, suggesting the importance of understanding why certain strains and serotypes differentially influence biofilm formation. A key contributor to increased biofilm formation was the upregulation of biofilm formation by epithelial cell factors.
Article Reference Ultrasound imaging and characterization of biofilms based on wavelet de-noised radiofrequency data.
The ability to non-invasively image and characterize bacterial biofilms in children during nasopharyngeal colonization with potential otopathogens and during acute otitis media would represent a significant advance. We sought to determine if quantitative high-frequency ultrasound techniques could be used to achieve that goal. Systematic time studies of bacterial biofilm formation were performed on three preparations of an isolated Haemophilus influenzae (NTHi) strain, a Streptococcus pneumoniae (Sp) strain and a combination of H. influenzae and S. pneumoniae (NTHi + Sp) in an in vitro environment. The process of characterization included conditioning of the acquired radiofrequency data obtained with a 15-MHz focused, piston transducer by using a seven-level wavelet decomposition scheme to de-noise the individual A-lines acquired. All subsequent spectral parameter estimations were done on the wavelet de-noised radiofrequency data. Various spectral parameters-peak frequency shift, bandwidth reduction and integrated backscatter coefficient-were recorded. These parameters were successfully used to map the progression of the biofilms in time and to differentiate between single- and multiple-species biofilms. Results were compared with those for confocal microscopy and theoretical evaluation of form factor. We conclude that high-frequency ultrasound may prove a useful modality to detect and characterize bacterial biofilms in humans as they form on tissues and plastic materials.
Article Reference Minimal biofilm eradication concentration of antimicrobial agents against nontypeable Haemophilus influenzae isolated from middle ear fluids of intractable acute otitis media.
Nontypeable Haemophilus influenzae (NTHi) makes the clinical course of acute otitis media (AOM) intractable by forming a biofilm that may hamper the clearance of the bacteria from middle ear cavity. In this study, we evaluated the minimum biofilm eradication concentration (MBEC) of antimicrobial agents against biofilm-forming NTHi strains. Twelve NTHi strains isolated from middle ear fluids of Japanese children with intractable AOM before antimicrobial treatment were evaluated for MBEC of fluoroquinolones in comparison with those of β-lactams and macrolides. AMPC and CDTR required much higher concentration, i.e., high MBECs, to suppress the biofilm formation of NTHi. In contrast, fluoroquinolones followed by macrolides showed lower MBECs. MBEC would be a good parameter to infer the efficacies of antimicrobials against NTHi in biofilm.
Article Reference QseC controls biofilm formation of non-typeable Haemophilus influenzae in addition to an AI-2-dependent mechanism.
Non-typeable Haemophilus influenzae (NTHi) is a common pathogen associated with diseases such as acute otitis media or exacerbations in patients with chronic obstructive pulmonary disease. The biofilm-forming capability substantially contributes to the persistence of NTHi. However, the regulation of biofilm formation is not completely understood. Quorum sensing regulated by autoinducer-2 produced by luxS is until now the only described regulatory mechanism. In this study, we show that the two-component signalling system QseB/C is involved in the biofilm formation of NTHi in vitro. An isogenic NTHi mutant of qseC (Hi3655KR2) showed a significant decrease in biofilm formation under static and semi-static conditions as assessed by crystal violet staining. In addition, under constant flow conditions, Hi3655KR2 formed less biofilm after 48 h. The biofilm defects were irrespective of autoinducer-2 levels. Hence, here we suggest for the first time a regulatory circuit in NTHi, which controls biofilm formation by mechanisms other than or in addition to luxS-dependent factors.
Article Reference Quorum signaling and sensing by nontypeable Haemophilus influenzae.
Quorum signals are diffusible factors produced by bacteria that coordinate communal responses. For nontypeable Haemophilus influenzae (NTHi), a series of recent papers indicate that production and sensing of quorum signals are determinants of biofilm formation/maturation and persistence in vivo. In this mini-review I will summarize the current knowledge about quorum signaling/sensing by this organism, and identify specific topics for additional study.
Article Reference Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections.
Like many pathogens inhabiting mucosal surfaces, nontypeable Haemophilus influenzae (NTHi) forms multicellular biofilm communities both in vitro and in various infection models. In the past 15 years much has been learned about determinants of biofilm formation by this organism and potential roles in bacterial virulence, especially in the context of chronic and recurrent infections. However, this concept has not been without some degree of controversy, and in the past some have expressed doubts about the relevance of NTHi biofilms to disease. In this review, I will summarize the present information on the composition and potential role(s) of NTHi biofilms in different clinical contexts, as well as highlight potential areas for future work.
Article Reference Dps promotes survival of nontypeable Haemophilus influenzae in biofilm communities in vitro and resistance to clearance in vivo.
Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within surface-attached biofilm communities. In this study, we tested the hypothesis that bacterial stress-responses are activated within biofilms. Transcripts for several factors associated with bacterial resistance to environmental stress were increased in biofilm cultures as compared to planktonic cultures. Among these, a homolog of the DNA-binding protein from starved cells (dps) was chosen for further study. An isogenic NTHi 86-028NP dps mutant was generated and tested for resistance to environmental stress, revealing a significant survival defects in high-iron conditions, which was mediated by oxidative stress and was restored by genetic complementation. As expected, NTHi 86-028NP dps had a general stress-response defect, exhibiting decreased resistance to many types of environmental stress. While no differences were observed in density and structure of NTHi 86-028NP and NTHi 86-028NP dps biofilms, bacterial survival was decreased in NTHi 86-028NP dps biofilms as compared to the parental strain. The role of dps persistence in vivo was tested in animal infection studies. NTHi 86-028NP dps had decreased resistance to clearance after pulmonary infection of elastase-treated mice as compared to NTHi 86-028NP, whereas minimal differences were observed in clearance from mock-treated mice. Similarly, lower numbers of NTHi 86-028NP dps were recovered from middle-ear effusions and bullar homogenates in the chinchilla model for otitis media (OM). Therefore, we conclude that Dps promotes bacterial survival within NTHi biofilm communities both in vitro and in chronic infections in vivo.
Article Reference SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus.
Non-typeable Haemophilus influenzae (NTHI) is a common commensal bacterium that resides in the human upper respiratory tract of healthy individuals. NTHI is also a known causative agent of multiple diseases including sinusitis, otitis media, as well as exacerbates disease severity of patients with cystic fibrosis and chronic obstructive pulmonary disease. We have previously shown that the Sap transporter mediates resistance to host antimicrobial peptides (AMPs) and import of the iron-containing compound heme. Here, we analyzed the contribution of the Sap structural ATPase protein, SapF, in these essential functions. In contrast to SapD, SapF was dispensable for NTHI survival when exposed to AMPs in vitro. SapF was responsible for heme utilization and recovery of depleted internal heme-iron stores. Further, a loss of SapF resulted in morphological plasticity and enhanced community development and biofilm architecture, suggesting the potential role of heme-iron availability in coordinating the complexity of NTHI biofilm architecture. SapF was required for colonization of the nasopharynx and acute infection of the middle ear, as SapF deficiency correlated with a statistically significant decrease in NTHI persistence in vivo. These data suggest that SapF is required for proper heme utilization which directly impacts NTHI survival. Thus, these studies further support a role for the Sap complex in the transport of multiple substrates and further defines substrate specificity for the two ATPase subunits. Given the multiple essential functions provided by the Sap transporter, this complex could prove to be an effective therapeutic target for the treatment of NTHI diseases.
Article Reference Respiratory syncytial virus promotes Moraxella catarrhalis-induced ascending experimental otitis media.
Otitis media (OM) is a polymicrobial disease wherein prior or concurrent infection with an upper respiratory tract virus plays an essential role, predisposing the middle ear to bacterial invasion. In episodes of acute bacterial OM, respiratory syncytial virus (RSV) is the most commonly isolated virus and thus serves as an important co-pathogen. Of the predominant bacterial agents of OM, the pathogenesis of disease due to Moraxella catarrhalis is the least well understood. Rigorous study of M. catarrhalis in the context of OM has been significantly hindered by lack of an animal model. To bridge this gap, we assessed whether co-infection of chinchillas with M. catarrhalis and RSV would facilitate ascension of M. catarrhalis from the nasopharynx into the middle ear. Chinchillas were challenged intranasally with M. catarrhalis followed 48 hours later by intranasal challenge with RSV. Within 7 days, 100% of nasopharynges were colonized with M. catarrhalis and homogenates of middle ear mucosa were also culture-positive. Moreover, within the middle ear space, the mucosa exhibited hemorrhagic foci, and a small volume of serosanguinous effusion was present in one of six ears. To improve upon this model, and based on epidemiologic data, nontypeable Haemophilus influenzae (NTHI) was included as an additional bacterial co-pathogen via intranasal administration four days before M. catarrhalis challenge. With this latter protocol, M. catarrhalis was cultured from the nasopharynx and middle ear homogenates of a maximum of 88% and 79% animals, respectively, for up to 17 days after intranasal challenge with M. catarrhalis. Additionally, hemorrhagic foci were observed in 79% of middle ears upon sacrifice. Thus, these data demonstrated that co-infection with RSV and NTHI predisposed to M. catarrhalis-induced ascending experimental OM. This model can be used both in studies of pathogenesis as well as to investigate strategies to prevent or treat OM due to M. catarrhalis.