An infant mouse model of influenza-driven nontypeable Haemophilus influenzae colonization and acute otitis media suitable for preclinical testing of novel therapies.

Nontypeable Haemophilus influenzae (NTHi) is a major otitis media (OM) pathogen, with colonization a prerequisite for disease development. Most acute OM is in children <5 years old, with recurrent and chronic OM impacting hearing and learning. Therapies to prevent NTHi colonization and/or disease are needed, especially for young children. Respiratory viruses are implicated in driving the development of bacterial OM in children. We have developed an infant mouse model of influenza-driven NTHi OM, as a preclinical tool for the evaluation of safety and efficacy of clinical therapies to prevent NTHi colonization and the development of OM. In this model, 100% of infant BALB/cARC mice were colonized with NTHi, and all developed NTHi OM. Influenza A virus (IAV) facilitated the establishment of dense (1 × 105 CFU/mL) and long-lasting (6 days) NTHi colonization. IAV was essential for the development of NTHi OM, with 100% of mice in the IAV/NTHi group developing NTHi OM compared with 8% of mice in the NTHi only group. Histological analysis and cytokine measurements revealed that the inflammation observed in the middle ear of the infant mice with OM reflected inflammation observed in children with OM. We have developed the first infant mouse model of NTHi colonization and OM. This ascension model uses influenza-driven establishment of OM and reflects the clinical pathology of bacterial OM developing after a respiratory virus infection. This model provides a valuable tool for testing therapies to prevent or treat NTHi colonization and disease in young children.

The effects of the dietary compound L-sulforaphane against respiratory pathogens.

L-sulforaphane (LSF) is an isothiocyanate derived from cruciferous vegetables that has long been known for its anticarcinogenic, antioxidant and anti-inflammatory effects. LSF also possesses antimicrobial properties, although the evidence for this is limited. Respiratory pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and respiratory syncytial virus (RSV), are leading global causes of illness and death among children aged under five years, particularly in resource-poor countries where access to vaccines are limited or, in the case of S. pyogenes and RSV, vaccines have not been licensed for use in humans. Therefore, alternative strategies to prevent and/or treat these common infectious diseases are urgently needed. This study was conducted to investigate the antimicrobial effects of LSF against common respiratory pathogens, S. pneumoniae (serotypes 1 and 6B), H. influenzae type B (HiB), non-typeable H. influenzae (NTHi), S. pyogenes and RSV in relevant human cell-based models. LSF significantly inhibited the growth of H. influenzae, but not S. pneumoniae or S. pyogenes. LSF did not improve opsonophagocytic capacity or killing by human phagocytic cell lines (HL-60s and THP-1 macrophages) for S. pneumoniae yet showed some improved killing for H. influenzae species in THP-1 macrophages. However, LSF significantly reduced RSV infection in human lung epithelial cells, associated with increased expression of cyclin D1 (CCND1) gene as well as the antioxidant genes, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX-1). Overall, LSF represents an exciting avenue for further antimicrobial research, particularly as a novel therapy against H. influenzae species and RSV.

The novel biphasic medium for transport, culture and conservation at an ambient temperature of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae.

Bacterial meningitis remains a very important disease worldwide, and the major causative pathogens were Neisseria meningitidis (N. meningitidis), Streptococcus pneumoniae (S. pneumoniae) and Haemophilus influenzae (H. influenzae). In our context, the technical difficulties encountered in the routine practice were associated with the fragility of these bacteria, the high rates of negative culture and the demanding transport conditions. That's why the need to look for a solution to its technical problems and to propose a new proper solution with the local situation. The aim of this study was to develop, perform and evaluate a novel biphasic medium used for the transport, culture and conservation at an ambient temperature of N. meningitidis, S. pneumoniae and H. influenzae. The results showed that this biphasic medium provided more, novels and easy nutriments through the addition of liquid phase and solid phase medium and it was found to be conducive to the growth and conservation of N. meningitidis, S. pneumoniae and H. influenzae at an ambient temperature of a minimum of 40 days. And the ingredients used in the medium are readily available at a low cost as well as the components prepared in large quantities, they could be stored at + 4 ± 1 °C for 2 years without significantly altering their growth and conservation supporting their potential. The survival and recovery for the fastidious bacteria on the biphasic medium and the other media used for comparison in this study were significantly different (P < 0.05). In addition, the Sensitivity, Specificity, Positive and Negative Predictive Value of biphasic medium showed highest among the three bacteria at least 40 days of storage at room temperature in this study. In conclusion, we found the biphasic medium to be low cost and suitable for previously mentioned bacteria from suspected meningitis patients, offering an optimal condition and an increase in the viability of the isolates at ambient temperature. And it was concluded that this biphasic medium could be used as a technical solution in laboratories for the management of meningitis.

Differences in Pneumococcal and Haemophilus influenzae Natural Antibody Development in Papua New Guinean Children in the First Year of Life.

Background: Development of vaccines to prevent disease and death from Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi), the main pathogens that cause otitis media, pneumonia, meningitis and sepsis, are a global priority. Children living in low and lower-middle income settings are at the highest risk of contracting and dying from these diseases. Improved vaccines with broader coverage are required. Data on the natural development of antibodies to putative vaccine antigens, especially in high-risk settings, can inform the rational selection of the best antigens for vaccine development. Methods: Serum IgG titres to four pneumococcal proteins (PspA1, PspA2, CbpA, and Ply) and five NTHi antigens (P4, P6, OMP26, rsPilA and ChimV4) were measured in sera collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age using multiplexed bead-based immunoassays. Carriage density of S. pneumoniae and H. influenzae were assessed by quantitative PCR on genomic DNA extracted from nasopharyngeal swabs using species-specific primers and probes. All data were log-transformed for analysis using Student's unpaired t-tests with geometric mean titre (GMT) or density (GMD) calculated with 95% confidence intervals (CI). Results: Serum -pneumococcal protein-specific IgG titres followed a "U" shaped pattern, with a decrease in presumably maternally-derived IgG titres between 1 and 4 months of age and returning to similar levels as those measured at 1 month of age by 24 months of age. In contrast, NTHi protein-specific IgG titres steadily increased with age. There was no correlation between antibody titres and carriage density for either pathogen. Conclusion: This longitudinal study indicates that the waning of maternally- derived antibodies that is usually observed in infants, after infants does not occur for NTHi antigens in Papua New Guinean infants. Whether NTHi antigen IgG can be transferred maternally remains to be determined. Vaccines that are designed to specifically increase the presence of protective NTHi antibodies in the first few months of life may be most effective in reducing NTHi disease. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01619462.

Effectiveness of antibacterial agents against cell-invading bacteria such as Streptococcus pyogenes and Haemophilus influenzae.

BACKGROUND: Recurrent tonsillitis is one of the most common otolaryngological disorders caused by cell-invading bacteria, such as Streptococcus pyogenes (S. pyogenes) and Haemophilus influenzae. The aim of this study was to investigate the effect of antibacterial agents against cell-invading bacteria. METHODS: The intracellular invasion of Detroit 562 cells by five strains of nontypeable Haemophilus influenzae (NTHi) and four strains of S. pyogenes was investigated. The antibacterial agents used were garenoxacin (GRNX), clarithromycin (CAM), amoxicillin (AMPC), cefditoren pivoxil (CDTR-PI), and levofloxacin (LVFX). RESULTS: Both NTHi and S. pyogenes fully invaded Detroit 562 cells in 6 h and were less sensitive to CAM. GRNX, CAM, and LVFX were effective against bacteria invading the cells, but AMPC and CDTR-PI were not effective. GRNX was the most effective. CONCLUSION: GRNX was the most effective agent against bacteria invading cells.

Non-typeable Haemophilus influenzae chronic colonization in chronic obstructive pulmonary disease (COPD).

Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes to the pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

Long term storage of fastidious bacteria (Neisseria spp. and Haemophilus spp.) with swab preservation at -80 °C.

Preservation of strains is important to further studies and characterization; we have shown a technique of long-term preservation of Neisseria spp. and Haemophilus spp. by freezing cotton tip swabs impregnated with fastidious organisms at -80 °C without the addition of cryoprotectants.

Nasal Delivery of a Commensal Pasteurellaceae Species Inhibits Nontypeable Haemophilus influenzae Colonization and Delays Onset of Otitis Media in Mice.

Nasopharyngeal colonization with nontypeable Haemophilus influenzae (NTHi) is a prerequisite for developing NTHi-associated infections, including otitis media. Therapies that block NTHi colonization may prevent disease development. We previously demonstrated that Haemophilus haemolyticus, a closely related human commensal, can inhibit NTHi colonization and infection of human respiratory epithelium in vitro We have now assessed whether Muribacter muris (a rodent commensal from the same family) can prevent NTHi colonization and disease in vivo using a murine NTHi otitis media model. Otitis media was modeled in BALB/c mice using coinfection with 1 × 104.5 PFU of influenza A virus MEM H3N2, followed by intranasal challenge with 5 × 107 CFU of NTHi R2866 Specr Mice were pretreated or not with an intranasal inoculation of 5 × 107 CFU M. muris 24 h before coinfection. NTHi and M. muris viable counts and inflammatory mediators (gamma interferon [IFN-γ], interleukin-1ß [IL-1ß], IL-6, keratinocyte chemoattractant [KC], and IL-10) were measured in nasal washes and middle ear tissue homogenate. M. muris pretreatment decreased the median colonization density of NTHi from 6 × 105 CFU/ml to 9 × 103 CFU/ml (P = 0.0004). Only 1/12 M. muris-pretreated mice developed otitis media on day 5 compared to 8/15 mice with no pretreatment (8% versus 53%, P = 0.0192). Inflammation, clinical score, and weight loss were also lower in M. muris-pretreated mice. We have demonstrated that a single dose of a closely related commensal can delay onset of NTHi otitis media in vivo Human challenge studies investigating prevention of NTHi colonization are warranted to reduce the global burden of otitis media and other NTHi diseases.

Characterization of biofilm formation and induction of apoptotic DNA fragmentation by nontypeable Haemophilus influenzae on polarized human airway epithelial cells.

Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within biofilm communities in vivo. Biofilm studies so far are mainly based on assays on plastic surfaces. The aim of this work was to investigate the capacity of clinical NTHi strains to form biofilm structures on polarized Calu-3 human airway epithelial cells and primary normal human bronchial epithelial cells and to characterize the biofilm architecture. Formation of adherent NTHi biofilms post colonization of host cells at multiple time-points was evaluated using confocal laser scanning microscopy and electron microscopy. NTHi biofilms were analyzed in terms of biofilm height and presence of extracellular matrix components, and their apoptotic effects on epithelial cells were measured by TUNEL assay. Strain Fi176 was observed to form robust biofilms on airway epithelia over time, while disrupting the integrity of Calu-3 monolayer by 72 h of co-culture. NTHi biofilms were observed to induce apoptotic DNA fragmentation in host cells at 24 h post infection. Biofilm formation on cell monolayers by Fi176ΔpilA strain was markedly reduced compared to WT strain. Biofilm inhibition and disruption assays by crystal violet staining indicated that DNA and proteins are part of NTHi biofilms in vitro. Our findings highlight critical stages of NTHi pathogenesis following host colonization and provide useful biofilm models for future antimicrobial drug discovery investigations.

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae.

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.

Inorganic particulate matter modulates non-typeable Haemophilus influenzae growth: a link between chronic bacterial infection and geogenic particles.

Australian Aboriginal populations have unacceptably high rates of bronchiectasis. This disease burden is associated with high rates of detection of pathogenic bacteria, particularly non-typeable Haemophilus influenzae (NTHi). While there is evidence to suggest that exposure to inorganic particulate matter (PM) is associated with worse respiratory infections, no studies have considered the direct effect of this PM on bacterial growth. Nine clinical isolates of pathogenic NTHi were used for this study. Isolates were exposed to two common iron oxides, haematite (Fe2O3) or magnetite (Fe3O4), or quartz (SiO2), as the main constituents of environmental inorganic PM. NTHi isolates were exposed to PM with varying levels of heme to identify whether the response to PM was altered by iron availability. The maximal rate of growth and maximum supported growth were assessed. We observed that inorganic PM was able to modify the maximal growth of selected NTHi isolates. Magnetite and quartz were able to increase maximal growth, while haematite could both increase and suppress the maximal growth. However, these effects varied depending on iron availability and on the bacterial isolate. Our data suggest that inorganic PM may directly alter the growth of pathogenic NTHi. This observation may partly explain the link between exposure to high levels of crustal PM and chronic bacterial infection in Australian Aboriginals.

In Vitro Derivation of Fluoroquinolone-Resistant Mutants from Multiple Lineages of Haemophilus influenzae and Identification of Mutations Associated with Fluoroquinolone Resistance.

Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to ß-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzae isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and ΔGlu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae In conclusion, H. influenzae clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than ß-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.

Electronic cigarette vapour increases virulence and inflammatory potential of respiratory pathogens.

INTRODUCTION: Bacteria have been extensively implicated in the development of smoking related diseases, such as COPD, by either direct infection or bacteria-mediated inflammation. In response to the health risks associated with tobacco exposure, the use of electronic cigarettes (e-cigs) has increased. This study compared the effect of e-cig vapour (ECV) and cigarette smoke (CSE) on the virulence and inflammatory potential of key lung pathogens (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa). METHODS: Biofilm formation, virulence in the Galleria mellonella infection model, antibiotic susceptibility and IL-8/TNF-α production in A549 cells, were compared between bacteria exposed to ECV, CSE and non-exposed bacteria. RESULTS: Statistically significant increases in biofilm and cytokine secretion were observed following bacterial exposure to either ECV or CSE, compared to non-exposed bacteria; the effect of exposure to ECV on bacterial phenotype and virulence was comparable, and in some cases greater, than that observed following CSE exposure. Treatment of A549 cells with cell signaling pathway inhibitors prior to infection, did not suggest that alternative signaling pathways were being activated following exposure of bacteria to either ECV or CSE. CONCLUSIONS: These findings therefore suggest that ECV and CSE can induce changes in phenotype and virulence of key lung pathogens, which may increase bacterial persistence and inflammatory potential.

Reprioritization of biofilm metabolism is associated with nutrient adaptation and long-term survival of Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) is a human-restricted pathogen with an essential requirement for heme-iron acquisition. We previously demonstrated that microevolution of NTHI promotes stationary phase survival in response to transient heme-iron restriction. In this study, we examine the metabolic contributions to biofilm formation using this evolved NTHI strain, RM33. Quantitative analyses identified 29 proteins, 55 transcripts, and 31 metabolites that significantly changed within in vitro biofilms formed by RM33. The synthesis of all enzymes within the tryptophan and glycogen pathways was significantly increased in biofilms formed by RM33 compared with the parental strain. In addition, increases were observed in metabolite transport, adhesin production, and DNA metabolism. Furthermore, we observed pyruvate as a pivotal point in the metabolic pathways associated with changes in cAMP phosphodiesterase activity during biofilm formation. Taken together, changes in central metabolism combined with increased stores of nutrients may serve to counterbalance nutrient sequestration.

Uptake of Sialic Acid by Nontypeable Haemophilus influenzae Increases Complement Resistance through Decreasing IgM-Dependent Complement Activation.

Although nontypeable Haemophilus influenzae (NTHi) is a human-specific nasopharyngeal commensal bacterium, it also causes upper respiratory tract infections in children and lower respiratory tract infections in the elderly, resulting in frequent antibiotic use. The transition from symbiotic colonizing bacterium to opportunistic pathogen is not completely understood. Incorporation of sialic acids into lipooligosaccharides is thought to play an important role in bacterial virulence. It has been known for more than 25 years that sialic acids increase resistance to complement-mediated killing; however, the mechanism of action has not been elucidated thus far. Here, we provide evidence that growth of NTHi in the presence of sialic acids Neu5Ac and Neu5Gc decreases complement-mediated killing through abrogating the classical pathway of complement activation by preventing mainly IgM antibody binding to the bacterial surface. Therefore, strategies that interfere with uptake or incorporation of sialic acids into the lipooligosaccharide, such as novel antibiotics and vaccines, might be worth exploring to prevent or treat NTHi infections.

Metagenome - Inferred bacterial replication rates in cystic fibrosis airways.

Bacterial replication rates were determined from metagenome sequencing of nasal lavage, throat swabs and induced sputa collected from healthy subjects and individuals with COPD or cystic fibrosis. More than 90% of peak-to-trough coverage ratios of major clones were above 1.4 indicating that the most abundant bacterial species in the microbial communities were replicating in the airways including common inhabitants such as Prevotella and Streptococcus species as well as the cystic fibrosis pathogens Staphylococcus aureus and Pseudomonas aeruginosa. The populations of P. aeruginosa and S. aureus were replicating their pool of chromosomes more slowly than the populations of the common inhabitants of a healthy airway microbial flora. The assessment of growth dynamics in microbial metagenomes could become a decision-making tool for the diagnosis and management of bacterial infections in cystic fibrosis.

Safety and Immunogenicity of Pneumococcal Conjugate Vaccines in a High-risk Population: A Randomized Controlled Trial of 10-Valent and 13-Valent Pneumococcal Conjugate Vaccine in Papua New Guinean Infants.

BACKGROUND: There are little data on the immunogenicity of PCV10 and PCV13 in the same high-risk population. METHODS: PCV10 and PCV13 were studied head-to-head in a randomized controlled trial in Papua New Guinea in which 262 infants received 3 doses of PCV10 or PCV13 at 1, 2, and 3 months of age. Serotype-specific immunoglobulin G (IgG) concentrations, and pneumococcal and nontypeable Haemophilus influenzae (NTHi) carriage were assessed prevaccination and at 4 and 9 months of age. Infants were followed up for safety until 9 months of age. RESULTS: One month after the third dose of PCV10 or PCV13, ˃80% of infants had IgG concentrations ≥0.35µg/mL for vaccine serotypes, and 6 months postvaccination IgG concentrations ≥0.35 µg/mL were maintained for 8/10 shared PCV serotypes in > 75% of children vaccinated with either PCV10 or PCV13. Children carried a total of 65 different pneumococcal serotypes (plus nonserotypeable). At 4 months of age, 92% (95% confidence interval [CI] 85-96) of children vaccinated with PCV10 and 81% (95% CI 72-88) vaccinated with PCV13 were pneumococcal carriers (P = .023), whereas no differences were seen at 9 months of age, or for NTHi carriage. Both vaccines were well tolerated and not associated with serious adverse events. CONCLUSIONS: Infant vaccination with 3 doses of PCV10 or PCV13 is safe and immunogenic in a highly endemic setting; however, to significantly reduce pneumococcal disease in these settings, PCVs with broader serotype coverage and potency to reduce pneumococcal carriage are needed. CLINICAL TRIALS REGISTRATION: NCT01619462.

Cellular content plays a crucial role in Non-typeable Haemophilus influenzae infection of preinflamed Junbo mouse middle ear.

Non-typeable Haemophilus influenzae (NTHi) is a major pathogen causing acute otitis media (AOM). The relationship between the cellular content of the middle ear fluid (MEF) during AOM and infection of NTHi is poorly understood. Using the Junbo mouse, a characterised NTHi infection model, we analysed the cellular content of MEF and correlated the data with NTHi titres. The MEF of the Junbo mouse was heterogeneous between ears and was graded from 1 to 5; 1 being highly serous/clear and 5 being heavily viscous/opaque. At seven-day post-intranasal inoculation, NTHi was not found in grade-1 or 2 fluids, and the proportion of MEF that supported NTHi increased with the grade. Analyses by flow cytometry indicated that the cellular content was highest in grade-4 and 5 fluids, with a greater proportion of necrotic cells and a low-live cell count. NTHi infection of the middle ear increased the cell count and led to infiltration of immune cells and changes in the cytokine and chemokine levels. Following NTHi inoculation, high-grade infected MEFs had greater neutrophil infiltration whereas monocyte infiltration was significantly higher in serous noninfected low-grade fluids. These data underline a role for immune cells, specifically monocytes and neutrophils, and cell necrosis in NTHi infection of the Junbo mouse middle ear.